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We identified 134 consecutive patients with clinically diagnosed msa whose brains were sent to the mayo clinic brain bank between 1998 and 2014 from 37 states and 1 province of canada . Brain autopsies were obtained after consent of the legal next of kin and are considered exempt from human subject research . The mayo clinic brain bank operates under protocols approved by the mayo clinic institutional review board . Most patients were white; 7 were asian, 1 was pacific islander, and 1 was african american . Dlb = dementia with lewy bodies; msa = multiple system atrophy; pd = parkinson disease; psp = progressive supranuclear palsy; opca = predominantly olivopontocerebellar involvement type of multiple system atrophy; snd = predominantly striatonigral involvement type of multiple system atrophy; snd / opca = equally severe striatonigral and olivopontocerebellar involvement type of multiple system atrophy . All cases underwent a standardized neuropathologic assessment for alzheimer - type and lewy - related pathologies as previously reported . Braak neurofibrillary tangle (nft) stage and thal amyloid phase were assigned to each case based upon thioflavin s fluorescent microscopy . Immunohistochemistry for -synuclein (nacp; 1:3,000) was used to establish neuropathologic diagnosis of msa . Msa was subclassified as msa with predominantly striatonigral involvement (msa - snd), msa with predominantly olivopontocerebellar involvement (msa - opca), and msa with equally severe involvement of striatonigral and olivopontocerebellar systems (msa - snd / opca). Lewy - related pathology was assessed in cortex, amygdala, basal forebrain, and brainstem, and classified as brainstem, transitional, or diffuse lewy body disease . Lewy body subtype and degree of alzheimer - type pathology were used to classify cases as low, intermediate, or high likelihood of dementia with lewy bodies (dlb) according to the third consortium on dementia with lewy bodies (cdlb) recommendations; a pathologic diagnosis of dlb was assigned to cases with intermediate or high likelihood of cdlb . A pathologic diagnosis of parkinson disease (pd) required moderate to severe neuronal loss in the substantia nigra and cdlb scores of low likelihood . A neurologist (s.k .) Abstracted the following information from medical records collected throughout the course of disease and entered it into a database: sex, age at symptomatic onset, age at death, family history of neurologic disease, initial and final clinical diagnoses, signs and symptoms during the disease course and their timing, and neurologic findings as documented by a neurologist or movement disorder specialist . For each patient, a particular clinical symptom or sign if clinical symptoms or signs were not described, then for the purpose of analysis, they were considered to be absent, except in the case of levodopa responsiveness . The following symptoms and neurologic signs were abstracted from medical records: orthostatic hypotension, syncope, dizziness, urinary incontinence, constipation, erectile dysfunction, asymmetry of parkinsonism, resting tremor, bradykinesia, axial / limb rigidity, falls, early falls (defined as occurring within 1 year of symptomatic onset), gait ataxia, limb ataxia, nystagmus, vertical gaze palsy, pyramidal signs (spasticity, hyperreflexia, and babinski sign), cognitive impairment, visual hallucinations, and rem sleep behavior disorder (rbd). Orthostatic hypotension was considered to be positive if there was documented blood pressure drop of at least 30/15 mm hg (according to msa criteria) or patients were medicated for orthostatic hypotension (e.g., fludrocortisone, midodrine). Patients were considered to have cognitive impairment if at least short - term memory loss, disorientation, or executive dysfunction were diagnosed by a physician, or there were recorded complaints of these symptoms by the patient or their family members . Rbd was positive if found on polysomnography or if it was clinically suspected based upon behavioral descriptions of the bed partner and noted by a physician . The degree of levodopa responsiveness was recorded as no response, partial response, or good response . The information on symptoms was gathered from a combination of medical records, pathology records summarizing clinical history, or a brain bank questionnaire filled out by a close family member . The questionnaire included the clinical diagnosis, age at onset of symptoms, family history, initial symptoms, clinical symptoms (disorientation, agitation, hallucinations, tremors, stiffness, difficulty walking, fluctuating course, violent outbursts, eating disorder, wandering, weight loss, sleep disorder, visual problems, delusions, falls, personality changes, and other noteworthy symptoms), hand dominance, specialty of the physician (neurology, psychology, or psychiatry), and medications . All patients were retrospectively assigned a diagnosis of probable or possible msa from available clinical information according to the second consensus criteria of msa . Given the retrospective nature of the study, the quality of available medical records was variable, and a score was devised to provide a means to assess possible bias that might be related to differential completeness of clinical information with respect to pathologic diagnostic groups: 0, inadequate clinical records; 1, only the brain bank questionnaire; 2, clinical records from general practitioners; 3, clinical records from neurologists; 4, clinical records from movement disorder specialists . To assess mri findings, the following features were abstracted from both radiology reports and interpretations of the physician of record: atrophy of cerebral cortex, cerebellum, brainstem, and putamen, abnormal signal intensity in putamen, and specific description suggesting a certain diagnosis such as a hot cross bun sign and a hummingbird sign . For the subset of patients evaluated at mayo clinic, digitized scans were reviewed . All statistical analyses were performed in sigmaplot 11.0 (systat software, san jose, ca). Analysis of variance (anova) on ranks, followed by dunn post hoc test, or one - way anova, followed by post hoc holm - sidak test, were used for analyses of continuous variables as appropriate . P values <0.05 were considered statistically significant . To adjust for age at death, multivariable logistic regression models were built for each combination of the pathologic groups using the significant pathologic variables from univariate analyses . We identified 134 consecutive patients with clinically diagnosed msa whose brains were sent to the mayo clinic brain bank between 1998 and 2014 from 37 states and 1 province of canada . Brain autopsies were obtained after consent of the legal next of kin and are considered exempt from human subject research . The mayo clinic brain bank operates under protocols approved by the mayo clinic institutional review board . Most patients were white; 7 were asian, 1 was pacific islander, and 1 was african american . Dlb = dementia with lewy bodies; msa = multiple system atrophy; pd = parkinson disease; psp = progressive supranuclear palsy; opca = predominantly olivopontocerebellar involvement type of multiple system atrophy; snd = predominantly striatonigral involvement type of multiple system atrophy; snd / opca = equally severe striatonigral and olivopontocerebellar involvement type of multiple system atrophy . All cases underwent a standardized neuropathologic assessment for alzheimer - type and lewy - related pathologies as previously reported . Braak neurofibrillary tangle (nft) stage and thal amyloid phase were assigned to each case based upon thioflavin s fluorescent microscopy . Immunohistochemistry for -synuclein (nacp; 1:3,000) was used to establish neuropathologic diagnosis of msa . Msa was subclassified as msa with predominantly striatonigral involvement (msa - snd), msa with predominantly olivopontocerebellar involvement (msa - opca), and msa with equally severe involvement of striatonigral and olivopontocerebellar systems (msa - snd / opca). Lewy - related pathology was assessed in cortex, amygdala, basal forebrain, and brainstem, and classified as brainstem, transitional, or diffuse lewy body disease . Lewy body subtype and degree of alzheimer - type pathology were used to classify cases as low, intermediate, or high likelihood of dementia with lewy bodies (dlb) according to the third consortium on dementia with lewy bodies (cdlb) recommendations; a pathologic diagnosis of dlb was assigned to cases with intermediate or high likelihood of cdlb . A pathologic diagnosis of parkinson disease (pd) required moderate to severe neuronal loss in the substantia nigra and cdlb scores of low likelihood . A neurologist (s.k .) Abstracted the following information from medical records collected throughout the course of disease and entered it into a database: sex, age at symptomatic onset, age at death, family history of neurologic disease, initial and final clinical diagnoses, signs and symptoms during the disease course and their timing, and neurologic findings as documented by a neurologist or movement disorder specialist . For each patient, a particular clinical symptom or sign if clinical symptoms or signs were not described, then for the purpose of analysis, they were considered to be absent, except in the case of levodopa responsiveness . The following symptoms and neurologic signs were abstracted from medical records: orthostatic hypotension, syncope, dizziness, urinary incontinence, constipation, erectile dysfunction, asymmetry of parkinsonism, resting tremor, bradykinesia, axial / limb rigidity, falls, early falls (defined as occurring within 1 year of symptomatic onset), gait ataxia, limb ataxia, nystagmus, vertical gaze palsy, pyramidal signs (spasticity, hyperreflexia, and babinski sign), cognitive impairment, visual hallucinations, and rem sleep behavior disorder (rbd). Orthostatic hypotension was considered to be positive if there was documented blood pressure drop of at least 30/15 mm hg (according to msa criteria) or patients were medicated for orthostatic hypotension (e.g., fludrocortisone, midodrine). Patients were considered to have cognitive impairment if at least short - term memory loss, disorientation, or executive dysfunction were diagnosed by a physician, or there were recorded complaints of these symptoms by the patient or their family members . Rbd was positive if found on polysomnography or if it was clinically suspected based upon behavioral descriptions of the bed partner and noted by a physician . The degree of levodopa responsiveness was recorded as no response, partial response, or good response . The information on symptoms was gathered from a combination of medical records, pathology records summarizing clinical history, or a brain bank questionnaire filled out by a close family member . The questionnaire included the clinical diagnosis, age at onset of symptoms, family history, initial symptoms, clinical symptoms (disorientation, agitation, hallucinations, tremors, stiffness, difficulty walking, fluctuating course, violent outbursts, eating disorder, wandering, weight loss, sleep disorder, visual problems, delusions, falls, personality changes, and other noteworthy symptoms), hand dominance, specialty of the physician (neurology, psychology, or psychiatry), and medications . All patients were retrospectively assigned a diagnosis of probable or possible msa from available clinical information according to the second consensus criteria of msa . Given the retrospective nature of the study, the quality of available medical records was variable, and a score was devised to provide a means to assess possible bias that might be related to differential completeness of clinical information with respect to pathologic diagnostic groups: 0, inadequate clinical records; 1, only the brain bank questionnaire; 2, clinical records from general practitioners; 3, clinical records from neurologists; 4, clinical records from movement disorder specialists . To assess mri findings, the following features were abstracted from both radiology reports and interpretations of the physician of record: atrophy of cerebral cortex, cerebellum, brainstem, and putamen, abnormal signal intensity in putamen, and specific description suggesting a certain diagnosis such as a hot cross bun sign and a hummingbird sign . For the subset of patients evaluated at mayo clinic all statistical analyses were performed in sigmaplot 11.0 (systat software, san jose, ca). Analysis of variance (anova) on ranks, followed by dunn post hoc test, or one - way anova, followed by post hoc holm - sidak test, were used for analyses of continuous variables as appropriate . P values <0.05 were considered statistically significant . To adjust for age at death, multivariable logistic regression models were built for each combination of the pathologic groups using the significant pathologic variables from univariate analyses . Brains of 134 patients with a clinical diagnosis of msa were received by the brain bank in the time frame of the study, and 83 (62%) met pathologic criteria for msa (figure). The breakdown of the 51 misdiagnosed patients by pathologic diagnosis is as follows: dlb in 19 (37%), progressive supranuclear palsy (psp) in 15 (29%), pd in 8 (15%), and other disorders in 9 (18%) (including 2 corticobasal degeneration and 2 vascular parkinsonism, as well as 5 miscellaneous disorders). The proportion of patients included in final clinicopathologic analyses after exclusion of those with inadequate medical records was similar for the 4 major pathologic groups (i.e., msa, dlb, pd, and psp). The diagnostic accuracy was not different between general neurologists (33/53, 62%) and movement disorder specialists (35/56, 63%). After retrospective assessment of clinical features, 49 patients were judged to fulfill the criteria for probable msa, 35 for possible msa, and the remaining 41 were not assigned to levels of diagnostic certainty due to lack of adequate clinical information (e.g., levodopa responsiveness). Correctly diagnosed patients with msa had a younger age at onset and age at death than patients with pd or psp, but duration of symptoms did not differ . Demographic and pathologic features of pathologically diagnosed msa compared with non - msa although the brain weights did not differ among the 4 groups, braak nft stage in both dlb and pd and thal amyloid phase in dlb were higher than in msa (table 1). In a multiple logistic regression analysis adjusting for age at death, the difference for thal amyloid phase was higher in dlb than in msa (odds ratio 1.5, 95% confidence interval 1.052.26, p = 0.028), but differences in braak nft stage in dlb and pd were not significant . The breakdown of lewy - related pathology and pathologic variants of msa is summarized in table 1 . Table 2 lists the frequency of clinical features in autopsy - confirmed msa, dlb, pd, and psp . Comparing msa and dlb, urinary incontinence, limb ataxia, nystagmus, and pyramidal signs were more frequent in msa . Cognitive impairment and visual hallucinations were more frequent in dlb . Comparing msa and pd, urinary incontinence was less frequent and visual hallucinations were more frequent in pd . Comparing msa and psp, urinary incontinence, constipation, orthostatic hypotension, and rbd were more frequent in msa . Frequency of levodopa responsiveness and average mini - mental state examination score were not different among the groups . Clinical features of pathologically diagnosed msa compared with non - msa to clarify the factors that led to misdiagnosis, we summarized initial diagnosis, final diagnosis, reasons for diagnosing msa, and pathologic features in 34 pathologically confirmed patients (18 dlb, 6 pd, and 10 psp) with the best medical documentation (i.e., scores 34), since records with quality scores of 2 or less usually did not describe the rationalization for diagnosing msa (table 3). The most frequent reason for misdiagnosing dlb as msa was autonomic failure . Seventeen of 18 patients with dlb presented with autonomic failure, which was specifically mentioned as the reason for reaching a clinical diagnosis of msa in 14 patients . Seven patients were given a diagnosis of msa as an initial diagnosis because of autonomic failure . Similar to dlb, autonomic failure was the most frequent reason for misdiagnosing pd as msa . It is worth noting that 3 patients with pd with severe autonomic failure early in the disease course were diagnosed with msa . In contrast to dlb and pd, the most frequent reason for misdiagnosing psp as msa was the presence of cerebellar ataxia . Three patients with psp presented with cerebellar ataxia as the initial clinical feature, and 4 other patients developed ataxia (limb ataxia in 6, gait ataxia in 6, and ataxic speech in 2) during the course of the disease . Eight patients with psp also had signs or symptoms of autonomic failure, and 7 patients had vertical gaze palsy . Clinical and pathologic features of 34 patients masquerading as msa we chose patients with at least moderate quality medical records (i.e., scores 24) and compared mri findings in msa, dlb, pd, and psp (table 4). Although the frequency of cerebellar atrophy was lower in dlb than in msa, the frequency of brainstem atrophy, cerebral atrophy, and abnormalities in the putamen (e.g., hyperintensity or hypointensity in lateral putamen on t2-weighted images) were not different among the 4 groups . The duration between performance of mri and death was shorter in dlb and pd than in msa (1.9 vs 3.8 years). Hot cross bun sign was noted in 1 patient with msa, and a hummingbird sign was noted in 1 patient with psp . In this unselected referral autopsy series of patients with antemortem diagnoses of msa, the diagnostic accuracy was about 62%, which is within the range of other autopsy series . This study confirms that msa can be difficult to differentiate from dlb, pd, and psp not only in early stages, but also at late stages of the disease process . One of the most intriguing results from the present study is that patients with atypical presentations (e.g., ataxia in psp) or uncommon clinical features (e.g., dysautonomia in dlb and pd) of dlb, pd, and psp can be misdiagnosed as msa . Other studies of clinical and autopsy studies have demonstrated that autonomic failure can be a feature of dlb, but this fact does not seem to be widely appreciated in clinical practice . Indeed, 6 patients with dlb were initially diagnosed with pd, but the diagnoses were changed to msa because of developing autonomic failure . Furthermore, 4 patients initially presenting with autonomic failure (orthostatic hypotension in 3 patients) and later developing parkinsonism were diagnosed as msa . Similar to dlb, 5 patients with pd were misdiagnosed with msa because of autonomic failure . Three of them had autonomic failure as an initial symptom, adding further evidence that dysautonomia can present another premotor feature of pd . Until now, severe dysautonomia in early stages of pd has been considered an exclusion criterion for pd . In addition to the autonomic failure, some atypical features in pd (e.g., short duration of symptoms and levodopa unresponsiveness) may also contribute to misdiagnosis as msa . One patient with dlb and 2 patients with pd developed limb ataxia, usually slight dysmetria on finger - to - nose testing and not severe ataxia seen in msa . Two patients with ataxia had sensory neuropathy, and sensory ataxia may be the etiology . In this unselected autopsy series of patients with clinically diagnosed msa, absent or mild cognitive impairment limited correct diagnosis of dlb defined as intermediate or high likelihood cdlb . This contrasts with findings in prospectively studied cohorts recruited from memory disorder clinics where the cdlb neuropathologic criteria are highly correlated with the dlb clinical syndrome . The results of this study suggest that a subset of patients in a nonspecialty setting with intermediate to high likelihood of dlb pathology (i.e., limbic or diffuse cortical lewy bodies and minimal alzheimer - type pathology) may have an atypical parkinsonian syndrome with minimal cognitive impairment that can be misdiagnosed as msa . In this autopsy series, only 4/18 patients with dlb underwent formal neuropsychological evaluations, and cognitive impairment might have been overlooked . While cognitive impairment was more frequent in dlb than in msa, the degree of cognitive impairment in patients with dlb thought to have msa was not sufficient to diagnose dementia and pathologic analyses showed minimal alzheimer - type pathology (median braak nft stage iii and thal amyloid phase 3). These results suggest that pathologically pure dlb can masquerade as msa because of absent or mild cognitive impairment in combination with features of autonomic failure or limited response to levodopa . Although the presence of prominent, early cerebellar symptoms is an exclusion criterion for clinical diagnosis of psp, 7 patients with psp in our series had cerebellar ataxia . These patients may fit with an atypical form of psp with cerebellar ataxia (psp - c). Our findings suggest that when cerebellar ataxia is present in a patient with features of an atypical parkinsonian disorder, physicians should consider psp in addition to msa . A recent study has shown that older onset, early falls, and vertical gaze palsy without dysautonomia may differentiate psp - c from msa - c . Although patients with psp in our cohort had frequent autonomic failure, older age at onset and the combination of vertical gaze palsy and early falls might be useful in the differential diagnosis of psp and msa (33% in psp vs 4% in msa). Even with mri studies, clinical diagnosis of msa is challenging . In this retrospective series, 38% of patients with msa in which imaging results were available had no abnormal mri findings, and only one had a typical hot cross bun sign . The reason for the low frequency of abnormal findings may be explained by the timing of the mri . In most cases it was performed relatively early in the disease course, with no clinical indication to repeat scans as the disease progressed . While longitudinal mri is frequent in movement disorder research clinics, such is not the case in routine clinical care . In addition, some abnormal findings might have been overlooked because patients were evaluated by general radiologists, whose focus is often on cerebrovascular, traumatic, neoplastic, or other acute processes . Patients with psp had abnormal findings on mri at a similar frequency as patients with msa . Even when a characteristic finding, such as the hummingbird sign, was noted on antemortem mri, patients with psp were still misdiagnosed with msa . Only a few patients with pathologically confirmed dlb had cerebellar atrophy, brainstem atrophy, or abnormality in the putamen, suggesting that mri may be helpful in differentiating dlb from msa . Taken together, the results suggest that mri is helpful in some patients, but is not reliable for diagnosis of msa if performed too early in the disease course and not repeated later as the disease progresses . First, it is a retrospective analysis and is not based on standardized prospective clinical evaluations . Hyposmia is an important preclinical sign of pd, but it was not assessed in our study because it was not described in most patients . Although other modalities such as [i]-mibg myocardial scintigraphy are useful for differentiating msa from other parkinsonian disorders, it is not widely available in clinical practice . Third, the timing of the clinical examinations and autopsy varied among patients . Patients have different clinical features early compared to late in the disease course, and depending upon the records available, some features at either end of the clinical spectrum may have been missed . Although our scoring system of the quality of the medical records does not reflect these issues, records scored in the 3 or 4 range tended to be written later in the disease course . An inherent limitation of any study using autopsy samples is selection bias, with atypical patients being more likely to come to autopsy than typical patients, as shown for parkinsonian syndromes . A notable strength of our study is that many of the patients were derived from the community setting rather than specialty clinics, and therefore, our findings may better represent the state of diagnostic accuracy of msa in general clinical practice . Another strength is that pathologic diagnostic evaluation used the latest methods for detecting -synuclein and tau pathologies, and the most current pathologic classification systems for msa, dlb, pd, and psp . The results serve as a powerful reminder that the misdiagnosis rate can be high in msa, and that dlb can be a key culprit in causing this confusion, along with psp and pd . This has implications not only for patient care, but also for research studies that do not have pathologic confirmation . Shunsuke koga: conceptualization, execution of the statistical analysis, execution of the project, writing of the first draft . Dennis w. dickson: conceptualization, organization of the research project, review and critique of the manuscript . R. uitti receives research support from the nih / national institute of neurological disorders and stroke (p50-ns072187) and nih / national institute of neurological disorders and stroke (r01-ns057567), research funding from advanced neuromodulation systems, inc./st . Jude medical, and a gift from carl edward bolch jr, and susan bass bolch . Uitti is an editorial board member of neurology. J. van gerpen receives research funds from the mayo clinic cr program and nih / national institute of neurological disorders and stroke (p50-ns072187). This research received no specific grant from any funding agency in the public, commercial, or not - for - profit sectors . W. cheshire is consultant for american academy of neurology, neuro sae examination writer, 2013; and receives support from nih, autonomic rare diseases clinical research consortium, 6102, principal investigator, 20112013; mayo clinic program in professionalism & ethics, ppe-5 grant, principal investigator, 20132014; and is an editorial board member of autonomic neuroscience . K. josephs receives research support from the nih (r01-dc010367, r01-dc012519, r01-ag037491) and the alzheimer's association . Wszolek serves as co - editor - in - chief of parkinsonism and related disorders, associate editor of the european journal of neurology, and on the editorial boards of neurologia i neurochirurgia polska, the medical journal of the rzeszow university, and clinical and experimental medical letters; holds and has contractual rights for receipt of future royalty payments from patents re: a novel polynucleotide involved in heritable parkinson's disease; and receives royalties from publishing parkinsonism and related disorders (elsevier, 2013, 2014) and the european journal of neurology (wiley - blackwell, 2013, 2014). J. langston reports receiving consulting and lecture fees from teva and consulting fees from merck serono, lundbeck, and iperion, as well as grant support from the michael j. fox foundation, department of defense, the california institute for regenerative medicine (cirm tri-01246 and ttii-019665), and nih (u54-es012077). Langston serves as co - editor - in - chief of journal of parkinson's disease . D. dickson receives research support from the nih (p50-ag016574, p50-ns072187, p01-ag003949) and curepsp: foundation for psp cbd and related disorders . Dickson is an editorial board member of acta neuropathologica, annals of neurology, brain, brain pathology, and neuropathology, and he is editor - in - chief of american journal of neurodegenerative disease and international journal of clinical and experimental pathology.
The medullar layer is responsible for catecholamine production, and the cortical layer produces mineralocorticoids, glucocorticoids and sex hormones, respectively for each zone . Adrenal diseases can be accompanied by medullar hyperfunction (as in pheochromocytoma) or cortical hyperfunction (as in primary hyperaldosteronism, cushing's syndrome and congenital adrenal hyperplasia). Insufficiency of the adrenal cortex, or addison's disease, is a separate entity . In developed countries, it can also be caused by tuberculosis, metastases to both adrenal glands and destruction by a neoplastic process (lymphoma). This group of pathologies should include the most common incidentally detected adrenal tumors, called incidentalomas . They include a range of histopathological entities but cortical adenomas without hormonal hyperfunction are the most common . The most frequent sources of metastases to the adrenal glands (via vascular spread) are lung and kidney cancers . The diagnostic triad in adrenal diseases includes: interview and physical examination, biochemical tests and imaging . Imaging techniques used in the diagnosis of adrenal pathologies are presented in tab . 1 . Contemporary imaging modalities applied to the diagnostics of adrenal diseases magnetic resonance imaging positron emission tomography catheterization of the adrenal veins biopsy guided by imaging each abdominal ultrasound examination of a child or adult should include the assessment of the suprarenal areas . Ultrasonography is a method of choice in the assessment of the adrenal glands in neonates and young children . Owing to the availability, cost and non - invasiveness, it is often used for the assessment of the suprarenal areas in adolescents and adults with extra - adrenal tumors . Moreover, it is recommended in patients with arterial hypertension or hyperadrenalism or adrenal insufficiency, and in the monitoring of adrenal tumors diagnosed in computed tomography or magnetic resonance imaging as benign adenoma - like neoplasms . This examination enables the differentiation between a solid tumor and adrenal cysts, which are rarely encountered . Although literature reports claim that normal adrenal glands can be visualized in most examinations in adults (80%), it is not that easy in practice . This is because of the fact that the echogenicity of the adrenal glands is similar to that of the retroperitoneal fat . Moreover, a number of other factors have to be taken into account, such as the acoustic window, quality of equipment and examiner's experience . Because of these limitations and difficulties in visualizing certain slight diffuse or focal adrenal lesions, and due to the impossibility to differentiate between adrenal tumors (with few exceptions), the methods of choice in the assessment of adrenal pathologies are computed tomography and magnetic resonance imaging (with appropriate indications, including the detection of a solid mass in the adrenal area on an ultrasound examination). Examinations of adrenal glands in neonates and young children are conducted with the use of broadband transducers of high frequency (up to 10 mhz) and a scanner equipped with the sensitive color or power doppler option (diagnosis of neuroblastoma tumors). In adolescents and adults, examinations of the adrenal glands are conducted with the use of convex, broadband transducers with the frequency of 25 mhz, which are used in abdominal scans . In patients of slight constitution the preparation for an examination is the same as for an abdominal scan . Of new ultrasound techniques to assess the suprarenal areas, harmonic imaging or spatial compound imaging are deemed useful and recommended . In numerous cases, these techniques improve the contrast between an adrenal tumor and adjacent tissues, and enable better visualization of tumor margins (fig . 1 and 2). Harmonic imaging in particular enables better visualization (better contrast) of even normal adrenal glands, which can allow their measurements to be taken . Three - dimensional scans enable more thorough assessment of tumor sizes and better correlation with computed tomography (fig . 3d us enables easier and more careful interpretation of complex anatomic relationships . A three - dimensional examination can be particularly useful for the visualization of tumors in the left adrenal gland since, because of the anatomic conditions, they are more difficult to visualize than tumors of the right adrenal gland (fig . 3). In some cases, however, even the usage of the aforementioned new ultrasound techniques does not allow small adrenal tumors to be detected, particularly in the left gland . A. conventional ultrasound in the b - mode b. b - mode with harmonic imaging . Compared with image a, fewer artefacts clearer tumor margins and clearer contrast with surrounding structures pheochromocytoma of the right adrenal gland (arrow). A. conventional ultrasound in the b - mode b. b - mode with spatial compound imaging . Compared with image a, fewer artefacts, smoother image (lower image speckles) clearer tumor margins and clearer contrast with surrounding structures slight adenoma of the left adrenal gland three - dimensional ultrasound axial image (a). This slight tumor of the left adrenal gland was not detected in a real - time 2d examination . 3d ultrasound enables the lesion to be visualized and its size to be compared with ct (b) b - mode ultrasound imaging is useful in differentiating between rare cysts and solid tumors of the adrenal glands . This information is particularly significant if the diagnosis of adrenal adenoma in computed tomography has been established only on the basis of its low density (approximately 0 hounsfield units) without the administration of iodinated contrast agent . Tomography enables the differentiation between these lesions based on the observation of contrast enhancement . As for ultrasound imaging, if a hypoechoic lesion does not present all features of a simple cyst in a b - mode examination, a new dynamic elastography technique, called shear wave elastography, can be used . This technique allows convex probes for abdominal scans to be used by holding them motionlessly (by contrast with static elastography, no compression is applied). The analysis of this phenomenon is possible by using the method of rapid ultrasound image generation, with the velocity of approximately 20,000 images per second . Tissue elasticity is presented in real - time as a color map placed on a b - mode image . By contrast with solid tumors, cysts do not present a colored elastographic signal since shear waves do not propagate through fluids (fig . 4). Recurring lymphatic cyst of the right adrenal gland of the maximum size of 10 cm: shear - wave elastography (swe) upper image, b - mode lower image . In the upper swe image, there is no colored signal since shear waves do not propagate through fluids . Inside the lesion at its periphery, an artefact - like colored signal can be seen, however indicating low stiffness values (approximately 4 kpa) contrast - enhanced ultrasound examinations have not demonstrated any features that would enable certain differentiation between benign and malignant adrenal tumors . The assessment of the vascularization pattern in the group of benign adrenal lesions has demonstrated that there are differences between nodular hyperplasia and adenomas . In nodular hyperplasia, the vascularization begins at the periphery of a lesion, which can be observed in parametric images of the contrast agent inflow (arrival) time . Adenomas, however, usually present mixed or central vascularization pattern (fig . 5 and 6). Nodular hyperplasia of the adrenal cortex with two tumors masses of the right adrenal gland (arrows) with the mean diameter of 23 mm and 10 mm . The parametric image of the time of contrast agent (sonovue) inflow (arrival) (on the left) shows vascularization of both tumors, starting from the periphery adenoma of the adrenal cortex (arrows) with the mean diameter of 16 mm . The parametric image of the time of contrast agent (sonovue) inflow (arrival) (on the left) shows vascularization of the tumor, starting from the center and periphery at the same time the adrenal gland is a paired organ located cephaloanterio- medially from the upper renal poles . The right suprarenal area borders the inferior vena cava anteriorly; the upper and lateral border is the liver; the diaphragm is located medially and the kidney is positioned below it . The left suprarenal area borders the pancreatic tail, stomach and intestine anteriorly; the upper and lateral border is the spleen; the diaphragm and aorta are located medially and the kidney is positioned below it . Both adrenal glands are located in the perirenal adipose tissue surrounded by the gerota's fascia . Adrenal scans in neonates are usually conducted with a probe applied laterally in coronal, axial and intermediate planes . This is associated with large sizes of adrenal glands compared with kidneys, low amount of the retroperitoneal fat tissue and short distance from the probe, which enables the usage of high - frequency linear transducers with higher resolution . The right adrenal gland can be visualized in 97% of neonates, and the left one in 83% . The cephalocaudal length of the adrenal gland in this group of patients ranges from 936 mm, mean 15 mm . The thickness of the adrenal limb ranges from 25 mm . In a neonate, there can be a hyperechoic center corresponding to the medullar layer and a hypoechoic edge that corresponds to the cortical layer . An examination of adrenal glands is usually conducted in a supine position by applying the transducer laterally in the coronal sections along the long renal axis and in axial sections with the evaluation of the area located anteriorly, medialy and above the kidney . When assessing the left suprarenal area, the right lateral position of the patient can cause the displacement of the stomach and intestine, thereby improving the its visualization . In this position, the probe can be applied more posteriorly so that intestinal gas does not conceal the suprarenal area . Depending on the conditions, sometimes, this position enables one to observe focal lesions in the adrenal glands that cannot be detected from the lateral approach . The assessment of the suprarenal areas as well as the visualization of the adrenal glands and focal lesions can be difficult or impossible in the cases of considerable fatty liver (normal liver is a good acoustic window in the assessment of the right suprarenal area), large amount of gas in the intestine and stomach (left suprarenal area) and obesity (worse translucency for ultrasound wave). In adults, normal adrenal glands are visualized as longitudinal organs that consist of the body and limbs and assume the shape of the letter v, y or, a comma or triangle . By contrast with schematic figures presented in various anatomical atlases, the adrenal gland is a narrow organ with the limb diameter of 49 mm (5 1 mm) and the body diameter of up to 10 mm . Its length reaches up to 50 mm . In an ultrasound examination, this organ is usually hypoechoic or its echogenicity resembles that of the perirenal fat tissue, which might make its visualization more difficult (fig . A focal lesion in the suprarenal area should be visualized in two perpendicular planes; three perpendicular maximum dimensions should be marked (adrenal tumors can grow irregularly in three directions). Sometimes, ultrasonic artefacts within the adipose capsule of the kidney can mimic focal lesions but their analysis in two perpendicular planes should enable the proper classification of the image . Ultrasound b - mode image of the normal right adrenal gland with marked measurements of the thickness of the limbs and body (calipers). The echogenicity of the adrenal limb ends is similar to the echogenicity of the retroperitoneal fat tissue . B. longitudinal section through the adrenal gland . In this view, the adrenal gland is of shape . It is only slightly hypoechoic in relation to the retroperitoneal fat tissue . C. longitudinal section with harmonic imaging: increased contrast between the hypoechoic adrenal gland and the retroperitoneal fat tissue (compared with fig . Harmonic imaging is recommended in the visualization of normal adrenal glands and their focal lesions in the monitoring of adrenal masses, it is necessary to correlate findings with maximum dimensions found in computed tomography or magnetic resonance imaging (axial sections and height). An ultrasonologist should be familiar with previous ultrasound, computed tomography or magnetic resonance images of evaluated adrenal lesions . This should help to visualize them in ultrasound examination and enable better assessment of their growth dynamics (fig . When examining adults using a convex probe with the frequency of 25 mhz, the mean diameter of adrenal lesions that can be visualized exceeds 10 mm . However, the literature mentions that focal lesions with the diameter of 5 mm can be visualized via the abdominal access and those with 23 mm in diameter can be detected in endoscopic ultrasound . Nevertheless, some tumors with their maximum diameter below 20 mm in the left adrenal gland can remain invisible for transabdominal ultrasonography . This depends on their localization in the gland as well as on anatomic and physiological conditions . The suprarenal areas can also include objects that are normal structures (splenic vessels, lobulated spleen, accessory spleen), tumors or cysts of surrounding organs (kidneys, liver, pancreas), called adrenal pseudotumors . If a focal lesion is detected in the suprarenal area, the remaining abdominal organs must also be assessed on ultrasound examination . It must be emphasized that some forms of adrenal diseases, such as certain forms of adrenal hyperplasia, may remain invisible in sonography or even with modalities of choice in adrenal imaging, such as computed tomography or magnetic resonance imaging . In neonates, the description of the examination should include the size of the gland and the presence of possible focal lesions . When describing focal lesions of the adrenal glands, the following should be taken into account: dimensions (two perpendicular maximum dimensions in the axial section and the third dimension height), echogenicity, homogeneity and vascularization in color and power doppler examinations in order to differentiate between adrenal hematoma and neuroblastoma tumor . The description of examinations conducted in adolescents and adults should also include possible enlargement of the adrenals and presence of focal lesions . When describing focal lesions of the adrenal glands, the following should be taken into account: dimensions (two perpendicular maximum dimensions in the axial section and the third dimension height), shape (regular, irregular), margins (well - circumscribed, uneven) echogenicity, homogeneity and nature (solid or cystic). The differential diagnosis should include the organ from which a lesion visible in the suprarenal area may arise . If focal lesions in adrenal glands are visualized, other possible lesions in other abdominal organs should be described as well . If a previously non - reported, incidental solid focal lesion exceeding 1 cm (incidentaloma) is detected in the suprarenal area, computed tomography or magnetic resonance imaging should be conducted to confirm its presence and for differentiation and the tumor functional status should be determined . If the tumor does not grow, the next follow - up scans should be conducted every 6 months for 2 years, and subsequently once a year . The authors do not report any financial or personal connections with other persons or organizations, which might negatively affect the contents of this publication and/or claim authorship rights to this publication . The publication has been created thanks to the grant of the polish ministry of science and higher education no n n402481239, carried out within 20102014.
Diabetic ketoacidosis (dka) is considered to be a common presentation of type 1 diabetes mellitus (t1 dm) and occasionally, type 2 diabetes mellitus (t2 dm) in children and adolescents . Insulin stops the use of fat as an energy source by inhibiting the peptide hormone glucagon . Without insulin, glucagon levels rise resulting in the release of free fatty acids from adipose tissue, as well as amino acids from muscle cells . The biochemical criteria for dka diagnosis include hyperglycemia (blood glucose [bg] higher than 11 mmol / l or 200 mg / dl) with a venous ph of <7.3 and/or a bicarbonate (hco3) level of <15 mmol / l; ketonemia and ketonuria . Although not universally available, blood -hydroxybutyrate concentration should be measured whenever possible, and a level of 3 urine ketones, of moderate or large size (typically 2 +), are also indicative of dka . The clinical signs of dka include dehydration (may be difficult to detect), tachycardia, tachypnoea (may be mistaken for pneumonia or asthma), deep sighing (kussmaul) respiration with a typical smell of ketones in the breath (variously described as the odor of nail polish remover or rotten fruit), nausea, vomiting (may be mistaken for gastroenteritis), abdominal pain (may mimic an acute abdominal condition), confusion, drowsiness, progressive reduction in level of consciousness, and eventually loss of consciousness . Risk factors for dka in newly diagnosed cases include younger age (<2 years), delayed diagnosis, and lower socioeconomic status with limited access to medical services . Risk factors for dka in patients with known diabetes include insulin omission, poor metabolic control, previous episodes of dka, acute gastroenteritis with persistent vomiting and inability to maintain hydration, psychiatric (including eating) disorders, challenging social and family circumstances, peripubertal and adolescent girls, those with limited access to medical services and insulin pump therapy failure . The severity of dka is categorized by the degree of acidosis: mild dka is characterized by a venous ph of <7.3 and/or a hco3 level of <15 mmol / l, moderate dka is characterized by a venous ph of <7.2 and/or a hco3 level of <10 mmol / l and in severe dka, venous ph is <7.1 with or without a hco3 level <5 mmol / l . Diabetic ketoacidosis results from a deficiency of circulating insulin and increased levels of the counter regulatory hormones: glucagon, catecholamines, cortisol and growth hormone . Relative insulin deficiency occurs when the concentrations of counter - regulatory hormones markedly increase in response to stress, infection or insufficient insulin . The combination of absolute or relative insulin deficiency and high counter - regulatory hormone concentrations results in an accelerated catabolic state with increased glucose production by the liver and kidney (via glycogenolysis and gluconeogenesis), and simultaneously impaired peripheral glucose utilization, which combine to result in hyperglycemia and hyperosmolarity; increased lipolysis and ketogenesis leading to ketonemia and metabolic acidosis . Hyperglycemia together with hyperketonemia cause osmotic diuresis, dehydration, and obligatory loss of electrolytes . Thus dka leads to a vicious life - threatening cycle of events ranging from hyperglycemia, hyperketonemia, osmotic diuresis, severe vomiting, dehydration, and subsequently obligatory loss of electrolytes, greater stress hormone production, and thus more severe insulin resistance . If not interrupted by exogenous insulin, fluid and electrolyte therapy, it would lead to fatal dehydration, hypoperfusion, and ultimately metabolic acidosis . Ideally, dka can be managed in any hospital / private unit or in a pediatric inpatient ward in case of children, with adequately trained nursing and medical personnel where lab services are available 24 h throughout the week . However, an intensive care unit (icu)/pediatric icu is required for children <2 years of age and in case of severe dka characterized by compromised circulation, coma, and an increased risk of cerebral edema . Diagnosis of dka should be done accurately due to a possibility of a confusing clinical picture such as dehydration, meningitis, acute abdomen, pneumonia, etc . Emergency assessment can be done by following the general guidelines of pediatric advanced life support . Initial immediate assessment or investigation includes evaluation of the severity of dehydration, level of consciousness through glasgow coma scale, body weight and height if the person is mobile . Baseline investigations involve the measurement of bg levels, beta - hydroxybutyric acid, serum electrolytes and renal functions . During physical examination, physician may look for signs of dehydration, acidosis, and electrolyte imbalance, including shock, hypotension, acidotic breathing, central nervous system (cns) status, etc . The essential principles of dka treatment include careful replacement of fluid deficits, correction of dehydration, correction of acidosis and hyperglycemia via insulin administration, maintenance of glucose levels at a normal range, correction of electrolyte imbalance and treatment of any precipitating cause . Successful management of dka requires constant clinical and biochemical monitoring and timely adjustment of insulin dose, fluid and electrolyte status . However, hco3 administration is shown to have no clinical benefit and may cause paradoxical cns acidosis . Fluid therapy is initially used for the treatment of dka, followed by insulin therapy if required . The main objectives of fluid therapy are: restoration of circulating volume, replacement of electrolytes, improvement of glomerular filtration and clearance of glucose and ketones from the blood . Before starting fluid therapy, the physician should check if the child was treated earlier before the current admission . During fluid therapy, water and salt deficit are replaced using 0.9% normal saline and a 10 - 20 ml / kg normal bolus may also be used for approximately 1 - 2 h. if the patient is in shock, several boluses may be given . Normal saline or ringer lactate is used over a period of 4 - 6 h. consequently, maintenance fluids are used . Usually, half normal saline (0.45%) with potassium chloride is given depending on the state of hydration and electrolyte levels . Fluid therapy is usually planned for a period of 48 h. however, a child may improve earlier than 48 h. normal circulation is often achieved in 12 6 h. when the child becomes stable, fluids can be given orally, and subsequently insulin can be given subcutaneously . In cases of mild dka, no bolus is needed . The main principle of fluid therapy is to never infuse fluids more than 1.5 - 2 times the normal daily requirement . Potassium replacement therapy is used when the total body potassium deficit is nearly ~3 - 6 mmol / kg . If the patient is hypokalemic, start potassium replacement at the time of initial volume expansion and before starting insulin therapy . Otherwise, start replacing potassium after initial volume expansion and concurrent with starting insulin therapy . If the patient is hyperkalemic, defer potassium replacement therapy until urine output is documented . In dka, rehydration alone reduces bg . Insulin therapy is used to restore normal metabolism, to suppress lipolysis, ketogenesis and normalize bg . A low dose of intravenous (iv), insulin infusion is considered to be safe and effective . Insulin infusion should be initiated 1 - 2 h after starting fluid replacement therapy; that is after the patient has received initial volume expansion . The dose of insulin should usually remain at 0.05 - 0.1 unit / kg / h, at least until resolution of dka viz . Ph <7.30, serum hco3 levels <15 mmol / l and beta - hydroxybutyrate levels <1 mmol / l . No iv bolus is required to be given because it may worsen hypokalemia or precipitate cerebral edema . As long as possible, the physician should minimize the time on iv insulin infusion, and optimal doses of insulin should be used to avoid severe hypokalemia . Bg should be gradually lowered at a rate of 50 - 100 mg / dl . When bg level falls to 250 mg / dl, 5% glucose is added to iv fluid . Furthermore, 10% or 12.5% glucose may be needed while continuing insulin infusion to correct metabolic acidosis . Furthermore, 2 hourly subcutaneous or intramuscular short - acting insulin may also be used if facilities for iv infusion are not available . However, for successful dka management, meticulous monitoring of the patient's clinical and biochemical response using a flow chart is essential . Neurological observations, for warning signs and symptoms of cerebral edema, and capillary bg concentration should be measured on an hourly basis . Every 2 - 4 h serum electrolytes, blood gases, and beta - hydroxybutyrate should be measured . Restlessness, irritability, increased drowsiness, cranial nerve palsies, abnormal pupillary responses, headache, slow heart rate (hr), rising blood pressure (bp) or recurrence of vomiting . Risk factors include initial ph of <7.1, abnormal baseline mental status, newly diagnosed patients who are <5 years old, patients suffering from dehydration and severe acidosis with lower partial pressure of carbon - dioxide, rapid rehydration (> 50 cc / kg in the first 4 h), insulin given before or within 1 h of fluid initiation, persistent hypernatremia and high blood urea at presentation . Diagnostic criteria of cerebral edema include abnormal motor or verbal response to pain, decorticate or decerebrate posture, cranial nerve palsy, and abnormal respiratory patterns (grunting, tachypnoea, apnoea, cheyne - stokes respirations). Major criteria include altered sensorium, sustained deceleration of hr (decrease by> 20 beats / min) and age - inappropriate incontinence . Minor criteria include vomiting, lethargy, diastolic bp <90 mm hg, headache, and age <5 years . G / kg of mannitol should be administered intravenously over 10 - 15 min, and should be repeated if there is no initial response in 30 min to 2 h. hypertonic saline (3%) with suggested dose of 2.5 - 5 ml / kg, administered over 10 - 15 min, may be used as an alternative to mannitol, especially if there is no initial response to mannitol . Diabetic ketoacidosis is a common presentation of both t1 dm and t2 dm in children and adolescents . Dka arises due to lack of adequate insulin in the body . For successful dka management, meticulous monitoring of the patient's clinical and biochemical response using neurological observations, for warning signs and symptoms of cerebral edema, and capillary bg concentration should be measured on an hourly basis . Every 2 - 4 h electrolytes, blood gases, and beta - hydroxybutyrate should be measured.
The effects of exogenous administration of neurotrophic molecules on neurorestoration of lesioned central nervous system have been largely evaluated . Pigment epithelium derived factor (pedf) has emerged as a potential candidate to promote behavioral gain and neurorestorative events in the spinal cord injury because pedf receptor is concentrated in ventral motor region of the spinal cord and the molecule is highly expressed and it is able to trigger trophic actions to motor neurons [1, 2]. The potential capacity of pedf to promote motor restoration may involve its ability to modulate neurotrophic / neuroplasticity events and its capacity to interfere with endothelial and glial reactions to injury, cells that are also important actors in the scenario of neurorestoration [25]. Despite the great effort to understand the inhibitory nature of neuronal environment to regenerating fibers, recent results have pointed out neuronal plasticity in the lesioned spinal cord as the major event leading functional recovery in that pathological condition [610]. In fact, studies have identified and characterized molecular signals related to axonal growth and functional target innervation in the lesioned spinal cord . Much of the functional restoration observed in the experimental models of spinal cord lesion has been triggered however by the neuroplasticity - related molecules that are expressed by rescued neurons nearby injury, especially in the regions of the central pattern generators (cpg) [1114]. Furthermore, the apoptotic and neurotrophic responses in neurons that are not enrolled by primary lesion as well as the paracrine glial mechanisms may modify the functional outcome [1517]. Those events occurred in several rostral / caudal spinal levels in the subsequent periods after lesion [18, 19]. Molecular signaling plays a central role in the cell communication - mediated neuroplasticity in the lesioned nervous system, especially those that are involved in neuroprotection, neovascularization, glial activation, and extracellular matrix regulating neuronal fiber growth . In that context, neurotrophin 3 (nt-3), glial derived neurotrophic factor (gdnf), brain derived neurotrophic factor (bdnf), fibroblast growth factor 2 (fgf-2), and pedf have been described as proteins that are able to exert autocrine / paracrine trophic actions to spinal cord neurons and also able to modulate wound repair events that are triggered by nonneuronal cells [5, 16, 20]. Furthermore, the ephrin system may also play important action on neurorestoration, remarkably in the lesioned spinal cord, due to its actions on multiple cellular regenerative events . For instance, ephrins could modulate axonal guidance [2123], target reinnervation, synaptic plasticity [2426], neurotrophic factor signaling - induced neuroprotection / neuroplasticity, and endothelial / glial reaction - induced wound repair [2729]. The large range of actions of ephrin system is favored by the spread distribution of the receptor tyrosine kinase eph and its membrane - bound ligand ephrin in astrocytes, endothelial cells as well as in presynaptic and postsynaptic neurons [3032]. Remarkably, the above mentioned intercellular signaling events that mediate the outcome of a spinal cord injury are modulated by molecules of the extracellular matrix specially the members of the chondroitin sulfate proteoglycan (cspg) family . Two molecules, which were revealed by immunoblot analysis after chondroitinase treatment, are in the context of neuroregeneration . The 70 kda chondroitin sulfate / dermatan sulfate hybrid chain and also the 150 kda neurocan - derived c - terminal product, a proteolytic and biological activity fraction of the full - length neurocan [34, 35], seem to play a role in neurotrophic factor binding and neuronal fiber growth . Among the vast, complex, and imbricate cellular and molecular events driving neurorestorative functional outcome, the local ischemia is a pathological condition that appears not only as a consequence of lesion reaction to a spinal cord injury but also as a disorder that is able to interfere with local wound repair and neurotrophic / neuroplasticity mechanisms [36, 37]. The effects of a pedf injection in the epicenter of an ischemic lesion applied in a low thoracic level of the rat spinal cord were investigated behaviorally and also at cellular and molecular levels . Regulations of molecules related to neuroplasticity, neurotrophism, and neovascularization and also those of the growth cone inhibitor chondroitin sulfate proteoglycans (cspg) family and ephrin system were analyzed in the lumbar spinal cord cpg . Specific pathogen - free adult male wistar rats from the university of so paulo school of medicine (n = 42, weighting 350400 g) were used in the present study . Rats were kept under controlled temperature and humidity conditions with a standardized light / dark cycle (light on at 7: 00 a.m. and off at 7: 00 p.m.) with free access to food pellet and tap water . The study was conducted according to protocols approved by the animal care and use ethic committee at the university of so paulo and in accordance with the guide for the care and use of laboratory animals adopted by the national institutes of health . Rats were anesthetized intraperitoneally with a mixture of ketamine chlorhydrate (62.5 mg / kg) and xylazine chlorhydrate (10 mg / kg). . Rose bengal dye (aldrich chemical, 40 mg / kg, diluted in 0.9% nacl in a final concentration of 20 mg / ml) was administered slowly in the rat dorsal vein (n = 30). The rat back was shaved and disinfected with an iodopovidone solution . Immediately after dye injection, a midline longitudinal incision was made over the thoracic 8- to the lumbar l1 (t8-l1) vertebrae; the skin was retracted and paravertebral muscles were dissected . The spinal process and the vertebral lamina of thoracic 12 (t12) spinal cord level were removed with a complete exhibition of circular regional dura mater (laminectomy). A 5 mm diameter - fiber - optic linked to a xenon lamp device (schott kl 1500, mainz, germany), which produces a 560 nm wavelength irradiation, was placed on the surface of the dura - exposed spinal cord (thoracic level) illuminating it for 20 minutes . The fiber - optic was attached to a stereotaxic device (kopf, usa), attempting to maintain a 2 mm distance between the fiber - optic and the dorsal spinal cord to be lesioned . The procedure induced an excitation of the systemically injected dye, triggering a local thrombosis and ischemia [38, 39]. Sham operated rats (n = 12) were submitted to microsurgical procedures and light exposure, without receiving the dye intravenous injection . Immediately after spinal cord ischemia or sham surgery, the animals received a stereotaxical injection of the neurotrophic factor pedf (10 l, 100 ng / ml; hs300210, kindly supplied by dr . Joyce tombran - tink, university of missouri - kansas city) or solvent (10 l, phosphate buffered saline, ph 7.4) at the epicenter of the lesion . The spinal injections were made with 100 m outer diameter glass needles, which were obtained by means of a pipette puller (kopf). The needle was connected to a hamilton syringe (50 l) and to a stereotaxical apparatus adapted with a spinal cord unit (kopf) in order to promote the mechanical injection, which lasted 35 minutes . Were submitted to a sham surgery and a local injection of solvent (n = 12). In the saline group rats received an ischemic lesion and a local injection of solvent (n = 14). In the pedf group rats received an ischemic lesion and a local injection of pedf (n = 16). At the end of the procedure the animals received preventive antibiotic therapy (ceftriaxone, 40 mg / kg, i m . Daily) for 15 days and also accompanied for bladder evacuation using the crede method until bladder functional recovery . Animals submitted to photothrombotic spinal cord injury or sham operation were evaluated behaviorally using the inclined plane test 24, 48, and 72 hours after surgery and then weekly until the end of week 6 . Animals were placed head down on an adjustable inclined plane covered by a rubber mat . The angle of the plane was increased from 0 to the point where the rat could not maintain its position for 5 seconds . Normal uninjured rats remained on the plane to an angle of 50 to 60 inclination . The inclined plane test was chosen because the performance on the inclined plane correlates with the integrity of the rubrospinal tract and other nonpyramidal pathways that could be compromised at week 6 after ischemic injury . The test can be also used as an index of animal strength and it has been shown to be a sensitive and reliable test for clip compression injury . Half of the animals in each group (n = 68) were processed for immunohistochemistry six weeks after spinal cord injury . The rats were anesthetized with sodium pentobarbital and euthanized by a transcardiac perfusion with 100 ml isotonic saline at room temperature, followed by 500 ml of fixation fluid (4c) over a period of 6 minutes [20, 43, 44]. The fixative consisted of 4% paraformaldehyde (w / v, merck, darmstadt, germany) in 0.1 m phosphate buffer, ph 6.9 . The spinal cords were removed, kept in the fixative solution at 4c for 90 minutes, and then rinsed in 10% sucrose (merck) dissolved in 0.1 m phosphate - buffered saline (pbs), ph 7.4, for 48 hours . The spinal cords were cut into pieces of 0.8 cm long at the lumbar intumescence level . The segments were then frozen in dry ice - cooled (40c) isopentane (sigma, milwaukee, wi) and stored at a 70c freezer until use . Adjacent serial 14 m thick frozen sections were obtained with a cryostat (leica, cm3000, germany) from the spinal cord lumbar segment (l2l5), containing the cpg region . Series in a cranial - caudal order including every 100th sections were used for tissue labeling . Thus, three sampling sections / animal were submitted to each immunolabeling procedure (see below). Immunoreactivity was detected by the avidin - biotin peroxidase technique [46, 47]. Sections were washed for 2 10 minutes in pbs and incubated with 5% normal goat serum for 30 minutes at room temperature . The series of sections were then incubated for 48 hours at 4c with the mouse monoclonal microtubule associated protein-2 antibody (map-2, diluted 1: 2,500, sigma). The antibody was diluted in pbs containing 0.5% triton x-100 (sigma) and 1% bovine serum albumin (sigma). The sections were washed again in pbs (2 10 minutes) and incubated with biotinylated horse anti - mouse immunoglobulins (vector, burlingame, ca, usa; diluted 1: 250) for 2 hours . After rinsing in pbs, sections were incubated with an avidin - biotin peroxidase complex (both diluted 1: 125, vectastain, vector, usa) for 45 minutes . Immunoreactivity was visualized using 3 - 3-diaminobenzidine tetrahydrochloride (sigma) as a chromogen and h2o2 (0.05%, v / v, sigma) for 8 minutes . To standardize the immunohistochemical procedure, we used a dilution of the primary antibody, a dab concentration, and an incubation time, all adjusted so that the darkest elements in the spinal cord sections were below saturation . To further analyze the specificity of the immunostainings, sections were incubated with the solvent of the primary and secondary antibodies or with the solvent of the avidin - biotin solution and processed at the same time with the experimental sections . The sections submitted for measurements were selected from those three of sampling regime described above that represented better the cranial (l2-l3) and caudal (l4-l5) regions of the lumbar rat spinal cord . The means of the data of the two regions were presented because there is no description of functional differences between two regions of the cpg . The map-2 immunoreactivity was measured in two spinal cord sections per rat by means of semiquantitative morphometric image analysis . The measurements were done in the ventral horn of lumbar intumescence (caudal to the injury site), where the lumbar cpg is located and the spinal cord showed a preserved morphology . The image analysis procedures, implemented on a kontron - zeiss ks400 image analyzer (germany), have been described previously [19, 43, 46, 4850]. Briefly, a television camera from the microscope (40 objective) acquired the image . The mean gray value (mgv) and s.e.m . Of gray matter in areas of the spinal cord devoid of specific labeling (background, bg) were measured . Gray values darker than bgmgv, 3 s.e.m . Were considered as belonging to specific labeling and thus discriminated . The specific (sp) mgv was subsequently defined as the difference between the bgmgv value and the mgv of discriminated profiles . The procedure was repeated for each section to correct every measurement of specific labeling for its background value . The morphometric and microdensitometric (spmgv) measurements indicate the amount of immunoreactive cell profiles and the intensity of immunolabeling in the sampled fields, respectively . The map-2 immunoreactive dendritic and cell body profiles were quantified individually by means of a stereological method . The point intercepts stereological tool was employed to obtain the areal fraction in the sampled region of the ventral horn of the spinal cord rats, bilaterally, as described elsewhere [20, 43, 44]. A point - grid with an area per point of 400 m was used to estimate the area of the counted profiles . The aa was calculated [20, 43, 44] (aa = pstructure/psection). A 40x oil - immersion objective was used to acquire the images that were used to quantify the profiles in the gray matter sampled fields . All map-2 immunoreactive processes that emerged from the cell perikarya (first level processes) were not considered in the discrimination procedures so that axons and axon hillock were not included in the measurements . The sections were initially washed for 3 10 minutes in pbs and then were incubated for 48 hours at 4c with a mixture of one of the antibodies to receptor eph or ephrin subtypes (the descriptions of the antibodies and the employed dilutions are shown in table 1) and one of the following antibodies to neuronal and astroglial markers: mouse monoclonal neurofilament-200 antibody (nf-200, diluted 1: 200, sigma), rabbit polyclonal glial fibrillary acidic protein antibody (gfap, diluted 1: 200, sigma), or goat polyclonal gfap antibody (diluted 1: 50, dako). The antibodies were diluted in pbs containing 0.5% triton x-100 (sigma) and 1% bovine serum albumin (sigma). After the incubation of the primary antibodies, sections received two washes of 10 minutes in pbs and were incubated for 2 hours in the dark at 37c with a mixture of fluorescein isothiocyanate (fitc) and texas red (both diluted 1: 40, jackson, west grove, usa). The sections were rinsed in pbs and were examined in an ax70 olympus epifluorescence photomicroscope . The other half of the animals (n = 68) were euthanatized by decapitation and processed to western blot and real - time polymerase chain reaction (rt - pcr) techniques . The spinal cords were rapidly removed and a 0.8 cm fragment was cut from the lumbar intumescence as described previously . The ventral part (motor area) was carefully separated from the dorsal part (sensory area) in each fragment . The caudal portion was then submitted to western blot technique and the cranial one to the rt - pcr procedures . The material of rt - pcr was frozen in dry ice and stored at 70c freezer until processing . The samples were homogenized in lyses buffer containing 1% np40 (sigma), 0.5% sodium deoxycholate (sigma), 1% sodium dodecyl sulfate (biorad), 1 mm ethylenediaminetetraacetic acid (sigma), 1 mm ethylene glycol tetraacetic acid (sigma), and 1% protease inhibitor cocktail (sigma), diluted in phosphate buffered (ph 7.4). After centrifugation (14,000 rpm) for 20 min at 4c, as described previously, the supernatants were transferred into new tubes and stored at 70c until use . The samples (60 g of protein / lane) were separated on a 12% sodium dodecyl sulfate (sds) polyacrylamide (biorad) gel electrophoresis at 100 v for 1 h. proteins (120 g) were transferred to polyvinylidenefluoride (pvdf) membrane at 100 v during one hour . Membranes were then blocked with 10% milk diluted in tbs - t (mixture of tris - buffered saline and 0.05% tween 20) for 30 minutes under slight agitation at room temperature . Then, all membranes were incubated overnight at 4c with the respective antibodies: a mouse monoclonal antibody to map-2 (1: 1,000; novus biological), a mouse monoclonal antibody to brain core protein of cspg (1: 500, millipore), a rabbit polyclonal antibody to laminin (1: 1,000; sigma), and a rabbit polyclonal antibody to b - cell lymphoma protein-2 (bcl-2, diluted 1: 1,000; santa cruz). Membranes were also submitted to specific labeling of subtypes a and b of eph receptors and ephrins . The concentration used and the descriptions of the antibodies are shown in table 1 . Membranes were washed 2 times for 10 minutes in tbs - t and incubated at room temperature for 1 hour with anti - mouse (1: 6,000; ge), anti - rabbit (1: 10,000; ge), or anti - goat (1: 2,000; ge) igg ecl conjugated secondary antibodies . In the sequence, after final washes, the membranes were incubated with western lightning chemiluminescence reagent plus (perkinelmer life science, usa) for 1 minute . The membranes were exposed to an x - ray film for imaging (hyperfilmtm ecl, ge healthcare, usa) to visualize protein bands . The membranes of every blot were then washed and incubated individually with a mouse monoclonal antibody to alpha - tubulin (1: 30,000; sigma) diluted in tbs - t containing 1% bsa for 1 hour at room temperature and developed as described previously . The densitometry of the bands was quantified by means of a computer assisted image analyzer and a software developed by imaging research (brock university, canada) as described elsewhere . Dividing the density of the protein signal by the correspondent alpha - tubulin signal value performed data normalization . Total rna from spinal cord fragments was extracted with rnaspin mini kit (ge healthcare, uk) according to the manufacturer's instructions . Rna quantity and integrity were assessed by spectrophotometry (nanodrop) and microfluidics - based electrophoresis (bioanalyzer, agilent 2100), respectively . Only rna samples with od 260/280 1.8 and rin (rna integrity number)> 7 were used for quantitative rt - pcr . For rt - pcr reactions, total rna was converted into cdna in the presence of reverse transcriptase (multiscribe, applied biosystems) and random hexamer primers (applied biosystems), according to manufacturers' instructions . Quantitative pcrs reactions were performed on a step one plus sequence detection system (applied biosystems) using taqman universal pcr master mix (applied biosystems) in a total volume of 20 l . The gene expression of the following molecules was analyzed: the neurotrophic factors nt-3 (rn00579280_m1), gdnf (rn00569510_m1), bdnf (customized, forward 5tggttatttcatacttcgg ttgcatga3, reverse 5tgtccgtggacgtttgctt3, and probe 5ctgcgcccatgaaag3), and fgf-2 (rn00570809_m1), the eph receptors epha6 (rn01474859_m1) and ephb2 (rn01181017_m1), and the small gtpase rhoa (rn04219610_g1). The amplification protocol included 3 minutes at 95c, followed by 45 cycles of 10 seconds at 95c for denaturation and 45 seconds at 60c for annealing and extension . Statistical analysis for the behavioral data consisted of a two - way analysis of variance and followed the bonferroni posttest . A second analysis was performed including only the saline and pedf groups, in order to obtain data related to motor recovery of the injured animals . In the western blot, immunohistochemistry and rt - pcr analyses, the one - way anova was applied with tukey's multiple comparisons posttest to identify statistical significances between groups . Data were presented as means s.e.m . And significance level was set at p <0.05 . The motor evaluation of animals submitted to ischemic spinal cord injury was performed by the inclined plane test . The statistical analysis by the two - way anova showed effects of time (p <0.0001; f = 101.4), treatment (p <0.0001; f = 32.99), and interaction time / treatment (p <0.0001; f = 7.516) when the three groups were analyzed together, as well as when only the saline and pedf groups were included in the evaluation (effects of time p <0.0001, f = 99.14; of treatment p = 0.002, f = 9.543; and of interaction time / treatment p = 0.004, f = 5.80). Therefore, differences between the saline and pedf groups (p <0.001) were seen at week 4 to week 6 of assessment, according to the bonferroni posttest (figure 1). Furthermore, although sham and saline groups differed at all evaluated periods, sham and pedf groups were different only at 13 days time - interval (figure 1). The effects of photothrombotic ischemic injury and treatment with pedf on the neuroplasticity in the ventral horn of the lumbar spinal cord were analyzed by means of map-2 detection . Western blot revealed an increase (87.78%) of map-2 protein level in the ventral region of lumbar spinal cord of pedf treated group compared to sham (p <0.05, figure 2(a), also illustrated in figure 2(c)). Moreover, map-2 immunoreactivity was elevated (p <0.001) in the ventral horn of lumbar spinal cord in rats of pedf group compared with the sham (35.99%) and saline (33.81%) groups (figure 2(a)). The qualitative analysis showed an increased map-2 immunoreactivity in the neuronal cell bodies and fibers of the spinal cord ventral horn of pedf rats, compared to saline rats (figure 2(b)). Furthermore, the stereological tool point intercepts revealed that pedf injection elevated (11.71%, values expressed as areal fraction) the amount of map-2 immunoreactive dendritic processes in the ventral horn of the lumbar spinal cord in relation of saline injected rats (figure 3). The stereological method revealed no alterations in the areal fraction of the map-2 immunoreactive perikarya among experimental groups . The values of areal fraction of map-2 immunoreactive perikarya were 31.69 1.61, 34.79 2.17, and 38.94 4.39 of sham, saline, and pedf groups, respectively (mean s.e.m ., p> 0.05). The increased map-2 immunoreactive dendritic profiles in the ventral horn of pedf treated rats are illustrated in higher magnification microphotographs of representative rats of the three studied groups (figure 4). The stereological tool, named point intercepts (red frame), that was projected on the digital image of map-2 immunoreactive profiles for quantification of neuronal dendrites and cell bodies of ventral horn were illustrated in figure 4 . It should be mentioned that the sections incubated with the solvent of the primary or secondary antisera as well as with the solvent of the avidin - biotin solution showed no reaction (data not shown). Rt - pcr for relative gene expression of neurotrophic factors in the ventral region of lumbar spinal cord revealed a decrease of nt-3 in the pedf group compared to sham (38.52%, p <0.05, table 2). Moreover, gdnf gene expression was found to be elevated in the pedf group compared to sham (38.21%, p <0.05) and also to saline (94.0%, p <0.01) groups (table 2). No differences were seen in the analyses of bdnf and fgf-2 gene expression (table 2). No differences were observed among the experimental groups at the postoperative periods performed in this study (data not shown), thus representing no autocrine regulation of that neurotrophic factor in the present experimental conditions . Western blot of the growth cone inhibitor cspg levels in the ventral region of lumbar spinal cord showed two bands of molecular weights 70 and 150 kda (figures 5(a) and 5(b)). The chondroitinase treatment allowed the identification of a 70 kda chondroitin sulfate / dermatan sulfate hybrid chain and also a 150 kda neurocan - derived c - terminal product [34, 35]. Decreases in the cspg level were found in the 70 kda band (p <0.05) of the pedf (12.92%) and also saline (18.33%) groups compared to sham rats (figure 5(a), also illustrated in figure 5(b)). Laminin and bcl-2 proteins were used as markers for angiogenesis and antiapoptosis regulator, respectively, and were quantified by means of western blot in the ventral region of lumbar spinal cord of the rats (figures 6(a) and 6(b)). Laminin level was elevated (66.23%) in the saline group compared to sham (p <0.01) and a decrease (24.97%) in the level of laminin was found in the pedf group compared to saline (p <0.5). Regarding the bcl-2 analysis, no changes were demonstrated among groups at the postoperative studied periods (figure 6). The protein levels of subtypes a and b of the eph receptors and ephrins were quantified by western blot in the ventral region of lumbar spinal cord of the rats (figure 7). The epha4 level increased in the saline (122.16%) and pedf (127.93%) groups compared to sham (p <0.01), and no changes were observed in protein levels of the receptors epha2, a3, a5, and a7 (figure 7(a)). Regarding the levels of type - a ephrins, ephrin a2 increased (64.28%) in the pedf group compared to sham (p <0.05), and no changes were seen in the ephrins a1, a3, a4, and a5 (figure 7(b)). The representative bands of ephs a and ephrins a illustrated the effect described above (figures 7(c) and 7(d)). Furthermore, regarding the ephs b1, b4, and b6 (figure 7(e)), no differences were found among the experimental groups . Moreover, increases of ephrin b1 levels were observed in the saline (71.78%) and pedf (53.51%) groups compared to sham (p <0.05, figure 7(f)). Ephrin b2 level was elevated in the pedf group compared to sham (49.67%) and saline (43.54%) groups (p <0.05), and the ephrin b3 level was increased in the pedf group (87.55%) compared to sham (p <0.05), (figure 7(f)). The representative bands of ephs b and ephrins b illustrated the effects described above (figures 7(g) and 7(h)). Some members of studied eph receptors, particularly the epha1, a6, a8, a10, b2, and b3, showed no specific signal bands at the western blot assay . Finally, the relative gene expression analyses of epha6 and ephb2 receptors as well as the small gtpase rhoa, performed by rt - pcr in an adjacent region, showed no differences among the experimental groups (table 2). The cellular analysis of the two - color immunofluorescence in ventral horn of the rat spinal cord showed a colocalization of the epha1, epha2, epha7, epha8, ephb1, ephb4, and ephb6 receptors, as well as the ephrin a5 with the nf-200 positive motor neurons of this region . The type b ephrins (b1, b2, and b3) are colocalized with astrocytes of that region (data not shown). The presence of ephrin b2 in astrocytes of ventral horn of pedf treated rat is illustrated in figure 8 . The qualitative analysis of immunofluorescence allowed the recognition of cellular component of the labeling signals but did not lead the identification of differences among groups . Spinal cord ischemia is considered the most relevant event present in a series of pathological situations, including trauma, tumor, infection, and circulatory disorders, contributing to the deleterious secondary mechanisms that amplify initial injury [5255]. Ischemia might also influence injury outcome and neurorestorative events due to its ability to modify neurotrophic and neuroplasticity responses of nearby lesion [56, 57]. Experimental models of spinal cord trauma are largely employed [5862]; however, analyses have failed to attempt the circumstances of related ischemia . This work performed a spinal cord injury photothrombotic ischemia using the rose bengal method, as first described by watson et al . Reproducible lesion with regard to its size, localization, and behavioral response is achieved because the method allows a precise control of dye concentration and irradiation settings thus leading, in rats, to a nonrecovery of blood flow, an absence of short term behavioral recovery, and a 3-month - old circumscribed lesion in the dorsal half of the cord as described previously [6668]. Importantly, the photothrombotic injury applied at the low thoracic levels of rat spinal cord of the present work preserved the lumbar segments caudally where rat lumbar cpg is located . Neuroplasticity in the lumbar cpg has been addressed as the major source of motor recovery, spontaneously or after treatments, following spinal cord lesion [7072]. The understanding of the neuroplasticity potential of the lesioned spinal cord would favor the translation of spinal cord regeneration to clinical practice (http://www.clinicaltrial.gov, nct00406016) [73, 74]. Neurotrophic factors have been tested on spinal cord injury experimentally, especially those with actions on several aspects of neurorestoration, for instance, wound repair, neuronal trophism, and neuroplasticity [7577]. Pedf has been pointed out as a potential candidate mainly in terms of motor recovery because the molecule is highly expressed and triggered trophic actions to motor neurons and because its receptor is concentrated in ventral motor region of the spinal cord [1, 2]. In fact, we described motor behavior improvements and neuroplasticity responses in the low thoracic lesioned rats after a pedf injection in the epicenter of the injury . The rats that were submitted to the experimental procedures were accompanied behaviorally by means of the inclined plane test, which evaluates muscle strength and endurance parameters required to keep the animal in a static position when its plane is submitted to a progressive increase of the inclination angle [78, 79]. The possibility of pedf injection in the epicenter of a spinal cord photothrombotic injury to trigger neuroplasticity events in the lumbar regions of the organ was evaluated by means of the responses of structural protein map-2 . The regulation of that neuronal microtubule protein in the lesioned nervous system has been associated with dendritic branching and synaptic plasticity . Western blot analysis revealed increases of map-2 levels in ventral regions of the lumbar spinal cord after the ischemic lesion applied cranially, remarkably in the rats treated with pedf, indicating that the neurotrophic factor is able to stimulate neuronal plasticity in the regions of the spinal cord distant to the wound . Quantitative microdensitometric image analysis of map-2 immunoreactivity revealed, at cellular level, increases of map-2 immunoreactivity in the perikarya and neuropil of large motoneurons in the ventral horn of the lumbar region, caudal to ischemic injury site, of rats treated with pedf . Image analysis is not able to separate map-2 positive dendrites from the cell bodies profiles . The stereological method however allowed us to show the neuroplasticity responses taking place in the dendritic process of map-2 positive neurons of the ventral horn of pedf treated rats . These results are in agreement with previous publication which treated a traumatic spinal cord injury with another neurotrophic factor, thus revealing the potential of spinal cord plasticity to lead a higher behavioral recovery after organ injury . Neuroplasticity is favored by a complex intracellular signaling that takes place in the rescued regions of the lesioned nervous tissue . In fact, the unaltered levels of the antiapoptotic factor bcl-2 [8183] in lumbar ventral region indicate the absence of local neuronal death, favoring further neuronal plasticity in that region . Neuroplasticity is a result of events specially addressed by the responsive neurons, their neighbor nonneuronal cells, and extracellular matrix molecules [8486]. Regarding that, pedf seems to be a promising candidate to spinal cord repair due to its actions on spinal cord neurons and ability of signaling to the endothelial and glial cells, the main actors of neurorestorative events in nervous tissue [3, 87]. Remarkable pedf actions include those that are inhibitory to angiogenesis and astrogliogenesis [87, 88], thus with substantial impact to extracellular matrix elements . Although neovascularization is required for neuronal fiber growth in regions close to injury acutely, to our knowledge, there is no publication referring to a long lasting possible interaction of vasculature regulation and neuronal plasticity in regions distant to lesion . Nevertheless, a recent publication correlated a local pedf expression to opposite responses of nerve fibber growth and neovessels in the cornea treated with the neurotrophic factor fgf-2, which is in agreement with our results on pedf - induced downregulation of laminin, a marker of small blood vessels, in lumbar spinal cord neuroplasticity responsive region that is distant from the injury . Inhibitory actions of pedf to astrocytes may have not interfered with ability of reactive astrocytes of the lesioned spinal cord to secrete cspg in the neuroplasticity responsive region caudally . Inhibitory influence of extracellular matrix molecules, especially cspg, to fiber growth close to the spinal cord injury site has been described [92, 93]; however, there is a lack of descriptions on the regulation of cspg in the neuroplasticity responsive spinal cord regions . The decreased level of 70 kda cspg in the motor ventral region of the lumbar levels, far from the photothrombotic injury site, in animals treated or not with pedf and at a chronic period after surgery (6 weeks) already emphasizes the ability of the lesioned spinal cord to last a permissive microenvironment for neuronal plasticity [94, 95]. All in all, pedf actions to nonneuronal actors of neurorestorative events seem to favor neuronal plasticity in the lesioned spinal cord . Additional feature of pedf is its capability to modulate the expression of other neurotrophic factors, thus amplifying neurorestorative events triggered by specific trophic molecules in the lesioned nervous system [9699]. There are actually several neurotrophic factors that play paracrine / autocrine actions on the spinal cord motor neurons . Based on that, we evaluated gene expression of nt-3, gdnf, bdnf, and fgf-2 by means of rt - pcr in the neuroplasticity responsive lumbar motor region of the spinal cord injury rats . We do not know whether photothrombotic ischemia in the low thoracic level of the rat spinal cord has triggered gene expression of studied neurotrophic factors in the neuroplasticity responsive lumbar region acutely; however, at the later week 6th time point, pedf treatment modulated the differentially nt-3 and gdnf gene regulation . The pedf - induced downregulation of nt-3 gene expression in the late period after injury might be related to a fine tuning of neuroplasticity responses in the cord [100, 101] by an action on the descending corticospinal motor fibers [102, 103]. Furthermore, such a regulation that was induced by pedf treatment might have favored the expression of a neurotrophic factor with stronger actions to postsynaptic spinal cord motor neurons . That would be actually the case for gdnf [5, 76, 97, 104]. The absence of a late gene regulation of bdnf and fgf-2 might be explained for their ability to modulate early events after injury like neuronal rescue, progenitor cell proliferation, glial reactivity, and wound repair [14, 105]. The bidirectional signaling system provided by the eph receptors and their ligands was recently mentioned as a key regulator of neuroplasticity in central nervous system . Ephrin signaling plays important actions regarding axonal guidance and synaptogenesis during development [107, 108]. The role of ephrin on the lesioned spinal cord has been the subject of recent investigation [29, 109]. The ephrin function is highlighted on neurorestorative events mainly because of its ability to prompt communication between neuronal and nonneuronal cells [110, 111]. We have analyzed at biochemical and cellular levels several members of the ephrin a and b families as well as the receptor subtypes of the eph a and b families that could play a role in the lesioned spinal cord . The epha6 and ephb2 signals that were not shown by western blot and immunohistochemistry were evaluated at molecular levels by employing rt - pcr . Upregulations of ephrins and their eph receptors have been described in reactive astrocytes close to rat spinal cord injury [29, 31, 113]. The regulation of ephrin system close to spinal cord injury has been associated with its ability to interfere with cell activation related to wound repair and also to inhibit neurite outgrowth and axonal regeneration close to a wound repair region [114, 115]. Remarkably, increases of epha4 in reactive astrocytes close to a scar region of the lesioned spinal cord were correlated to inhibition of axonal regeneration [116, 117], event that gained importance after the description of the improvement of axonal regeneration and reduction of glial scar in the epha4-deficient spinal cord injury mouse . Furthermore, epha4 antagonist was able to block retrograde axonal degeneration, to increase axonal regeneration, and to improve motor behavior in lesioned spinal cord rats [109, 116]. The present analysis demonstrated regulation of ephrin system in the neuroplasticity responsive lumbar motor region after a chronic spinal cord low thoracic injury, remarkably the ephrins a2, b1, and b3 and the receptor epha4 . The details of their contribution to neuronal plasticity in the lesioned spinal cord must be further evaluated . Nevertheless, changes in the levels of ephrin b system were correlated to neuropathic pain due to their ability to regulate neuronal excitability at spinal cord . Important finding of the present study was the upregulation of ephrin b2 in the neuroplasticity responsive region of pedf treated rats compared to nontreated lesioned animals, thus with a special relevance to motor recovery . In fact, the epha4/ephrin b3 bidirectional signaling in developing spinal motor neurons seems to be involved in locomotion function [119122]. Because ephrin b2 is specifically present in astrocytes of neuroplasticity responsive area, as demonstrated by the double immunofluorescence analyses, that ephrin signalling may contribute to well describe the ability of glial cells to promote neuronal plasticity . All in all, it is likely that long term spinal cord injury might employ ephrin signaling for spontaneous motor recovery, event that could be amplified by neurotrophic factor - induced regulation of specific ephrin molecules, as it is the case of pedf . Finally, long term ephrin regulation in the neuroplasticity responsive region of the lesioned spinal cord does not seem to require the transcription factor rhoa . Rhoa impairs neurite outgrowth, by means of growth cone collapse, an event that could be restricted to wound areas [124, 125] where upregulation of rhoa mrna and protein last on reactive astrocytes of the lesion site of a cord injury [126128]. Further analyses are required for a further understanding of the rhoa function in the neuroplasticity areas after a long period after injury . Pedf injection in the epicenter of a low thoracic rat spinal cord photothrombotic ischemic injury improved motor behavior and triggered neuroplasticity responses in the motor regions of lumbar levels far from the lesion site . Changes in the expression of extracellular matrix protein, neurotrophic factors, and molecules of the ephrin system may have favored neuroplasticity.
Bone mineral density (bmd) measurements are the main determinant for an osteoporosis diagnosis (1). The gold standard for bmd measurements is bone ash assessment, but its noninvasive substitute is dual - energy x - ray absorptiometry (dxa) (1, 2). Dxa can be performed locally or for the whole body (2); however, the usual method of bmd measurement is the assessment of specific skeletal regions . The best areas for bmd assessment are the hip, lumbar spine, and forearm (3). It has been previously recommended that the bmd of the femoral neck, trochanter, and total hip, as well as the lumbar spine, should be measured, with the lowest value selected as the basis for treatment planning (1, 4); however, some reports have advised against this method (5). Occasionally, bmd measurements of the spine or other regions of interest are not feasible or could yield false results . For example, bmd may be overestimated in patients with spondylosis (5, 6); this could be due to osteophyte formation, which falsely increases the average bmd, while the patient may have a lower bone density in other parts of the spine, predisposing him or her to fracture (6). Thus, some authors have proposed whole - body bmd measurements instead of local measurements . However, questions remain regarding the accuracy and validity of a whole - body bmd measurement as a guide for spinal bmd and the associated fracture risk . Some authors have shown a good correlation between whole - body and local bmds (2), while other authors have reported only a weak correlation (3). Bmd measurements of different parts of the skeleton could also give different results, as bone loss is not a homogenous process (2). For instance, it has been shown that the bmd of the spine varies, and estimating the fracture risk of a particular region according to bmd measurements of other regions could be misleading (1). In this study, we evaluated the correlation between whole - body and local bmd assessments of the lumbar spine, hip, and forearm, to determine if whole - body bmd measurements could be used instead of local bmd measurements . In this cross - sectional study, we evaluated all patients who were referred to a single rheumatology clinic for bmd measurements during the period of 2009 2010 . Patients who had bmd measurements taken of the hip, lumbar spine, forearm, and the whole body were included in the study . The study was approved by the ethics committee of shahid beheshti university of medical sciences . The exclusion criteria were: increasing or decreasing pelvic and spinal bmd due to osteoarthritis, metal artifact, local skeletal sclerosis, lytic or sclerotic lesions, paget s disease, fractures, vascular calcifications, the presence of contrast media, urolithiasis, scoliosis, or prior laminectomy . Standard bmd measurements were performed using a dxa hologic device (hologic delphi a; hologic, bedford, ma, usa). Pelvic measurements included the whole pelvic bone and the femoral neck, and forearm measurements were also performed . Bmd, z - score, and t - score were measured for all patients and all body regions . Normal bmd was defined as a t> -1 in post - menopausal women and patients over 50 years of age, and as a z <-2 in non - menopausal women or patients under age 50 . Osteopenia was defined as a t <-1 in post - menopausal patients or those over age 50, and as a z <-2 in non - menopausal women or patients under age 50 . In patients over age 50, a t -2.5 was considered to indicate osteoporosis . The following guidelines were used to categorize the patients (4): low bone mass (osteopenia): -2.5 sd <t <-1 sd osteoporosis: t -2.5 sd severe or established osteoporosis: a value for bmd of 2.5 sd or more below the mean for young adult females in the presence of one or more insufficiency fractures . The manufacturer s standard values were used for calculating the t - score and z - score . Normal data distribution and correlation of different bmd measurements were assessed by the kolmogorov - smirnov test and pearson s correlation coefficient, respectively . Local bmd estimations according to whole - body values were done using linear regression models . Agreement of different variables was assessed and interpreted by kappa coefficient of agreement (7). All analyses were performed using spss statistical software (version 16.0, spss inc ., chicago, il, usa). Normal data distribution and correlation of different bmd measurements were assessed by the kolmogorov - smirnov test and pearson s correlation coefficient, respectively . Local bmd estimations according to whole - body values were done using linear regression models . Agreement of different variables was assessed and interpreted by kappa coefficient of agreement (7). All analyses were performed using spss statistical software (version 16.0, spss inc ., chicago, il, usa). Of the 152 total patients, 148 were female (97.4%), 134 of whom were post - menopausal (90.5%). The mean patient age was 56.7 12.6 years (range 25 88 years), with 106 patients older than 50 years (69.7%). The respective mean whole - body bmd, z - score, and t - score were 1.1 0.12 (range 0.8 1.4), 0.72 1.2 (range 2.1 3.8), and -0.33 1.42 (range -3.5 2.9), respectively (table 1). There was no statistically significant difference between genders with regard to age, bmd, z - score, or t - score . Repeated anova showed that the mean bmds of the four different sites were significantly different . This significant difference was also observed between all pair - wise comparisons in the whole - body bmd test (all p <0.0001). Both the t- and the z - scores showed significant differences between measurements on the anova test (p <0.0001); however, this was not confirmed in the bonferroni correction (p = 0.37 and 0.23). Other pairwise comparisons, including comparisons of the whole - body bmd with the bmd from specific sites, were significantly different (p <0.007 and 0.01, respectively). The respective pearson correlation coefficients of the whole - body bmd with the spine bmd, hip bmd, and forearm bmd were 0.73, 0.75, and 0.77, respectively (all p <0.0001) (table 2). The correlation coefficients of the whole - body z - scores and t - scores related to local values are listed in table 2 . All correlation coefficients between local values were lower than 0.58, except for the coefficient of 0.66 for the correlation of t - scores of the forearm and hip . Abbreviation: bmd, bone mineral density as densitometry is more important in women over age 50, we divided the patients into two groups: those over 50 and those equal or under 50 . We further divided these two main groups into two subgroups: those with normal bmd, and those with abnormal bmd . The correlation coefficients of the whole - body indices with local parameters are listed in table 2 . The results from a univariate regression model for the prediction of each local densitometry index according to the relevant whole - body measurement in patients over age 50 are shown in table 3 . With regard to predictions of the hip t - score according to the whole - body t - score, abbreviation: bmd, bone mineral density values for standard error and b are expressed in order as constant and independent variable . As mentioned previously, subcategorization was done separately for the spine, hip, forearm, and whole - body bmd measurements in patients over age 50 . Patients with normal and abnormal t - scores were cross - tabulated using the mcnemar test to determine whether the frequency of patients with abnormal results for local measurements and whole - body measurements were equal . According to whole - body t - scores, 52 of 106 patients were osteopenic (49.1%); in comparison, the numbers of osteopenic patients according to site - specific t - scores for the spine, hip, and forearm were 68 (64.2%), 75 (71.4%), and 74 (69.8%), respectively (tables 4 and 5). Comparison of these percentages using the mcnemar test showed that compared with all local densitometries, the whole - body results underestimated the percentage of patients with abnormal bmd (table 5). A similar analysis was performed with a cutoff point of -2.5 for osteoporosis of all local and whole - body t - score measurements (table 4 and 5). Abbreviation: bmd, bone mineral density abbreviation: bmd, bone mineral density next, we classified the patients as normal, osteopenic, or osteoporotic based on their t - scores . As it was possible for a single patient to fit into multiple categories based on t - scores from different areas, the kappa agreements according to this categorization were measured (table 6). In all instances, based on the above mentioned analyses, it could be proposed that the considering similar cutoff points of -1 and -2.5 for whole body densitometry t - scores ensues in misleading assessment results . So in next step, we evaluated the diagnostic indices of whole body densitometry when we considered the t - score cutoff points of -1 for osteopenia and -2.5 for osteoporosis (exactly similar to cutoff points in site specific densitometry). For this assessment, we considered the results of each individual site as the gold standard, and generated the diagnostic indices of whole - body measurements for the diagnosis of an abnormal t - score (t <-1) and osteoporosis (t <in addition, kappa coefficients of agreement are presented in table 7, demonstrating more than 30% underestimation of abnormal results when using whole - body t - scores with similar cutoff points as opposed to site - specific t - scores (table 7). Abbreviation: tp, true positive; fn, false negative; tn, true negative; fp, false positive; sen ., specificity; ppv, positive predictive value; npv, negative predictive value; plr, positive likelihood ratio; nlr, negative likelihood ratio; bmd, bone mineral density; wb, whole body; ci, confidence interval . So we assessed the diagnostic accuracy of all whole - body indices for the diagnosis of abnormal densitometry and osteoporosis using receiver operating characteristic (roc) analysis yielded an area under the curve (auc) for each site . In this regard, we had six different situations for each site (spine, hip, and forearm). For each site, we considered the results of the densitometry as the gold standard and calculated the auc for bmd, z - score, and t - score (table 8). According to the auc results, the best measurement for diagnosis of site specific abnormal bmd and osteoporosis was the whole - body bmd in diagnosing low bmd and osteoporosis in hip based on t - score (auc=0.96 and 0.86, respectively). Abbreviation: bmd, bone mineral density; wb, whole body; auc, area under the curve . All numbers are aucs and their 95% confidence interval in parenthesis . In both of these rocs, we considered two cutoff points, -0.5 and -1, for calculating the diagnostic indices . At the cutoff point of -0.5, the sensitivity and specificity of the test for diagnosis of abnormal bmd were 91 and 90, respectively . When using the cutoff point of -1, the respective sensitivity and specificity were 96 and 58 for a diagnosis of osteoporosis (tables 9 and 10) (figure 1a and b). Abbreviation: tp, true positive; fn, false negative; tn, true negative; fp, false positive; sen ., specificity; ppv, positive predictive value; npv, negative predictive value; plr, positive likelihood ratio; nlr, negative likelihood ratio; bmd, bone mineral density; ci, confidence interval . We found a significant difference between the mean bmd values for the whole - body versus local measurements . The correlations of whole - body measurements with the corresponding variables for each individual location were good; however, the regression model predictions of variables for each location using the corresponding whole - body values were not reliable . Using the same t - score cutoff points for diagnosis of abnormal bmd and osteoporosis from whole - body readings markedly underestimated abnormal bmd and osteoporosis according to hip bmd results . The auc for diagnosing abnormal hip bmd from whole - body measurements was very promising; however, the profile for diagnosing osteoporosis was not as good . Different skeletal locations have been proposed for bmd assessment; some of these proposed sites are more generally recommended, while others are only recommended in certain circumstances, such as when the principal assessments are not feasible or could yield false results (4). Forearm measurements should be reserved for patients with hyperparathyroidism or morbid obesity, or in whom hip and/or spine measurements are not feasible or cannot be interpreted (4). Femoral neck assessment is preferred mainly because of osteoporotic femoral neck fracture morbidity, and the hip t - score is the best predictor of femoral fracture . In addition, the hip has the greatest relevance to the clinical and biological aspects of osteoporosis (8). As noted earlier, bmd measurements can be associated with false results; for example, the t - score will be falsely increased in osteoarthritis (4, 6). Thus, bmd assessment of other skeletal sites, such as the forearm and the whole body, has been proposed (4). The main issue is the validity and precision of whole - body bmd measurements for a diagnosis of osteoporosis in standard measurement sites, such as the lumbar spine and, especially, the hip and the femoral neck . Many studies have evaluated the concordance and discordance of bmd measurements of the hip and spine (9, 10). These studies have reported a high rate of discordance between the bmd values of the hip and spine, with findings of minor discordance (4%) and major discordance of up to 40% between the bmd measurements for these two sites . Using the same t - score cutoff points for determining osteoporosis could make a difference in the perceived prevalence of osteoporosis; that is, an over- or underestimation of osteoporosis prevalence could result (3). This could lead to some difficulty in decision - making, as the t - scores could fall into two different world health organization (who) categories . Some physiologic and/or pathologic risk factors for this phenomenon include older age, menopause, and obesity (9, 10). Another possible reason could be the performance or analysis of the dxa (11). This over - or underestimation of osteoporosis prevalence could also result from considering whole - body bmd measurements, as various skeletal regions with different dynamic properties and types (for example, long bones versus cancellous bones) will be considered as one region . To account for this, some authors have introduced different cutoff points for the whole - body bmd definition of osteoporosis and osteopenia (12). Discordance of t - scores between the hip and spine could be due to the fact that age - related bone loss is nonhomogeneous (2). For example, it has been reported that lumbar spine bmd loss occurs at a younger age than hip bmd loss (3). In addition, osteoarthritis of the lumbar spine at older ages could interfere with the estimation of the real t - score (3). In this study, we employed different statistical approaches to assess the concordance and correlation of whole - body measurements with local values . Regarding the mean values, there was a significant difference between the mean values for whole - body versus local measurements . The correlation of whole - body measurements with the corresponding variables at each individual location were good; however, when considering the regression model results, the prediction of each variable from each specific location using the corresponding whole - body value did not seem perfect (all r2 values were lower than 0.65). At best, using a regression model to predict the hip t - score from the whole - body t - score is associated with some incorrect classifications, which may interfere with choosing the correct treatment plan . Using the same t - score cutoff points for the diagnosis of abnormal bmd and osteoporosis from whole - body readings markedly underestimated abnormal bmd (by 32%) and osteoporosis (by 67%) according to hip bmd results; this is probably because all bones affect the measurement in whole - body densitometry, including the long, wide, and spongiform bones (long bones could increase the mean of the densitometry measurement). This means that the values of the whole - body scan are generally higher than those from local regions, so using the whole - body t - score cutoff for determining osteoporosis underestimates the true result . To resolve this problem, we used the roc curve analysis to assess the diagnostic performance of whole - body bmd measurements for the diagnosis of abnormal bmd and osteoporosis of the hip, as using other cutoff points may improve this misclassification . The auc for diagnosing abnormal hip bmd was very promising (0.96), and we found a good cutoff point of -0.5 (instead of -1). The use of this cutoff point could yield a sensitivity of 90% and a specificity of 91%, which seems to be acceptable . Similarly, the cutoff point of 0 yielded a sensitivity of 95% and specificity of 80% for diagnosing patients with abnormal hip bmd . The profile for diagnosing osteoporosis was not as good as for determining abnormal hip bmd; the auc for determining osteoporosis was 0.84 (lower than the auc of 0.96 for abnormal hip bmd), and the resulting diagnostic indices for this situation were insufficient for the diagnosis of abnormal bmd (sensitivity of 96% and specificity of 58% using the cutoff point of -1). Most previous studies assessing whole - body measurements have evaluated the correlation coefficient of whole - body versus local measurements (for example, the hip). The results of a study by franck and munz are in line with our study (2). However, our study yielded different results than boyanov s, who studied 132 women (mean age 51 years) and estimated the lumbar spine bmd by dxa in standard anteroposterior scans, as well as the subregional analysis of whole - body measurements (12). Using the roc method for the diagnosis of abnormal bmd and osteoporosis, boyanov reported respective aucs of 0.78 and 0.74, which were lower than the aucs in our study . At a sensitivity level of 90%, boyanov reported respective specificities of 83.5% and 70.5% for abnormal bmd and osteoporosis, which are again different from our findings . The possible reasons for these differences are that boyanov derived the whole - body lumbar spine value instead of the whole - body measurement, the regression models from the previous study yielded a better fit and r in comparison to our study (all> 0.75), and there was a different age - range of patients (our study mainly involved patients older than 50 years). In addition, the kappa coefficient of agreement was greater than 0.6 in the previous study, which was better than that in our study . Discordance has also been shown in many other studies between the bmd results for different local areas, such as between the hip and spine . However, the value of this discordance is lower than for whole - body compared with local measurements; for example, moayyeri et al . Conducted a study of about 4,300 patients and reported a discordance of 40% between the hip and spine measurements (9). Cole and larson reported a discordance of 5.4% when comparing the measurement of one hip to that of the contralateral hip (13). The limitations of this study were only minor . A larger sample size would have been favorable, and the chance to gather data from other clinics would have made the results more reliable . At the end, we can conclude slight underestimation of abnormal bmd and osteoporosis should be considered in cases in which whole - body measurements are used instead of local measurements . This underestimation could be reduced by choosing appropriate cutoff points for the diagnosis of abnormal bmd and osteoporosis (different from the classic t - score cutoff points of -1 and -2.5); however, this would still not eliminate possible resultant misclassification.
Bacterial isolates - fifty - five m. tuberculosis isolates obtained from culture stocks at -80c were tested by cvda . Eighteen were mdr isolates, nine isolates were only resistant to inh and the remaining 28 were sensitive to both inh and rif . In the present study, h37rv - atcc 25618 (susceptible to all drugs), atcc 35822 (resistant to inh) and atcc 35838 (resistant to rif) the bactec 460 tb system (becton & dickinson) was used as the reference method for the determination of susceptibility in the 18 mdr isolates, whereas the bactec mgit 960 system (becton & dickinson) was used as the reference method for the determination of susceptibility in the remaining isolates . Preparation of antibiotics and cv - inh, rif and cv were purchased from sigma . Stock solutions of inh and rif at 1,000 mg / l were prepared in sterile distilled water and methanol, respectively, and the stock solutions were stored at -20c until use . Stock solutions of cv at 25 mg / l were prepared with sterile distilled water, sterilised by filtration and stored at 4c until use . Preparation of bacterial inocula - five or six colonies of each the 55 isolates freshly grown on lj media was suspended in a physiological saline tube containing 10 - 15 glass beads and then vortexed for 30 sec . The tube was kept in a vertical position for 1 h at room temperature to allow for the sedimentation of aerosols and large particles . The turbidity of the supernatant was adjusted to a mcfarland standard of 1 (clsi 2011). Preparation of the medium - the bacteria were tested in middlebrook 7h9s broth (containing 0.1% casiton, 0.5% glycerol and 10% oleic acid, albumin, dextrose and catalase). Inh and rif were tested at critical concentrations of 0.25 mg / l and 0.5 mg / l, respectively (martin et al . The test was performed in three tubes, including two assay tubes and one growth control tube . The assay tubes contained inh or rif and the growth control tube was without antibiotics . Determination of the cv concentration - in this study, the concentration of cv was determined according to the value of tolerance described by jones and falkinham iii (2003). The appropriate test concentration of cv was determined by titration of the stock solution . The most suitable concentration was 100 l of 25 mg / l a stock solution in a 1-ml tube . Application of the test - an inh test tube (0.25 mg / l), an rif test tube (0.5 mg / l) and an antibiotic - free growth control tube were used for each bacterium . A bacterial suspension (50 l) that was adjusted to mcfarland standard 1 was inoculated into the three tubes and the tubes were incubated at 37c . On the seventh day of incubation, 100 l of cv (25 mg / l stock solution) was then added to the tubes and incubated for an additional 24 - 48 h. as cv (blue / purple) is decolourised by the growth of bacteria, the isolate was considered to be resistant to that drug if the colour of cv was lost (figs 1 - 3). A: negative control tube without drug or bacteria; b: positive control tube with bacteria without drug; c: test tube with rif (resistant to rif); d: test tube with isoniazid (inh) (susceptible to inh). Decolourisation - there is bacterial growth . A clinical multidrug resistant isolate - resistant to isoniazid (inh) and rifampicin (rif). A: negative control tube without drug or bacteria; b: positive control tube with bacteria without drug; c: test tube with inh (resistant to inh); d: test tube with rif (resistant to rif). Decolourisation - there is bacterial growth . A clinical isolate - susceptible to to isoniazid (inh) and rifampicin (rif). A: negative control tube without drug or bacteria; b: positive control tube with bacteria without drug; c: test tube with rif (susceptible to rif); d: test tube with inh (susceptible to inh). The sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and agreement for inh were found to be 92.5%, 96.4%, 96.1%, 93.1% and 94.5%, respectively . Two isolates were found to be sensitive to inh by cvda, though they were resistant according to the reference method . The sensitivity, specificity, ppv, npv and agreement for rif were 88.8%, 100%, 100%, 94.8% and 96.3%, respectively . Two rif - resistant isolates were found to be susceptible by cvda (table). Tablecomparing the results of crystal violet decolourisation assay (cvda) with those obtained with reference methoddrugcvdareference method 460tb / mgit sensitivity (%) specificity (%) ppv (%) npv (%) agreement (%) rs inhr18/70/192.596.496.193.194.5 s0/227 - ----rifr16/00/088.810010094.896.3 s2/00/37 - ---- a: automated systems included bactec 460 tb and mgit 960 were used as reference methods; inh: isoniazid; npv: negative predictive value; ppv: positive predictive value; r: resistant; rif: rifampicin; s: sensitive . A: automated systems included bactec 460 tb and mgit 960 were used as reference methods; inh: isoniazid; npv: negative predictive value; ppv: positive predictive value; r: resistant; rif: rifampicin; s: sensitive . There is increasing interest in the use of colourimetric methods for the susceptibility testing of m. tuberculosis isolates . These methods have certain advantages in that they are easy to perform, reproducible, reliable and readily available and have a very low cost . The present study is the first to assess cv decolourisation for the evaluation of m. tuberculosis antimicrobial susceptibility . Jones and falkinham iii (2003) showed that cv and malachite green were decolourised by water - borne pathogenic mycobacteria, a feature that is believed to be due to the properties of the bacterial cell wall . It was suggested that decolourisation could be due to the reduction of cv and its sequestration in the lipid fraction . In that same study, water - related mycobacterial pathogens were shown to be tolerant to cv concentrations of up to 15 mg / l (jones & falkinham iii 2003). Therefore, a concentration of less than 15 mg / l was used in the present study . As a result of titration, 100 l of a 25 mg / l stock solution of cv was added to the assay tubes; thus, the final concentration would be 2.5 mg / l, which the bacteria may be able to tolerate . Colourimetric methods, such as the resazurin microplate method, the nitrate reductase assay (nra), (2-methoxy-4-nitro-5-sulphophenyl)-2h - tetrazolium-5-carboxanilide, 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2h - tetrazolium hydroxide and the malachite green decolourisation assay, have been developed for the drug susceptibility testing of m. tuberculosis clinical isolates and these new colourimetric methods have yielded concordant results with reference methods (angeby et al . Nra requires three different compounds, including sulfanilamide, n-1-naphthylethylenediamine and concentrated hydrochloric acid for the griess reagent; in addition, freshly prepared griess reagent should be used for the test . In contrast, cv dye is inexpensive and is used for gram staining in clinical microbiology laboratories; therefore, it can be easily obtained by many laboratories . In this study, the critical concentration used was 0.5 mg / l for rif and the results from two isolates were inconsistent with the reference method: these isolates were sensitive according to cvda, but were resistant with the reference method . However, full agreement may be observed if the critical concentration is reduced by one or two dilutions . This new colourimetric method showed sensitivities, specificities and agreements of 92.5%, 88.8% and 96.4% and 100%, 94.5% and 96.3%, respectively, for inh and rif . However, the low number of tested resistant strains may have resulted in the low sensitivity of the test . For this reason, further studies with more resistant strains are needed before routine laboratory implementation . In conclusion, based on the accuracy reported here, cvda is a rapid, simple, inexpensive and potent method for the detection of inh and rif resistance in m. tuberculosis isolates, particularly in developing countries.
Postoperative uveitis associated with inflammatory membrane formation occurs in less than 3% of cases after uneventful cataract surgery and intraocular lens (iol) implantation . Complications from resultant membrane formation include iol displacement, pupillary block glaucoma, posterior capsule opacification, and side - effects from prolonged topical steroid treatment . Intracameral injection of recombinant tissue plasminogen activator (rtpa), a highly potent fibrinolytic protein used for systemic thrombolysis, has been shown to successfully lyse fibrin membranes [25]. Reported uncommon complications of intracameral rtpa include corneal oedema, band keratopathy, anterior chamber turbidity, and hyphaema, while iol opacification would not appear to have been previously reported [68]. We report 7 cases of iol opacification subsequent to rtpa treatment for postoperative inflammatory membranes following uneventful phacoemulsification and hydrophilic acrylic one - piece iol implantation (rayner c - flex 570c and superflex 620h). To the best of our knowledge, this is the first report of hydrophilic acrylic iol opacification following the use of intracameral rtpa . Three patients had type 2 diabetes and treated proliferative retinopathy, and one patient presented with phacomorphic glaucoma requiring urgent cataract surgery . Two patients had medically controlled systemic hypertension but none had abnormal albumin or serum calcium levels . All patients underwent uneventful cataract phacoemulsification and posterior chamber iol implantation under local anaesthesia between august 2002 and september 2009 . Two percent hydroxypropylmethylcellulose (coatel) and balanced salt solution (bss) were used in all cases . The rayner c - flex 570c iol was implanted in 4 patients and the rayner superflex 620h was implanted in 3 patients . No remaining viscoelastic material or soft lens matter was observed at the end of all procedures . Inflammatory membrane formation was noted within 1 week in three patients and between 2 and 4 weeks in the remaining 4 patients . Intracameral rtpa (actilyse) was prepared under sterile conditions using 50 mg vials of rtpa diluted with 50 ml of sterile water to create a 1 mg / ml solution . 1050 l of this solution was injected into the anterior chamber using an insulin syringe with a 30-gauge needle . Slit lamp examination and intraocular pressure measurement were performed 2 and 24 hours after rtpa treatment . The frequency of dexamethasone 0.1% was increased to be hourly by day, together with chloramphenicol 6 hourly and cyclopentolate 1% 8 hourly . Further reviews were scheduled at weeks 1 and 3 and at 6 months after treatment . Opacified iols were viscodissected with sodium hyaluronate (healon), with care taken to avoid manipulation of the opacified portion . One patient required bisection of the iol and anterior vitrectomy due to severe adhesion between the iol and lens capsule . Explanted iols were placed in a sterile container with neutral buffered formalin 10% before they were sent for laboratory analysis . Two explanted iols were sent to the laboratories for ophthalmic devices research, sullivan's island, south carolina, usa, for light microscopy and histochemical analysis . Another iol was sent for light microscopy, scanning electron microscopy, and x - ray spectrometry at the international vision correction research centre, department of ophthalmology, university of heidelberg, germany . Detailed techniques for preparation and staining of explanted iols for calcium have been described elsewhere . Briefly, iols were photographed under a light microscope and were subsequently treated with special stains for calcium (von kossa 0.5% and alizarin red 1%). The iols were reexamined and photographs were again taken . Full - thickness sections were made through the opacified portion and the iols were restained for cross - sectional examination . Scanning electron microscopy (sem) and energy - dispersive x - ray spectrometry (eds) for elemental analysis were also separately performed . Table 1 summarises the preoperative comorbidities, procedures performed, and subsequent treatment for the 7 patients . Inflammatory membranes developed between 1 and 4 weeks postoperatively, and resolution of inflammatory membrane occurred within 24 hours after intracameral rtpa in all cases . Iol exchange was carried out in 3 patients with a mean final bcva of 0.20 logmar . Three other patients declined iol exchange and 1 patient was deemed unsuitable for further surgery due to poor visual potential secondary to neovascular glaucoma . Macroscopic and microscopic findings on all explanted iols were highly comparable . On macroscopic examination, fine granular whitish opacities were observed in the central part of all explanted iol optics (figure 1). Light microscopy revealed diffuse fine granular deposits on as well as below the anterior surface of the nonencapsulated optic . These deposits stained positively with von kossa and alizarin red and were linearly distributed parallel to the anterior iol surface, diminishing towards the periphery and posteriorly . Cross - sectional sem of opacified iol showed crystalline deposits distributed close to the surface of the iol (figure 2). Analysis with eds confirmed the presence of calcium and phosphate (figure 2(d)) within the compound . Iol opacification is an infrequent complication after uneventful cataract surgery, resulting in symptoms of visual loss or glare, necessitating iol exchange in some cases . To our knowledge, this is the first report of iol opacification due to calcification following the use of rtpa for postoperative fibrinous membranes after cataract surgery . A number of cases of iol opacification have previously been reported; almost all were involving hydrophilic acrylic iols from various manufacturers [915]. Reported 2 cases of surface calcification of hydrophilic acrylic rayner c - flex 570c implants related to inflammatory membrane formation after combined vitrectomy and cataract surgery . Similar to their report, we found localised calcification on the anterior and subsurface within the capsulorhexis area on both the c - flex and the superflex iols . Such calcification is independent of the manufacturing and packaging process and is thus classified as secondary calcification relating to environmental causes . Iol calcification has been associated with systemic disease, intraocular surgery, inflammation, or drug administration [5, 9, 1618]. Breakdown of blood - aqueous barrier (bab) and disruptions to aqueous calcium homeostasis are thought to lead to dystrophic calcium - phosphate precipitation on the iol, although the precise mechanism remains unclear . Reported a case series of 15 patients with hydrophilic acrylic iol opacification following descemet stripping automated endothelial keratoplasty (dsaek), in which they identified air injection for dsaek graft rebubbling as a significant risk factor . Analysis of the opacified iols demonstrated findings identical to those observed in our study, using similar methodology . In our series, all cases had postoperative inflammatory membranes that were successfully treated with rtpa . In our unit, we are not aware of any other pseudophakic patients who had received intracameral rtpa injections . Thus it would be suggested that intracameral rtpa injection is an absolute risk factor for iol opacification . In response to our findings, we stopped further usage of intracameral rtpa in cases of postoperative inflammatory membrane formation, and we have not observed any cases of iol opacification since . These cases were also reported to the medicines & healthcare products regulatory agency in the united kingdom . We hypothesise that rtpa contributes to iol opacification through its action on aqueous calcium - phosphate homeostasis and possible disruption of the blood - aqueous barrier (bab). Intracameral rtpa lyses fibrinous inflammatory membranes and in doing so releases sequestered calcium from the fibrinous matrix . It also introduces phosphate ions contained in its buffer solution, which may potentiate abnormal calcium precipitation . A case report of band keratopathy formation after recent laboratory model has also shown that calcification occurred when the iol was exposed to a supersaturated solution of calcium and phosphate ions, and the reaction is initiated within the iol towards the anterior iol surface due to ionic diffusion . This coincides with our observation and may explain the preferential calcification on the anterior subsurface / surface of the hydrophilic iol . Similar to air injection in dsaek graft rebubbling, rtpa injection may also lead to further disruption of the bab . This may be due to the induction of nonplasminogen intracellular signalling pathways [22, 23] or the mechanical effect of the injection itself . Although intraocular inflammation could be solely responsible for iol opacification, it fails to explain the strong association between iol opacification and the use of rtpa . The dosages of rtpa used in our patients were within the range reported by previous authors (325 g) in all except 2 cases [24, 24]. It is plausible that the higher doses in the 2 cases may have exacerbated the extent of iol opacification, although this has not been formally quantified . Cases of iol opacification occurred during a period when our unit exclusively used rayner iols . C - flex and superflex differ in the size of the optic (5.75 mm and 6.25 mm in diameter, resp .) And the range of diopter power (+ 8.0 to + 34.0d and 10.0 to + 22.0d, resp . ), but they are both hydrophilic acrylic one - piece monofocal iols . However, there was no evidence of any manufacturing defects on the explanted iols in our histopathological analysis . This study highlights that intracameral injection of rtpa for the treatment of postoperative inflammatory membranes could lead to iol opacification requiring iol exchange . The limitations of this study were its retrospective nature and the small number of cases involved . The intraocular effects of rtpa may be more extensive than previously thought, and further laboratory investigations on this are needed . Caution should be exercised in using rtpa to treat inflammatory membranes following intraocular surgery, especially in the presence of hydrophilic iols.
Colorectal cancer (crc) is a leading cause of cancer - related deaths in the world . It is the second and third - most commonly diagnosed cancer in females and males respectively, and more than 1.2 million patients are diagnosed with crc every year . Currently, crc patients with lymph node metastasis (tnm stage iii) are treated with adjuvant chemotherapy that includes cytotoxic drugs such as 5-fluorouracil (5-fu) and oxaliplatin, following surgical resection of the cancer . Large proportion of patients receiving chemotherapy finally became metastatic and chemo - resistant, and this has been a key barrier to the efficacy of crc treatment [57]. Thus, finding new diagnostic and prognostic targets will be indispensable for developing effective therapy for crc patients . Recently, a number of adjuvant strategies have also been developed to further enhance the response and survival rates . Despite the advances in diagnostic methods, such as fecal occult blood testing and stool dna tests, early diagnosis for crc still remains difficult and the overall survival rate of crc patients has not changed dramatically . At present, more and more researchers focus on non - invasive biomarkers, and one or a cluster of specific marker is urgently needed for increasing the early detection rate pf crc and decreasing the mortality rate of crc . Predictive biomarkers are better if they are blood - based, as blood is easily available and provides the chance to monitor chemotherapy response . Therefore, it is important to identify blood markers that predict a patient s responsiveness to chemotherapy, which may allow for the development of targeted therapies for overcoming chemoresistance . Micrornas (mirnas) are increasingly recognized to be key regulators of gene expression in several biological systems, including cancer . They regulate gene expression primarily via their interaction with the 3utrs of target mrnas, resulting in mrna decay or translational repression . Mirnas sometime exert a role as oncogenes or tumor suppressor genes through affecting the response to various therapeutic regimens . In recent years these findings suggest that mirna can act as important regulators during chemoresistance among different cancers . Previous reports showed that mir-429 was important for predicting clinical outcome in gastric cancer patients through suppressing tumor cell proliferation and inhibiting tumor metastasis . However, another study revealed that mir-429 was an oncogene and predicted poor survival in ovarian cancer patients . These findings suggest that mir-429 may be a oncogene or tumor suppressor in different conditions . Recently, cantini et al . Found that mir-429 has prognostic value in crc, but its role in crc chemotherapy was still unknown . In this study, we intended to determine the mir-429 expression levels in primary tissues and serum from crc patients, and further analyzed its association with pathologic factors . Moreover, the potential diagnostic and prognostic value in patients receiving 5-fu - based chemotherapy was also investigated . The results indicated that mir-429 expression was significantly downregulated in crc patients and can serve as a diagnostic and prognostic factor in crc patients receiving first - line 5-fu - based treatment . For clinical parameter analysis, 78 crc tissues and paired normal tissues were collected at the second people s hospital of hefei city . Fresh surgical specimens were immediately frozen in liquid nitrogen and were then stored at 80c until further use . At the same time, pre - operative blood samples were collected from another 45 patients with crc, as well as from 45 healthy volunteers for further analysis . Immediately after collection, the blood samples were processed for isolation of cell - free nucleic acids to prevent contamination from cellular nucleic acids . Serum samples were then stored at 80c until further processing . For the chemo - response study, 116 primary tissues were collected from the patients who received standard 5-fu - based chemotherapy at the second people s hospital of hefei city between 2009 and 2013 . All the patients were pathologically confirmed as crc patients and the clinical samples were collected before chemotherapy was started . Patients were classified according to the who criteria and staged according to the tumor - node - metastasis (tnm) classification . Tumor response status was evaluated according to the response evaluation criteria in solid tumors (recist) version 1.0 criteria and was assigned to patients with complete or partial response (cr and pr, respectively) and stable or progressive disease (sd and pd, respectively) in tumor measurements confirmed by repeat studies performed no less than four weeks after the criteria for response was first met . Overall survival was updated on february 1, 2012 and was defined as the time from inclusion in the study until death for any reason . All patients received standardized follow - up including ct - scans of the chest and abdomen at 12 months and 36 months after surgery . Written informed consent was obtained from all patients according to local ethical regulations of the ethics committee of the second people s hospital of hefei city . Total rna of crc tissues and adjacent tissues was extracted by using trizol (invitrogen, usa); and plasma rna was extracted by using acid phenol according to the manufacturer s instructions . The samples with a260 nm / a280 nm ratios between 1.8 and 2.0 were used for further experiments . For primary crc tissues, the cdna was synthesized from 200 ng extracted total rna using the primescript rt reagent kit (takara bio company, shiga, japan) and amplified by qrt - pcr with sybr green kit (takara bio company) on 7500 realtime pcr system (applied biosystems). For serum mir-429 detection, we treated the total rna with a reverse transcription kit (bioteck, beijing, china) according to the manufacturer s protocol to obtain cdna and then qrt - pcr was performed by using 7500 realtime pcr system (applied biosystems). The 2 method was used to determine the relative quantification of gene expression levels and u6 was used as a housekeeping gene . All reactions were performed in triplicate . For crc serum versus healthy control, and crc tissue versus adjacent non - tumor tissue, differences in mean expression the association between tissue and plasma mirna levels was analyzed using the spearman s correlation coefficient . The survival curves of crc patients were estimated via the kaplan - meier method and the difference in survival curves was estimated using log - rank testing . Statistical analyses were undertaken using graphpad prism version 5.01 (graphpad software, san diego, ca, usa). For clinical parameter analysis, 78 crc tissues and paired normal tissues were collected at the second people s hospital of hefei city . Fresh surgical specimens were immediately frozen in liquid nitrogen and were then stored at 80c until further use . At the same time, pre - operative blood samples were collected from another 45 patients with crc, as well as from 45 healthy volunteers for further analysis . Immediately after collection, the blood samples were processed for isolation of cell - free nucleic acids to prevent contamination from cellular nucleic acids . Serum samples were then stored at 80c until further processing . For the chemo - response study, 116 primary tissues were collected from the patients who received standard 5-fu - based chemotherapy at the second people s hospital of hefei city between 2009 and 2013 . All the patients were pathologically confirmed as crc patients and the clinical samples were collected before chemotherapy was started . Patients were classified according to the who criteria and staged according to the tumor - node - metastasis (tnm) classification . Tumor response status was evaluated according to the response evaluation criteria in solid tumors (recist) version 1.0 criteria and was assigned to patients with complete or partial response (cr and pr, respectively) and stable or progressive disease (sd and pd, respectively) in tumor measurements confirmed by repeat studies performed no less than four weeks after the criteria for response was first met . Overall survival was updated on february 1, 2012 and was defined as the time from inclusion in the study until death for any reason . All patients received standardized follow - up including ct - scans of the chest and abdomen at 12 months and 36 months after surgery . Written informed consent was obtained from all patients according to local ethical regulations of the ethics committee of the second people s hospital of hefei city . Total rna of crc tissues and adjacent tissues was extracted by using trizol (invitrogen, usa); and plasma rna was extracted by using acid phenol according to the manufacturer s instructions . The samples with a260 nm / a280 nm ratios between 1.8 and 2.0 were used for further experiments . For primary crc tissues, the cdna was synthesized from 200 ng extracted total rna using the primescript rt reagent kit (takara bio company, shiga, japan) and amplified by qrt - pcr with sybr green kit (takara bio company) on 7500 realtime pcr system (applied biosystems). For serum mir-429 detection, we treated the total rna with a reverse transcription kit (bioteck, beijing, china) according to the manufacturer s protocol to obtain cdna and then qrt - pcr was performed by using 7500 realtime pcr system (applied biosystems). The 2 method was used to determine the relative quantification of gene expression levels and u6 was used as a housekeeping gene . For crc serum versus healthy control, and crc tissue versus adjacent non - tumor tissue, differences in mean expression were determined using mann - whitney u test . The association between tissue and plasma mirna levels was analyzed using the spearman s correlation coefficient . The survival curves of crc patients were estimated via the kaplan - meier method and the difference in survival curves was estimated using log - rank testing . Statistical analyses were undertaken using graphpad prism version 5.01 (graphpad software, san diego, ca, usa). Seventy - eight patients who provide primary tissue samples were enrolled in this study . Among these patients, there were 49 males and 29 females; 35 patients were older than 60 years of age, and 43 patients were younger than 60 years of age with the median age of 56 years old, range (3179 years). According to the seventh edition of the tumor node metastasis (tnm) staging criteria of crc, 8 cases were considered stage i, 34 cases were stage ii, 29 cases were stage iii, and 7 cases were stage iv . Qrt - pcr was used to examine the mir-429 levels in 78 cancerous and paired noncancerous tissues, and our results indicated that the expression of mir-429 was significantly increased in tumor tissues compared with paired normal tissues (p<0.001, figure 1a). Moreover, the mir-429 expression in 60.3% (47 of 78) cases were 2-fold higher than in adjacent tissues (figure 1b). As shown in table 1, mir-429 expression was positively correlated with tnm stage, lymph node metastasis, distant metastasis and tumor size, while no correlations were observed between mir-429 expression and age, sex, tumor location, and differentiation . These results suggest that high expression of mir-429 may participate in the carcinogenesis of crc . To further verify the clinical value of mir-429 in diagnosis of crc another independent set of 45 serum samples from cancer patients (32 males and 13 females) were enrolled with the median age of 58 years old (range 4072 years). The corresponding data were following: stage i (5 cases), ii (19 cases), iii (18 cases) and iv (3 cases); well differentiation (18 cases), moderately differentiation (17 cases) and poor differentiation (10 cases). Forty - five serum samples from healthy individuals (27 males and 18 females) were used as controls, and the median age of controls was 47 years old (range 2569). As shown in figure 1c, the serum mir-429 expression levels in crc patients were also statistically significantly higher than healthy controls (p<0.01). Different from the primary tissues, serum mir-429 level was significantly correlated with tnm stage, but not correlated with other factors such as age, sex, tumor location, tumor size, local invasion, lymph node metastasis, differentiation, and distant metastasis (table 2). We next sought to validate the diagnostic role of mir-429 of primary crc tissues by using the receiver operating characteristic (roc) curve analysis, and compared it with the traditional crc marker, cea . As shown in figure 2a and 2b, the area under the curve (auc) of mir-429 were 0.775 (95% ci: 0.7020.838). The auc of mir-429 was higher than cea, while its auc was 0.742 (95% ci: 0.6660.808), and diagnostic sensitivity and specificity were 70.51% and 64.10%, respectively . We divided these patients into a high and a low expression group by using the median value (4.89) of 78 primary crc tissues . The 5-year survival rate of the crc patients whose tumors expressed high levels of mir-429 was 35.9% (14/39), which was significantly lower than that of the patients whose tumors expressed low levels of mir-429 (66.7%, 26/39). More importantly, the kaplan - meier analysis indicated that patients with high expression of mir-429 was associated with shorter overall survival compared with the low expressing crc patients (p=0.0021, figure 2d). We next sought to validate the association between mir-429 and response to treatment in 116 primary tissues . Patients were divided into responding (cr+pr) and non - responding (sd+pd) groups according to recist criteria . The expression level of mir-429 was significantly higher in patients who did not respond to treatment (n=42) compared with patients responding to treatment (n=74) (p<0.001, figure 3a). To exclude the influence from different chemotherapy methods, mir-429 expression was determined in patients receiving different 5-fu - based regimens, and the results showed that no difference was found between different regimens, including folfox (5-fu+oxaliplatin+leucovorin); folfiri (5-fu+leucovorin+irinotecan) and 5-fu / lv (5-fu+leucovorin) (p=0.1191, figure 3b). Then, a receiver operating characteristic (roc) curve analysis was performed to investigate the potential significance in predicting the patients response to chemotherapy . The area under the curve of mir-429 were 0.721 (95% ci: 0.6300.800, figure 3c), and the diagnostic sensitivity and specificity reached 52.70% and 85.71%, respectively . These results indicated that mir-429 was associated with 5-fu - based chemo - response among crc patients . Finally, we aimed to explore the prognostic value of mir-429 for chemotherapy . When we stratified patients into a low (n=46) and a high (n=70) groups with the previously established optimal cut - off value of relative mir-429 level (3.08), the proportion of patients not responding to chemotherapy was significantly higher in the high mir-429 expressing group than in the low group (p<0.01, figure 4a). The 5-year survival rate of the crc patients whose tumors expressed high levels of mir-429 was 21.4% (15/70), which was significantly lower than that of the patients whose tumors expressed low levels of mir-429 (52.2%, 24/46); this difference was statistically significant (p=0.0011). Besides, the kaplan - meier survival analysis also showed that patients with a low level of mir-429 expression had a significant longer overall survival than did those with a high level of mir-429 expression (p=0.0001, figure 4b). Furthermore, we performed cox regression multivariate analysis to identify whether mir-429 was an independent indicator for overall survival of crc patients who received 5-fu chemotherapy . As expected, a high mir-429 level was significantly correlated with poorer survival in an independent manner (table 3). Taken together, these results indicated that mir-429 was an independent predictor for chemo - response to 5-fu - based treatment in crc patients . Seventy - eight patients who provide primary tissue samples were enrolled in this study . Among these patients, there were 49 males and 29 females; 35 patients were older than 60 years of age, and 43 patients were younger than 60 years of age with the median age of 56 years old, range (3179 years). According to the seventh edition of the tumor node metastasis (tnm) staging criteria of crc, 8 cases were considered stage i, 34 cases were stage ii, 29 cases were stage iii, and 7 cases were stage iv . Qrt - pcr was used to examine the mir-429 levels in 78 cancerous and paired noncancerous tissues, and our results indicated that the expression of mir-429 was significantly increased in tumor tissues compared with paired normal tissues (p<0.001, figure 1a). Moreover, the mir-429 expression in 60.3% (47 of 78) cases were 2-fold higher than in adjacent tissues (figure 1b). As shown in table 1, mir-429 expression was positively correlated with tnm stage, lymph node metastasis, distant metastasis and tumor size, while no correlations were observed between mir-429 expression and age, sex, tumor location, and differentiation . These results suggest that high expression of mir-429 may participate in the carcinogenesis of crc . To further verify the clinical value of mir-429 in diagnosis of crc, we tested the expression level of mir-429 in serum of crc patients . Another independent set of 45 serum samples from cancer patients (32 males and 13 females) were enrolled with the median age of 58 years old (range 4072 years). The corresponding data were following: stage i (5 cases), ii (19 cases), iii (18 cases) and iv (3 cases); well differentiation (18 cases), moderately differentiation (17 cases) and poor differentiation (10 cases). Forty - five serum samples from healthy individuals (27 males and 18 females) were used as controls, and the median age of controls was 47 years old (range 2569). As shown in figure 1c, the serum mir-429 expression levels in crc patients were also statistically significantly higher than healthy controls (p<0.01). Different from the primary tissues, serum mir-429 level was significantly correlated with tnm stage, but not correlated with other factors such as age, sex, tumor location, tumor size, local invasion, lymph node metastasis, differentiation, and distant metastasis (table 2). We next sought to validate the diagnostic role of mir-429 of primary crc tissues by using the receiver operating characteristic (roc) curve analysis, and compared it with the traditional crc marker, cea . As shown in figure 2a and 2b, the area under the curve (auc) of mir-429 were 0.775 (95% ci: 0.7020.838). The diagnostic sensitivity and specificity was 71.790% and 62.820%, respectively . The auc of mir-429 was higher than cea, while its auc was 0.742 (95% ci: 0.6660.808), and diagnostic sensitivity and specificity were 70.51% and 64.10%, respectively . And we divided these patients into a high and a low expression group by using the median value (4.89) of 78 primary crc tissues . The 5-year survival rate of the crc patients whose tumors expressed high levels of mir-429 was 35.9% (14/39), which was significantly lower than that of the patients whose tumors expressed low levels of mir-429 (66.7%, 26/39). More importantly, the kaplan - meier analysis indicated that patients with high expression of mir-429 was associated with shorter overall survival compared with the low expressing crc patients (p=0.0021, figure 2d). We next sought to validate the association between mir-429 and response to treatment in 116 primary tissues . Patients were divided into responding (cr+pr) and non - responding (sd+pd) groups according to recist criteria . The expression level of mir-429 was significantly higher in patients who did not respond to treatment (n=42) compared with patients responding to treatment (n=74) (p<0.001, figure 3a). To exclude the influence from different chemotherapy methods, mir-429 expression was determined in patients receiving different 5-fu - based regimens, and the results showed that no difference was found between different regimens, including folfox (5-fu+oxaliplatin+leucovorin); folfiri (5-fu+leucovorin+irinotecan) and 5-fu / lv (5-fu+leucovorin) (p=0.1191, figure 3b). Then, a receiver operating characteristic (roc) curve analysis was performed to investigate the potential significance in predicting the patients response to chemotherapy . The area under the curve of mir-429 were 0.721 (95% ci: 0.6300.800, figure 3c), and the diagnostic sensitivity and specificity reached 52.70% and 85.71%, respectively . These results indicated that mir-429 was associated with 5-fu - based chemo - response among crc patients . When we stratified patients into a low (n=46) and a high (n=70) groups with the previously established optimal cut - off value of relative mir-429 level (3.08), the proportion of patients not responding to chemotherapy was significantly higher in the high mir-429 expressing group than in the low group (p<0.01, figure 4a). The 5-year survival rate of the crc patients whose tumors expressed high levels of mir-429 was 21.4% (15/70), which was significantly lower than that of the patients whose tumors expressed low levels of mir-429 (52.2%, 24/46); this difference was statistically significant (p=0.0011). Besides, the kaplan - meier survival analysis also showed that patients with a low level of mir-429 expression had a significant longer overall survival than did those with a high level of mir-429 expression (p=0.0001, figure 4b). Furthermore, we performed cox regression multivariate analysis to identify whether mir-429 was an independent indicator for overall survival of crc patients who received 5-fu chemotherapy . As expected, a high mir-429 level was significantly correlated with poorer survival in an independent manner (table 3). Taken together, these results indicated that mir-429 was an independent predictor for chemo - response to 5-fu - based treatment in crc patients . Despite recent chemotherapeutic regimens that have significantly increased survival in metastatic disease, invariably, nearly all crc patients finally become chemo - resistant . It is urgent to find new diagnostic methods and prognostic targets for crc . In this study, we validated the upregulation of mir-429 among crc specimens and serum samples, and found that enhanced mir-429 expression was correlated with tumor progression in crc . Moreover, we found that high mir-429 expression was negatively associated with chemotherapy response in crc patients receiving 5-fu - based chemotherapy . Micrornas, acting as post - transcriptional regulators of gene expression, can regulate 30% of the protein coding genes in the human genome . Several studies have identified mir-429 as an oncogene that is involved in the development in early stages of malignancies . It is reported to promote tumor progression in pancreatic cancer; and downregulation of mir-429 was found to significantly inhibited cell growth of endometrial tumors . Recently, cristobal et al . Reported that deregulation of mir-429 indicated its potential relevant role in patients with colorectal cancer liver metastasis, which further indicating the functional role of mir-429 in crc . Consistent with previous studies, our data showed that the mir-429 levels increased statistically significantly in crc specimens compared with corresponding adjacent non - tumorous tissue . We also found that enhanced mir-429 expression was associated with tumor progression, such as tnm stage . Besides, the roc curves revealed a comparative diagnostic value of mir-429 when compared with cea, a traditional biomarker in crc . It is widely accepted that the effective ways to improve the recovery rate and the prognosis of cancer patients are early detection and early treatment . Therefore, it is very important to search for cell - free markers for early diagnosis and prognosis evaluation . Utilizing mirna expression level in peripheral blood to diagnose tumors early is effective and deserves to be explored further because mirna is very stable in blood plasma and serum . Thus, we wondered whether mir-429 is also highly expressed in peripheral blood of crc patients . Our results indicated that mir-429 levels were also significantly increased in serum from crc patients compared with healthy controls, which further validated the potential function of mir-429 in crc patients . 5-fluorouracil is an antimetabolite used as the first - line chemotherapeutic agent for various cancers . However, patients who respond to chemotherapy initially will eventually acquire resistance to these treatments, and the mechanisms underlying such acquired chemoresistance remain unclear . Elucidating the mechanisms of chemoresistance is critical for development of effective therapeutic strategies . The identification of cancer - specific mirnas is critical for understanding the roles of mirnas in tumorigenesis and may be important for defining novel therapeutic targets . Revealed that enhanced mir-129 expression in crc promoted cell death and improved chemosensitivity to 5-fu treatment . Moreover, liu et al . Found that mir-429 was an oncogene regulated by evodiamine and berberine in human crc . These studies indicated that mir-429 might play important roles during chemotherapy in crc . In our research, our novel results indicated that mir-429 was significantly correlated with 5-fu response in crc patients, and exerted its important function in distinguishing the patients with a response to 5-fu treatment from the patients with no response . Moreover, high mir-429 level was positively associated with objective response in crc patients receiving 5-fu - based chemotherapy . These results revealed a diagnostic value of mir-429 for 5-fu - based chemotherapy in crc patients . Crc patients who expressed high mir-429 levels showed a shorter 5-year survival rate than patients with low mir-429 levels . These data, together with further multivariate analyses, suggest that high expression of mir-429 might be a significant independent predictor of poor prognosis for crc patients . Additionally, the survival analysis indicated that overall survival of crc patients with high level mir-429 expression who received 5-fu - based chemotherapy was significantly lower than that of patients with low - level mir-429 expression . Thus, it was concluded that overexpression of mir-429 might be involved in 5-fu resistant phenotypes of human crcs . A limitation of the current study is a lack of a large cohort of samples to establish a strong correlation between the expression of mir-429 and chemo - response to 5-fu - based treatments among crc patients . Future studies with a large cohort of samples based on a multi - center, randomized controlled trial are needed to help identify potential clinical applications of mir-429 in crc patients . Besides, this article focused only on individual phenomena, and further explanation of the underlying mechanism (e.g., mirna target genes) or mechanistic pathways are needed in future studies . In conclusion, this study showed that high mir-429 expression levels in crc tissues and serum was associated with enhanced malignant potential and poor prognosis of crc patients . Furthermore, mir-429 could affect the chemo - sensitivity of crc patients to 5-fu and was associated with poor response to 5-fu - based chemotherapy in crc patients . Thus, mir-429 may be a novel prognostic biomarker and therapeutic target for crc patients . Suppression of mir-429 could be a future direction to enhance chemo - sensitivity to 5-fu - based chemotherapy regimen.
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Stromal fibroblast activation occurs early prior to cancer development . In human pancreatic cancer and mouse models of pancreatic cancer, cafs are present surrounding the high - grade dysplastic lesions in the pancreas and even to a lesser extent in the low - grade dysplastic lesions . Similar findings in hepatocellular carcinoma and oral squamous cell carcinoma and their dysplastic precursor lesions have been found . These data suggest that cancer is not necessary for the transformation of caf, but rather myofibroblast activation occurs earlier in the process of neoplastic progression when dysplasia is present . Palladin appears to play a key role in fibroblast transformation in some cancers, including pancreatic cancer and breast cancer . It is a cytoskeletal protein that acts as a scaffold and serves to crosslink the components of stress fibers, actin bundles, z discs, focal adhesions and other subcellular structures . Palladin is upregulated in the leading edge of wounds and in the cancer - associated fibroblasts of metastatic cancers . Interestingly, palladin has also been detected in expression screens for invasion - specific genes in pancreatic and breast cancer . It is overexpressed in the stromal fibroblasts immediately surrounding low and high - grade dysplasia . The expression of palladin closely correlates with the expression of -sma in these pre - cancerous lesions . Our recent studies to unravel the role of palladin in fibroblast activation in cancer reveal that simply co - culturing a normal human fibroblast next to a pancreatic cancer cell is sufficient to impart myofibroblast properties to the fibroblast; this process occurs in a palladin - dependent fashion (fig . 1). Because myofibroblasts can be detected early in tumorigenesis, we tested whether the initiating event in pancreatic ductal adenocarcinoma, e.g., a k - ras mutation in an epithelial cell, was sufficient to cause transformation of an adjacent resting fibroblast into a myofibroblast . Transwell experiments involving normal fibroblasts co - cultured with normal epithelial cell expressing wild - type or mutated k - ras were performed: activated k - ras (wild - type or mutated) paracrine signaling is sufficient to induce the adjacent, but non - touching, quiescent fibroblasts to become myofibroblasts . Abrogation of palladin, using sirna, causes loss of the myofibroblast phenotype, including loss of common myofibroblast markers such as -sma, and loss of myofibroblast function, such as migration and invasion . Normal human dermal fibroblasts were grown adjacent to pancreatic ductal cells in a transwell plate . Left: fibroblasts grown adjacent to pancreatic cancer ductal cells caused upregulation of palladin (stained red) in the fibroblasts . Right: k - ras expression (either wild type or mutated k - ras) in a normal pancreatic ductal cell was sufficient to upregulate palladin in the adjacent normal fibroblast . Scale bars indicate 20 m . Curiously, palladin - expressing fibroblasts appear to be primed but not activated . The palladin - expressing fibroblasts have the phenotype of a myofibroblast: an elongated shape and expression of -sma and vimentin; however, an inflammatory or wounding signal is required for the myofibroblasts to become migratory and invasive . In absence of the inflammatory signal, the palladin - expressing myofibroblasts remain dormant, with diminished capability for migration or invasion . This finding might be one reason for the underlying inactivity of some indolent tumors, if inflammation is absent the fibroblast - led invasion cannot not occur . In summary, while a palladin - expressing fibroblast is primed, expressing all of the proteins one would expect in a myofibroblast, it does not yet act as a leading partner for cancer cell invasion without the inflammatory signal . In the clinical setting, such inflammation could be driven from environmental factors such as smoking, infections or inflammatory cytokines associated with obesity . Myofibroblasts can produce tracks within the extracellular matrix, which in effect, create tunnels for the carcinoma cells to follow . More recently, we have identified the mechanism of how this fibroblast - led cancer invasion occurs . Upregulation of palladin causes the fibroblast to develop a fusiform / mesenchymal shape with apparent invadopodia feet that contain proteolytic enzymes (fig . 2). To identify the contents of the feet of palladin - activated myofibroblasts, we ensnared the myofibroblasts in the act of invasion in a sieve that was large enough to let the feet through, but too small for a whole cell to pass through . Proteomic analysis performed on the ensnared and isolated feet revealed the overexpression of proteolytic enzymes such as metalloproteinases and cathepsin, invadopodia proteins and proteins associated with poor prognosis in cancer . Functional studies demonstrated that, in the setting of an inflammatory or wounding signal, the palladin - activated fibroblasts can both rip and destroy the extracellular matrix literally creating tunnels through which the cancer cells follow . Fibroblasts without palladin expression have markedly diminished capacity to create tunnels and cancer cells do not follow them (fig . 3). Remarkably, once the activated myofibroblasts escort the cancer cells through tunnels in the organ of origin, labeling studies have shown that the cancer cell and myofibroblasts invade together through blood vessels and implant in metastatic sites . In support of these studies, we found palladin - expressing fibroblasts adjacent to cancer cells in lymph node and liver metastases . Figure 2 . Normal human fibroblasts transfected with an empty vector (ev) remained boxy in appearance and had no effect on collagen or matrigel when exposed to wounding media (a and b). In contrast, fibroblasts transfected with wild - type palladin (wt) became elongated with mesenchymal features (c), caused destruction of the collagen matrix (d) with apparent clumping of the collagen edges . Additionally, palladin - expressing fibroblasts created tunnels in matrigel (stained red) when exposed to wounding media in 3d invasion cultures (e). The cartoon in (a) depicts the 3d invasion culture chamber: two wells, one containing fibroblasts and cancer cells (right) and the other filled with chemoattractant egf (left) are separated by a chamber filled with matrigel . Fibroblasts without palladin (b) and with palladin (c) were co - cultured with pancreatic cancer ductal cell line, panc-1, over a period of 72 h. fibroblasts were stained with white q - dots and cancer cells were stained pink . Note in (c), the palladin - expressing fibroblasts tunneled through the matrigel and were followed by the pink cancer cells (arrow heads) as the cells moved toward the egf . In (b), pancreatic cancer cells remained at the baseline and did not invade in the 3d cultures when the fibroblasts did not express palladin . Elegant studies by rhim et al ., using lineage tracing in a pancreatic cancer engineered mouse model, revealed that mutant ductal cells undergo epithelial mesenchymal transformation (emt), invade into the blood stream, and lodge into metastatic sites such as the liver prior to histologic evidence of cancer . The invasion of these mutant epithelial cells occurs in 2.7% of all panin 2 (low - grade dysplasia) and 6.8% of panin 3 (high - grade dysplasia) lesions, but never in the setting of panin 1 (hyperplasia). Not surprisingly, cox-2, an inflammatory mediator, is increasingly overexpressed between panin lesions and malignant pancreatic tissues . In the studies by rhim, if dexamethasone was added as an anti - inflammatory agent, the dissemination of mutated circulated pancreatic epithelial cells was abolished . Even more amazing was the loss of panin lesions and associated myofibroblasts within the pancreatic parenchyma in the setting of dexamethasone taken on a daily basis: the pancreata return to a normal appearance, while the control mice proceed to get pancreatic adenocarcinoma . Taken as a whole, this work implicates the invasion of mutated cells earlier than originally thought in cancer and would help explain the very lethal nature of some cancers, such as pancreatic, even when the tumors are quite small . The early activation of fibroblasts into tunneling myofibroblasts by k - ras mutated epithelial cells fits in mechanistically with the model of earlier invasion of epithelial cells prior to cancer formation . Because of the interdependent behavior of cancer cells and stromal fibroblasts, the latter have become a target of interest for oncologists . Pancreatic cancer cells have increased resistance to gemcitabine, in part due to direct activation of the hedgehog pathway resulting from cross - talk between myofibroblasts and adjacent cancer cells . Recent chemotherapy using a hedgehog inhibitor results in a significant loss of the tumor - associated fibroblasts in pancreatic cancer and prolonged survival in mouse models . This finding, combined with the negative outcome of a recent human phase iii clinical trial testing the efficacy of chemotherapy and hedgehog pathway inhibition, suggests that compensatory pathways may exist if only one pathway in the targeting of caf is abrogated . This is particularly of issue because there are usually myofibroblasts remaining at the surgically resected pancreatic cancer margins and the sources for tumor stromal fibroblasts may be derived from both local and potentially non - local sources . Other methods of directly targeting the caf have included use of monoclonal antibodies, drugs, and vaccines . A novel monoclonal antibody targeting fibroblast activation protein (fap), a cell surface protease of activated tumor fibroblasts, has been shown to induce long - lasting inhibition of tumor growth and complete regression in xenograft models of lung, pancreas, and head and neck cancers . Vaccination against stromal fibroblasts targeting fap has also shown some promise in mouse models . While no current therapy targets palladin expression in fibroblasts, we have performed preliminary studies suggesting that reagents that target anti - sma and regulate hepatic stellate cell activation, (such as ppar agonists and metformin) are useful when used in combination to block palladin expression in myofibroblasts and human caf . These combined palladin - targeting therapies concomitantly block -sma expression and the myofibroblast phenotype (unpublished data). Further investigation using small molecular and high - throughput drug screening is required to identify which drugs are most effective in blocking palladin and whether these drugs are effective in the early and the late stages of cancer in mouse models . Therapies to decrease the inflammatory component of carcinogenesis have included use of aspirin, cox2 inhibitors such as celcoxib, nfb inhibitors such as curcumin and ppar agonists such as troglitazone . Some of these anti - inflammatory drugs have been used in human phase ii trials with mixed success, where many of the patients had later stage tumors . With our current knowledge of the role of inflammation in driving forth the early dissemination of myofibroblast - aided cancer cells, it is possible that the inflammation needs to be treated earlier in the neoplastic progression before the cancer cells have escaped . In keeping with this concept, cross - talk between fibroblasts and cancer cells is essential to invasion and potentially chemotherapeutic agents that disrupt this process could be effective . Inflammatory cytokines and signaling molecules including tnf, il-6, il-1/, nfb and tgf- play key roles in paracrine signaling between tumor cells and fibroblasts, as outlined in an excellent review by bhomick and moses . Therapeutics designed to modulate these molecules are described elsewhere; in general most of these therapies are relatively new and thus trials of some of these agents are just being undertaken in humans . Figure 4 summarizes steps and mechanism of myofibroblast activation and the partnership these cells play in cancer invasion . Although difficult, targeting the stromal fibroblasts remains an attractive strategy in the fight of aggressive cancers because of the interdependence of the caf and the cancer cells . In addition, it may be valuable to determine when during the neoplastic process targeting the caf is most effective: in the initiating stages of tumorigenesis or whether the strategy can be effective in an established, even metastatic cancer . K - ras activation in ductal cells leads to paracrine signaling that is sufficient to induce palladin - associated myofibroblast transformation of the adjacent fibroblasts . This event occurs early in tumorigenesis, when epithelial cells are dysplastic, and increases with neoplastic progression . In the setting of a wounding signal, the palladin - activated fibroblasts develop cellular protrusions (feet) that express invadopodia proteins, proteases and enhance the capacity for invasion . The palladin - activated fibroblasts create tunnels through the matrix, assisting the escape of cancer cells into the neo - vasculature . The activated fibroblasts appear to accompany the cancer cells to their metastatic niche in breast and pancreatic cancer models . The arousal of the stroma is a key and transformative event in the invasive stages of tumorigenesis in many solid tumors . Stromal fibroblasts can be transformed through paracrine signaling of adjacent k - ras overexpressing epithelial cells . The fibroblast then undergoes phenotypic change into a myofibroblast that is mediated through palladin, a cytoskeletal protein essential in cell motility . However, this change is insufficient to cause fibroblast - assisted cancer cell invasion and migration . For the latter events to occur, the presence of these three events (overexpression of k - ras, palladin - expression in the fibroblasts and inflammatory signal) instigates the dynamic relationship between the stroma and the mutated epithelial cell . These three events are sufficient for the activated myofibroblast to tunnel through the extracellular matrix and provide avenues for the dysplastic and cancerous epithelial cells to follow . Future studies will help elucidate the role of epithelial - mesenchymal transition to enhance the migration of cancer cells through the fibroblast - created tunnels and the potential for chemotherapeutic targeting of the initiating events in cancer invasion.
Trauma is no more considered accidental but a preventable epidemic with a unique pattern of host, agent, and environment working in unison to produce injury . Traumatic injuries are associated with significant morbidity and mortality and are of particular relevance currently with technological sophistication in all spheres of life and upsurge in armed conflicts globally . An injury is said to be a bodily lesion at organic level resulting from acute exposure to energy in amounts that exceeds the threshold of physiologic tolerance or the absence of such essentials as heat or oxygen . Injuries accounted for 16% of the global burden of disease and an estimated 5.8 million deaths in 1998 with injury - related mortality predicted to increase by 40% between 2002 and 2030 . Mortality statistics, in isolation, do not adequately depict the magnitude of injury as for each death there are many more injuries that resulted in hospitalization, treatment in emergency departments, treatment by practitioners outside the formal health sector, or never received treatment at all . To accurately characterize the burden of injury, nonfatal outcomes are also measured by using disability - adjusted life years (dalys) which combine the number of years of life lost from premature death with the loss of health from disability among persons with nonfatal injuries . Road traffic accidents, falls, assaults, firearm injuries, burns, sports injuries, animal bites, and industrial accidents are some causes of of trauma . Deaths from road traffic accidents are predicted to increase from 1.2 million in 2002 to 1.9 million in 2020 globally, to become the third leading cause of dalys losses . Increasing waves of terrorism and civil conflicts are expected to lead to a surge in violence - related injuries . Trauma care in most developing nations is at infancy level despite the fact that about 90% of injury deaths occur in these nations . Dearth of trauma centers, nonexistent ambulance services and prehospital care, and unavailability of data on trauma are some of the impediments to trauma management in nigeria . Trauma has been recognized to be preventable over the past few decades and methods for the scientific study of injury prevention have been established . However, the development of effective trauma prevention measures depends on reliable and detailed information on the characteristics and pattern of injury . The aim of this study is to elucidate the pattern and characteristics of trauma at benue state university teaching hospital (bsuth), makurdi, nigeria . This was a hospital - based study of trauma patients of all age groups and gender who presented to the accident and emergency (a and e) department of bsuth, makurdi, nigeria from january to december 2013 . Bsuth is a tertiary care and teaching hospital for the benue state university and is located along the shores of river benue . There is no trauma center in the city as such most seriously injured patients are referred to the facility . It provides trauma care for inhabitants of the city and surrounding areas of north central nigeria . The case records of all patients seen with injuries at the a and e department during the study period were retrieved . Demographic data, types of injuries sustained, causes and circumstances of injuries, as well as outcome of treatment were extracted from the case files and entered onto a computerized questionnaire . Data were analyzed using the software statistical package for social sciences for windows version 15.0 (spss inc; chicago, illinois). Descriptive statistics were used to display single variable quantities using means and standard deviations (sd) for continuous variables or proportions for categorical variables unless otherwise stated . This was a hospital - based study of trauma patients of all age groups and gender who presented to the accident and emergency (a and e) department of bsuth, makurdi, nigeria from january to december 2013 . Bsuth is a tertiary care and teaching hospital for the benue state university and is located along the shores of river benue . There is no trauma center in the city as such most seriously injured patients are referred to the facility . It provides trauma care for inhabitants of the city and surrounding areas of north central nigeria . The case records of all patients seen with injuries at the a and e department during the study period were retrieved . Demographic data, types of injuries sustained, causes and circumstances of injuries, as well as outcome of treatment were extracted from the case files and entered onto a computerized questionnaire . Data were analyzed using the software statistical package for social sciences for windows version 15.0 (spss inc; chicago, illinois). Descriptive statistics were used to display single variable quantities using means and standard deviations (sd) for continuous variables or proportions for categorical variables unless otherwise stated . There were 203 (81.2%) males and 47 (18.8%) females with a male to female ratio of 4.3:1 . Their ages ranged from 3 to 74 years with a mean of 32 13.1 years . Sex distribution of patients with respect to age groups unintentional injuries were the predominant form of trauma (n = 209, 83.6%). Road traffic accidents were the most common cause (n = 180, 72.0%). This was followed by gunshot injuries (n = 21, 8.4%) and assault (n = 20, 8.0%). Etiologies of trauma with respect to age groups the extremities were the most commonly injured body region (n = 148, 43.5%). This was followed by the head (n = 112, 32.9%), chest (n = 46, 13.5%), abdomen (n = 16, 4.7%), spine (n = 10, 2.9%), and pelvis (n = 8, 2.4%). Open wounds (abrasions and lacerations) were the most common injury type sustained (n = 95, 28.2%). Isolated injuries occurred in 166 (66.4%) patients, while 84 (33.6%) patients were multiply - injured . One hundred and twenty - one patients (48.8%) sustained severe / profound injuries . Distribution of injuries sustained by patients the majority of patients (n = 138, 55.2%) were admitted and treated in the a and e, 110 (44.0%) in the general / pediatric surgical and orthopedic wards and two (0.8%) in the intensive care unit . Of the 250 patients, 142 were treated surgically of which surgical wound debridement (n = 90, 63.3%) was the most common procedure carried out . Length of hospital stay ranged between 1 and 160 days with a mean duration of about 8 days . Mortality pattern of traumatized patients majority of patients (n = 133, 53.2%) were treated and discharged without permanent disability . Eleven (4.4%) were discharged with permanent disability (paraplegia, limb loss, etc . ), 56 (22.4%) opted for discharge against medical advice, while 12 (4.8%) were referred . There were 203 (81.2%) males and 47 (18.8%) females with a male to female ratio of 4.3:1 . Their ages ranged from 3 to 74 years with a mean of 32 13.1 years . Unintentional injuries were the predominant form of trauma (n = 209, 83.6%). Road traffic accidents were the most common cause (n = 180, 72.0%). This was followed by gunshot injuries (n = 21, 8.4%) and assault (n = 20, 8.0%). Etiologies of trauma with respect to age groups the extremities were the most commonly injured body region (n = 148, 43.5%). This was followed by the head (n = 112, 32.9%), chest (n = 46, 13.5%), abdomen (n = 16, 4.7%), spine (n = 10, 2.9%), and pelvis (n = 8, 2.4%). Open wounds (abrasions and lacerations) were the most common injury type sustained (n = 95, 28.2%). Isolated injuries occurred in 166 (66.4%) patients, while 84 (33.6%) patients were multiply - injured . One hundred and twenty - one patients (48.8%) sustained severe / profound injuries . Distribution of injuries sustained by patients the majority of patients (n = 138, 55.2%) were admitted and treated in the a and e, 110 (44.0%) in the general / pediatric surgical and orthopedic wards and two (0.8%) in the intensive care unit . Of the 250 patients, 142 were treated surgically of which surgical wound debridement (n = 90, 63.3%) was the most common procedure carried out . Length of hospital stay ranged between 1 and 160 days with a mean duration of about 8 days . Majority of patients (n = 133, 53.2%) were treated and discharged without permanent disability . Eleven (4.4%) were discharged with permanent disability (paraplegia, limb loss, etc . ), 56 (22.4%) opted for discharge against medical advice, while 12 (4.8%) were referred . The burden of trauma in this study is mostly borne by young males, a finding similar to those from other studies . Male predominance is likely to be due to the fact that males are more involved in risk - taking activities and more exposed to the elements being the main revenue - earners in families . Involvement of the active and productive segment of the society suggests huge losses to the economy . The impact of trauma on this group may be reduced by incorporating preventive measures into children's school curriculum so as to particularly target boys in their formative years . Road traffic injuries are a major cause of death and disability globally, with a disproportionate number occurring in developing countries . They were the leading cause of injury in this study in a similar manner to a number of studies and accounted for over 70% of the mortality . Developing countries accounted for more than 85% of all deaths due to road traffic crashes globally in 1998 . Reasons put forward for this high burden of road traffic accidents in developing countries include growth in motor vehicle numbers, poor enforcement of traffic safety regulations, and poor access to health services . Passengers and pedestrians constituted the majority of road traffic injured patients in this study in keeping with studies from other developing countries . This is in contradistinction to findings from the united states where drivers are the majority of the injured . This may be because of frequent crashes involving multi - passenger vehicles like buses and trucks as well as nonexistent pedestrian walkways on most roads in low - income countries . Firearms are the most destructive of readily available weapons in modern society and injuries resulting from them are associated with a high morbidity and mortality . Firearm injuries were the second leading cause of trauma in this study constituting over 20% of overall causes . The high rate of gunshot injuries in this study is likely to be due to an armed conflict between nomadic herdsmen and local farmers over rights to grazing land in the study area . Designating special grazing zones along the seasonal migration route of the herdsmen and establishing open channels for conflict resolution may go a long way to help mitigate future occurrences . Snake bites constituted the most fatal of all the causes of trauma in this study with about 40% mortality rate . Most patients got bitten while working on farms in remote areas and late presentation to hospital prevented early institution of treatment . Provision of personnel and facilities to administer anti - snake venom at primary healthcare centers in the countryside before referral to tertiary centers may help improve the outcome of treatment . Use of approved safety helmets by at - risk individuals like construction workers and motorcyclists is known to reduce the incidence traumatic head injury . In fact, severe head injury among motorcyclists is said to be a reflection of low usage of helmets . The high mortality from head injuries in this study may be due to the culture of poor usage of safety helmets that is prevalent in developing countries . Trauma in makurdi is a predominantly young adult male occurrence with road traffic accidents being the leading etiological factor.
The number of people with disabilities is globally increasing, mainly because of their higher survival rates due to advances in medical and social care services . As a consequence of the increased number of people with disability, research has shown that people with disabilities, in general, and intellectual disabilities, in particular, experience poor health and inferior access to high quality health services compared to the normal population . Individuals with intellectual disabilities may suffer more from the consequences of oral disease than those without any intellectual disabilities due to their inability to accurately communicate their symptoms and their caregivers inability to appraise their oral health condition . In addition to causing physical pain and discomfort, oral disease often results in negative consequences regarding self - esteem, quality of life, and general health . Studies have shown that individuals with disabilities, including those with intellectual disabilities, are at a higher risk of experiencing poor health and increased age - adjusted mortality compared to the normal population, highlighting the presence of significant health inequalities . While the epidemiology of dental disease in people with an intellectual disability has not been extensively studied, several studies have indicated high rates of dental disease in this population, reportedly being the most unattended health problem . Assessments of oral health in the intellectually disabled individuals (i d) residing in developing countries are extremely scarce, however, the situation in lebanon in particular has received no research attention whatsoever . Despite the presence of a growing population of more than 27000 i d individuals in lebanon, data regarding their oral health conditions is lacking . The aim of this study is to compare the oral health of the institutionalized i d lebanese population with existing data on the normal lebanese population (nlp). A total of 652 institutionalized lebanese individuals with intellectual disabilities residing in the 5 main lebanese governorates were assessed as part of a national cross - sectional study that was carried out between november 2015 and april 2016 . Participants belonged to four age groups, i.e. 6, 12, 15, and 3544 years old . Ethical approval was provided by the ethical research committee at the faculty of medical sciences of the lebanese university in addition to the ministry of social affairs in lebanon . All participants parents or legal guardians provided permission in the form of written consent prior to enrolment in the study . Individuals who were absent or whose cooperation prevented adequate data collection were excluded from the study . The sample size was not calculated a priori because the aim was to collect data from all institutionalized individuals . All registered institutions for the mentally disabled across the lebanese territory approved their participation except one located in mount lebanon . Housing a total of 703 individuals belonging to the targeted age groups, a total of 689 guardians provided consent (98% response rate), but 14 disabled individuals were absent and 37 were uncooperative, resulting in a final sample of 652 participants (final response rate of 92.7%). Clinical information was collected through an oral examination, and data on background characteristics and behavioral factors were collected via a questionnaire filled by each participant's primary caretaker [annex1]. The questions explored background characteristics (age, sex, and governorate of residence), arrangement with the institution, characteristics of the primary caretaker (relation to the disabled person and education), the type of disability and severity, oral hygiene practices, and dietary habits (intake of sugar and unhealthy foods). During oral examination, carious indices and periodontal disease were recorded according to the methods outlines in the world health organization's (who) oral health surveys (1997). The number of decayed, missing, and filled permanent teeth (dmft) was recorded and the periodontal condition was assessed using the community periodontal index for treatment needs (cpitn). All oral examinations were performed by one examiner (hd) in appropriate lighting conditions, using plane mirrors, explorers, and the who periodontal probe . The comparison population was derived from the national oral health survey carried out in lebanon in 1994, which represents the most recent epidemiological study of the oral health of the normal lebanese population that provides data on 1877 individuals from all age groups targeted by this study . Means and standard deviations were generated for the five outcomes (per individual): the number of (1) decayed (d); (2) missing (m); and (3) filled teeth (f); in addition to (4) the total dmft score and (5) the cpitn score . The outcomes for the number of decayed and filled teeth in addition to the total number of dmft include pooled data for both primary and permanent teeth, however, the number of missing teeth refers to missing permanent teeth only . For the comparison population, data was available on the mean values (standard deviations not reported) for the separate caries indices and the overall dmft score for ages 6, 12, 15, and 3544 years, mean dmft score across genders and governorates for all ages except 6 years, and periodontal indices for all ages of interest . The statistical package for the social sciences software was used to carry out all data computations (ibm spss statistics 20.0, usa). Clinical information was collected through an oral examination, and data on background characteristics and behavioral factors were collected via a questionnaire filled by each participant's primary caretaker [annex1]. The questions explored background characteristics (age, sex, and governorate of residence), arrangement with the institution, characteristics of the primary caretaker (relation to the disabled person and education), the type of disability and severity, oral hygiene practices, and dietary habits (intake of sugar and unhealthy foods). During oral examination, carious indices and periodontal disease were recorded according to the methods outlines in the world health organization's (who) oral health surveys (1997). The number of decayed, missing, and filled permanent teeth (dmft) was recorded and the periodontal condition was assessed using the community periodontal index for treatment needs (cpitn). All oral examinations were performed by one examiner (hd) in appropriate lighting conditions, using plane mirrors, explorers, and the who periodontal probe . The comparison population was derived from the national oral health survey carried out in lebanon in 1994, which represents the most recent epidemiological study of the oral health of the normal lebanese population that provides data on 1877 individuals from all age groups targeted by this study . Means and standard deviations were generated for the five outcomes (per individual): the number of (1) decayed (d); (2) missing (m); and (3) filled teeth (f); in addition to (4) the total dmft score and (5) the cpitn score . The outcomes for the number of decayed and filled teeth in addition to the total number of dmft include pooled data for both primary and permanent teeth, however, the number of missing teeth refers to missing permanent teeth only . For the comparison population, data was available on the mean values (standard deviations not reported) for the separate caries indices and the overall dmft score for ages 6, 12, 15, and 3544 years, mean dmft score across genders and governorates for all ages except 6 years, and periodontal indices for all ages of interest . The statistical package for the social sciences software was used to carry out all data computations (ibm spss statistics 20.0, usa). For all comparisons subgroups (gender, governorate, and overall), mean dmft scores exhibited a general increasing trend with increasing age in both the i d and the nlp [table 1]. For all age groups, overall dmft score was larger in the i d than in the comparison group . The difference was smallest in the adult group (mean dmft = 12.71 in i d and 14.68 in nlp) and largest for the 15 year olds (mean dmft = 4.80 in i d compared to 8.09 in nlp). When comparing males and females, scores were generally similar in both the i d and the nlp with only minor differences . Some exceptions were noted; in the i d group aged 15 years, females had larger dmft scores (5.46 4.40) compared to males (4.27 4.56); adult males in the i d group had larger dmft scores (13.58 7.77 and 11.92 7.08, respectively), and adult females in the nlp had larger dmft scores than adult males (15.44 compared to 13.84, respectively; table 1). Dmft scores compared between the institutionalized intellectually - disabled and the normal lebanese population similar to the i d group, dmft scores in the glp were generally the highest in the south, although at age 15 the dmft scores among the i d were similar or higher for all governorates except for mount lebanon [table 1]. Similarly, among 12-year - old children with i d, dmft scores were highest in beirut rather than in the south (5.69 3.23 and 5.04 4.35, respectively). Six - year - old children with i d had lower numbers of primary decayed teeth (3.28 4.45) but more filled teeth (0.22 0.98) than the nlp (4.90 and 0.1, respectively; table 2). For the remaining ages, the mean numbers of permanent decayed, missing and filled teeth were lower among the i d than the nlp with the following exceptions: similar numbers of missing teeth were recorded at age 15 and larger mean numbers of filled and missing teeth were recorded among the i d at age 3544 years (6.24 7.02 missing teeth and 3.31 4.56 filled teeth compared to 4.98 and 2.50, respectively, in the nlp; table 2). Separate caries indices compared between the institutionalized intellectually - disabled and the general lebanese population the mean numbers of missing and filled teeth showed similar patterns across ages in the i d and nlp; slowly increasing up to the age of 15 and then exhibiting a sharp increase at the age of 3544 years [table 2]. This trend was similar to that observed for the overall dmft score, which increased gradually up to age 15 in both i d and the nlp and then increased sharply at age 3544 years (12.71 7.43 in i d and 14.68 in nlp; table 1). The number of decayed teeth, on the other hand, displayed a gradual increase from age 6 (mean = 4.90) to 3544 years (mean = 7.20) in the nlp but showed no such trend in the i d [table 2]. In the disabled population, the number of carious teeth was lowest in the adult group (3.17 4.11) and was highest among 15 year olds (4.01 4.28). In both the populations, cpitn scores worsened with age [table 3]. There were some differences, however, in the distribution of periodontal disease between ids and nlps . Among the nlp, the highest prevalence was for the presence of calculus, both at age 15 (42.7%) and at 3544 years (51.0%). The prevalence of health periodontium and bleeding on probing was larger in the i d group than the nlp (30.9% compared to 24.4% and 34.2% compared to 29.5%, respectively), however, the prevalence of pockets of 45 mm depth was also larger in the i d population (11.0% compared to 4.6% for the overall populations). This difference is especially apparent for 15 year olds where the prevalence of pocketing (45 mm) was 7.2 in the i d compared to 3.2% in the nlp . With respect to deep pocketing greater than or equal to 6 mm, there were almost similar proportions within each population, though the proportion in the i d group (overall) was slightly higher (3.7% compared to 1.5%, respectively; table 3). Estimations suggest there are over a billion people living with disability, representing approximately 15% of the world's populations . In the majority of cases, pitied by the society, disparaged, ignored, isolated, and hidden away in institutions, they are often deprived of basic and essential healthcare services . Oral health has been reported to be the most unattended health need among the disabled, the provision of which remains a challenge in the 21 century . Individuals with intellectual disability have been found to suffer from dental disease up to seven times as frequently as the general populations, particularly with respect to periodontal disease, oral mucosal pathology, and malocclusion . In a population study of adults with an intellectual disability, beange et al . (1995) noted that 86% of the participants suffered from dental disease, representing the most frequent health problem for this group . The data of this study represents the first assessment of oral health in the i d population in lebanon and is the results of an exhaustive survey that was conducted in an attempt to include all registered institutionalized handicapped persons in the country . Several studies have highlighted higher prevalence rates and severity of oral disease among the i d populations when compared to the general population . When compared to healthy individuals of the same age, most studies show that individuals with intellectual disability have poor oral hygiene and higher prevalence of gingivitis and periodontitis . While some studies have shown higher disease prevalence in individuals with disability, other studies have reported similar or even lower decay rates in healthy individuals of the same age . Shaw et al . (1986) reported very similar dmft scores for disabled and nondisabled children at the ages of 8, 12, and 15 years . Choi et al . (2003) even reported lower dmft / dft scores and a higher prevalence of gingivitis among the disabled than that in nondisabled persons . Our study similarly illustrated lower mean dmft / dft scores among the institutionalized i d participants in comparison to the normal population for all age groups assessed . However, the comparison study used as a reference to represent the normal lebanese population was undertaken in 1994, two years after the end of the civil war which started in april 1975 and lasted 17 years . This war left significant detrimental impact on the economic and social situation as well as on the health sector . It has been suggested that the elevated caries indices reported for the nlp are reflective of the war circumstances . It may, therefore, be argued that the results of the 1994 study may not be reflective of the situation in lebanon today . This study is nonetheless the most recent study to assess both dmft scores and the periodontal condition among children and adults comparable to those assessed by our studies . Although scarce, few other studies have since assessed some aspects of oral health in various age groups . Shortly after the national oral health survey, doughan et al . Reported even higher dmft scores among adults examined in the year 1996 at 16.3 teeth . The high score was mainly explained by the lack of a fluoridation program at the national level and poor dietary habits . In another similar based on data collected in 2000, dmft scores were 3.42 and 5.44 in 12 and 15 year olds, respectively, lower than that in 1994 and closer to the values obtained in the i d population in our study . The authors of this study explain the drop in dmft scores either due to improved oral hygiene or changes in the examination criteria, having followed closely the who criteria, recorded only obvious lesions, and avoided the use of sharp dental explorers to explore potential cavities . These procedures are more in line with those utilized in this study and the results are therefore more comparable . Following more than a decade lacking research on the status of oral health in lebanese children and adults, hanna et al . (2015) reported mean dmft scores of 7.30 and 3.50 in 611-year - old children attending public and private schools, respectively . Unfortunately, neither overall dmft score nor data by age are reported, thereby limiting comparisons . In a recent unpublished thesis, dmft scores reported for adolescents aged 1218 were 5.83 and 4.08 in public and private schools, respectively . Although the wide age group limits comparability to our data, the values reported for this adolescent sample are somewhat similar to the scores recorded among our 12 and 15-year - old i d population . Apart from the overall dmft scores, i d children aged 12 and 15 years received less restorative treatment (filled teeth) than those of the same age in the comparison study from the nlp . In addition, the number of missing teeth was larger in the i d population than that in the nlp for adults . These results support increasing research pointing to the reduced access of disabled persons to the needed oral health care services due to various barriers such as the disabled person's inability to communicate their symptoms and poor cooperation with treatment . Compared with representative data on the oral health of german adults, individuals with intellectual disabilities had more extracted teeth than normal adults, and the same was reported among german athletes with i d . In other countries, it was also observed that, due to the reduced cooperation, the dental treatment for persons with intellectual disabilities consisted mostly of extractions . This phenomenon may be the result of the tendency for emergency extractions (because of delayed seeking of treatment) or due to difficulties in behavioral management during dental procedures favoring measures that are less technique sensitive such as the extraction of teeth rather than their restoration . Despite lower dmft scores among the assessed i d population when compared to the historical comparison, the results nonetheless represent poor oral health compared to who standards and compared to the global situation . Among the assessed i d population, both the mean dmft score of 4.28 estimated at age 12 and the mean score of 12.71 estimated at age 3544 years are considered at the higher end of a moderate dmft score, the cut - off values for severe being 4.4 and 13.9 for 12 and 3544 year olds, respectively . Using the age of 12 as a benchmark for comparisons of global dmft, studies have shown a definite trend of decreasing dmft scores globally from 2.43 in 1980 to 1.86 in 2015 . The dmft score estimated in the i d lebanese population at age 12 is higher even compared to estimates in the eastern mediterranean region (dmft = 1.64). Adolescents and adults with i d in our study had more severe periodontal disease and reduced levels of calculus compared to the nlp . This is unusual because in the normal population calculus is usually associated with severe periodontal disease . However, lower levels of calculus for i d individuals have previously been reported, especially in individuals with down syndrome . Research has also shown that people with down syndrome and other disabilities are more susceptible to periodontal disease and have a greater need for oral hygiene compared to the general population . Therefore, the reason behind the comparatively lower proportion of participants with only calculus in the i d population may simply be explained by greater progression to more severe periodontal diseases and is supported by the greater prevalence of pocketing among the i d than the nlp . Reduced manual dexterity and subsequent difficulties in tooth brushing may be the cause of poor health condition in this group . In fact, they often depend on other people such as parents or caregivers for oral health hygiene practices, in contrast to individuals without disabilities who usually take care of their own oral health . Studies are increasingly demonstrating the ability to achieve lower dmft scores in the disabled than that in the normal population in the presence of adequate preventive measures, assisted care, and public dental health services . Our results may or may not support this observation because definite conclusions are limited due to the greater than 20-year difference between our i d study population and the comparison group . If the results of the first national oral health survey hold true today, they may indicate that the institutionalized i d population in lebanon is receiving some form of oral hygiene instruction comparable to or even better than the normal population (though still lacking compared to the global standards). This, however, gives no indication regarding the oral health of the i d outside facilities, an area of research that needs to be planned as part of future comprehensive assessments of oral health in various segments of the lebanese population . It is also possible that the data from 1994 is reflective of a specific social, economic, and political post - war context, and is in fact worse that the current situation, in which case oral health among the institutionalized i d may be similar to or worse than the nlp . Again, the lack of information warrants future national surveys conducted in the same age groups to produce meaningful data that will allow more accurate comparisons with historical data on the nlp and with our work on this vulnerable segment of the population . Although our data cannot be generalized to the entire i d population (residing outside of institutions) and cannot also be generalized to age groups other than those assessed, our study is the first to assess the oral health in the i d in lebanon and to attempt comparison with the nlp . The lack of recent data on the nlp is reflective of the inadequate allocation of resources towards the assessment of oral health in the lebanese population in general, and in vulnerable populations such as the i d, in particular . The elevated levels of poor periodontal health recorded in the i d population, even when compared with information on the nlp from a vulnerable post - war period, warrant public health interventions that raise awareness on the importance of adequate oral hygiene practices among the i d population . Parent and caretakers poor understanding of the importance of health management and their crucial role in its provision, the use of anticonvulsant medications that affect gum health, and the fear of dental procedures are all factors that promote poor oral health among individuals with disabilities . Family members and caretakers are especially crucial in the oral hygiene process of dependent i d individuals both in the provision of assisted brushing and in monitoring the need for professional dental care because i d individuals are often unable to communicate their oral health needs or express their symptoms accurately . Caretakers must be encouraged regarding involvement in preventative oral health measures including tooth brushing, flossing, mouth rinsing with 0.2% chlorhexidine solution, and frequent prophylactic dental care . Practicing and future dentists must also be educated regarding the proper management of this vulnerable population through continuing education course and the curriculum changes in dental schools . Poor oral health indices and particularly poor periodontal conditions were highlighted in the i d population in lebanon . If adequate services are to be provided, policy changes and implementation must comply with the findings of this study . Future prospective studies should be conducted to attempt comprehensive assessments of the oral health of the normal lebanese population in addition to vulnerable populations to provide comparable data that is crucial for the policy changes and resource allocation.
The cardiac syndrome of isolated, left ventricular noncompaction is characterized by a persistence of the embryonic pattern of a highly trabeculated myocardium in the left ventricle.1 this condition can be familial or sporadic and can be due to a variety of mutations in any one of the following proteins: mitochondrial, cytoskeletal, z - line or sarcomeric.1 chin et al2 were the first to describe the entity and the complications of heart failure, ventricular arrhythmias and thromboembolic events . An association with wolff - parkinson - white syndrome was also noted.2 this condition can present during early life as fetal hydrops1 or as sudden infant death syndrome due to neonatal heart failure and/or ventricular fibrillation.1,36 we present a case report of a three month old male infant who presented with sudden infant death syndrome . This three month old non - caucasian died suddenly and unexpectedly at his day care centre . He did not have any known medical problems and no surgical procedures were ever performed . No known allergies were present and no known family history of sudden, unexpected death were present . Unfortunately the family members were lost to follow up, before electrocardiographic and echocardiographic screening could be performed to assess the risk for sudden unexpected death . Postmortem examination of the heart revealed numerous apical trabeculations of the left ventricle (see fig . The left ventricular wall thickness measured 1 cm and the histological assessment was perfectly normal . No abnormalities were detected in any other organs during the postmortem examination and no thrombi were detected in the arterial system or the left ventricle . Current consensus on the mechanism of left ventricular noncompaction is that an arrest of myocardial maturation occurs during embryogenesis.7 before the eighth week of fetal life the myocardium consists of a network of fibres, washed by deep recesses, communicating with the left ventricular cavity.1 the reason for this is that there is no coronary vasculature yet, therefore the trabeculations of myocardial tissue increases the myocardial surface area in contact with the ventricular lumen, as the myocardium is nourished directly from the endoventricular column of blood at this stage.1 the coronary vasculature develops during the fifth to eighth week of fetal life and after this the mesh - work of myocardial fibres will become compacted.1 this compaction process advances from the base of the heart to the apex and from the epicardium to the endocardium as this is the direction of coronary arterial development.1 various echocardiographic criteria have been proposed for the diagnosis of left ventricular noncompaction.2,8 in reaction to these proposed echocardiographic criteria stllberger et al1,9,10 named this peculiar left ventricular phenotype left ventricular hypertrabeculation and defined the condition as the presence of more than three trabeculations in the left ventricle in a location distal (apical) to the papillary muscles . A postmortem study by boyd et al1,11 in 474 normal human hearts have shown that while 68% of these hearts displayed prominent trabeculations, only 4% of these hearts had more than three and none had more than five trabeculations . Left ventricular noncompaction / hypertrabeculation can occur in a sporadic or familial form and both types can be due to a variety of mutations in various sarcomere, mitochondrial, z - line or cytoskeletal proteins.1,1215 figure 1 clearly demonstrates more than three trabeculations in the left ventricle in a location apical to the papillary muscles . According to boyd et al11 this case which presented as a sudden infant death syndrome thus fulfills the criterion for left ventricular hypertrabeculation . The association of hypertrabeculation / noncompaction with various sarcomere mutations supports the concept that this entity is a cardiomyopathy and furthermore, that there is a spectrum from hypertrophic cardiomyopathy, especially apical hypertrophic cardiomyopathy, dilated cardiomyopathy and hypertrabeculation / noncompaction.14 the natural history of left ventricular hypertrabeculation / noncompaction varies widely . Presentation with heart failure, arrhythmias and/or thromboembolism is described in patients of all ages.15 mild cases may remain asymptomatic . We propose that this case of sudden infant death syndrome is due to an episode of fatal arrhythmia due to underlying left ventricular hypertrabeculation and that the presence of more than three trabeculations apical to the papillary muscles should be specifically excluded during postmortem examination in cases of sudden infant death syndrome . We acknowledge the possibility that the left ventricular hypertrabeculation may be an incidental finding as this entity may be found in asymptomatic, healthy individuals, but due to the observation that arrhythmias may occur at any age15 we propose that this may be an underrecognized cause of sudden, unexpected death, mimicking sudden infant death syndrome.
Noroviruses (novs) and rotaviruses (rvs) are the major causative agents of pediatric acute gastroenteritis (age) in children worldwide . There is no licensed vaccine for nov available, and despite efficacious live rv vaccines currently in use, there is a need for nonlive rv vaccines that could be safer, more affordable, and more efficacious in developing countries [46]. Our group has developed a subunit combination vaccine candidate against nov and rv gastroenteritis, consisting of nov virus - like particles (vlps) and rv vp6, aiming to confer protection from both leading etiological agents of severe age [79]. The double - stranded (ds) rna genome of triple - layered rv particle is surrounded by core protein vp2 . The outermost layer, composed of vp7 capsid glycoprotein and the spike protein vp4, contains most epitopes for rv neutralizing antibody interaction . The intermediate layer is formed by the major internal structural protein vp6 that represents 51% of the virion mass . Vp6 trimers are organized into hexagons and packed into higher order structures, for example, nanotubes, nanospheres, or sheets when expressed in vitro in baculovirus (bv) or bacterial expression systems [1216]. The variable morphology of polymeric vp6 protein expressed in vitro depends on biochemical composition, mainly on ph and ionic strength . Recombinant vp6 has been considered as a nonlive next generation vaccine candidate against rv by us and others, being the most abundant, highly conserved, and immunogenic rv protein [2, 7, 8, 18]. B - cell - mediated immune responses, especially iga seroconversion following rv vaccination and natural infection, are mostly directed against vp6 . Even though the inner capsid protein vp6 cannot elicit classical neutralizing antibodies, it induced heterotypic protective immunity against live rv challenge in mice that correlated with postchallenge vp6-specific serum iga . Vp6-specific polymeric iga inhibits rv replication intracellularly during iga transcytosis, a phenomenon termed intracellular neutralization [2123]. We have recently reported that vp6 protein provides dose sparing effect on nov vlps in vivo and acts as both th1 and th2 type adjuvant on immune response induced by nov vlps . Other investigators have shown that when vp6 is used as an antigen carrier or delivery platform for foreign antigens, the response to the antigen was improved [25, 26]; however, the mechanism was not investigated . Particulate nature and characteristics of an antigen, such as size, shape, surface charge, and receptor interactions, are generally considered important for antigen presenting cell (apc) targeting . Moreover, the antigen size influences development and quality of immune responses [28, 29]. Even though vp6 nanotubes and rv double - layered (dl) 2/6-vlps have been shown to be equally immunogenic in vivo, there have also been indications of improved uptake of vp6 nanotubes compared to spherical forms of rv dlvlp . We hypothesized that due to their size and morphology vp6 nanotubes may be potent inducers of apc activation and maturation . Apcs are known to be critical for initiating and modulating antigen - specific immune responses [32, 33] and therefore are likely to have a key role in vp6 exerted adjuvant effect [24, 31]. In here, we investigated the effect of vp6 nanotubes on two commonly used apc lines, raw 264.7 macrophages and jawsii immature dendritic cells (dcs), aiming to improve understanding of the immunostimulatory and immunomodulatory mechanism of vp6 oligomeric protein . Adherent murine immortalized raw 264.7 macrophage cell line, originating from blood monocyte / macrophages (h-2d) (cat . Tib71), and jawsii immature dcs, derived from bone marrow of h-2b c57bl/6 p53-knockout mouse (cat . Crl-11904), were obtained from the american type culture collection (atcc, manassas, va). The cells were grown at 37c in a 5% co2 air humidified atmosphere and used for the treatments after at least 5 but less than 20 passages in our laboratory . Raw 264.7 cells were cultured in dulbecco's modified eagle's minimum essential medium (dmem, cat . D6546), supplemented with heat inactivated 10% fetal bovine serum (fbs, cat . F9665), 100 units / ml penicillin, 100 g / ml streptomycin (cat . G7513) (all from sigma, st . Louis, mo). The jawsii cells were maintained in minimum essential medium eagle, alpha minimum essential medium (mem) supplemented with ribonucleosides, deoxyribonucleosides (cat . S8636, sigma), 20% fbs, 4 mm l - glutamine, and 5 ng / ml recombinant murine granulocyte - macrophage colony - stimulating factor (gm - csf, cat . Adherent caco-2 cell line was cultured in a complete culture medium consisting of dmem medium, 10% fbs, 100 units / ml penicillin, and 100 g / ml streptomycin . Caco-2 cells were subcultured when they reached ~80% confluency and were not allowed to form tight monolayer or to differentiate before being used for the internalization analyses . Human rv strain wa (g1p1a) was propagated in fetal rhesus monkey kidney (ma104) cells as described elsewhere . Human rv vp6 protein (database accession number gq477131) and nov gii.4 (reference strain accession number af080551) vlps were produced in a bv - insect cell expression system according to the previously described procedures [7, 8, 35]. The proteins were highly purified with demanding multistep chromatographic procedures and the concentration, purity, integrity, and morphology of the proteins were verified as described in detail elsewhere [7, 24]. In brief, pierce bca protein assay (thermo scientific, cat . 23227) was used for quantification of the total protein . The integrity of rv vp6 nanotubes (figure 1(a)), trimeric vp6 (figure 1(b)), and nov gii.4 vlps (figure 1(c)) was verified under electron microscopy (em). The purity was confirmed by quant - it dsdna broad - range assay kit (invitrogen, cat . Q33120; <10 ng dsdna/10 g of protein) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds - page) and densitometric analysis after silver staining (pagesilver silver staining kit, fermentas, cat . Infectious bv and endotoxin level were determined with bacpak rapidtiter kit (clontech laboratories, cat . 631406; 0 pfu live bv / ml) and limulus amebocyte lysate assay (lonza, cat . 244 n184 - 25; <0.1 endotoxin units/100 g of protein). Crude purified vp6 preparation containing impurities related to the expression system, for example, residual bvs (10 plaque forming units / ml), was used in parallel with pure vp6 in internalization inhibition experiments as described below . The proportion of vp6 in impure vp6 preparation was calculated after the densitometric analysis of page with alphaease fc software (alpha innotech, san leandro, ca), as described previously . Uptake of rv vp6 nanotubes and rv wa was tested in mouse raw 264.7 macrophages, jawsii dcs, and human colorectal epithelial caco-2 cell lines . Each cell line was plated to cell - culture treated multidish wells and was allowed to attach overnight in the cell - type - specific culture medium . The culture medium was replaced with the medium containing 100 g / ml of highly purified vp6 nanotubes, 1: 10 diluted rv wa, or fresh culture medium (for untreated control cells). After the incubation period of 4 h for raw 264.7 cells only or 24 h for all three cell types, the internalization of nov gii.4 vlps and cointernalization of gii.4 vlps with rv vp6 nanotubes were carried out using the raw 264.7 cells and 24 h incubation . G / ml of gii.4 combined with 100 g / ml of vp6 . The internalization of gii.4 vlps was analyzed by intracellular staining of nov vlp and flow cytometry . Vp6 mediated activation and maturation were explored by determining the change in the expression of cell surface markers and upregulation of cytokine secretion . After overnight incubation of raw or jawsii cells on the plates, the cell - culture medium was changed and cells were exposed to rv vp6 nanotubes (10 g / ml or 50 g / ml), trimeric (denatured) vp6 (10 g / ml or 50 g / ml), gii.4 vlps (10 g / ml or 50 g / ml), rv wa (1: 10 dilution), or 1 g / ml of bacterial lipopolysaccharide (lps) (cat . L6143, sigma) and incubated further for 24 h or 48 h. supernatants of all cultures were collected, centrifuged, and stored at 80 until cytokine analysis . The cell surface expression of costimulatory and maturation markers cd80, cd86, cd40, and major histocompatibility complex (mhc) class ii molecules was analyzed by flow cytometry as described below . Raw cells were plated and allowed to attach before adding the inhibitors at predetermined nontoxic concentrations . Cells were preincubated in cell - culture medium supplemented with 15 m chloroquine, 2 mm mcd (without serum), or 2 m cytochalasin d for 60 min before exposing cells to 50 g / ml of vp6 nanotubes and incubating them further for 17 h. in addition, the concentration of 50 g / ml of crude purified vp6 nanotubes containing impurities related to the expression system, for example, residual bvs, was used as a control . The cells were harvested and washed with cold pbs + 3% fbs . Prior to staining or permeabilization the cells were blocked with rat anti - mouse cd16/cd32 (fc block, clone 2.4g2, becton dickinson, san jose, ca) on ice for 10 min followed by washing with pbs + 3% fbs . For intracellular staining of the internalized rv vp6 or nov gii.4 vlps, the bd cytofix / cytoperm plus kit (becton dickinson, san jose, ca) was used according to manufacturer's instructions . For detection of vp6, permeabilized and fixed cells were stained intracellularly with rabbit polyclonal rotavirus group a antibody (cat . Gwb-459fc9, genway biotech inc .) Followed by fluorescein isothiocyanate- (fitc-) conjugated goat anti - rabbit ig (bd pharmingen). For gii.4 vlp staining, a human serum highly positive for anti - gii.4 nov antibodies was used at 1: 2000 dilution, followed by polyclonal anti - human igg (fc -specific) phycoerythrin (pe) conjugate (ebioscience) for detection . Internalization was examined by overlaying the histograms of treated and untreated cells and by comparing mean fluorescence intensity (mfi) of each population . For the cell surface molecules expression analysis, raw 264.7 cells were stained on ice for 30 min with anti - mouse monoclonal antibodies percp - cy5.5-conjugated cd80 (16 - 10a1), pe - cy7-conjugated cd86 (gl1), pe - conjugated cd40 (3/23), and alexa fluor 647-conjugated i - a / i - e (m5/114.15.2) (mouse mhc class ii). Bd compbeads used for compensation and conjugated cell surface marker monoclonal antibodies were purchased from bd biosciences . The flow cytometry analyses were performed on a facscanto ii (becton dickinson, san jose, ca) flow cytometer, using the facsdiva version 6.1.3 software (becton dickinson). The data analysis was carried out with flowjo software version 10 (three star inc . Quantities of tumor necrosis factor alpha (tnf-), interleukin-6 (il-6), and interferon - alpha (ifn-) cytokines in raw and jawsii cell supernatants were determined by commercial enzyme - linked immunosorbent assay (elisa) kits: mouse tnf- duoset (cat . Victor 1420 multilabel counter (wallac, perkin elmer) plate reader was used for optical density reading (od) of the plate . For each assay the background signal from the blank wells (wells without supernatant) was subtracted from all of the od readings on the plate . Standard curves were plotted and used for calculating the cytokine concentration of each sample (pg / ml). Briefly, raw 264.7 cell - culture supernatants were collected after 24 h stimulation with 100 g / ml vp6 nanotubes or left untreated . The membranes precoated with 40 different cytokine / chemokine - specific capture antibodies were blocked and incubated with 1: 5 diluted supernatants overnight at + 4c . After washing, the detection antibody cocktail was added for 30 min, followed by streptavidin - horseradish peroxidase and chemiluminescent detection reagent . The immunoblot images were captured and visualized using a bio - rad chemidoc mp system and the intensity of each spot in the captured images was analyzed using a imagej 1.50b software . The data were analyzed by ibm spss statistics software version 22.0 and p value of <0.05 was considered statistically significant . After incubating raw 264.7, jawsii, or human colorectal epithelial caco-2 cells with 100 g / ml of vp6 or rv wa, the entry of the rv protein was examined by flow cytometry and compared to cells incubated with culture medium only (untreated cells). Vp6 nanotubes were shown to be internalized by macrophages (figure 2(a)) and dc (figure 2(b)) but not by caco-2 epithelial cells (figure 2(c)). Within 24 h of incubation of raw cells with vp6, the rv - specific intracellular staining increased by 202256% (range of percent increase of mfi for three independent experiments) compared to untreated cells . A 120% increase was observed in raw cells exposed to rv wa (figure 2(a)). Vp6 entry to raw cells was observed already at 4 h but at a much lower quantity than after 24 h (data not shown). Vp6 was also internalized by jawsii cells within 24 h incubation but less efficiently than by raw macrophages (figure 2(b)). Compared to untreated cells, vp6 treatment induced a moderate 4960,9% increase in rv - specific intracellular staining . In contrast to these results, a control cell line for internalization, human colorectal epithelial caco-2 cells highly susceptible to rv infection, did not take up vp6 alone, but a 282311% increase in mfi was detected after rv wa exposure (figure 2(c)). After 24 h and 48 h stimulation of raw and jawsii cells with 50 g / ml of vp6 nanotubes, 1: 10 diluted rv wa, or 1 g / ml of lps, the change in the expression of cell surface molecules cd80, cd86, cd40, and mhc ii was examined by flow cytometry . As shown in figures 3(a) and 3(b), vp6 stimulation upregulated mfi of all four markers compared to untreated raw cells already in 24 h, which further increased within 48 h. lps, used as a positive control, upregulated strongly all markers already within 24 h as expected (figure 3(b)). Viral control (rv wa) also induced high surface marker upregulation similarly to lps (figures 3(a) and 3(b)). Similar pattern of cell surface molecules expression upregulation with all three stimuli was observed with jawsii cells but the increases were not as prominent as with the raw cells (data not shown). After 24 h or 48 h incubation of raw and jawsii cells with vp6 nanotubes, gii.4 vlps, nonpolymeric denatured vp6 (10 g / ml and 50 g / ml each protein), rv wa, or lps, supernatant was collected and tnf-, il-6, and ifn- were quantified by elisa (figure 4(a)). Nonpolymeric trimeric vp6 lacking conformational structures (figure 1(b)) was used as a control protein . Both raw and jawsii cells secreted tnf- in a dose - response manner after 10 and 50 g / ml vp6 nanotube exposure (figure 4(a)). However, gii.4 vlp did not induce considerable tnf- in jaws cells (74 57 pg / ml, resp .) As did vp6 nanotubes at the same concentration (713 296 pg / ml). Tnf- production was significantly higher in vp6 nanotube exposed cells in both raw and jawsii cells compared to untreated cells (p <0.05). These two cell lines exhibited different cytokine expression profiles upon stimulation, the primary cytokine secreted by raw cells being tnf-, while jawsii dcs produced higher quantities of il-6 (figure 4(a)). Interestingly, vp6 nanotubes and rv wa induced similar tnf- secretion in raw cells (p> 0.05), but only vp6 stimulated significant production of tnf- in jawsii cells . A tubular structure of vp6 was essential for the stimulatory effect of the protein, as more tnf- was secreted after exposing raw cells to 10 g / ml (1756 135 pg / ml) of vp6 nanotubes, compared to the equal concentrations of nonpolymeric vp6 control (411 75 and 539 14 pg / ml, resp . ). The constitutive il-6 secretion by jawsii cells was remarkably stimulated by vp6 nanotubes, whereas only subtle changes were detected in the supernatants of the cells incubated in the presence of gii.4 vlp (figure 4(a)). Rv wa had a moderate effect on il-6 secretion by jawsii cells, whereas the positive control lps elicited high il-6 secretion . Only small quantities of il-6 were produced by raw cells stimulated with vp6 and rv wa but not at all with gii.4 vlps . As expected, antiviral cytokine ifn- secretion was detected only after rv wa stimulation in both cell lines but not when exposed to protein products or lps . The cytokine profile of raw supernatant stimulated with 100 g / ml vp6 for 24 h was further examined by cytokine array, indicating a strong activation of the cells (figures 4(b) and 4(c)). Several cytokines and chemokines were upregulated (figure 4(c)), including interferon gamma - inducible protein (ip-10) and macrophage inflammatory protein (mip-) 1 and mip-1, when compared to supernatant of untreated cells (cm only). However, tnf-, granulocyte - colony - stimulating factor (g - csf), mip-2, il-1ra, and rantes were only detected in vp6 stimulated cells . As il-6 cytokine production by raw cells at 24 h was not detected by elisa (figure 4(a)) and it was not detected by the cytokine array either, corroborating the findings (data not shown). The effect of chemical inhibitors mcd, cytochalasin d, and chloroquine on the vp6 nanotube internalization and vp6-induced tnf- secretion by raw 264.7 cells was investigated . Both highly purified vp6 nanotubes and the vp6 nanotubes known to contain bv - related impurities, which assumingly employ different entry pathways into the cells, were used in these experiments . After mcd treatment, a 22 11% decrease in tnf- secretion was observed in raw cells stimulated with pure vp6 (figure 5), whereas mcd showed an increasing effect (43 2%) when impure vp6 was used . Other two chemical inhibitors, cytochalasin d and chloroquine, did not decrease tnf- production in raw cells induced by pure vp6 nanotubes . On the contrary, tnf- production induced by impure vp6 was almost completely blocked (94 0.1%) by chloroquine treatment (figure 5), indicating that the two protein preparations have different stimulatory mechanism . This observation was further supported by ~26% inhibition of impure vp6 internalization by chloroquine treatment, while the internalization of pure vp6 did not significantly change with any of the treatments (data not shown). The coadministration of vp6 nanotubes with gii.4 vlps improved uptake of gii.4 vlps to raw 264.7 cells (figure 6(a)). When raw cells were incubated for 24 h with 100 g / ml gii.4 vlps, 47% of the cells were positive for nov gii.4 when analyzed by intracellular staining . Coadministration of gii.4 vlps with vp6 nanotubes further increased the number of nov - positive cells by 30% (from 47% to 61%, resp . ). A twofold increase in tnf- secretion was observed in cells incubated with gii.4 + vp6 combination (11490 741 pg / ml) (figure 6(b)), indicating an additive effect of the two proteins on the cytokine secretion . Furthermore, improved maturation of raw macrophages was achieved with coadministration, as illustrated in figure 6(c). Nov gii.4 vlps + vp6 upregulated cell surface molecules cd40, cd80, cd86, and mhc ii expression compared to gii.4 vlps alone . Interestingly, mhc ii downregulation was repeatedly observed when incubating raw cells with gii.4 vlps only . We have recently shown in vivo adjuvant effect of vp6 nanostructures on the immunogenicity of nov vlps, where coadministration of vp6 nanotubes with suboptimal doses of nov gii.4 vlps increased the level of type - specific and cross - reactive nov - specific immune responses . Therefore, in the present work, we investigated the effect of vp6 on activation and maturation of mouse raw 264.7 macrophages and jawsii immature dcs . Macrophages and dcs are professional apcs with high endocytic capacity known to be central for mounting effective t- and b - cell immune responses to foreign antigens in vivo [3740]. Extremely pure recombinant vp6 nanotubes were efficiently internalized by the raw macrophages and to less extent by jawsii dcs . Human epithelial caco-2 cells were used as a control cell line as, unlike apcs, they are not efficient in uptake of larger particles but are susceptible to human rv wa infection . Accordingly, no significant uptake of vp6 was detected in caco-2 cells, while rvs were readily internalized . These results are congruent with the report by rodrguez et al ., who showed that vp6 nanotubes were favorable for macrophage uptake . Along with being internalized by macrophages and immature dcs, vp6 nanotubes induced activation and maturation of apcs by upregulating cell surface costimulatory and antigen presenting molecules cd40, cd80, cd86, and mhc ii . Signaling through cd80 and/or cd86 on mature apcs, which bind to cd28 on t cells [41, 42], is a key costimulatory pathway for antigen - specific t cell activation . A bacterial activator lps, a toll - like receptor (tlr) 4 agonist, was used as a control stimulus, as it is well known that it induces activation of apcs, including raw 264.7 cells [43, 44]. The raw 264.7 and jawsii activation state was also determined by release of broad panel of cytokines and chemokines . An increase in the expression pattern of cell activation markers following vp6 exposure was accompanied by increase in proinflammatory cytokine secretion, primarily tnf- by raw cells and il-6 by jawsii cells . In raw cells, 50 g / ml vp6 nanotubes induced tnf- at a similar level to rv wa and gii.4 vlps stimulation . Interestingly, only vp6 nanotubes but not gii.4 vlps or rv wa stimulated remarkably high tnf- and il-6 response in jawsii dcs . Reported that even if rv dlvlps were internalized by dcs, they could not trigger their activation . A key proinflammatory cytokine, tnf-, recruits and activates apcs at the site of inflammation (antigen delivery) and facilitates apc migration to the lymph nodes, thereby improving the antigen uptake and presentation to mhc i and mhc ii restricted t cells . In addition, tnf- and il-6 have important roles in the generation of b - cell functions like proliferation and antibody secretion . Overall, quantification of tnf- and il-6 showed that vp6 nanotubes are more potent in inducing these cytokines than nov vlps or rv . In addition, a nonpolymeric vp6 protein was not able to induce cytokine secretion near to the vp6 nanotubes . These results underline the importance of intact high order tubular structure conformation of vp6 in activation of apcs . Furthermore, changes in the levels of different cytokines and chemokines were evaluated in untreated or vp6 stimulated raw 264.7 cells by cytokine array . The results suggested that vp6 induces multiple cytokines (tnf-, g - csf, and il-1ra) and chemokines (ip-10, mip-1, mip-2, and rantes) secretion into the supernatants of stimulated cells . G - csf is a multifunctional cytokine that is required for modulating macrophage and dc responses and t cell responses following antigen stimulation . Rantes, ip-10, il-8, mip-1, and monocyte chemoattractant protein-1 (mcp-1) have been suggested to be the key host factors in gastrointestinal immunity during rv infection [50, 51]. Employing highly purified protein preparations is of high importance when investigating adjuvant properties of a protein, eliminating the immunostimulatory effect of impurities related to the protein expression system on apcs . The results with vlp preparations containing residual bv particles have strongly suggested that bv contaminants can trigger an innate immunity, inducing inflammatory cytokine production such as tnf-, il-6, and ifn- [44, 52, 53]. Therefore, we tested raw 264.7 and jawsii cells for secretion of ifn-, an antiviral cytokine induced upon live virus or virus - derived dsrna stimulation of the cells through tlr3 [45, 53, 54]. Only rv wa stimulation but not any other stimuli including highly purified vp6 nanotubes, gii.4 vlp, or lps induced significant release of the cytokine by these cell lines showing that the recombinant proteins used to stimulate the apc in our assays were free of bv contamination . The size from 0.2 m to 1.5 m and the morphology of vp6 nanotubes are favorable for macrophages and dc recognition and uptake resembling those of microorganisms [30, 31, 55]. Antigen acquisition by apcs is mediated by several uptake mechanisms such as receptor - mediated endocytosis, macropinocytosis, and phagocytosis, depending of the nature of the antigen [5659]. Smaller particles (20200 nm) larger particles are taken up by macrophages, b cells, langerhans cells, and dc, through receptor - independent macropinocytosis (> 0.5 m) or receptor - mediated phagocytosis (0.55 m). Bluetongue virus nonstructural ns1 protein tubular structures, which are similar in size (up to 1 m long and app . 50 nm in diameter) and morphology to vp6 nanotubes, were suggested to be internalized by macrophages and dc via endocytosis and macropinocytosis . We used chemical inhibitors mcd, chloroquine, and cytochalasin d to study the possible uptake and activation pathways of vp6 nanotubes . Tnf- secretion was used for evaluating inflammatory apc activation and impure vp6 preparation containing production related impurities, including bv, was included as a control . Our results indicated a difference in the mechanism of raw 264.7 cells uptake and activation by the two rv vp6 protein preparations . Mcd treatment depletes cholesterol which is a reversible mechanism that inhibits the formation of cholesterol - rich microdomains termed lipid rafts important in the lipid raft - mediated endocytosis . Interestingly, when pure vp6 nanotubes were used, mcd partially inhibited tnf- secretion (22%), whereas impure vp6-induced tnf- secretion was blocked almost completely by clathrin - dependent endocytosis inhibitor chloroquine . In support to our findings, rodrguez et al . Furthermore, abe et al . Observed that treatment with chloroquine inhibited bv - induced innate immune system activation and tnf- secretion through bv gp64-mediated membrane fusion and tlr9/myd88-dependent pathway . Cytochalasin d, which inhibits actin - dependent cellular processes such as phagocytosis, was not observed to have an effect on tnf- secretion or vp6 protein uptake, unlike the published results where bv expression system - derived human immunodeficiency virus vlps entry to apcs was blocked . Altogether, the results with chemical inhibitors indicate that vp6 entry and activation of raw cells require host cholesterol and may be partially mediated by lipid rafts . Due to the size range of vp6 nanotubes (~0.21.5 m,), several entry pathways are likely to be involved and reaching total blocking is challenging . Adjuvants generally work to spare the dose of particular vaccine antigen, to broaden the immune responses, and to prolong the duration of the response . The results of this study support our earlier findings of vp6 adjuvant effect on nov vlps immunogenicity in vivo and show that vp6 nanotubes are efficiently internalized by apcs and induce activation and maturation of apcs . Induction of proinflammatory cytokines and chemokines by vp6 stimulated apcs, as shown in here, indicates efficient activation of the innate immune system, a major mechanism of adjuvant action . A depo effect is yet another important mechanism of adjuvant action that facilitates and improves the uptake of antigens by apcs . In here, a 30% increase in nov gii.4 vlp uptake by apcs was observed when nov vlps were codelivered with rv vp6, compared to gii.4 vlp alone . In addition, vp6 coadministration showed increased activation and maturation of apcs measured by tnf- production and upregulation of cell surface markers expression compared to nov vlps alone . Moreover, the exposure of raw cells to gii.4 vlps caused downregulation of mhc ii expression, which was corrected by addition of vp6 . Vp6 nanotubes activate and mature apcs and increase the uptake of nov vlps by the apc . In the present study, we show that recombinant rv inner core capsid vp6 nanotubes are efficiently internalized by professional apcs probably by a clathrin - independent endocytosis pathway involving cholesterol - rich lipid rafts, but likely other internalization routes are also involved . Moreover, vp6 nanotubes efficiently activate and mature macrophages and dcs as shown by upregulation of costimulatory molecules and mhc ii expression and release of inflammatory mediators such as tnf-, thus augmenting the action of cells of both innate and adaptive immune system . Furthermore, vp6 nanotubes facilitate the apc uptake of codelivered antigen, nov gii.4 vlps . These results provide insights into the mechanism of adjuvant action of vp6 nanotubes and confirm the immunostimulatory and immunomodulatory potential of vp6 observed in vivo.
Adult cardiovascular disease is associated with metabolic and physiological aberrations that occur during fetal development . Epidemiological studies provide evidence that intrauterine growth retardation increases the risk of hypertension and cardiovascular disease in adult life . Such findings led to the fetal programming hypothesis that exposure to a suboptimal intrauterine environment can predispose to adult noncommunicable disease . Animal models setup to test the hypothesis demonstrates the onset of cardiovascular pathologies by limiting fetal and neonatal growth through global maternal nutrient restriction, protein [4, 5], or micronutrient restriction . The well - established maternal low - protein rat model produces offspring that develop elevated blood pressure which persists throughout adult life [7, 8]. Hypertension is a major independent risk factor for the development of ischemic cardiovascular conditions such as myocardial infarction and stroke . Our laboratory has recently reported that male rats exposed to protein restriction in utero showed impaired cardiac contractile recovery to ischemia - reperfusion (ir) injury compared to offspring from control fed dams . This sex - specific effect concurs with previous findings in rats in which intrauterine growth restriction as a result of either hypoxia or undernutrition caused cardiac remodelling and impaired recovery to ir in adult male offspring . The catecholamines adrenaline and noradrenaline activate myocardial cell surface -adrenergic receptors . 1 and 2-ar are the principal -receptor subtypes in the heart making up approximately 7080% and 3020% of the -ars expressed, respectively . This stimulates adenylyl cyclase activity and generation of camp- and pka - mediated activation of the contraction - relaxation cycle [12, 14]. These effects are more tightly regulated following 2-ar activation which acts in an antagonistic fashion by coupling gi leading to the attenuation of camp - mediated activation of pka . The receptors then translocate from the plasma membrane into vesicular cytosolic compartments and either targeted for degradation or dephosphorylation and recycled back to the membrane . The interaction between ir injury and -ar signalling in the heart has been widely investigated and established that myocardial ischaemia is commonly associated with an increase in membrane expression of -ars and that their overexpression prior to ischaemia increases ir - injury [1519]. However, the effects of ir on -ar signalling in developmental programming are yet to be explored . This study therefore aims to examine the capacity of isolated adult rat hearts from protein - restricted offspring to recover from ir injury in relation to alterations in shuttling of -ar and g protein signalling between the sarcolemma and cytosolic compartments of the myocardium . Twenty - four virgin wistar rat dams (harlan ltd, belton, leicestershire, uk) weighing approximately 250 g were mated in the animal facilities at the university of nottingham . The appearance of a semen plug on the cage floor confirmed that mating and rats were allocated either a control (con) diet (180 g casein / kg, n = 12) or a low - protein diet (90 g casein / kg, n = 12, mlp), as described previously in detail . The pregnant rats were then fed the isoenergetic semisynthetic diets throughout gestation until birth at 22 days of gestation . All mothers were then transferred to standard laboratory chow diet (b&k universal ltd, hull, uk) and each litter culled to a maximum of 8 pups to minimise variation in the nutrition of the offspring during suckling . At 4 weeks of age, all offspring were weaned onto chow diet, to ensure that litters from control and low - protein fed rat dams only differed in terms of their prenatal nutrition . All experimental procedures were performed in accordance with the home office guidance on the operation of the animals (scientific procedures) act, 1986 . At 6 months of age, male and female rats from each con and mlp litter were randomly selected (n = 9 or 10), anaesthetised using 3% isofluorane in 2 litres o2/min and killed by cervical dislocation . The heart was rapidly excised and cannulated via the aorta to langendorff perfusion apparatus within 90 seconds (ad instruments, oxford, uk) and perfused with krebs henseleit buffer (118 mm nacl, 4.7 mm kcl, 1.2 mm kh2po4, 1.2 mm mgso4, 25 mm nahco3, 11 mm glucose, and 1.25 mm cacl2 (ph 7.4)) bubbled with o2-co2 (95: 5, v / v) in a coronary retrograde fashion . Perfusion pressure was maintained at a constant pressure of 60 mmhg, with perfusate warmed to 37.4c, and the heart immersed in a water - jacketed temperature - controlled glass chamber set at 37.4c, therefore ensuring normothermia throughout the perfusion protocol . Contractile function was monitored by the careful insertion of a saline filled latex balloon (linton instruments) into the left ventricle which was adjusted to an end diastolic pressure of 510 mmhg . Left ventricular and perfusion pressure were continuously monitored through precalibrated physiological pressure transducers (senso - nor 844, ad instruments). Data recording was not started until all variables were stable (1530 min) after which the following 30 min were defined as baseline . Ischemia was then induced by switching off the coronary perfusion apparatus for a 30-minute period . Coronary perfusion was then reinstated for a further 60-minute period to assess cardiac responses during reperfusion . Data for left ventricular developed pressure (lvp), heart rate (hr) and left ventricular first derivative (dp / dtmax) were collected and processed using the powerlab data acquisition system (ad) instruments . To determine the effect of ir on myocardial expression of -ar, hearts from con or mlp offspring that underwent either (i) 30 min baseline perfusion alone (n = 9 - 10), (ii) 30 min baseline perfusion followed by 30 min ischemia (n = 9 - 10) or (iii) 30 min baseline perfusion followed by 30 min ischemia and 60 min reperfusion (n = 9 - 10) on the langendorff apparatus were collected and snap frozen for western blot procedures . Hearts collected for western blot analysis from the langendorff study were used to prepare cardiac membrane and cytosolic fractionated samples as described by fernandez - twinn and colleagues . Frozen whole heart tissue was ground to a powder in liquid nitrogen and homogenised briefly for 30 s in ice cold buffer containing 5 mm tris ph 7.4, 2 mm edta, and protease inhibitor cocktail (calbiochem). Homogenates underwent centrifugation at 1000 g for 15 min at 4c to pellet nuclear material . The supernatant fraction was then spun at 100,000 g for 1 h 20 min at 4c in an ultracentrifuge to generate a pellet and supernatant which corresponded to the plasma membrane and cytosolic fractions, respectively . The pellet was then resuspended in lysis buffer (50 mm hepes ph 8, 150 mm nacl, 1% triton - x-100, 1 mm na3vo4, 30 mm naf, 10 mm na4p2o7, 10 mm edta, and protease inhibitor cocktail). Protein concentrations of the supernatant and resuspended pellet fractions were determined using the bio - rad protein assay system (bio - rad, hemel hempstead, uk) according to the manufacturer's instructions . Samples were standardised to a concentration of 3 mg / ml with laemmli's sample buffer (62.5 mm tris ph 6.8, 2% sds, 10% glycerol, 0.02% bromophenol blue, 150 mm dithiothreitol) and boiled for 3 min before equal protein quantities of each sample were separated by sds page . Proteins were transferred to nitrocellulose membrane (hybond - c extra, amersham bioscience) for probing with antibodies to 1-ar (affinity bioreagents; rabbit polyclonal raised against a synthetic peptide corresponding to residues 394408 of mouse / rat 1-ar), 2-ar (abcam; rabbit polyclonal raised against against residues 1100 of human 2-ar), g stimulatory protein (santa cruz gs (k-20): sc-823), and g inhibitory protein (santa cruz gi-3 (c10): sc262). Membranes were incubated in blocking solution (5% dried skimmed milk in tbs with 1% tween 20) prior to incubation with primary antibodies . Horseradish peroxidise secondary antibody conjugated to rabbit igg was used at a working concentration of 1: 5000 (ge healthcare, amersham, uk). Bands were developed on high performance chemiluminescence film (hyperfilm ecl, amersham) using ecl plus reagent (ge healthcare, amersham, uk). Densitometric analysis of band intensity across gels was performed using a biorad gel doc xr imaging system and quantity one 1d analysis software . Three replicates of a pooled extract were included on every gel and used as a standard to facilitate gel - to - gel comparisons . Rats at 5 weeks, 10 weeks, and 6 months of age were housed in metabolic cages for 24 hours and urine collected as previously described . Urine concentrations of adrenaline, noradrenaline, and dopamine were determined using the dld diagnostika catecholamine elisa kit (dld diagnostika gmbh, hamburg, germany), following the manufacturer's instructions . All data were analysed for homogeneity of variance, and if heterogeneous transformed by square root or logarithmic conversion . For the analyses, ischemia - reperfusion data were split into three distinct time bins: (1) 30 min baseline, (2) recovery 1 (030 min of reperfusion), and (3) recovery 2 (3160 min of reperfusion). The effects of maternal diet on the recovery in lvp, hr, and dp / dtmax relative to baseline were analysed using a one - way repeated measure anova . Western blot data were analysed by three - way anova considering maternal diet, sex, and ischemia - reperfusion treatment as fixed factors . Elisa data were analysed by three - way anova considering maternal diet, sex, and age as fixed factors . Measurements from related animals from the same litter were grouped in all anova analyses and assigned as a random factor . The effect of maternal diet on the recovery of contractile function relative to baseline in the first (r1) and second (r2) 30 min periods of reperfusion was measured in langendorff - perfused hearts from male and female rats . Maternal diet had no effect on lvp, hr, or dp / dtmax baseline indices in either sex (table 1). Similarly, there was no significant effect of maternal diet on the recovery of female indices of cardiac function (lvp, dp / dtmax, hr) to ischemia - reperfusion insult (table 1). However, significant differences in the capacity of male hearts to recover from ischemia - reperfusion injury were observed between con- and mlp - exposed offspring . Interestingly the recovery of lvp in mlp offspring during r1 and r2 phases of reperfusion was 2-fold higher than the recovery seen in con males (p <0.01) (table 1). A similar result was observed with dp / dtmax where mlp males exhibited almost a 2-fold greater recovery during the r1 phase (p <0.05) and a trend (p = 0.06) for an improved recovery during the r2 phase in comparison to con hearts (table 1). In contrast, there was no significant effect of maternal diet on recovery of heart rate (table 1). Adrenergic signalling is a central mechanism involved in the maintenance of cardiac inotropic and chronotropic function and has been shown to play a key role in recovery of the heart from ir . Accordingly, we quantified levels of the two main -ar isoforms, 1-ar and 2-ar, as well as levels of the stimulatory and inhibitory g proteins gs and gi, respectively, in isolated hearts exposed either to baseline perfusion (b), baseline perfusion plus ischemia (i), or baseline perfusion plus ischemia plus reperfusion (ir). Expression was measured in both membrane (figure 1) and cytosolic (figure 2) fractions prepared from each heart . Baseline membrane or cystolic expression of all proteins was not significantly altered by gender or maternal diet; however differential levels of expression were observed following ischemia and ischemia - reperfusion . 1-ar levels at the membrane increased following ischemia and returned to baseline levels by the end of 60 min reperfusion (table 1; p <0.05). This correlated with a decline in 1-ar in the cytosol by the end of the ischemic period which had declined further by the end of reperfusion (figure 2(a); p <0.001). A significant interaction between diet and sex (p <0.05) was also observed for membrane expression of 1-ar indicating that the overall level of receptor expression in females was lower in the mlp group when compared to con, an effect not observed in males . Cardiac membrane expression of 2-ar revealed a significant interaction between maternal diet, sex, and ischemia - reperfusion treatment (figure 1(b); p <0.05). 2-ar decreased in con females following ischemia which was in complete contrast to all other groups where receptor expression levels were unchanged from baseline levels . Following reperfusion, 2-ar expression in con females remained unchanged from levels observed after ischemia . Hearts from con males showed decreased membrane expression of 2-ar after reperfusion similar to the decreased expression observed in con female hearts following ischemia (figure 1(b)). However, hearts from mlp females and males maintained baseline levels of 2-ar expression throughout ischemia and reperfusion . 2-ar expression in cardiac cytosolic fractions was found to increase after ischemia and increased further following reperfusion (figure 2(b); p <0.001) which was in direct contrast to 1-ar cytosolic expression . These findings suggest that ischemia - reperfusion initiates a differential response in membrane trafficking of 1-ar and 2-ar from the cytosol to the cardiac membrane . Despite changes in -ar isoform within the cardiac membrane and cytosol, no overt changes were observed in the levels of gs and gi (figure 1(d) and figure 2(d)) proteins within either of these cellular locations . Concentrations of the catecholamines adrenaline, noradrenaline, and dopamine were measured in the urine of rats at 5 weeks, 10 weeks, and 6 months of age . No significant differences were observed in the concentration of adrenaline at each stage of development (table 2). Urine levels of noradrenaline however were significantly affected by maternal diet where greater levels were observed in con compared to mlp rats (table 2; p <0.02). In addition, a significant effect of age was observed where noradrenaline concentrations were significantly higher at 5 and 10 weeks of age respectively (p <0.02). An interaction between age and sex was also evident (p <0.01) where urine concentrations of noradrenaline in females were highest at 5 weeks of age with a concentration of 15 ng / ml before dropping to 8 ng / ml at 10 weeks of age, and remaining at this level when aged 6 months . For males, noradrenaline concentration at 10 weeks of age had risen from 10 ng / ml observed at 5 weeks to 14 ng / ml before dropping by 60% to 8 ng / ml at 6 months of age . Dopamine concentrations were significantly reduced by an mlp diet (table 2; p <0.01), and an interaction between sex and age was also observed (table 2; p <0.04). Female rats had a significantly reduced urinary excretion of dopamine at 6 months of age compared to both 5 and 10 weeks of age . In contrast, urinary excretion of dopamine in male rats increased significantly with age . At 6 months of dopamine concentrations the key finding from the present study is that hearts from mlp male offspring demonstrated improved recovery in cardiac function following ir when compared to con males, whereas ir recovery in females was unaffected by maternal diet . This study verifies that sex is an important factor in the capacity of the heart to recover from ir injury [10, 15, 2224]. This contrasts starkly with our previous finding where con male rats demonstrated improved contractile recovery to ir compared to con females or mlp rats of either sex . Introduction of restraint prior to langendorff perfusion may have conditioned the con male rat hearts' response to stress and enhanced their capacity to recover from ir injury . Stressors, such as hyperthermia, hypo- and hyperoxia, and dexamethasone treatment, have been observed to precondition and protect hearts from subsequent ischemic injury [2528]. If true, hearts from either con females or mlp of either sex were not receptive to preconditioning . This is consistent with the cardioprotective effects of ischemic preconditioning only observed in male and not female mouse hearts . Interestingly, there was no significant difference in cardiac recovery following ischemia in mlp rats between the present and previous study . Mlp rats consistently develop elevated blood pressure which persists throughout adulthood and may prevent cardiac preconditioning in these animals . Previous work has shown that protein restriction programmes changes in the hypothalamic - pituitary - adrenal axis, which is likey to have a major impact on the response of mlp rats to stress . To investigate how cardiac function during ir relates to -ar signaling, we determined protein expression of the -ar isoforms, and the gs and gi proteins in baseline - perfused ischemia or ischemia - reperfused hearts . 1-ar expression was associated with increased expression within the plasma membrane after ischemia that returned to baseline levels following reperfusion . This suggests that the 1-ar expression is upregulated in the membrane in response to ischemia and declines throughout reperfusion . This agrees with strasser et al ., who identified that sarcolemmal 1-ar levels increase following 50 min ischemia that was associated with decreased adenylyl cyclase activity . More recently, 1-ar protein expression in isolated rat hearts has also been demonstrated to increase at the plasma membrane following 30 min ischemia but with no change in adenylyl cyclase activity . Further agreement with this study is that 1-ar density at the plasma membrane decreases following 60 min reperfusion compared to levels after ischemia which was associated with a doubling in adenylyl cyclase activity . It is important to concede that the changes in 1-ar expression following ischemia and reperfusion in the present study may not be reflective of downstream changes at the level of adenylyl cyclase . Protein expression of 2-ar in the cardiac membrane decreased following ischaemia in con females and following reperfusion in con males and mlp females . These findings demonstrate subtle sex - specific responses in downregulation of 2-ar at the cardiac membrane to ischaemia and reperfusion . To our knowledge, this is the first time sex - specific effects of ir on 2-ar abundance have been reported . A previous study focussing solely on male hearts reported that 2-ar density at the plasma membrane was unaffected following 30 min ischemia but decreased with 60 min reperfusion, thus agreeing with the present findings in con males . In contrast 2-ar protein expression in the cardiac membrane of mlp males did not change throughout ischemia and reperfusion from baseline levels . Consequently, 2-ar levels were higher in mlp males compared to all other treatment groups following reperfusion . All these changes in bar expression at the membrane occurred against a background where neither gs nor gi expression levels changed, suggesting that -ar density rather than g protein content at the cardiac membrane is the rate limiting component mediating cardiac contractility . Alterations in expression of -ar isoforms through transgenic mouse models show profound effects on haemodynamic function of the heart [15, 3335]. Recent work has focussed on the role of 2 rather than the 1-ar activation in cardiac pathology due to its specific capacity to enhance ca homeostasis and protection against apoptosis [3538]. However, transgenic mouse models have shown that these therapeutic effects cannot be recapitulated by simply overexpressing 2-ar of the heart [16, 34, 35]. Despite increased inotropic and chronotropic activity, cardiac 2-ar overexpression led to progressive cardiac hypertrophy and delayed onset cardiomyopathy . Moreover, 2-ar overexpression exacerbated ischemic injury but in the hearts of male mice only . Female mice overexpressing 2-ar were protected from damage following ir and linked to the female sex hormone oestrogen acting through endothelial nitric oxide synthase to prevent ca dysregulation in the tissue . Such findings suggest that the contractile function of the female heart is less sensitive to 2-ar expression changes . This may explain why cardiac -ar expression levels observed between con and mlp groups in the present study resulted in differences in haemodynamic parameters in male rats only . The cardiac pathology observed in transgenic mouse models is possibly an artefact of such high 2-ar overexpression and does not represent a moderate increase in 2-ar expression . Comparisons of 2-ar overexpression have shown that 100360-fold increases in 2-ar expression resulted in the onset of cardiomyopathy, whereas a moderate 60-fold increase had no overt pathological consequence . Indeed recent studies have presented evidence supporting the putative protective role of 2-ar signalling against cardiac pathology [3739]. Long - term 2-ar agonist treatment in conjunction with a 1-ar blocker was found to improve survival and contractility in a rat model of dilated cardiomyopathy [36, 37]. Furthermore treatment with the 2-ar agonist clenbuterol alone was sufficient to exert a cardioprotective effect in rat hearts through a reduction in myocardial apoptosis and oxidative stress and improving diastolic function following ir injury . This cardioprotective effect of 2-ar activation is thought to occur via stimulation of gi rather than gs - mediated signalling mechanisms which enable ca regulation to be maintained and apoptosis to be inhibited . Our key finding is that mlp male hearts do not downregulate 2-ar protein expression within the cardiac membrane following ir, which is in complete contrast to con males . We propose that downregulation of 2-ar in response to ir is the cause of impaired contractile recovery in con male hearts . Cardiac membrane expression of 2-ar also decreased after reperfusion in con and mlp female hearts to a level comparable to con males but despite this decrease showed no impairment in contractile recovery following ischemia . This may result from the protective role of oestrogen in maintaining a favourable ca microenvironment within female hearts during reperfusion that limits tissue damage and counteracts any changes in -adrenergic signalling activity that would otherwise impair contractile recovery after ischemia . Measurements of urinary catecholamine levels indicate that by 6 months of age there is no overt difference in either adrenaline or noradrenaline levels in these animals . However, we did observe an increase in dopamine levels in males compared to female rats at 6 months of age . We did not see any evidence to indicate that this sex - specific difference in dopamine had any impact on basal levels of expression of any of the -adrenergic signalling components measured in this study or on baseline activity of the hearts prior to ischemic reperfusion . Further work is required to explore other potential receptor targets of dopamine which may alter cardiac function in response to ischemia reperfusion . Urine excretion of noradrenaline and dopamine was significantly lower in mlp compared to con animals and may offset the ischaemic damage observed in con males . Evidence to support this is the fact that depletion of catecholamines by surgical denervation causes a redistribution of -ars, decreasing 1 and increasing the 2 subtype although total -ar content remained the same . Furthermore, suppression of noradrenaline turnover in the rat heart protects against myocardial ir injury . We propose that the contractile dysfunction in male con hearts following ir occurs as a result of increased catecholamine production and decreased 2-ar expression at the cardiac membrane . Likewise, protein expression of 2-ar at the cardiac membrane decreased in response to ir in female con and mlp hearts . However they displayed an improved cardiac recovery compared to con males suggesting specific protective mechanisms in female hearts that counteract alterations in -adrenergic receptor expression . Our findings indicate that in male offspring prenatal protein restriction maintains cardiac membrane expression of 2-ar during ir which improves cardiac contractile recovery . In contrast, female hearts were resistant to these effects suggesting subtle sex differences in the molecular mechanisms controlling cardiac membrane expression of -ar in response to ir.
Painful legs and moving toes (plmt) is the descriptive term given to an unusual syndrome first recognized by spillane and colleagues in 1971 . The main features of this syndrome are involuntary and irregular movements of the toes associated with moderate to severe diffuse pain felt deeply in the foot and leg . The pathophysiology of this syndrome is poorly understood and there are no current guidelines on its treatment . The second case also responded favorably to pregabalin, but the drug had to be discontinued because of side effects . A 64-year - old hispanic male, a former boxer, with a past medical history of diabetes mellitus, early alzheimer's dementia and spinal stenosis, presented with a seven - month - history of right foot pain and involuntary jerky movements of his toes . These symptoms did not change with rest, walking, or any other activity, nor the time of the day . He was on glipizide, lisinopril, hydrochlorothiazide, donepezil, ferrous sulfate, simvastatin, and atenolol . The neurological examination revealed intermittent, irregular flexion and extension movements of all the toes on the right foot . Light touch, pinprick, vibration, and cold sense were normal with no hyperalgesia, hyperpathia, or allodynia . Laboratory revealed a mild anemia; otherwise hemoglobin a1c, thyroid stimulating hormone, hepatic enzymes, vitamin b-12 level, lipid panel, and complete blood cell count were normal . Brain mri with and without contrast showed a few non - specific scattered non - enhancing white matter lesions . Lumbar spine mri showed moderate to severe canal stenosis at l2 - 3, l3 - 4, and l4 - 5, but no nerve root compression . The patient's baseline pain score was 9/10 that decreased to a 4/10 after addition of pregabalin . He also reported an improvement in activities of daily living, increased duration of exercise, and quality and duration of uninterrupted sleep . An increase of pregabalin up to 300 mg twice daily showed a further improvement in quality of life with good tolerance . However, the movement of the toes persisted despite adequate pain control for six months . He was subsequently lost to follow up . A 77-year - old white male presented with left heel paresthesias, left great toe pain, and involuntary constant movements of the left great toe, which was worse at night . He was previously diagnosed with restless leg syndrome, but had failed multiple drug trials including with carbidopa / levodopa, ropinirole, and tramadol . Motor exam showed normal strength, but showed an involuntary non - suppressible flexion and extension movement of his left great toe . Gait was steady and slow with a mild limp caused by chronic left hip pain . The patient was initiated on pregabalin and titrated to 150 mg orally twice a day . In a follow up visit one month later, he reported almost complete cessation of great toe movements and paresthesias with significant improvement in pain . However, because of worsening of lower extremity edema, which is a known side effect of this medication, pregabalin dose was reduced and subsequent discontinuation was required . At a later date, the patient was re - challenged on a lower dose of pregabalin-50 mg orally twice daily that helped reduce but not eliminate his toe movements or pain . Further pregabalin dose increases were poorly tolerated with significant exacerbation of leg edema, which resulted in pregabalin discontinuation . A 56-year - old white male presented with inability to sit still with an aching sensation throughout both legs, worse from the feet to the knees with tingling of the soles of the feet . He was previously diagnosed with restless leg syndrome (rls) two years prior to initial neurology visit . He had been taking ropinirole since diagnosed with rls, which was initially helpful but was unable to tolerate increased doses . Neurological examination was completely normal including mental status, cranial nerves, motor, sensory, coordination, reflexes, and gait . He was noted to have involuntary toe movements of the second and third toes of both feet, worse on the left . Initial emg / ncs was normal, but a follow up emg / ncs two - and - half - years later revealed an axonal sensorimotor polyneuropathy . Seven months later while on same dose of pregabalin revealed a significant improvement in pain, and almost complete resolution of his toe movements . A 64-year - old hispanic male, a former boxer, with a past medical history of diabetes mellitus, early alzheimer's dementia and spinal stenosis, presented with a seven - month - history of right foot pain and involuntary jerky movements of his toes . These symptoms did not change with rest, walking, or any other activity, nor the time of the day . He was on glipizide, lisinopril, hydrochlorothiazide, donepezil, ferrous sulfate, simvastatin, and atenolol . The neurological examination revealed intermittent, irregular flexion and extension movements of all the toes on the right foot . Light touch, pinprick, vibration, and cold sense were normal with no hyperalgesia, hyperpathia, or allodynia . Laboratory revealed a mild anemia; otherwise hemoglobin a1c, thyroid stimulating hormone, hepatic enzymes, vitamin b-12 level, lipid panel, and complete blood cell count were normal . Brain mri with and without contrast showed a few non - specific scattered non - enhancing white matter lesions . Lumbar spine mri showed moderate to severe canal stenosis at l2 - 3, l3 - 4, and l4 - 5, but no nerve root compression . The patient's baseline pain score was 9/10 that decreased to a 4/10 after addition of pregabalin . He also reported an improvement in activities of daily living, increased duration of exercise, and quality and duration of uninterrupted sleep . An increase of pregabalin up to 300 mg twice daily showed a further improvement in quality of life with good tolerance . However, the movement of the toes persisted despite adequate pain control for six months . A 77-year - old white male presented with left heel paresthesias, left great toe pain, and involuntary constant movements of the left great toe, which was worse at night . He was previously diagnosed with restless leg syndrome, but had failed multiple drug trials including with carbidopa / levodopa, ropinirole, and tramadol . Motor exam showed normal strength, but showed an involuntary non - suppressible flexion and extension movement of his left great toe . Gait was steady and slow with a mild limp caused by chronic left hip pain . The patient was initiated on pregabalin and titrated to 150 mg orally twice a day . In a follow up visit one month later, he reported almost complete cessation of great toe movements and paresthesias with significant improvement in pain . However, because of worsening of lower extremity edema, which is a known side effect of this medication, pregabalin dose was reduced and subsequent discontinuation was required . At a later date, the patient was re - challenged on a lower dose of pregabalin-50 mg orally twice daily that helped reduce but not eliminate his toe movements or pain . Further pregabalin dose increases were poorly tolerated with significant exacerbation of leg edema, which resulted in pregabalin discontinuation . A 56-year - old white male presented with inability to sit still with an aching sensation throughout both legs, worse from the feet to the knees with tingling of the soles of the feet . He was previously diagnosed with restless leg syndrome (rls) two years prior to initial neurology visit . He had been taking ropinirole since diagnosed with rls, which was initially helpful but was unable to tolerate increased doses . Neurological examination was completely normal including mental status, cranial nerves, motor, sensory, coordination, reflexes, and gait . He was noted to have involuntary toe movements of the second and third toes of both feet, worse on the left . Initial emg / ncs was normal, but a follow up emg / ncs two - and - half - years later revealed an axonal sensorimotor polyneuropathy . A follow up visit seven months later while on same dose of pregabalin revealed a significant improvement in pain, and almost complete resolution of his toe movements . Painful legs and moving toes is a rare syndrome with involuntary and irregular movements of toes and moderate to severe pain in the foot and leg . The pain is often continuous and throbbing in nature and its intensity may vary from constant discomfort to a living torment . The presenting age of onset is from second to seventh decade of life, although the mean age of onset is the sixth decade of life . Plmt can be idiopathic or associated with several disorders, with peripheral neuropathy, trauma, and radiculopathy among the most common etiologies . Medical conditions associated with plmt the diagnosis of plmt is based on history and physical examination . Electrodiagnostic tests (nerve conduction studies and emg) may show a peripheral neuropathy or root lesions . Emg may show spontaneous repetitive and irregular bursts lasting from 50 milliseconds to 1 second with a frequency of 2 to 200 hz in association with the movement . Polysomnography may shows disruption of the sleep patterns with predominance in light sleep, reduction in slow wave sleep, and fragments of rapid eye movement (rem). Lesions of peripheral nerves and/or central nervous system are possibly implicated in the genesis of spontaneous inappropriate electrical impulses . Posterior nerve roots and afferent nerve fibers lesions generate spontaneous frequent discharges that may lead to reorganization of segmental or suprasegmental efferent motor pathways with pain arising as a central effect and abnormal movement from activation of motor neurons . The differential diagnosis of plmt includes restless leg syndrome, polyneuropathy, akathisia, and nocturnal leg cramps . Table 2 shows the main similarities and differences between plmt and its most common imitators . Plmt differential diagnosis rls restless leg syndrome; epc epilepsy partialis continua; pd parkinson disease there is currently no treatment for plmt . These include baclofen, benzodiazepines (diazepam, clonazepam), anticonvulsants (carbamazepine, gabapentin), tricyclic antidepressants, beta blockers, corticosteroids, lumbar sympathetic blockade, transcutaneous electrical nerve stimulation with or without vibratory stimulation, and analgesics . Case 1: the patient responded successfully to pregabalin at doses of 150 mg bid, even better at 300 mg bid, and the effect was sustained for six months . Case 2: the patient responded to pregabalin (at a dosage of 150 mg bid) but developed exacerbation of lower extremity edema prompting reduction and then discontinuation of this medication . Case 3: the patient responded successfully to pregabalin dose 75 mg bid, which was sustained at seven - month follow up . Pregabalin binds to the alpha 2-delta subunit of voltage - dependent calcium channels in the central nervous system reducing presynaptic end neuronal calcium currents . This reduces the calcium influx at the nerve terminals and subsequently reduces the release of neurotransmitters (glutamate, substance p, and noradrenaline) involved with the ascending neuropathic pain transmission pathway at the level of the dorsal horn neurons . Pregabalin is effective in the therapy of different chronic pain disorders such as painful diabetic polyneuropathy, post herpetic neuralgia, and fibromyalgia . Pregabalin is well tolerated with low incidence of adverse events; drowsiness and dizziness are the most frequent dose - limiting events . Gabapentin, another gabaergic agent structurally related to pregabalin, has also shown benefit in plmt . One study showed improvement in pain and involuntary movement with gabapentin at 300 mg three times per day for three months, at which time the dose was doubled to further control pain and movement . In another study, partial relief was achieved with gabapentin 200 mg three times daily, with more complete control of pain at 700 mg per day . However, toe movement continued in both studies . Combination of gabaergic agents, such baclofen and clonazepam, are potentially effective in plmt syndrome . A patient who developed plmt after taking the antipsychotic drug molindone was successfully treated with combination of baclofen and clonazepam . A second case reported benefit in pain and toe movements with combination of baclofen and clonazepam, but the benefit was transient, lasting only eight months . Clonazepam affects gaba - a type receptor, whereas baclofen affects gaba - b type receptor . Chronic use of these drugs can theoretically lead to possible downregulation of gaba receptors with reduced therapeutic value . A gabaergic agent, progabide, used in europe as antiepileptic, showed similar response to clonazepam and baclofen . Progabide is a gaba agonist that has an effect on gaba - a and gaba - b receptors . In summary, we report three patients with plmt syndrome who favorably responded to pregabalin . We recommend pregabalin in the therapy of plmt syndrome, although more studies are necessary to confirm these observations . Painful leg moving toe syndrome is a rare syndrome, poorly understood, presenting with abnormal involuntary movement of the toes and moderate to severe pain of the foot and leg.
Digital gene expression sequencing, namely dge - seq, refers to the use of high - throughput sequencing technologies to sequence cdna in order to get information about rna content of a sample (1). It can provide researchers with a powerful tool to obtain unbiased and unparalleled information about gene transcripts 2, 3 . Currently, computational methods are being developed to identify and annotate these transcripts with alternative splice forms 4, 5 . Although most dge - seq studies have identified expression outside of known loci (in intronic or intergenic regions) 6, 7, 8, 9, 10, few attempts have been made to ab initio define the read - enriched regions (rers) in detail and compare them with known gene models . Here, we present gene2dge, a free perl software package for rer detection and gene model update . This novel method consists of rer definition based on read clustering followed by annotation comparison with known gene models . The input of gene2dge is the file of mapped reads from rna - seq data and a gene annotation file of the corresponding genome . In addition, a cmap file needs to be prepared for application to different species to correct the chromosome numbers . The output of gene2dge includes a text file containing a set of rers and a series of text files containing annotated information of the eligible rers . The gene2dge package is freely available at http://bighapmap.big.ac.cn/. We developed gene2dge as an ab initio tool to annotate the transcriptome using mapped reads from the solid platform (applied biosystems) and annotation information downloaded from ensembl . First, we filter the uniquely mapped reads from the aligned results of the solid whole transcriptome pipeline . A uniquely mapped read is defined as one with a max scoring alignment to the genome scoring at least 24 and at least four higher than any of the other alignments of that read to the genome (11). Considering the restrictions of computer memory, this process will be performed for each chromosome in order, so it is relatively convenient for use on any personal computer . Second, based on uniquely mapped reads, we construct the rers by grouping overlapped reads with a number greater than a threshold (at least four reads) (12). We list each rer including start position, end position and the number of mapped reads . Maximal distance between rers, defined by the start positions of rers minus the start positions of the first one upstream . It can be customized according to the specific requirement of the experiment (the default value is 50 bp). Finally, we compare the rers to existing gene models, and generate a catalogue of candidate genes with new annotation information, including exon extension, possible additional exons, and novel genes . The file of existing gene models in gtf format can be downloaded from the ensembl website for the candidate species . We picked out eligible rers and then checked their overlap with known gene models . As a result, a series of annotated files will be output and then can be used in further analysis . We applied gene2dge to the rna - seq data from the mouse blastomere dataset obtained from a single - cell whole transcriptome (13). The mrna - seq short reads were analyzed using whole - transcriptome software tools (applied biosystems, http://www.solidsoftwaretools.com/). The reads generated were mapped to the mouse genome (mm9, ncbi build 37). We got more than 6.6 million reads that could be uniquely aligned to the mouse genomic reference (uniquely mapped reads). Based on ensembl annotation (ncbi m37.61), 89% reads (5.9 million) were mapped to annotated regions in exons including coding sequences (cds) and untranslated regions (utr), which is significantly higher than those mapped to intronic (0.3 million, 5%) and intergenic (0.4 million, 6%) regions (figure 1a). Across the mouse genome, 98,532 rers were identified and each contained more than 4 reads . A total of 62.3% of rer boundaries were within 10 bp of the ends for the corresponding exons (figure 1b). Meanwhile, we identified 2,217 exon ends with remarkable extension into un - annotated regions (> 50 bp), suggesting that the mouse transcriptome was more complex than we expected . The transcript levels for rers overlapping with known exons (exonic rers) were significantly higher than those of novel rers (mann - whitney u test, p<10). We detected 12,277 expressed transcripts (with at least 1 rer across the genic region), in which 11,261 (92%) transcribed genes contained at least one exonic rer . For the 72,628 (74% of all 98,532 rers) exonic rers, we found that the known gene models were well defined by the ab initio method . For example, read distribution on chr 7 (56900000 - 57200000) revealed sharp boundaries of rer regions (figure 2). Furthermore, we detected a certain proportion of rers (25,904, 26% of all rers identified), which are located outside of the annotated regions in this transcriptome dataset . Among them, 13,291 intronic rers (about 13% of all) were identified in 4,449 genes, indicating possible additional exons . Interestingly, about 1,016 genes (23%) only had transcripts detected in the intronic regions but not in the exonic regions . As a result, we renewed 4,788 gene models, which account for 39% of 12,277 transcribed genes in total . The remaining 12,613 rers are located in the intergenic regions, suggesting possible presence of novel genes outside of the existing gene models . We developed gene2dge as an ab initio tool to annotate the transcriptome using mapped reads from the solid platform (applied biosystems) and annotation information downloaded from ensembl . First, we filter the uniquely mapped reads from the aligned results of the solid whole transcriptome pipeline . A uniquely mapped read is defined as one with a max scoring alignment to the genome scoring at least 24 and at least four higher than any of the other alignments of that read to the genome (11). Considering the restrictions of computer memory, this process will be performed for each chromosome in order, so it is relatively convenient for use on any personal computer . Second, based on uniquely mapped reads, we construct the rers by grouping overlapped reads with a number greater than a threshold (at least four reads) (12). We list each rer including start position, end position and the number of mapped reads . In addition maximal distance between rers, defined by the start positions of rers minus the start positions of the first one upstream . It can be customized according to the specific requirement of the experiment (the default value is 50 bp). Finally, we compare the rers to existing gene models, and generate a catalogue of candidate genes with new annotation information, including exon extension, possible additional exons, and novel genes . The file of existing gene models in gtf format can be downloaded from the ensembl website for the candidate species . We picked out eligible rers and then checked their overlap with known gene models . As a result, a series of annotated files will be output and then can be used in further analysis . We applied gene2dge to the rna - seq data from the mouse blastomere dataset obtained from a single - cell whole transcriptome (13). The mrna - seq short reads were analyzed using whole - transcriptome software tools (applied biosystems, http://www.solidsoftwaretools.com/). The reads generated were mapped to the mouse genome (mm9, ncbi build 37). We got more than 6.6 million reads that could be uniquely aligned to the mouse genomic reference (uniquely mapped reads). Based on ensembl annotation (ncbi m37.61), 89% reads (5.9 million) were mapped to annotated regions in exons including coding sequences (cds) and untranslated regions (utr), which is significantly higher than those mapped to intronic (0.3 million, 5%) and intergenic (0.4 million, 6%) regions (figure 1a). Across the mouse genome, a total of 62.3% of rer boundaries were within 10 bp of the ends for the corresponding exons (figure 1b). Meanwhile, we identified 2,217 exon ends with remarkable extension into un - annotated regions (> 50 bp), suggesting that the mouse transcriptome was more complex than we expected . The transcript levels for rers overlapping with known exons (exonic rers) were significantly higher than those of novel rers (mann - whitney u test, p<10). We detected 12,277 expressed transcripts (with at least 1 rer across the genic region), in which 11,261 (92%) transcribed genes contained at least one exonic rer . For the 72,628 (74% of all 98,532 rers) exonic rers, we found that the known gene models were well defined by the ab initio method . For example, read distribution on chr 7 (56900000 - 57200000) revealed sharp boundaries of rer regions (figure 2). Furthermore, we detected a certain proportion of rers (25,904, 26% of all rers identified), which are located outside of the annotated regions in this transcriptome dataset . Among them, 13,291 intronic rers (about 13% of all) were identified in 4,449 genes, indicating possible additional exons . Interestingly, about 1,016 genes (23%) only had transcripts detected in the intronic regions but not in the exonic regions . As a result, we renewed 4,788 gene models, which account for 39% of 12,277 transcribed genes in total . The remaining 12,613 rers are located in the intergenic regions, suggesting possible presence of novel genes outside of the existing gene models . Here we have developed an exploratory tool, gene2dge, which can be employed to determine the rers and improve genome annotation . The package and methods can be applied to analyze other sources of any mapped short read counts from rna - seq data, such as results of sequencing by ab solid platform and illumina solexa platform . Moreover, gene2dge can be used on any personal computer with a low requirement for computer memory capacity, since data are processed for all chromosomes in order with one chromosome at a time . In this study, we provide an example of gene2dge usage to illustrate its application in transcriptome analysis . Gene2dge has also been applied to analyze datasets from other mouse tissues or tissues from other species (data not shown). All these results indicate that gene2dge is a suitable tool for the renewal of known gene models by ab initio prediction in transcriptome dissection . Dz, jl, yw, qz, xl and gl collected the dataset and participated in data analysis and visualization . Dz, jl, yw, qz, xl and gl collected the dataset and participated in data analysis and visualization.
Stem cells, their developmental function, and their potential roles in disease, particularly cancer, have been under intense investigation over the past decade . The / mammary stem cell is self - renewing and capable of functionally reconstituting the entire mammary epithelial cell lineage [13]. Mammary stem cells are potential contenders for the origin and recurrence of some mammary tumors [1, 4]. Thus, cancer therapies targeting the subset of cancer stem cells or tumor initiating cells (tics) may be most advantageous in treating these breast cancers and reducing the likelihood of recurrence . A major hurdle in the mammary stem cell field has been to develop methods to identify and isolate the small subset of normal or cancer stem cells in epithelial tissue . Initial work to classify the stem cell population relied on lineage tracing and cell division activity in image studies of whole tissue and in serial transplantation analyses [57]. Subsequently, several groups published methods to identify cell surface markers of mammary gland stem and progenitor cells using flow cytometry and further linked these protein markers to in vivo transplantation stem cell activity [811]. More advances in the quest to identify and analyze mammary stem cells in vitro came when dontu and colleagues described a nonadherent mammosphere assay (for review see as well as clarke and colleagues in this issue) which is an adaptation of the previously established neurosphere assay [13, 14]. In the original reports on mammospheres, human mammary epithelial cells were taken from reduction mammoplasties and grown over multiple passages in nonadherent conditions [12, 15]. These mammospheres maintained tripotentiality when allowed to differentiate in vitro by growth on either a collagen substratum or embedded in matrigel in the presence of the pregnancy hormone, prolactin . Mammospheres derived from reduction mammoplasties were found to be enriched for mammary epithelial stem cells which could produce successful outgrowths after transplantation of as few as 500 mammospheres into nod / scid mice . Moreas and colleagues also showed that mammospheres derived from primary mouse mammary epithelial cells were capable of regenerating complete mammary epithelial trees upon transplantation . The mammosphere assay has been widely utilized to measure in vitro stem / progenitor cell frequency in normal primary mammary epithelial cell preparations as well as frequency of cancer stem cells or tics derived from malignant mammary tissue [12, 17]. Although the gold standard stem cell assay is the in vivo transplantation assay, first described by deome et al ., the mammosphere assay has given investigators an in vitro assay that is less time consuming and more cost effective then the in vivo transplantation assay [18, 19]. Although the mammosphere assay does not measure actual in vivo stem cell frequency, it can serve as a relative measure of stem and progenitor cell frequency in a mixed cell suspension, between varying experimental groups in vitro . Recently the stem cell field has been turning to a method popularized initially in immunology research, known as limiting dilution analysis (lda). This quantitative approach incorporates the use of a limiting dilution cell culture assay and established statistical analysis . Several investigators have applied the quantitative lda to evaluate the stem cell population in the neurosphere assay [20, 21]. Currently, similar to the neurosphere assay, sphere number is scored in a multi - sphere culture . However, this method is prone to subjectivity in sphere identification as well as culture artifacts (i.e. Sphere aggregation / fusion). In the mammary gland field, many in vivo transplantation studies estimating stem cell frequency have successfully utilized limiting dilution to measure stem cell frequency [16, 22]. In this article, we discuss use of the quantitative lda for analyzing stem / progenitor cell frequency in secondary and tertiary mammosphere cultures, and, hereafter, refer to this analysis as a sphere limiting dilution analysis (slda). Use of the slda has the advantage that it reduces investigator bias, can be used for both human and mouse mammary epithelial cell populations, and requires a smaller number of cells than the traditional sphere assay . The slda operates on the assumption that a single cell can generate a sphere and that the variation within the experiment from well to well follows a poisson distribution . When conducting research on a mixed population of cells such as mammospheres we have found this analysis useful to evaluate the frequency of sphere formation as an indication of the presence of stem or early progenitor cells, and observe it to be consistent with sphere forming frequency derived from the traditional sphere counting approach . To perform the slda, the cells are plated into a 96 well plate over a range of densities from high to low (fig . 1). Plating density should be optimized, such that at the highest density, most wells will be positive for sphere formation, whereas the lowest density should have less then 1 cell per well, so the majority of wells are negative for sphere formation . The slda measures the probability of an event happening, i.e. Sphere formation, and assumes that a percentage of wells at the lowest plating density will be non - responsive and not form spheres . Therefore, the line plotted from the experiment should pass through the origin (x = 0, y = 0) when graphing density versus probability of sphere formation or the fraction of non - responsive wells . To determine the probability of sphere formation the natural log fraction of the negative wells (non - responding) is plotted on a linear scale versus the density . The density can be expressed as cells / cm, cells / ml or cells / well . For this review and for our experiments we choose cells / cm because it does not vary with volume of media in the well . A linear regression curve is then fit to the data points and the lower the density at which a population forms a sphere the higher its frequency for formation, by the equationfigure 1schematic showing strategy for limiting dilution plating of dissociated epithelial cells in 96 well plate for slda . The + refers to a well containing one or more spheres and a refers to a well without any spheres . The figure shows the cells being plated in a limiting dilution fashion across a 96-well plate . The amount of wells negative for sphere formation will increase as the plating density decreases as depicted\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\matrix {{{\text{p}} = {1} - {{\text{e}}^{\text{md}}}\left ({{\text{p}}\;{\text{is}}\;{\text{probability}},\;{\text{m}}\;{\text{is}}\;{\text{slope}}\;{\text{and}}\;{\text{d}}\;{\text{is}}\;{\text{density}}} \right),\;{\text{arranged}}} \\ {{\text{ln}}\left ({{1} - {\text{p}}} \right) = {\text{md}}\;{\text{where}}\;{\text{y}}\;{\text{is}}\;{\text{ln}}\left ({{1} - {\text{p}}} \right)\;{\text{and}}\;{\text{x}}\;{\text{is}}\;{\text{d}}.} \\} $$\end{document} schematic showing strategy for limiting dilution plating of dissociated epithelial cells in 96 well plate for slda . The + refers to a well containing one or more spheres and a refers to a well without any spheres . The figure shows the cells being plated in a limiting dilution fashion across a 96-well plate . The amount of wells negative for sphere formation will increase as the plating density decreases as depicted according to the assumed poisson distribution and as adopted from lefkovits, the natural log of the fraction of non - responding wells is linearly proportional to the mean number of cells with sphere forming potential . When plotting data using the form of the equation shown here, the value of x at the y = 1 intercept corresponds to the frequency of sphere formation and represents 37% non - responding wells . This value for different experimental groups can be compared to evaluate the frequency of sphere forming cells between varying conditions . The frequency can be calculated at any point on the line by using the slope (frequency = 1/slope) or by using the y = 1 intercept (for detailed theory see lefkovits) however for ease of interpretation we prefer to use the y = 1 intercept for directly comparing precursor frequency of different populations . Determining the frequency of sphere formation in culture assays can provide valuable information for interpreting the composition of the mixed population of starting cells . If used on cultures after treatment with a growth factor or receptor inhibitor, for example, the effects of the treatment on the percentage of sphere forming cells present in the population can be evaluated . Similarly, the consequence of genetic alterations on stem / progenitor cell number can be evaluated using slda . Similar to neurosphere cultures which enrich for mulitpotential progenitor cells over passage, the percentage of bipotential progenitor cells in nonadherent mammosphere cultures increases after several passages [12, 15]. Thus, using the slda on secondary or tertiary spheres allows one to study a more highly enriched bipotent progenitor population versus a more heterogeneous population in the primary spheres . We have used the slda to evaluate sphere formation in primary mammary epithelial cells (mecs) isolated from mammary glands expressing an mmtv - dominant negative insulin - like growth factor-1 receptor (dnigf-1r) transgene to determine if defective igf-1r signaling affected numbers of mammary stem / progenitor cells . The secondary spheres were dissociated with accutase for 5 min at 37 c and mechanically triturated until a single cell suspension was achieved . Cells were then plated at densities ranging from 1000 to 0.00012 cells (optimal range of cells for the slda in our mammosphere conditions; should be optimized for different cell populations and investigator conditions) across two 96 well plates in 200 l of pro - n media with 8 replicates for each dilution and then evaluated for tertiary sphere formation after 7 days in culture . We scored each well for the absence () or presence (+) of sphere growth to determine the fraction of negative wells (fig . 1, schematic). The plot shows natural log transformation for the fraction of non - responding wells (y - axis) versus plating density (x - axis) (fig . 2). As discussed, the probability of forming a sphere is determined by the x intercept (cell density) when y = 1 . The sphere forming frequency of the dn - igf-1r tertiary spheres (y = 1 intercept, x = 526), can be expressed as 1 in 526 cells capable of forming a tertiary sphere compared to the wild - type control cells that had a tertiary sphere forming frequency of 1 in 127 cells . Thus, we conclude that igf-1r signaling is required for survival and/or sphere formation and may therefore be important for stem and/or progenitor cell growth . This result is consistent with sphere growth measures using a visual counting method in a multi - sphere culture.figure 2slda comparing stem cell frequency in tertiary spheres between dnhigf-1r and fvb (wt) primary mammary epithelial cells grown in pro - n media in 96 well ultra - low adherent plates (corning). Plating density ranges from 1000 to 0.00012 cells / well, which was converted to cells / cm in this graph . Tertiary sphere forming frequency for dn - igf-1r is 1 in 526 cells versus 1 in 127 cells for the wild type control . Pro - n media contained egf / bfgf (20 ng / ml), bovine insulin 25 g / ml (sigma), d - biotin 10 ng / ml (sigma), progesterone 20 nm (sigma), putrescine 100 m (sigma), selenium 5 ng / ml (sigma), apo - transferrin 50 g / ml (sigma), gentamycin 50 g / ml (invitrogen - gibco), hydrocortisone 0.5 g / ml (sigma). Linear (fvb) and linear (dn) indicate best fit line for linear regression analysis slda comparing stem cell frequency in tertiary spheres between dnhigf-1r and fvb (wt) primary mammary epithelial cells grown in pro - n media in 96 well ultra - low adherent plates (corning). Plating density ranges from 1000 to 0.00012 cells / well, which was converted to cells / cm in this graph . Tertiary sphere forming frequency for dn - igf-1r is 1 in 526 cells versus 1 in 127 cells for the wild type control . Pro - n media contained egf / bfgf (20 ng / ml), bovine insulin 25 g / ml (sigma), d - biotin 10 ng / ml (sigma), progesterone 20 nm (sigma), putrescine 100 m (sigma), selenium 5 ng / ml (sigma), apo - transferrin 50 g / ml (sigma), gentamycin 50 g / ml (invitrogen - gibco), hydrocortisone 0.5 g / ml (sigma). Linear (fvb) and linear (dn) indicate best fit line for linear regression analysis although, it is clear that mammospheres are enriched for multipotent cells, it remains somewhat ambiguous as to what passage number (i.e. Primary, secondary, or tertiary) the spheres should be analyzed for the number of stem / multipotent progenitor cells in the population . It is generally accepted that number of spheres formed is equivalent to the combined number of stem and progenitors in a given population of mammary epithelial cells . The passage number (i.e. Primary, secondary, etc .) And plating density, however, at which the mammospheres are analyzed has varied between investigators and could have an influence on the number of stem and progenitor cells present . Another limitation of the sphere assay relates to whether this assay properly identifies the frequency of in vivo quiescent stem cells as opposed to measuring cells that adapt or can act as a proliferating mammary stem cell in vitro . The validity of using the sphere assay as a functional test to measure frequency of in vivo stem cells was recently addressed in a review considering the correlation between neurosphere forming cells and number of neural stem cells in vivo . Concluded that the neurosphere assay does not properly identify numbers of quiescent neural stem cells in vivo . Furthermore, until these quiescent stem cells can be definitively elucidated using cell markers, the frequency of these cells in vivo will remain an enigma . The same caveats are applicable for the mammosphere assay; it is still unclear whether the mammosphere assay truly identifies quiescent stem cells in the mammary gland . Also, multi - potential progenitor cells can give rise to sphere growth as well as stem cells . In addition, although stem cell growth and survival factors such as wnt and hedgehog have been identified, optimal growth conditions have not been standardized for mammosphere growth; therefore, the mammosphere assay is likely not a true measure of physiological stem cell frequency . Mammosphere cultures, like neurosphere cultures, however, do enrich for epithelial cells that behave like mammary stem cells in vitro and are capable of forming all three lineages when transplanted into cleared fat pads . Within the limitations of the mammosphere assay, by standardizing methods in quantifying the mammosphere assay using the slda, furthermore, using the slda to quantify mammospheres in secondary and subsequent passages, due to their known enrichment for multipotent cells, will allow for greater confidence in interpretation that the calculated number of sphere forming cells correlates with number of multipotential progenitors or stem cells . The use of the slda on primary mammospheres should be avoided if possible when measuring multipotent cell frequency because primary cultures are more heterogenous then are secondary and tertiary sphere cultures as discussed by dontu and colleagues (2005), however, in circumstances where a genetic alteration or treatment condition prevents subsequent passaging of mammospheres, it would still be useful to assess primary sphere forming frequency . An example of this was seen with constitutive activation of smoothened in mammary epithelial cells . Because the slda takes multiple plating densities (from clonal to higher densities) into account, it allows for a more comprehensive method of quantifying sphere formation and sphere size in a given population of cells . Sphere size may be a reflection of a mitogenic response to specific treatment conditions and progenitor expansion, however, due to sphere aggregation, emphasis has been placed on analyzing sphere size at clonal densities . In the slda, sphere size can accurately be evaluated at the lower densities (i.e. 1 cell per well). In summary, the slda is an advantageous tool in quantifying sphere forming frequency in a population of mammary epithelial cells . The slda does not require a large amount of cells so can be utilized when sample quantities are limited . Due to low density plating finally, the slda method reduces investigator bias and can lead to a more standard method for quantifying stem / progenitor cell frequency in mammospheres.
A 40-year - old male presented to the emergency department with complaints of irrelevant talk, abnormal behavior, and disorientation for four days . He had multiple generalized tonic clonic convulsions two days back . On admission he was afebrile, anxious, restless and confused with doubtful neck rigidity since he kept himself stiff and was non - cooperative during examination . He had tremors in both hands, pulse 106/min, blood pressure 146/96 mm of hg and respiratory rate of 20/min . History revealed him to be an alcoholic for 12 years with psychological and physical dependence, with abstinence for five days . He had no past history of any medical or surgical illness except frequent cough and occasional fever for few weeks for which he took cough syrups and antipyretics directly from chemists without any consultation with doctor . He was a salesman in a grocery shop with difficulties in saving his job because of frequent absenteeism . His wife reported him to be a happy type of person during the initial three - four years of starting alcohol with friends . He gradually became alcohol - dependent, irritable, abusive and frequently aggressive to the wife and both children of 10 and 12 years of age . There was no history of any promiscuous behavior or sexually transmitted disease . On mental status examination (mse) he had altered sensorium with poor concentration, disorientation to time and place, short - term memory impairment, slurring of speech, increased psychomotor activity and irritable mood . On the basis of history and mse a diagnosis of alcohol withdrawal delirium was made and patient admitted . He was started on chlordiazepoxide 25 mg four times a day with adequate fluids and multivitamins . His hemoglobin was 12.5 gm / dl, total leucocyte count 6700/mm with 80% polymorphonuclear cells and 20% lymphocytes . Erythrocyte sedimentation rate was 48 mm per h and random blood sugar was 124 mg / dl . Serum bilirubin total was 1.8 mg / dl and direct was 0.9 mg / dl, alanine amino transferase alt was 58 and aspartate amino transferase (ast) was 97 renal function test, serum electrolytes, thyroid function test, lipid profile, hiv test, electrocardiogram and computed tomography scan of head were unremarkable . During second and third day of admission he developed rigidity of limbs, mutism with apathetic look, open eyes and fixed gaze but no emotional responsiveness . Injection lorazepam 4 mg twice a day was added to reduced dose of chlordiazepoxide 25 mg twice a day considering his condition as unusual catatonic manifestation of alcohol withdrawal . Reviewing his temperature charts revealed that he had been having an evening rise of temperature during all these days . On reexamination physician reference was sought and the case was transferred to the medical ward with a clinical diagnosis of alcohol withdrawal with coexistent meningoencephalitis . On further investigation his chest x - ray showed bilateral miliary shadows and cerebrospinal fluid (csf) examination revealed protein 150 mg%, csf sugar 60 mg%, tlc- 60 cells / high power field, differential leucocyte counts- 100% lymphocytes, adenosine deaminase 18 u / l . Diagnosis was then revised to tubercular meningoencephalitis and treatment switched to antituberculous therapy and injection dexamethasone followed by oral steroids . After three days of starting antitubercular treatment he showed significant improvement and was discharged on category one antitubercular treatment with advice to follow up treatment from his nearby directly observed treatment supervised (dots) centre as the patient was from a remote area . He kept his commitment of fortnightly counseling sessions up to three months with complete abstinence and then was lost to follow - up . Delirium is commonly encountered by psychiatrists in alcohol withdrawal states, but coexisting medical illness may be contributory to it which should be carefully looked for, specially electrolyte abnormalities, hypoglycemia, change in fluid status (dehydration or volume overload), uncontrolled pain, etc . As per diagnostic and statistical manual of mental disorders, 4 edition (dsm-4) criteria a diagnosis of delirium due to multiple etiologies should be made if patient has disturbance of consciousness, change in cognition, disturbance developing in a short period of time with fluctuating course and evidence from history, physical examination or laboratory finding that delirium has more than one etiology i.e. A general medical condition plus substance intoxication or withdrawal . This case initially presented with alcohol withdrawal delirium and partially responded to conventional treatment but after that he developed catatonic symptoms which are not found in alcohol withdrawal states . Although catatonic states are uncommon in both tubercular meningoencephelitis and alcohol withdrawl, but between these two former has more probability of having an association with catatonia than later . Similar cases showing association of catatonia with meningoencephalitis have been reported by ito et al ., and by carroll et al . In a case report by caroll et al ., the patient had delirium with episodes of catatonic excitement alternating with stupor and was diagnosed to have meningoencephalitis . History of alcohol withdrawal, partial response to chlordiazepoxide, emergence of catatonic symptoms and good response to treatment of meningoencephalitis indicate that meningoencephalitis might have contributed to the delirium which justifies diagnosis of delirium due to multiple etiologies . Physicians and psychiatrists are advised to be cautious about two things, firstly, the secondary causes of delirium, and secondly, the potentially serious organic illnesses which may underlie the catatonic syndrome.
To specifically delete mature osteolineage cells, the oc - cre strain (from dr . Thomas l. clemens, johns hopkins university, md, usa), which expressed cre under the osteocalcin promoter was crossed to the idtr strain . Temporally controlled cell ablation was achieved upon injection of diphtheria toxin into the ocncre;idtr strain . To study the effect of ocn cells on hematopoiesis, ocncre injected with diphtheria toxin or ocncre;idtr injected with pbs served as controls while ocncre;idtr injected with diphtheria toxin were used as mutants . For most experiments, 25 ng diphtheria toxin in pbs / g of body weight was injected daily into both control and mutant animals for 28 days from 4 weeks of age to achieve an acute deletion of specific osteolineage populations . Mice were harvested for analysis the next day after the last dose of diphtheria injection . For all experiments, littermates were used as controls . All animal usage and procedures performed to specifically delete mature osteolineage cells, the oc - cre strain (from dr . Thomas l. clemens, johns hopkins university, md, usa), which expressed cre under the osteocalcin promoter was crossed to the idtr strain . Temporally controlled cell ablation was achieved upon injection of diphtheria toxin into the ocncre;idtr strain . To study the effect of ocn cells on hematopoiesis, ocncre injected with diphtheria toxin or ocncre;idtr injected with pbs served as controls while ocncre;idtr injected with diphtheria toxin were used as mutants . For most experiments, 25 ng diphtheria toxin in pbs / g of body weight was injected daily into both control and mutant animals for 28 days from 4 weeks of age to achieve an acute deletion of specific osteolineage populations . Mice were harvested for analysis the next day after the last dose of diphtheria injection . For all experiments, littermates were used as controls . All animal usage and procedures performed before sacrifice, peripheral blood was collected from each mouse and subjected to complete blood cell count . For each mouse, tibiae, femurs, iliac crests, spines, ulnae, radii, and humeri were collected for bone marrow cells . We routinely stain 5 10 cells per sample for the hematopoietic stem population, and 1 10 cells per sample for each progenitor and mature population . Lineage cocktail consists of biotinylated b220, cd3e, cd4, cd8a, cd19, cd11b, gr1, ter119, cd11c, and nk1.1 antibodies . The following antibody combinations were used to recognize ly6d clp (lineage - pacific orange, sca - pacific blue, ckit - apc - cy7, cd127-pe - cy7, thy1.2-fitc, ly6d - apc). Ly6d clps were sorted from 6 to 8 weeks old ocncre;idtr mutants and controls using the facsaria following daily dt treatment for 4 weeks . Rna was extracted using the trizol (invitrogen) according to the manufacturer's instructions . Samples were processed by the nugen ovation v2 laboratory process in the microarray core facility of dana - farber cancer institute . Briefly, first stand cdna was prepared from total rna using a dna / rna chimeric primer and a reverse transcriptase . The resulting double stranded cdna with a unique heteroduplex at the 5' end of the antisense strand were amplified using spiatm amplification, a repeated process of spiatm dna / rna primer hybridization, dna replication, strand displacement and rna cleavage which resulted in a rapid accumulation of cdna with sequence complementary to the original rna . The spiatm amplified cdna was purified using the zymo research dna clean & concentrator system . The purified cdna was fragmented through a chemical and enzymatic process and labeled via enzymatic attachment of a biotin - labeled nucleotide to the 3-hydroxyl end of the fragmented cdna . The biotinylated cdna was added to a hybridization solution containing several biotinylated control oligonucleotides (for quality control), and hybridized to the mouse430a microarray chip overnight at 45 c . The chips were then transferred to a fluidics instrument that performs washes to remove cdna that has not hybridized to its complementary oligonucleotide probe . The bound cdna was then fluorescently labeled using phycoerythrin - conjugated streptavidin (sape); additional fluors were then added using biotinylated anti - streptavidin antibody and additional sape . Each cdna bound at its complementary oligonucleotide was excited using a confocal laser scanner, and the positions and intensities of the fluorescent emissions were captured . Standard qa / qc analyses involved chip analysis with the assayqualitymetrics bioconductor package and found no significant quality issues with any of the chips, as determined by (among other methods) visual inspection, intensity distributions or rna degradation plots . The data was background corrected and normalized with rma (robust multichip average) using the affy bioconductor package . The selected database comprises 6 expression measurements of 41,345 genes, 3 in the first condition and 3 in the second . The recorded expression values ranged between 4.023 and 10,198.29 . Supervised learning or class prediction methods were used for molecular classification and pattern recognition . This analysis involves selecting the features (genes) most correlated with a phenotypic distinction of interest . These features or marker genes are biologically interesting in themselves but they can also be used as the input of a classification algorithm that uses existing labeled samples to build a model to predict the labels for future samples . Genes correlated with a binary class distinction, for example a morphological or clinical phenotype, is directly identified and selected by using a distance metric, for example t - test statistic = (ma mb) (s2a + s2b) [m and s are the means and std . Dev . Per class] or signal to noise ratio = (ma mb) / (sa + sb) [m and s are the means and std . Dev . Per class]. 1 is a color map illustrating 30 genes overexpressed in mutant ly6d clps compared to control ly6d clps . Table 1 shows the mean, standard deviation, score, and fold change of the 30 genes.
Despite a gradual reduction in the pediatric population in korea due to low birth rates, the number of children's visits to emergency departments (eds) each year has been increasing1). According to the national emergency department information system (nedis) data from 2008, the percentage of pediatric patients who visited eds was about 31.2% of total ed patients (fig . This figure is larger even considering a proportion of pediatric aged population (<19 years) in korea (23.8%) and higher than that of us data3). However, a significant number of children who visit an ed do not receive appropriate level of care and are pushed back on the priority list . This is because their symptoms are usually relatively mild compared to that of adult patients, and the care of children in ed is considered very time - consuming and cumbersome . Recently, due to a decreased number of pediatric residents, who are responsible for the care of the majority of pediatric patients in ed, the neglect of pediatric patients in ed has become an increasing concern . This situation is also exacerbated due to a lack of sufficient numbers of emergency physicians having adequate knowledge and skill to care of children . The results of a survey by the korean society of pediatric emergency medicine in april 2010 revealed a lack of preparation for pediatric emergency care throughout the country4). Among 81 eds that responded to the survey, only a quarter were equipped with pediatric emergency carts, and just about 60% of the eds had pediatric endotracheal tubes for every age . Intraosseous needles which are essential for vascular access in critically - ill children were prepared only in 40% of the eds . Moreover, the nighttime or weekend consultations to pediatric departments were mainly addressed by relatively inexperienced residents (first- and second - year). Monitoring services for children sedated for radiologic studies were not available in 53.1% of the eds . The survey showed that a huge unpreparedness exists in current pediatric emergency service in korea . An analysis of the nedis data for 3 years, from 2006 to 2008, provides us an estimate of the point of preparedness for pediatric emergency care in korea2). For children younger than 9 years, fever was a major complaint, while abdominal pain and headache were the most common symptoms for children over the age of 10 . Admission to the hospital was required in 15% of patients, and the admission rate was above 20% in infants younger than 1 year . During the 3 years, about 6,000 patients were critically ill or injured so as to require care in intensive care units (an average of 16 patients per day). Originally, the emergency medical system was initiated to provide proper care for adult patients, and the need for pediatric emergency care has not been met adequately for a long time . The resources that need to be established for pediatric patients who visit the ed are different from those needed for adult patients . For adequate care for children, experienced staff, who understand the anatomical and physiological differences between adults and children is needed . Considering the growth and development of children, emergency medical equipment and appliances of various appropriate sizes must also be prepared . Moreover, a child - friendly environment, preferably separated from adult patients, is strongly recommended in order to reduce the psychological trauma and discomfort to the child . A comprehensive and regionalized system should be prepared, in addition to proper preparation for basic pediatric emergency care . Without specialized pediatric emergency and critical care,, it is impossible that all eds can be made ready to support high - quality pediatric emergency care . The occurrence of serious pediatric emergency cases is not only relatively low compared to adult cases, but also the pool of experts for pediatric advanced care is limited . Especially in korea, the facilities and human resources for the pediatric field are greatly limited . Considering both cost - effectiveness and efficiency, a critically - ill or severely - injured child should be transferred to a specified referral center, which can take a charge of the definite treatment7). In addition, a child who needs a special procedure such as air - reduction or endoscopic foreign body removal, should be directed to a center with adequate personnel and equipment without delay . The term " regionalization " in healthcare refers to the development of a structured system of care " to improve patient outcomes by directing patients to facilities with optimal capabilities for a given type of illness or injury"8). Historically, the landmark report' accidental death and disability: the neglected disease of modern society'9) has been a powerful driving force for regionalization in the united states (us) since the 1970s . Regionalization improves patient outcomes through 2 primary mechanisms: specialized care at high - volume, high - specialty centers and increased coordination of care within a given geographic area10). There is substantial evidence that regionalization of services to designated hospitals with greater experience improves outcomes and reduces costs of care for severely injured patients11,12). Although the number of studies addressing pediatric experience of trauma centers is limited compared to adult trauma centers, the main results are that regionalized pediatric trauma system reduced the mortality of pediatric patients with severe damage13,14). All hospitals cannot be prepared to house neonatal intensive care units (nicus) for a fraction of complex preterm neonate . With regard to consideration of efficiency, the us and european countries have operated a regionalized nicu, which is invested in facilities and personnel . Substantial data from nicus in these regions showed that nicu regionalization programs contributed to a low infant mortality15,16). The 2006 institute of medicine (iom) report " emergency care for children: growing pains " concluded that pediatric emergency care is uneven in the us17). Fifty percent of all us eds see fewer than 10 pediatric patients per day . In the us, a comprehensive and definite care for critically - ill or injured pediatric emergency cases has been performed mainly at 226 children's hospital and 170 trauma centers with pediatric capabilities . As a categorization model for the regionalization of pediatric emergency care, the emergency department approved for pediatrics (edap) model was established in los angeles county in 198518). In this model, only those hospitals meeting requirements as an edap in los angeles county could receive children brought in by emergency medical services (ems). However, parents or guardians usually transport their child directly, by themselves, rather than by using ems system and they tend to visit the ed nearest to their home, and not necessarily one with pediatric capabilities . This is referred to as one of the barriers to developing the regionalized network of pediatric emergency care in the us10). Based on previous reports8,10,17), there are some suggestions for success of the regionalization of pediatric emergency care in korea . First of all, the appropriate categorization should be achieved in all areas in pediatric emergency care . While a transfer of patient by ems must be achieved according to a level of patient's acuity, properly assessed at pre - hospital field, a critically - ill child should be transferred to an ed with staffed with certified personnel and equipment . Training and continuing education of pediatric emergency care for ems providers should be introduced as an essential ems education course and the classification of pediatric patients must be made using well - established protocols . Information from emergency information center should also be properly matched with the patient's severity level, along with efforts to better inform the community about the level of critical care and services available for pediatric emergency care at the ed within a region . The categorization of eds according to their capability for pediatric emergency care is essential for a successful regionalization program . To eliminate controversies, further, as the iom report recommended, the coordination of all elements of child healthcare from an ems system to hospital care, should be governed by legislation and be followed as a matter of policy . A process of accreditation, to dedicate and approve a healthcare facility as a specialized provider of pediatric care from a certifying authority, is also required . In this respect, the current " pediatric exclusive emergency department projects " conducted by ministry of health and welfare should be reviewed . The role of the pediatric exclusive ed in the current project is not a comprehensive regional pediatric emergency center for resource - intensive care of critically - ill children, but rather just a well - equipped pediatric ed without a higher level function . Rather, a flock of children with low acuity to the center could make a delay of adequate care for critically - ill patient . This program has been initiated without a sufficient thought and long - term planning, and is focused only the facilities and equipment supply . Actually, it does not provide a plan for managing the human resource, which is the most important factor for the success of pediatric emergency care system . One of the concerns surrounding regionalization of pediatric emergency care is that transfer of too many patients from community hospitals would reduce the chance of appropriate pediatric care, raising the concern that there may be an eventual degradation of the quality of pediatric emergency care in whole community . However, the basis for regionalization is that all emergency center scaring for pediatric patients must be prepared to some baseline extent . Further, regionalized systems need a back - transport of patients to non - specialty hospitals after they have been stabilized and their condition improved . Parents or guardians may be reluctant to back - transport, because of their comfort and belief in the high level hospital19). However, if not supported by a re - distribution system, the referred hospital could become crowded with patients awaiting treatment . Other aspects to be addressed in establishing regionalization are a specialist on - call system and financial incentives . It may be difficult to get the on - call personnel to come to the ed, even though their privacy has been compromised by calling them directly, since financial incentives for their participation in emergency care cannot be provided easily under current korean health insurance policies . For these reasons, it has become increasingly difficult for eds to find specialists who will be ready for on call for the ed, and the resulting shortage of on - call specialists in pediatric emergency care leads to sometimes tragic results . Currently, the korean society of pediatrics and the korean society of emergency medicine have been carrying out some efforts to increase medical insurance fees for emergency and pediatric areas . In addition to an introduction of appropriate medical fees, a special compensation and/or financial incentives must be offered to regional pediatric emergency centers to attract highly qualified pediatric emergency care personnel . We took a first step towards understanding the basic status of pediatric emergency care and for determining its preparedness in providing adequate pediatric emergency care . We need to take some different regionalization strategies in consideration of the different circumstances in medical practices in korea . To do so, medical personnel, resources, and facilities that are closely related to pediatric emergency care should be identified and adequately categorized before implementation of a regionalization system . Based on this, information in all phases of emergency medical systems, including data from patients, paramedics, and hospitals, should be collected and share efficiently for building an information system in regionalization . A win - win strategy, to create a bi - directional flow of the patient between referral center and referring community hospitals that participate in the regionalization should be established . Efforts to set up a pediatric emergency care regionalization program based on the national healthcare system are urgently needed . The first step to building a safe and effective pediatric emergency system will be made possible through regional networks of health care.
The identification and enumeration of circulating tumor cells is an important tool for evaluation of the disease progression in especially breast cancer [13] and is also under consideration as a diagnostic tool in various other types including lung and colorectal cancer [46]. The type of ctcs found also serves as a potential marker for changes in the chemotherapy resistance of a cancer . The extreme rarity of ctcs in patient blood, typically one ctc per 10 blood cells, makes both collection and detection of these cells extremely challenging . The collection of ctcs from peripheral blood is in a majority of studies done by antiepithelial - cell - adhesion - molecule (epcam) antibody - coated isolation systems [5, 9], but also other types of immunomagnetic devices [10, 11], density gradient centrifugation, and membrane filtration are used for ctc enrichment . The detection of ctcs after collection is done by immunocytological staining or polymerase chain reaction (pcr). In the case of immunocytological staining the standard method of ctc enumeration is manual counting either at the microscope or from microscopy images [15, 16]. However, progress was lately made in using machine learning techniques for the detection of ctcs from fluorescence microscopy images [17, 18]. In these studies, as well as in any study applying classifiers to data, manual labelling the use of computational methods, in this case machine vision, makes the screening of the vast amounts of data that is readily available today quicker and more efficient . Instead of having a highly trained expert performing the time - consuming task of looking at numerous images, even if the computer is not able to completely take over the manual analysis it can at least screen the image data for regions of interest and provide a second opinion in difficult cases . This paper builds on the previous result in enumerating ctcs in images using image analysis techniques combined with support vector machines (svms) and nave bayesian classifiers (nbcs). Data for the study was collected with a functionalized and structured medical wire (fsmw) that is a ce - certified medical device for the isolation of ctcs . Human carcinoma cells expresses the epithelial cell adhesion molecule (epcam) on their surface while this molecule is absent from the surface of haematological cells [2022]. The fsmw is functionalized with anti - epcam antibodies and was inserted into the cubital vein of a patient through a standard 20 g intravenous cannula, where it was left for 30 minutes collecting ctcs from the blood that flows past . After cell collection the fsmw was fluorescently stained and microscopy images were made in which we aim to enumerate ctcs . Ideally only ctcs should adhere to the fsmw but because of the many blood cells compared to ctcs, even the unlikely event of catching a blood cell occurs regularly . The first step in the analysis was to identify regions of interest (rois) which are candidates as ctcs but may in fact also be a blood cell, some kind of debris or a staining artifact . In the previous study we concluded that both svms and nbcs achieved an accuracy of ctc detection in the range of 8590% after rois were identified . In that study, the annotation used for evaluation of classifier performance and training of the classifiers were based on the manual classification of the rois by one author (cms). The use of different machine learning and machine vision techniques is an active research field with the aim of making disease diagnosis more accurate and efficient . Especially in the diagnosis and treatment evaluation of different cancer types, including but not limited to prostate [24, 25] and colorectal cancer, automated algorithms are used . However, interobserver variability is a known issue in diagnostics of different cancer types and a disagreement of more than 15% is not uncommon when multiple observers, normally all trained experts, are interpreting patient image data of different types [2729]. When training and evaluating an automated classifier the labels provided by observers are of great importance as any inconsistencies will affect the performance of the classifier . In this study, we investigated how uncertainty in annotation, so called label noise, affects the performance of automated classification using a random forest (rf) and a svm and relate that to the performance of earlier studies [17, 18]. Interobserver variability for disease progression using ctcs is reported to be as low as 1% but is then related to the question if the patient has more than 4 ctcs per 7.5 ml blood . When considering the manual classification of images of possible ctcs, scholtens et al . Presented that observers disagree on approximately 15% of the data points . To investigate how this variability affects the estimated performance of the classifiers, we in this study carefully identified possible label noise through analysis of the manual annotation . Moreover, a consensus annotation was identified and training and testing of the classifiers with a controlled amount of label noise in both training and test sets was evaluated . The data set used for this study was the same as used in our earlier publication, where ctcs were captured using the fsmw both in vivo and in vitro . After collection the fsmw was fluorescently stained for cell nuclei (blue), epcam, or cytokeratins (green) and counterstained for cd45 (red) in order to differentiate between ctcs and blood cells that may have attached to the wire . Images were taken using a 10x ocular and 10x, 20x, or 40x objective resulting in 1.0 m, 0.5 m, or 0.25 m pixel resolution of the images . Ctcs are those cells that exhibit nuclear dye (blue) colocalized with the antibodies against cytokeratin and/or epcam (both green); see figure 1(a). Rois, for example, objects that may be ctcs and most likely at least some type of cell, were identified based on the blue signal that indicates positive staining of a cell nucleus . For full details of the collection, staining, imaging, and roi identification we refer the reader to earlier publications using this data set [18, 19]. The data points used for ctc classification were obtained by cutting out an image with area 100 100 pixels around the center of each identified roi resulting in 617 data points from 61 original microscopy images . Manual annotation was needed for both training and evaluation of the classifiers as well as for the determination of interobserver variability . The observers were instructed to determine if the most central object in each image cutout was a ctc or not . For an example of multiple objects occurring in the same cutout [18, 19], observers were instructed to count the object as a ctc if the nuclei (blue staining) were intact and the object showed positive staining for epcam or cytokeratin (green staining). The blue and green staining had to be distinguishable from each other; for example, the nuclei and the epcam staining should be structured . While it was required that the nuclei should be intact, it was allowed for ctcs to have irregular shapes or be clustered . Any object that showed positive cd45 staining (red) was not to be counted as a ctc; see figure 1(b). All nobs = 11 observers (with 5/6 male / female) had normal or corrected to normal eyesight and no one had any known issues with color vision . Cutouts were presented on individual laptops in one session to avoid different light conditions and without any time restrictions . The order of the data points was random and the observers were instructed not to confer . As the cutouts have been taken at different magnifications we first normalized the image matrix to cover a region of the size of 2500 m around the center of each image cutout . This means that the cutouts had 100 100, 71 71, or 50 50 pixels, depending on if they were from an image taken with a 40x, 20x, or 10x ocular . We then applied a gauss convolution filter with standard deviation = 1 pixel to the cutouts to reduce the effects of high frequency noise . The classifiers we used, svm and rf, both required inputs with fixed dimensions and therefore all cutouts were downsampled to 50 50 pixels using the raster package in r (https://cran.r-project.org/package=raster). The color space of the cutouts were red - green - blue (rgb) when read but for the classification we transformed them to hue - saturation - value (hsv) using the grdevices package (https://stat.ethz.ch/r-manual/). This was done as hsv has a natural division between color dimensions (h and s) and intensity (v), which is not present in rgb space . In the hsv space we dropped the v dimension as preliminary tests revealed that this factor is not decisive in the classification of cutouts containing a ctc or not . The image matrix was then vectorized so that each cutout is then represented by an array with 5000 entries with the hue and saturation values of the cutout . For the rest of this paper any reference to automated classification of a data point or cutout will mean that this vector containing the hue and saturation values of a cutout was presented to the classifier . The automated classifiers were implemented in r using the h2o interface (https://cran.r-project.org/package=h2o) for the rf and the kernlab package (https://cran.r-project.org/package=kernlab) for the svm . The svm with radial basis function (rbf) kernel had the parameters c = 2 and = 0.005, where c is the soft margin penalty and the inverted radius of the rbf . Parameters, number of trees as well as c and for the svm, were optimized to give the highest accuracy possible on a subset of the data . To get the classifier responses to the data, all data sets, that is, both the entire set of 617 cutouts and subsets that will be described, were divided into randomized folds . Training of the classifier was then performed on a number of folds and testing was then done on one or more folds that were not used for training . This was done iteratively with new folds chosen for training and testing until all data points were classified . For each subset of data the number of folds and how they were used for training and testing are described in the text where appropriate . In the n = 617 data points the observers found on average 300 ctcs with the median being 318, but the number varied largely as can be seen in table 1 . The lowest number of ctcs was found by the observer mtf with 221 ctcs and the largest number was 354 ctcs observed by st . The largest interobserver distance in an ordered list was between mb (223) and mp (281) with 48 ctcs, while the second largest distance was 17 between jp (330) and cms (347). The initial conclusion is therefore that two observers, mtf and mb, had a much more conservative opinion on what was to be considered a ctc than the other observers, thereby minimizing the risk for false positive ctc annotation . The rest of the observers have a range of detected ctcs that corresponds to approximately 10% of the total number of data points presented . We define the agreement between observers a and b as(1)pagra, b=11ni=1nabsaibi, where ai, bi indicates the annotation of image cutout i as ctc (1) or no ctc (0) by the respective observer . If two observers agreed on all data points their agreement is one, whereas total disagreement gives the value zero . In figure 2 it is worth noticing that the maximal agreement was 0.91 and that the average (median) agreement was 0.85 (0.87). This average agreement can be compared to the study by scholtens et al . That also had an interobserver agreement of 0.85, in that case across five observers . It should, however, be noted that the classification task in their study was not binary but objects were classified into one of five classes dividing the data into different types of ctcs and other objects including leukocytes . On the other hand, all observers in their study were referred to as experts, whereas in the present study the observers comprise experts as well as non - experts that were asked to identify ctc for the first time according to the criteria described in section 2 . In figure 3 we present the average agreement per observer against the average difference in identified ctcs between one specific observer and all other observers . The average agreement between observer a and the others is defined as(2)pagra=1nobs1bobserversapagra, band the mean difference in the number of ctcs found for observer a against all other observers is given by(3)ctca=1nobs1bobserversanctcanctcb.it can be seen from the clustering in figure 3 that the two observers avoiding false positives in their indication of ctcs are isolated from the rest of the observers in both dimensions . Even though all participants were given identical instructions, both written and orally, these two observers made a different interpretation on how to annotate the data compared to the other nine observers . While the majority of observers tried to make a guess on cases where they were unsure, the observers mtf and mb always went for no ctc when unsure . For the two observers which avoided false positive ctc annotation the difference in the number of found ctcs seems to be the underlying reason for the low agreement with the other observers . The difference or similarity in number of identified ctcs however does not uniquely predict observer agreement . As an example we consider the two observers sd and tl, who have indicated 307 and 303 ctcs, respectively, that have an agreement of 0.88 . On the other hand the observers mp and jp had an agreement of 0.89 although jp identified 49 more ctcs than mp . This emphasizes the need for a multidimensional and a multiobserver analysis regarding the interobserver agreement, rather than just looking at pairwise agreement and averages to identify observers with different biases . The average agreement between any pair of observers was 85%; that is, p(a = b) = 0.85, and the assumption that the probability of agreement would be equal for each image cutout can be inserted into the bernoulli distribution (4)pa = b=22p2=0.85,resulting in p=0.850.92 . Based on this value, we estimate that all eleven observers should agree in 617 p 259 of the cases . In our dataset, all eleven observers agreed on 365 data points and we refer to these data points as the total consensus data set . This implies in turn an average pairwise agreement of p(a = b) = 0.91, which is significantly different (p <10, student's t - test) from the measured agreements in figure 2 . From these considerations we can draw the conclusion that the probability for disagreement is not the same for all image cutouts . To exemplify this, we in figure 1 show a cutout for which all observers agreed of having a ctc (a) and one for which all agreed that there is no ctc (b). In the first case the conditions for a ctc are clearly fulfilled with the strong green staining and the clear integrity of the nucleus shown by the blue staining . The red staining identifies the object as a blood cell and all observers agreed that this is not a ctc . The third case, figure 1(c), shows an example where the decision was split six versus five . The staining intensity in this cutout is lower than for the other cutouts and it is therefore hard to verify the integrity of the nucleus . Furthermore, it is difficult to determine if the green staining is structured enough for a positive ctc classification . It is also quite possible that some observers did not see the green staining at all due to the low color intensity . The requirement that all observers should agree may be unnecessarily harsh as we may then discard data that a single observer made a mistake on . In studies where observers are not well supervised and possibly anonymous, as in the case of citizen science projects [32, 33], a single observer that misunderstands the task (or for some reason willingly gives false annotations) can severely damage the integrity of the data set . To determine how many observers we require to vote either ctc or no ctc, we defined a consensus limit, c, for which we say that consensus was reached . As the decision between ctc or no ctc is binary, we required that for the nobs = 11 observers(5)pbin=i=1nobsnobsc12nobs<0.05;that is, the probability that c observers by chance annotated the cutout as containing a ctc or not should be less than 5% . In our case this means that c = 9 observers had to agree that the cutout does or does not contain a ctc for consensus to be reached and in our data set consensus was reached in 502 of the 617 cutouts (81%). For the consensus data set the interobserver agreement was naturally higher with mean (median) of 0.93 (0.95). In figure 4 the agreement between observers for the consensus data set the observers that avoided false positive ctc annotation again had considerably lower number of ctcs than the other nine observers; see table 1 . Excluding the two observers with the no false positive bias (mb and mtf), the other nine observers are identified between 244 and 260 ctcs which gave a variation of around 3% of the total number of cutouts presented . Given the distinctly different number of ctcs (see table 1) identified by observers mtf and mb and their deviation from consensus (see figure 4) the hypothesis that these two observers had a different bias than the others however, instead of discarding the two observers as outliers, we decided that it may be rather interesting to see how annotations that arise from different biases affect the training of automated classifiers . In a setting where fewer observers are used it may not be possible to identify such differences in bias and it is also not sure that the differences in bias is restricted to a clear minority of observers . When evaluating automated classifiers different performance measures are used to show their agreement with an annotation considered to be ground truth . We have so far demonstrated that for certain data sets the annotation can vary strongly depending on the observer performing the annotation . The performance measures we use to evaluate the automated classifiers are defined with the help of correctly identified ctcs (tp), falsely identified ctcs (fp), objects correctly identified as not ctcs (tn), and ctcs that were not identified as such (fn). Our performance measures are then defined as accuracy acc:(6)acc = tp+tntp+fp+tn+fn, precision pre:(7)pre = tptp+fp, and recall rec:(8)rec = tptp+fn.here, accuracy quantifies the fraction of correctly classified data points relative to all data points, whereas a high precision (recall) indicates a low number of falsely identified ctcs (missed ctcs). In our earlier study, a support vector machine (svm) achieved accuracy acc = 0.89, precision pre = 0.87, and recall rec = 0.93 on the data set used here, given an annotation of data points performed by one observer (cms). In the same study, a nave bayesian classifier (nbc) was trained without the use of labels, also known as unsupervised learning, which achieved accuracy acc = 0.87, precision pre = 0.85, and recall rec = 0.92 . Our results from the interobserver variability study indicate that a different observer might have annotated the data quite differently . We divided the data set into five randomized folds, without any regard to whether the observers agreed on data points and train a random forest (rf) and a svm on 3 of those and test on 1 fold . We got the average performance measures acc = 0.86 0.04, pre = 0.83 0.06, and rec = 0.88 0.08 across observers for the rf and the performance measures acc = 0.86 0.03, pre = 0.85 0.05, and rec = 0.85 0.08 for the svm (see table 2). Thus, the performance of the svm and nbc in our previous study was within one standard deviation of the numbers found here, for both the rf and the svm . It should be noted that besides different implementations of the classifiers and the fact that only one annotation was used in, different features were also used . In our previous study, the features used were one - dimensional color histograms while in the present study the hue and saturation channels of hsv images were used . Taken together, we have used three automated classifiers (one rf, two svm implementations, and one nbc) that performed almost equal on the data set . To add to this, the average interobserver variability was conspicuously close the accuracy of the classifiers, strongly suggesting that the performance of the classifiers was strongly influenced by annotation ambiguities . To examine if and how differences in annotation affected the classifiers' performance, we split the data set into the total consensus data set that can be considered ground truth (gt) with 365 data points and a part with probabilistic annotation (probgt) containing the remaining 252 data points . From probgt different annotations can be generated by assigning the label for each data point from a randomly chosen observer . On average 81 data points will change label between two probabilistic annotations . For classifier evaluation, gt was in turn split into three folds and the probgt into two folds, giving in total five folds with approximately the same number of cutouts . To get prediction by the classifiers we trained on two folds and tested on a third fold . The test fold was always one of the gt folds as we were here trying to separate the effects of uncertain labels in the test set from uncertainty in training labels . Averages and standard deviations were obtained by 50 repetitions of the training and testing across the folds with new annotations drawn for probgt between each repetition . When training on only gt folds, which do not change any labels between repetitions, we repeated the procedure 10 times to check if any randomness originated in the training of the classifiers . In table 2 the performances of the classifiers are listed as we introduced different amounts of uncertainty in the training data . If training and testing were done only on the gt part of the data, the rf achieved performance measures acc = 0.98 0.00, pre = 0.98 0.00, and rec = 0.98 0.00, whereas the svm achieved performance measures acc = 0.96 0.00, pre = 0.95 0.00, and rec = 0.96 0.00 . The standard deviations were less than 1% confirming that both classifiers were stable between training runs and any deviations of this magnitude would originate from annotation changes in the probgt folds . The rf performances did vary in the order of 0.1%, which is due to the probabilistic build of the forest . Compared with the values achieved on the full data set this was a clear improvement when we tested and trained on noise - free data . If we, instead of training only on gt, took one fold from gt and one from probgt and then tested on one gt fold the rf achieved performance measures acc = 0.96 0.01, pre = 0.94 0.02, and rec = 0.98 0.01 and the svm achieved performance measures acc = 0.92 0.01, pre = 0.88 0.02, and rec = 0.95 0.01 . This means that the label noise during training generally decreased the performance with a stronger performance reduction for the svm than for the rf . The performances of both classifiers were still better than that recorded on the entire data set where testing was done against partly probabilistic annotation . Even when we trained the rf on the two probgt folds, which we know has a high degree of label noise and it can be assumed that the data in probgt is of a lower quality than in gt, its performance measures were acc = 0.94 0.02, pre = 0.89 0.05, and rec = 0.97 0.03 . An example of what we refer to as low quality data is the low color intensity cutout shown in figure 1(c). For this setting the performance of the svm clearly dropped to acc = 0.81 0.04, pre = 0.75 0.08, and rec = 0.84 0.07 . This nicely illustrates that the rf is more robust when faced with label noise than many other classifiers, as was shown in the comparison between rfs and decision trees by breiman . While the svm performed well in the pure gt case, its performance dropped more rapidly than the rf when uncertainty was introduced . When the training data contained at least 50% certain cases the svm still performed better than it did on the entire data set, but when only probgt was used the svm dropped to considerably lower levels . For the rf the performance level seen for the whole data set was mainly because the classifier is tested on unreliable annotation; that is, the training on unreliable labels did have an effect but that is fairly mild in comparison . To at least partly solve the issue of uncertain annotation affecting the performance of the automated classifiers we evaluated the classifiers against the consensus data set . As discussed earlier, it is reasonable that the consensus data set is defined as cutouts for which at least nine out of eleven observers agree with each other, because in this case the probability for random annotation of cutouts as containing a ctc or not is less than 5% . In the case of five observers it would be required that all five observers agree in order to satisfy this condition . When training and evaluating the classifiers against the consensus data set we split the data set of 502 consensus data points into four folds, trained on three of them and tested on the fourth . In figure 5 the performances of the manual observers, rf and svm versus the consensus labeling, are plotted . The svm had a bit better precision, whereas the rf had a somewhat better recall . The performance measures for the rf were acc = 0.94, pre = 0.96, and rec = 0.93, whereas the svm had performance measures acc = 0.94, pre = 0.95, and rec = 0.94 . In comparison with our earlier study, we found an increase of the accuracy by approximately 5%, a precision increase by around 9%, while recall remained unchanged . Given the uncertainty in annotation that has been demonstrated in this study these values are much more representative performance measures for the task of automated classification of fluorescently stained ctcs . The majority of observers had better performances than the automated classifiers, but it should be noted that each observer had a vote when determining the consensus, whereas the rf and svm did not . It should also be noted that none of the observers reached perfect performance in any of the measures . Hence, there exists not a subset of observers that could have served as a substitute for the consensus annotation . In summary, the use of a consensus data set for training and evaluation of automated classifiers turned out to be a good option for evaluation of automated classification . In combination with the resilience of the rf to label noise in the training data, it seems that especially the test set has to be carefully chosen to give a correct evaluation of how well the classifier performs . The issue remains to find a good consensus data set as the manual annotation of data is hard to come by and time - consuming for the observers . This is especially the case when the annotation requires expert knowledge and experience in interpreting, for example, radiology images [28, 29]. As the use of computational methods is growing in cell biology, both for classification and modeling of biological systems [17, 18, 34, 35], we have in this paper investigated the effect of label noise caused by uncertain or faulty annotation on the performance of automated classification tasks . In total, eleven observers were asked to manually classify 617 image cutouts that may or may not contain ctcs . The rarity of ctcs in patient blood can easily inflate the accuracy of any classifier due to the many true negatives (tn) that are normally present . The cutouts we used here were identified using a morphological classifier among approximately 35000 foreground objects found during initial image segmentation . The morphological classifier was designed for high recall so that very few ctcs were overlooked at the initial stage, for full details of the procedure see svensson et al . . For the 617 cutouts, it was revealed that observers agreed with probability 85% whether a ctc was present or not . This degree of agreement is comparable to the uncertainty often seen in manual assessment of medical image data [19, 2329]. When only considering cutouts on which all observers agreed, the classifiers rf and svm reached performance measures above 95% (see table 2). This is considerably higher than the previously reported performances when attempting to automatically classify images of ctcs using rf and svm [17, 18]. The rf turned out to be quite resilient to noise in the training data, even when using only uncertain data points in the course of training it performed better on the total consensus test set than classifiers in previous studies (see table 2). The svm was more sensitive to label noise in the training data and actually performed worse than it did when the whole data set was used for training and testing . These findings are in line with the findings of breiman that rfs are stable with regard to noise, although in that study rfs were only compared with decision trees . Going beyond that study, here we have demonstrated that they are also more stable than svms with a radial basis function (rbf) kernel . To test classifiers on data which is with a high probability incorrectly annotated or for which it cannot be uniquely decided on the actual class, as is the case for probgt, is of disadvantage for classifiers that cannot be corrected by machine learning algorithms . Any performance improvement above the uncertainty in annotation will be a type of overfitting and even if the achieved performance measures seem impressive the algorithm will most likely not perform well on other data sets . On the other hand, if the test data suffers from label noise it is of great importance to take this into consideration when evaluation any automated classifier . Two very pressing questions remain to be investigated: (i) how to determine what is good data to use for training and testing the classifiers and (ii) how to detect and treat data that may occur in a clinical setting that is not appropriate for classification using the automated classification . Regarding the first question, we have shown that the creation of a consensus data set is a valid approach, but this normally requires a considerable effort from many observers to make the consensus statistically sound . In many cases these observer also have to be experts, for example, trained physicians that may not be very motivated to annotate data for machine learning algorithms rather than dealing with patients . It can be imagined that machine vision could step in to provide additional observers supplementing human observers . In this case care must be taken that the automated classification can be interpreted as an independent observer that is not getting slaved by human observers . This study suggests that rfs may be a strong candidate for this issue, because we have shown that noise in training data does not strongly affect the rf's performance on a total consensus test set . Another possibility is to use generative models which can be trained without labels and which are therefore independent of the performance of the human observers . For the second question the ideal solution would be if the ctc imaging procedure would be (close to) perfect . In the case of ctc collection using fsmw, as done for the present data set, the cylindrical or spiral shape of the wire presents a considerable imaging challenge to get the entire surface in focus . Even assuming a close - to - perfect data collection technique, it can be expected that clinical use will regularly produce data of a type that was not seen in training of the classifiers . A human observer could in such cases easily conclude that this is an uncertain case, whereas an svm or rf will be forced to make a decision by design . In the machine learning literature there are methods for outlier detection and these may have to be implemented and developed to handle this task [36, 37]. For outlier detection to be efficient in this classification task, a further subgrouping of objects would probably be needed as the class representing objects that are not ctcs is a very inhomogeneous group of objects . Instead of simply enumerating ctcs, as done here, it is desirable to determine subgroups within the ctc population, for example, to distinguish between apoptotic and viable ctcs [7, 17]. In order to do this, new sets of features may have to be identified that complement or even replace the color content of the cutouts . Examples of possible features would be further morphological quantities and fourier - ring descriptors . To apply machine learning to the subgrouping task would require more data than used here and a more rigorous manual classification performed by experts . As scholtens et al . Demonstrated, we would in that case still be faced with a considerable interobserver variability that would require a handling along the lines presented in this study.
Malignancy and aortic aneurysms are common diseases, particularly among aging population . Nearly three quarters of malignant cases are diagnosed in people aged 60 years and over . Abdominal aortic aneurysms occur in about 7 - 8% of male population over the age of 65 years and is rare under the age of 55 years . The coexistence of malignant disease and aortic aneurysm presents a real management challenge, especially in establishing the therapeutic priorities and the ideal treatment approach . This study aims at reviewing the diagnosis modalities and treatment options and the changes that have taken place with the advances in the management of both cancer and aortic aneurysms, particularly with the development of minimally invasive procedures . The medline and highwire databases were searched using the terms (neoplasms[mesh terms] or neoplasms[all fields] or cancer[all fields]) and (aortic aneurysm[mesh terms] or (aortic[all fields]) and (aneurysm[all fields] or aortic aneurysm[all fields]). All abstracts from the english language articles and foreign language articles were examined by a single reviewer . Papers detailing relevant data were assessed for quality and relevance independently by two separate reviewers . All case series, review articles, and references of such articles were searched for additional relevant papers . Papers that outlined their relevant experience and outcome of their approach sufficiently to allow comparison were included; articles that failed to detail the effect of treatment approach on outcome were excluded . 75 were case series, 57 were case reports, and 5 were discussion papers on various aspects of aortic aneurysm and cancer and all were included in this paper . Although the true incidence of concomitant malignancy and aortic aneurysm is difficult to establish, most centers report a low incidence of intra - abdominal malignancy in patients with abdominal aortic aneurysm . Malignancies were found in 4% of aaa cases in one of the earlier reviews covering a period of 22 years (table 1). Some authors report a much higher incidence rate, with up to 14% of aaa cases associated with malignancy in one series published in japan (table 1). Based on the natural history of aaa simulated to match the age - specific prevalence rate for male subjects, aaa was estimated to coincidentally occur in 8.3% of patients with intra - abdominal malignancy . Some reports have found an association between the presence of cancer and mycotic aortic aneurysm . For instance, mycobacterium bovis was found in the wall of a ruptured abdominal aortic and femoral artery aneurysm following intravesical bacillus calmette - gurin (bcg) therapy for bladder cancer [14, 15]. Listeria monocytogenes infection was also found in the wall of a resected thoracic aneurysm two months before advanced rectal cancer was diagnosed . The authors believed that the rectal cancer predisposed the patient to the development of an arterial infection associated with l. monocytogenes . These findings do not establish a causal association but do raise the possibility of an infective aetiology in a very small number of aneurysms . Most synchronous abdominal aortic aneurysms and cancers are found incidentally during the investigation or treatment stages . Nevertheless, suspicion of the presence of cancer has been occasionally raised during the workup for aortic aneurysm and vice verse . Computerised tomography (ct) scan is the most common investigation reported to detect the presence of abdominal aortic aneurysms concomitantly with visceral malignancies and vice verse . Occasionally, malignancies are detected incidentally during laparotomy or following an endoscopic procedure such as bronchoscopy, upper gastrointestinal, or lower gastrointestinal endoscopy . Virtual colonography [3, 24, 25] or whole - body ct scanning was recommended by some as a more cost - effective strategy to use in population - based screening programmes but not widely accepted . There is no consensus on the best management approach for patients with simultaneous aortic aneurysm and malignancy . Several strategies have been considered, namely, to repair the aneurysm first and treat the malignancy later, to resect the malignancy first and repair the aneurysm later, to undertake both procedures simultaneously, and in some cases to treat the malignancy and manage the aneurysm conservatively . Aortic aneurysm repair is a prophylactic procedure and is worthwhile where the lifetime risk of rupture exceeds the risk from treatment . The perceived increase risk of aortic aneurysm rupture following cancer surgery, the significant delay in the treatment of cancer if aneurysm is treated first, and the risk of graft infection are the other important considerations in the management of concomitant aortic aneurysm and cancer . Table 3 summarizes the different approaches used in the treatment of different patient groups in case series and their outcome . The treatment of aaa with coexisting malignancy represents a challenging issue to the cancer and vascular specialists in terms of priority, timing, and expected outcome . Prospective randomized controlled trials with adequate statistical power have understandably not been done in this area . In the absence of such level of evidence, one has to combine the best available evidence with a sound clinical judgment applied on each individual case within a multidisciplinary setting . The management challenge was reflected on a survey of 46 general and vascular professors in the usa in 1985 who gave their responses as to which condition should receive priority of treatment . Excision of the carcinoma first was favored by about a third of them, repair of the aneurysm first was favored by another third, and the remaining third stated that they would withhold a decision until laparotomy was performed . The survey was undertaken in the mid - eighties and a similar survey in the current era would probably show different opinions, especially with the introduction of endovascular and laparoscopic approaches and the availability of advanced preoperative staging techniques and adjuvant therapy . There is some evidence that abdominal surgery increases the risk of aneurysm rupture, especially when the aaa diameter is over 5 cm . Baxter et al . From the mayo clinic reported two aaa ruptures in the immediate postoperative period following 20 crc (colorectal cancer) excisions (10% incidence rate). A similar phenomenon was noted by lin et al . From the michael e. debakey departement of surgery, with aaa rupture incidence rate of 6% following crc resection . Swanson et al . Noted this complication in ten previously asymptomatic patients with aneurysms within 36 days of a prior laparotomy, with a mean aaa diameter of 9.1 cm . On the other hand, durham et al . Prospectively studied 33 patients (29 men, 4 women) with a known abdominal aortic aneurysm who underwent 45 operations . The estimated risk of rupture was 3% of all patients undergoing major operation in this study . It was hypothesized that a reduction in the collagen contents of aortic wall following trauma may predispose the aortic aneurysm to rupture, through a mechanism mediated by collagenase and protease activation [40, 42]. The effect of delayed cancer treatment on the long - term outcome remains unclear . Ideally, any delay in cancer management should be avoided . A few months delay before initiating treatment of oesophageal cancer, for example, would have an impact on the stage of the cancer, and thereby on the patients' prognosis . Nevertheless, disease stage at the time of diagnosis is probably the most important influence on survival, and some delay in treatment may not strongly be associated with variation in survival . Delays occurring due to poor organization or the magnitude of the first operation requiring prolonged recovery before the second operation took place, need to be tackled as modern practice allows . Authors from many large centers (the mayo clinic, usa, the cleveland clinic foundation, usa, the university of naples federico ii and university of turin, italy [36, 37], the michael e. debakey department of surgery, usa, the keio university school of medicine, japan, the imperial college london, uk, the concord repatriation general hospital, australia, and others [16, 45]) agree that the most life - threatening (or symptomatic) lesion should be addressed first . Large abdominal aortic aneurysms, obstructing colonic cancers, or bleeding gastric cancers, for example, should be treated first where possible . The treatment modality may be a minimally invasive procedure (e.g. Stenting for obstructing crc) that can allow optimization and a better planned therapy . A one - stage operation to repair the aortic aneurysm and resect the malignancy has the advantage of avoiding a second major abdominal operation, avoiding the potential risk of ruptured aneurysm following the cancer resection stage, avoiding the potential delay in cancer treatment if the aortic aneurysm was addressed first, and avoiding the potential difficulty in dealing with adhesions resulting from an earlier laparotomy [3133]. Most studies have not shown any significant increase in the risk of graft infection following a one - staged operation (see table 3, [8, 13, 27, 28, 30, 31, 3337, 45]). Nevertheless, few reports have documented a possible correlation between graft infection and simultaneous open abdominal surgery [27, 46, 47]. The cumulative morbidity and mortality were significantly higher in one - staged operations when compared to two - staged operations in some reports (lin et al . ), and the one - staged option as a primary approach was unfavorable by some authors accordingly (lin et al . And hafez et al . ). Combined operation in cases where both lesions pose a life - threatening condition (e.g. Large aneurysm with advanced obstructing malignancy) is supported by most authors, providing a very high attention to details in place (thoughtful antibiotic coverage and possible irrigation of operative field with antibiotics, usage of antibiotic - bound grafts, good - risk patient selection, verified good colonic blood supply to avoid necrosis, possible use of omental wrap around the vascular graft [11, 32, 45], possible spray of fibrin glue around anastomosis, using extra - anatomic bypass instead of a grafted aorta, etc .) Some authors, however, remain in favor of a combined operation as a primary management approach to synchronous lesions [8, 13, 31, 32, 34], especially for fairly clean operations such as gastric [13, 34] or urological cancers [31, 37]. Staged operation (repair of aortic aneurysm first or resection of malignancy first) has the theoretical advantage of avoiding major longer operation and avoiding the risk of cross - contamination and consequent devastating graft infection . Nevertheless, many reports have shown a significant time delay before the second operation (especially when malignancy is treated first) [8, 11, 33, 35, 38, 48], a high incidence of catastrophic sequelae (ruptured aortic aneurysm in the case of treating the malignancy first) [11, 23, 27, 33], or a significant decrease in the long - term survival, especially when one pathology was treated eventually [31, 38, 45]. Despite these unfavourable side effects' of staged operations, this option (malignancy - first or aorta - first approach) remains the preference for some authors [6, 35, 36]. Few questions such as the impact of aneurysm repair in staged procedure on the timing of adjuvant therapy and the effect of radiotherapy on the evar stent need further well - controlled studies in the future . Endovascular aortic repair (evar) in the treatment of concomitant malignancy has attracted much attention more recently [27, 31, 33, 34, 36, 38]. As evar does not involve a laparotomy, patients recover quickly and in most cases would be able to undergo the cancer surgery within a couple of weeks if required . Drury and colleagues performed a systematic review and meta - analysis of randomized controlled trials on evar and showed a persistent reduction in 30-day mortality (1.6 versus 4.7%) and lower incidence of major complications (including cardiac, respiratory, and renal) after evar procedures when compared to open procedures . In line with these results, most authors dealing with synchronous aaa and malignancy have found a persistent decrease of the interval period between repairing the aortic aneurysm using evar and performing the malignancy operation in the two - staged approach [27, 33, 36], a persistent reduction in operative morbidity and mortality [27, 33] that was sustained up to 3 - 4 years of followup [27, 33], and a significant reduction in length of hospital stay (unless endoleak has to be ruled out [36, 38]), and in the intraoperative blood loss [27, 33, 36, 50]. However, recent prior colonic surgery may predispose patients to such a complication as reported in 2 patients who had right hemicolectomy (with confirmed patent i m a) followed by a staged evar (18% incidence rate) in one report . Lin and colleagues recommended using additional preoperative imaging to confirm the presence of sma / ima collateral flow before embarking on such staged operations [33, 38]. Endovascular aneurysm repair, where possible, followed by malignancy operation has become the initial preferred treatment choice in some centers [31, 33, 34, 38]. The risk of vascular graft infection is also likely to be less with an evar procedure, as there is no risk of direct contamination during the resection of abdominal malignancy . Patients with advanced / end - stage malignancy and large aortic aneurysm require sound ethical consideration and balanced clinical judgment [6, 8, 35, 45]. A good understanding of the expected outcome in each specific cancer type, as well as the presence of other comorbidities (age, physiologic well - being, etc . Aneurysm repair would be considered inappropriate if curative resection is not feasible, even if the evar procedure can be carried out with minimum morbidity . Patients who were deemed unfit for open aortic aneurysm repair were randomly allocated to best medical therapy alone or evar and best medical therapy . The trial was weakened by a significant number of patients that crossed over to undergo aneurysm repair . Nevertheless, the trial showed that patients with significant comorbidity do not show any survival benefits with aaa repair . These data can be relevant to cancer patients with poor prognosis who are, on a similar basis, unlikely to benefit from aaa repair . The need for adjuvant therapy for cancer treatment is another factor to consider when planning for a staged or simultaneous operation . Have raised concerns regarding chemotherapy treatment (associated with aggressive hydration and corticosteroids) in patients known to have aaa> 6 cm . Such treatment may result in enlargement of major arteries and may increase the risk of rupture . Chemotherapy administered to patients before or after evar had no consequences in some reports . Data on the risk of aneurysm rupture due to chemotherapy is limited and should not be an important consideration in management decision . It would be entirely reasonable to await the response of chemotherapy and get a better estimate of prognosis before undertaking the repair of aneurysm . The extent of nodal dissection has significant effect on the overall cancer prognosis, including the need for adjuvant therapy . No technical difficulties or specific limitation have been reported in this respect, including performing a d2 lymphadenectomy in gastric cancer or total mesorectal excision and full node dissection in rectal cancer . However, a one - staged operation appears to be correlated with more d2 dissection and less total gastrectomy rate when compared to staged operations in some series . The effect of paraaortic tissue dissection during open aortic aneurysm repair on the cancer staging or outcome (in terms of cancer dissemination or effect on chemoradiotherapy) or the effect of finding metastatic malignancy following the first stage of treatment (malignancy - first approach) on the cost - effectiveness of aortic - repair stage remains to be investigated further . Treatment priorities and patients' best interest should be considered when one of the two lesions is found incidentally intraoperatively . Treating the most immediate life - threatening condition and a two - staged operation might be a sensible option in such circumstances . It should be possible to diagnose aneurysms on ct in most patients preoperatively but malignancy may be occasionally overlooked on the preoperative ct scan for aneurysms . The increasing use of evar techniques means that most of these lesions will be detected on preoperative or postoperative surveillance scans and can be treated in the stages . The management decision for concomitant malignancy and aortic aneurysm needs to be made for each individual case based upon the best estimates of risks of aneurysm rupture, operative risks, prognosis of malignancy, and patient preference . There are multiple variables and it will be difficult to obtain high - level evidence for these patients . It is therefore essential to discuss such cases in a multidisciplinary setting to achieve a consensus opinion before gaining approval from the patient on one option or another.
The cuffed endotracheal tube (ett) has for long been considered the gold standard for securing airway, especially for procedures under general anesthesia . The disadvantages of laryngoscopy and tracheal intubation are concomitant hemodynamic responses and damage to the oropharyngeal structures . Laryngeal mask airway (lma) was used instead to obviate the shortcomings of ett . However, risk of gastric distension and inadequate ventilation with lma remained until the discovery of proseal lma (plma) in early 2000 . Plma has a dorsal cuff, in addition to the peripheral cuff of lma, which pushes the mask anterior to provide a better seal around the glottic aperture and permits use of high airway pressures without leak . The drain tube parallel to the ventilation tube permits passive drainage of regurgitated gastric fluid away from the airway and serves as a passage for gastric tube . Many studies have compared plma with ett for ease of insertion, hemodynamic changes, and postoperative airway complications . Idrees and khan observed comparable peak airway pressures (ppeak) with both lma and ett . Few have compared the ventilatory efficacy and changes in airway dynamics seen with use of plma and ett in positive pressure ventilation . Hence, this study was conducted with the aim of comparing ventilatory parameters and airway dynamics, needed to maintain a state of normocarbia and stable hemodynamics, and postoperative complications between plma and ett in adult patients under general anesthesia . A prospective, single blinded, randomized controlled clinical trial was undertaken and sample size was calculated by keeping power of study at 80% and confidence limits at 95% . To detect a 10% difference in tidal volume (vt), we enrolled 30 patients undergoing elective surgeries under general anesthesia, in each group for better authenticity of results . Patients were randomly allocated using numbers generated from www.random.org into group plma and group ett . Patients belonging to american society of anaesthesiologists physical status i and ii aged between 20 and 40 years of either sex were included . Patients with anticipated difficult airway, hiatus hernia, gastroesophageal reflux disease, body mass index> 35 kg / m, head and neck surgeries, patients suffering from respiratory pathology and patients undergoing laparoscopic surgeries were excluded from the study . All patients were premedicated with oral alprazolam 0.5 mg and ranitidine 150 mg on the night before surgery and were kept fasting overnight . After shifting the patients to operation theatre, pulse oximeter, electrocardiograph and noninvasive blood pressure monitors were connected and basal parameters recorded . Propofol 2 mg / kg and intubation / insertion of device was facilitated by inj . Airway was secured with respective airway devices . In group plma, either plma size 3 (weight 30 - 50 kg) or plma size 4 (weight 51 - 70 kg) was inserted . In group ett, trachea was intubated under direct laryngoscopy using 7.5 mm ett in female patients or 8.5 mm i d in male patients . Correct placement of device was confirmed by adequate chest movement on ventilation and square wave on capnography . Adequacy of tight seal was confirmed by absence of audible leak from drain tube (plma) at ppeak of 20 cm h2o in plma group . In ett group, train of four (tof), airway dynamics and ventilatory parameter monitors were connected following insertion of airway device . Ventilator settings consisted of respiratory rate 14 cycles / min, inspiratory: expiratory ratio 1:2, fresh gas flow 2 l / min . Vt was set at 6 ml / kg initially and adjusted subsequently with 1 ml / kg increments or decrements in vt if required, to maintain end tidal carbon dioxide (etco2) between 35 and 40 mm hg . Anesthesia maintained with 65% n2o in oxygen, isoflurane 1 - 1.5% to maintain entropy values between 40 and 60, inj . Atracurium 0.3 mg / kg bolus followed by infusion of 8 - 10 g / kg / min to maintain tof count <1 . Peak airway pressure, vt, compliance (compl . ), airway resistance (raw) were recorded every minute after insertion for first 5 min and later every 10 min . Other vital parameters such as heart rate (hr), systolic and diastolic bp, mean arterial pressure (map), oxygen saturation (spo2), etco2 were recorded after insertion of airway device, every minute for 1 5 min and then every 5 min till the end of surgery . Adequacy of ventilation was ensured by continuous monitoring of end tidal carbon di oxide and oxygen concentrations . All parameters were recorded on spirometry and gas analysis module of s5 avance datex ohmeda anesthesia workstation . Patients were excluded from study, if a difference of> 3% between set and inspired vts, or a leak fraction> 10% was observed . After removal of airway device, any complications such as coughing, laryngospasm, blood staining of airway device, tongue / dental trauma, and hoarseness of voice were observed . Data were analyzed using the spss version 17.0 chicago, il, usa statistical software . Demographic data of age, sex, weight and duration of surgery were comparable between the groups [table 1]. Mean time for insertion of airway device in group plma was 16.43 12.31s, and in group ett it was 13.66 8.04s (p = 0.308). Airway device was inserted in the first attempt in 28 patients in group plma as compared to 29 in group ett . One patient in either group needed second attempt for securing airway, and one patient of group plma required 3 attempts . Mean spo2 in group plma was 99.40 0.0695% and in group ett, it was 98.45 0.83% (p = 0.50). Mean etco2 was 35.11 2.82 mm hg in group plma and 37.12 3.17 mm hg in group ett (p = 0.656) [table 2]. Spo2, etco2, tidal volume and insertion details the vt initially set at 6 ml / kg was comparable in both groups (group plma-318 41.96 ml, group ett-329.67 43.98 ml p = 0.276). Mean adjusted vt to maintain etco2 between 35 and 40 mm hg was 317.68 51.90 ml in group plma as compared to 411.55 65.71 ml in group ett (p <0.01). The average difference between set vt and adjusted vt in group plma was 0.31 ml (p = 0.974) as compared to 81.89 ml (p <0.01) in group ett . In group ett the vt had to be increased significantly from set vt to maintain etco2 between 35 and 40 mm hg . The mean compl . In group plma was 29.02 4.54 ml / cm h2o while in group ett it was 33.52 2.89 ml / cm h2o . The values were statistically significant in the 1 5 min after insertion of airway device (p <0.05). Mean ppeak in group plma was 12.95 2.62 cm of h2 0 as compared to 16.02 2.59 cm of h2 0 in group ett (p = 0.003) and was statistically significant throughout the surgery . Mean raw in group plma was 10.65 2.88 cm h2o / l / s compared to 10.01 2.05 cm h2o / l / s in group ett (p = 0.256) [figure 1]. Graph showing comparison of compliance, peak airway pressures and airway resistance heart rate was significantly lesser in the 1 min after insertion in group plma (p = 0.043), but comparable subsequently throughout surgery in both groups . The systolic, diastolic and map s were lower in group plma at 1 and 2 min following insertion and after removal (p <0.05) [figure 2]. Graph showing comparison of heart rate (mean + standard deviation [s.d]) and mean arterial pressure (mean s.d) coughing was seen in one patient in group plma as compared to 10 patients in group ett, this was statistically significant (p = 0.006). Incidence of blood staining of device was comparable in both groups, 10 cases in group plma and 16 cases in group ett . In the present study, plma maintained adequate ventilation using lower vt and ppeak compared to ett . Unlike previous studies, where only ppeak was assessed, this study attempts to assess various additional parameters of respiratory mechanics, making it unique . Achieving adequate ventilation and maintaining in their review, observed that lower vt ventilation strategy had lesser incidence of developing acute lung injury in patients undergoing major abdominal, thoracic, and cardiovascular surgeries . In our study the average vt needed to achieve adequate ventilation, as evidenced by spo2 and etco2, was significantly lower in plma group . Berry and coworkers compared performance of lma with ett at different vts and noted that ppeak was lesser in lma group even at lower vt ventilation, along with significantly lower raw . The design of plma has some influence on resistive load of the device and was found to offer greater resistive load compared to classic lma . However, correct placement of the device and leak fraction play a crucial role in measurement of airway dynamics parameters . Natalini et al . Observed that in obese patients undergoing general anesthesia, the ppeaks were comparable between plma and lma . In the present study, the ppeak was significantly lesser in plma group throughout surgery while the raw was comparable in both groups . Though the internal diameter of airway tube of proseal lma is narrow compared to that of ett in the present study, the shorter length of proseal lma compared to that of ett might be the nullifying factor accounting for similar raw in both groups . All possible precautions were taken to minimize leak fraction and patients with leak fraction> 10% were excluded . Compared plma with laryngeal tube; it was observed that ppeaks were clinically comparable between both devices . Peripheral spo2 and etco2 monitoring have been considered as measures of adequate ventilation . Spo2 and etco2 were comparable in both groups in our study indicating adequate ventilation with both the devices . We did not notice a difference between inspired and expired oxygen concentration of> 5% in any of the patients . Higher ppeaks and large vts have been implicated as risk factors for development of barotrauma and volutrauma in critically ill mechanically ventilated patients . The plma maintained adequate ventilation at lower vts and airway pressures as compared with ett . Hence, possibility of barotrauma and volutrauma with plma may be low as compared to ett . Lower ppeaks noticed in patients receiving plma may be due to lower vts used during mechanical ventilation . Types of surgeries in both groups were comparable thus the possible effects of surgery on airway parameters are ruled out . Sympathetic stimulation by laryngoscopy and intubation is known to induce hemodynamic response during ett insertion . We observed that hemodynamic parameters were lower in plma group immediately after insertion of airway device and also soon after removal the same, though hr did not alter in post removal period in plma group . The incidence of coughing and hoarseness of voice was lower with plma as it is a supraglottic device . The patient who developed laryngospasm in group ett was reintubated and electively ventilated for 20 min with 40% oxygen, which was followed by uneventful recovery . There are limitations in the present study: the double blinding was not possible in this study.incidentally most of the surgeries were of short duration lasting for 40 - 60 min . Hence the implications of this study need to be extrapolated with caution, to situations requiring longer duration of mechanical ventilation.late postoperative complications such as sore throat were not included in observations since it was focused mainly on intraoperative airway dynamics . Incidentally most of the surgeries were of short duration lasting for 40 - 60 min . Hence the implications of this study need to be extrapolated with caution, to situations requiring longer duration of mechanical ventilation . Late postoperative complications such as sore throat were not included in observations since it was focused mainly on intraoperative airway dynamics . The plma maintains adequate ventilation at significantly lower vts and peak pressures without increasing raw, and has minimal hemodynamic variations compared to ett.
The biorefinery term pertains the production of biofuels, bioenergy, and valuable chemicals from renewable biomass sources and aims to substitute petroleum refineries which produce several fuels and products from petroleum . Biorefinery is the continuous processing of biomass into a range of profitable products (food, feed, materials, and chemicals) and energy (fuels, power, and heat). This conception is similar to that used within the petrochemical industry, but renewable biomass feedstocks are used instead of using oil as the feedstock . Renewable is a wide - ranging term, which could include any organic matter that becomes available on a continuous basis . This could include grasses, energy crops, agricultural feeds, or organic waste streams from animals and plants . Grass is one of the promising energy crops for biogas production in some eu countries, where grass is covering a wide areas of agricultural land with high yields compared to other crops in europe . The biorefinery products (i.e., fuels, therapeutics, food additives, or secondary chemicals) can be obtained using thermal, chemical, mechanical, enzymatic or microbial processes . Biorefinery is targeting the separation of all the added value from the biomass feedstock, with little or no waste . This will lower the total environmental impact, besides improving the economics so that these processes can contend with the petrochemical industry (figure 1). Using biomass in a biorefinery concept instead of oil for producing energy and chemicals . Uniformity of feedstock represents one of the common factors between the classic petrochemical refinery process and biorefinery . Biomass feedstocks could have a remarkable geographic and seasonal variations (ranging from simple sugars to complex polysaccharides such as starch, cellulose, and hemicellulose, as well as more complex sources such as lignin, triglycerides, lipids, and proteins). This variation could be viewed as a disadvantage, due to the variation of the consistency and yield of the end products . On the other hand, complexity could be a desirable trait in order to obtain a more expansive range of products, although it needs cautious optimization in relation to the input material . Moreover, one of the major disadvantages of biorefinery, compared to petroleum refinery, is in the miscellany of technologies required to obtain the end products . These include plant breeding and genetics, mechanical processes, sub- or supercritical fluid extractions, thermal treatments, chemical treatments, concurrent thermal and chemical treatment, enzymatic digestion, and biotransformations using microorganisms . Biorefineries are classified based on their system components, that is, platforms, products, feedstocks, and conversion processes . Platforms determine the complexity of the system in which they represent intermediates that link biorefinery systems and their processes (i.e., c5/c6 sugars, syngas, and biogas). Products may be energy, that is, bioethanol and biodiesel, or valuable chemicals (building blocks), that is, organic acids . Feedstocks can come from edible crops, agricultural residues, forestry residues and industrial or domestic wastes (bark, straw, paper mill black liquor, used cooking oils etc . ). These are thermochemical (e.g., pyrolysis), biochemical (e.g., fermentation), mechanical (e.g., size reduction), and (bio)chemical (e.g., esterification). The array of the most common terms that describe biorefineries are illustrated in table 1 . Crop based biorefineries have gained tremendous interest in recent years, several pilot - scale systems have been built and currently a lot of research is going on building full scale systems . As with any other technology, apart from the main products in this case ethanol or lactic acid, several side products and wastes are also generated . The main focus of research so far has been on treatment of these wastes . Also, most of the scientific literature is focused on treatment aspect only, but the focus has moved from treatment to valorization very recently . Coal displaced the biomass fuels, considering wood, as the main energy source during the industrial revolution . Since then a steady migration toward fossil fuels has continued, moving further away from biomass, not only for energy but also for sources of chemicals used to make everyday items . An outstanding pattern of this is furfural which can be produced from oat hulls . Until 1960s, dupont produced nylon from biologically derived furfural; however, nowadays it is being produced from fossil resources . The price of such fossil - fuel sources will be affected negatively in the near future due to the depletion of these oil reserves . Industry is therefore now being encouraged to take a more inventive approach, looking ahead of oil and identifying biobased systems as valuable stockroom of crucial chemical building blocks . These chemicals are the basics of our modern lifestyle and can be found in products as miscellaneous as foods and fabrics . Cereal grains have been a primary human food source since thousands of years ago, being one of the most vital source of calories for a large sector of the world population . The nature of grains produced worldwide relies on different factors, mainly being economic, cultural, and environmental . The availability of water and temperature are most likely the important environmental factors that determine the region in which these crops can be grown . The estimate for world cereal consumption in 2012/2013 has been increased to reach 2335 million tons (mainly use of maize), 5 million tons higher than reported in the first half of 2012 . The existing cereal biorefinery uses dry cereals such as wheat, rice, and maize as raw materials . Currently, some of these crops, that is, maize and wheat, are used to produce biofuels . Maize starch is the major source for ethanol production in the u.s ., where wheat feedstock is used for ethanol production in canada, australia, and in some european countries . All of these crops are the conventional ones used for the production of first - generation biofuels . Second - generation biofuels are produced from lignocellulosic crops, such as fast - growing trees and permanent grasses or from straw residues . Great interest has been directed toward producing second - generation ethanol, since the above - mentioned crops can be cultivated in secondary lands without the need to interfere with the fertilized lands generally used for food production . These crops can be cultivated in the food - unsuitable poor soils, with relatively low amounts of water, but their productivities are low and a lot of effort is needed to elevate their yields . Wide areas of land are required to cultivate the crops for biofuels production, together with irrigation and use of fertilizers, which may destroy wildlife habitats, as well as affect indigenous and rural poor communities around the world . Instead the byproducts of these crops should be considered as a source of these biofuels . The cereal grains used for human food are milled to eliminate the bran and germ, in order to meet consumers sensory expectations . Grains are striped by this process of key nutrients valuable to human health, that is, phenolics, dietary fiber, minerals, and vitamins, which also needs to be studied for their potential as feedstocks . Building economical cereal - based biorefineries as a replacement for petroleum refineries would require considerable improvements in existing cereal processing strategies by applying more proficient processing of cereal grains for the production of a range of value - added products, that is, biochemicals and bioenergy . The main schematic industrial processing steps of some cereals are illustrated in figure 2 . Wheat grains are separated into their constituents (bran, germ, and endosperm) by the milling process steps, in which wheat is ground into flour . Before milling, the wheat is passed into conditioning bins to produce uniform moisture content throughout the grain . The bran and germ are separated from the endosperm by a sequence of breaking and grinding processes and subsequently the endosperm is reduced to form a uniform particle size by separating operation . Schematic diagram of the main industrial processing of cereals illustrating products (green shade) and some byproducts (red shade). The cleaned grains for milling are first conditioned, after which barley is washed - out with diluted sulfur dioxide . Barley is then smashed through a pearling process to separate surface layers of the grain . The pearl barley is polished and flakes are made from them by steaming, flaking, and drying . Furthermore, barley could be processed by malting, in which barley kernels are allowed to grow by first steeping the grains, which are then transferred from the steep tank to the germination vessel . Germination is completed upon the full modification of the endosperm, after which the process is terminated by drying (kilning process). The kilning brittle rootlets byproduct are removed to be used as a high protein feed (75%). Also, milling is a crucial step in postproduction of rice . This process starts with the removal of the hard protective husk surrounding the grain, which is done by passing the rice through two spinning rubber roles and the resulted rice grain is packaged as brown rice healthy for body functions . The following step includes usage of fine milling to separate germ and bran layers from the grain and expose the white starch pulp, known as white rice . Corn is processed by a dry milling process (degerming) in which hull, germ, and endosperm are separated prior to milling . This process starts by drying and cleaning the corn, which is then conditioned to 20% moisture . The majority of the outer bran, germ, and tip cap are removed from the moist grains, leaving the endosperm, which is then dried, cooled, and sieved . Corn can also be processed by corn wet - milling, in which the precleaned grains are transferred to large steep tanks where they are soaked in a dilute sulfur dioxide solution . The corn germ is removed from the water soaked kernel, in which the germ is processed to recover the oil, and the remaining corn germ meal portion is collected for feed use . The corn nucleus is sieved to separate the bran leaving the starch and gluten protein . The gluten protein is concentrated and dried to form corn gluten meal with 60% protein feed . Similar to corn, sorghum grains are processed either by dry or wet milling processes . Additionally, sorghum could be milled for juice extraction by first being conditioned, then squeezed for juice extraction and produce bagasse byproduct which could be used as an excellent supplementation fiber for livestock or converted to a fuel . The filtered juice is precipitated and then the supernatant is evaporated and surface coagulated materials are removed to obtain syrup . Nowadays, there is much attention for universal pollution along with growing production cost, and limited availability of raw materials leading to a special highlight on the importance of recovery, recycling, and valorization of food processing byproducts . The amount of biodegradable wastes drained to landfills in eu countries in 2020 is estimated to increase by 35% of its level in 1995 . The european manufacturers of processed foods are obliged to meet the terms of eu environmental policy concerned with utilization or disposal of byproducts and the huge amounts of aqueous and solid wastes produced . Although these wastes represent serious economic and environmental challenges, they contain extensive amounts of potentially reusable materials and energy . One of the most contributing wastes to these challenges is cereal byproducts with biochemical oxygen demand (bod) and chemical oxygen demand (cod) levels that can reach 3.5 10 5 10 and 10 1.5 10 mg / l . Generally, as a result of the change in ratio of fibrous and nonfibrous carbohydrates, the energy values for the parent grains are higher than that of mill - feeds . In contrast, the protein contents for the parent grains are commonly lower than that of mill - feeds . Average percentage content of bioactive compounds of some cereal industrial processing byproducts; wheat bran, rice husk, sweet sorghum bagasse, corn dry distillers grain, corn cob, and brewer s spent grains . General terms, referring to the cereal milling byproducts, are used to depict related components for several grains, whereas, some of these terms are used exclusively to describe a byproduct of one grain . One of the most important milling byproducts is bran which includes the coarse outer shell of the seed with small amounts of flour and higher content of fiber and protein . The embryo of the cereal seed adds up to form the germ meal which is a byproduct rich in lipids and protein . After removal of the germ and the starch endosperm during wet milling, the primary substance remaining is the protein rich gluten . Moreover, grain screenings is a byproduct which contains a mixture of chaff, dust, broken grains, weed seeds, and all separated materials during cleaning and processing . The nutritional value of grain screenings varies with the relative proportion of each of the components . Grain screenings are produced from all types of milled cereals with a minimum of 70% grains and maximum of 6.5% ash . Grain hulls are the outer covering of the grain seed, commonly originating from oat and rice milling . Grain hulls are high in crude fiber and relatively low in energy and protein, whereas rice hulls are high in silica . Also, one of the major byproducts from malting industry is the barley malt sprouts which consist of roots, sprouts, and malt hulls . Malt sprouts are considered as a protein source with minimum crude protein content of 24% . The oat milling byproduct is oat meal, consisting of lower quality oat meal portions with maximum crude fiber content of 4% . The production of flour is accompanied by some byproducts such as middlings (abbreviated as midds) which contain bran, shorts, germ, flour, and tailings and mostly originate from rye and wheat industrial processing . The maximum levels of fiber for rye and wheat middlings are 8.5% and 9.5%, respectively . The main rice milling byproduct is rice polishing, which is a good source of thiamin and crude fat and relatively a poor source of crude fiber . Also, rice bran is another byproduct which has a high oil content and is a good source of b - complex vitamins, protein, and amino acids . The frequent corn byproducts of dry milling are corn flour, corn bran, and hominy feed, whereas the common byproducts of corn wet milling are liquefied corn product, starch molasses, germ meal, gluten, hydrolyzed corn protein, and condensed fermented corn extractives . Also, sorghum milling can produce some byproducts, that is, gluten and grits . The efficient and economic disposal of cereal processing byproducts is the main concern of industrial society, in order to fulfill the environmental regulations that regard them as wastes destined to be drained off in most cases . This trend may be due to the lack of information for different economic biomolecules availability and technical processes for extracting them, besides the lack of commercial knowledge concerning customer requirements . The value addition and adequate utilization of cereal processing byproducts is very important for the valorization technology of such byproducts . Moreover, if the valorization know - how is available for certain industrial byproduct in laboratory or pilot scale, the efficient scaling - up of this technique is limited by the standardization of the production process on the industrial scale . Current sugar - based platform biorefineries mainly focus on the valorization of cellulose and hemicelluloses, while lignin is generally considered as a low - value residue . This may be due to the complexity and polydispersity of lignin compared to sugars normally released as uniform monomeric carbohydrates, which limit lignin wide - scale use in biorefineries . Besides, recovery of lignin from their product streams is difficult, with different composition of lignin contained by the tissues of individual plants and according to its source . The efficient utilization of lignocellulosic materials requires its pretreatment prior to the enzymatic hydrolysis and fermentation, which results in opening up the cell wall structure by disturbing the lignin / hemicelluloses complex and exposing the cellulose to enzymatic hydrolysis . The pretreatment process is considered the most expensive step in the valorization of lignocellulosic byproducts, where it can contribute to about 30% of the total cost . Due to this problem, several methods of pretreatment processes have been investigated which include alkaline or acid hydrolysis, milling and grinding, gas treatment by ozone or sulfur dioxide, steam explosion, organic solvent treatment, liquid hot water, wet oxidation, ammonia fiber explosion, biological treatments, cellulose solvent pretreatment, and ionic liquid . On the other hand, some of these methods, that is, acid hydrolysis and steam explosion, produce some byproducts like furfural and hydroxyl methyl furfural (hmf) which negatively affect the fermentation process, and this requires a separate detoxification step, for example, ion exchange, addition of activated charcoal, and over liming, which in turn increase the overall process cost . Also, the expenditures are negatively affected by the power requirements for achieving the optimum reaction conditions for some pretreatments and costly downstream processing to remove extraneous chemicals that are added to the biomass . Moreover, the use of expensive industrial equipments are necessary to avoid corrosion normally associated with acid hydrolysis . All of this reflect the need for integrated technologies for efficient treatment and the maximal recovery of energy from lignocellulosic byproducts . Alternatively, the use of untreated lignocellulosic biomass as substrate cause several problems, that is, substrate agglutination and clogging, in addition to microbial degradation resistance . The substrate uniformity is one of the most significant factors that influence the energy poise and the total economics in the valorization process of lignocellulosic biomass . With high solid content, the sugar and resulting product concentrations will increase, which leads to a significant reduction in capital and production costs due to the compact equipment size and low energy consumption during heating, cooling, and distillation . On the other side, these high - solid loadings generate environmental problems due to the absence of free water in the pretreated material and difficulties to handle the slurry . Also, some cereal processing byproducts, that is, liquid stillage, must be dried because of their short shelf life which extends the durability, but increases the overall economics of the valorized product . Valorization is the transformation of byproducts to alternative fuels, energy and other useful chemicals, with specific attention for sustainability and environmental objectives . Generally, cereal based biorefinery substrates are chemically (i.e., naoh) or hydrothermally (i.e., steam explosion) and/or enzymatically (i.e., cellulase) pretreated before fermentation which could be through submerged, solid state, dark or photo fermentation . Submerged fermentation (smf) is any aqueous fermentation process occurring in the presence of liquid substrate, whereas, solid - state fermentation (ssf) is any fermentation process occurring in the absence or near - absence of free water by employing a natural substrate as a solid support . Dark (df) and photo (pf) fermentation is the bacterial conversion of organic substrate to biohydrogen through a complex process involving biochemical reactions, with the difference that pf is held by a diverse group of photosynthetic bacteria in the presence of light . The most recent valorization strategies for cereal based biorefinery wastes are illustrated in tables 2 and 3 and detailed in the following sections . Productivity is based on volume of product per weight of raw waste or volume per volume of culture . Productivity is based on volume of product per weight of raw waste or volume per volume of culture . Organic acids and their derivatives are widely used in food, pharmaceutical, leather, and textile industries . However, the high production cost becomes a limitation for their applications in more fields . Therefore, much attention has been paid to searching for cheaper sources for the fermentative production of organic acids . Lactic acid (la) is one of the most important organic acids extensively used in pharmaceutical, food, chemical, textile, leather, polymer, and cosmetic industries . The replacement of the costly pure raw materials usually used for la production by agro - industrial residues can provide an attractive substitute because of their low prices . These agro - industrial residues include brewer s spent grain (bsg) enzymatic hydrolyzate which was incorporated into culture medium, with glucose as carbon source, for the production of la by lactobacillus delbrueckii. (48) with this mixture, a yield of 0.99 g / g of bsg was obtained after 60 h of fermentation . This yield dropped to 0.45 g / g when wheat bran acidic hydrolyzate was used as the nitrogen source for la production by lactobacillus rhamnosus. (51) also, corn steep liquor (csl) was investigated as a sole and low cost nitrogen source in addition to some components to replace yeast extract for the cost - effective production of la . G / l was achieved in shake - flask scale, whereas the concentration of la in a bioreactor was 110 rice bran is one of the most abundant agro - industrial byproducts that was investigated in several studies for the production of la . In two different trials, l. delbrueckii(49) and l. rhamnosus(53) were used to produce la from enzymatically hydrolyzed rice bran which was used as carbon and nutrient source . L. rhamnosus (56 kg / m) was superior to l. delbrueckii (9 kg / m) and was 1.5 times higher in la production than the glucose and yeast extract traditional medium due to the high nitrogen content and the presence of rice bran oil rich in fatty acids which is an important factor for la production . Citric acid (ca) fermentation by aspergillus niger is one of the world s major industrial microbial processes . The same fungus was used to evaluate the economic ca production from the untreated bsg agro - industrial waste by solid state fermentation (ssf). The resulted ca yield was 11.8 g / kg after 120 h of fermentation . Among the vital organic acids is succinic acid (sa), which is conventionally manufactured from petrochemicals through expensive processes . Higher economic benefit could be achieved with the use of agro - industries byproducts as potential resources to produce petrochemically derived products . Two types of wheat milling byproducts were enzymatically pretreated and used to produce sa by actinobacillus succinogenes . The pretreatment process was achieved through integrated biorefinery concept by using some wheat milling byproducts (i.e., bran) as a substrate in ssf for aspergillus strains to produce glucoamylase and protease enzymes, which are used to pretreat wheat gluten or wheat bran . Also a. succinogenes was anaerobically used for sa production from acid pretreated corn fiber byproduct and 35.5 microorganisms are the most important and convenient sources for the production of commercial enzymes . They can be overexpressed under suitable growth conditions to produce abundant quantities of enzymes . With the initiation of a new era in biotechnology, the amylase enzyme family came forward with lot of industrial applications such as brewing, bread making, pharmacy, starch processing, paper, and textile industries . The untreated bsg was used for the production of -amylase by bacillus sp . In a submerged fermentation system . The highest tested concentration of bsg (5%, w / v) resulted in a 5-fold enhancement in the enzyme production . Another wheat milling byproduct, called gruel, was used by kammoun et al . As sole carbon source for the production of -amylase from aspergillus oryzae . The experimental conditions revealed an enhanced enzyme production of 151.1 u / ml . Also, ssf was applied for the production of this enzyme using paddy rice processing byproduct and the highest enzyme production was 211.5 u / gds (units per gram dried solid). Cellulases are another example of important industrial enzymes that are capable to degrade the most abundant cellulose biopolymer . A high yield (159.1 u / g) of -glucosidase was achieved from rice bran - based ssf by the penicillium citrinum fungus . Optimized the conditions of cellulase production by penicillium decumbens in ssf using rice bran as the substrate and obtained 5.76 iu / g, whereas lee et al . Used rice bran in combination with yeast extract to produce the same enzyme in smf by bacillus subtilis and 196.8 u / ml was obtained . Different agro - industrial residues were used for the production of this vital enzyme such as enzymatically treated wheat bran which exhibited 1.1 u / ml of the cellulase enzyme under smf by aspergillus flavus, or untreated corn cob which produced 5.25 iu / ml under smf by trichoderma reesei. (66) proteases are one of the most important categories of industrial enzymes, and the application of alkaline proteases has increased remarkably in a range of industrial processes including food, detergents, silk, and leather . Romero et al . Aimed at developing a process for protease production by bacillus licheniformis using corn distiller s dried grains with solubles (cddgs), a bioethanol industry byproduct, as the sole carbon / nitrogen source . The maximum protease production was 0.16 u / ml after 55 h of fermentation . Laccases are abundant in white - rot fungi, which are the only living organisms able to degrade whole wood components, particularly, the genus trametes which is the most efficient lignin degraders . The potential of the common brewing industry byproduct, barley bran, as a support - substrate for laccase production by trametes versicolor under ssf conditions was evaluated by rodrguez couto . Laccase activity was enhanced by 13-fold in relation to inert support cultures with initial ammonium concentration of 0.2 vanillin is used as flavoring agent in food industry, as intermediate in the herbicides industry, drugs or antifoaming agents, as component of household products such as floor polishes and air fresheners . Also it is used as food preservative because of its antimicrobial and antioxidant properties . The precursor of vanillin is ferulic acid which can be released from agricultural residues by physicochemical and/or enzymatic treatments . Di gioia et al . Explored the possibility of obtaining vanillin from the bioconversion of ferulic acid derived from enzymatically hydrolyzed wheat bran by an engineered e. coli strain . A vanillin concentration of 0.09 g / l was obtained, due to a high percent of reduction to vanillyl alcohol . Another trial was performed by torres et al ., in which corn cob was hydrolyzed by alkaline to obtain 1171 mg / l ferulic acid that was used as a medium for vanillin bioproduction by the engineered e. coli . A maximum vanillin concentration of 239 mg / l was obtained after 22 h. a different technology of converting ferulic acid, from the rice bran oil byproduct, into vanillin was developed by a combination of the fungal strains aspergillus niger and pycnoporus cinnabarinus, in which the filtrate of a. niger culture was concentrated and vanillic acid in the filtrate was fermented into vanillin by p. Cinnabarinus. (74) the concentration of vanillin reached 2.8 g / l . Biopolymers are important materials having applications in several industrial sectors like pharmaceutical, food and cosmetic industries . They can be categorized to polyesters (i.e., polyhydroxyalkanoates), polyalcohols (i.e., xylitol) and polysaccharides (i.e., pullulan). The biotechnological method of producing such polymers has high interest as an alternative to the chemical method because of the little energy required and specificity . However, the cost of these polymers is preventing their usage on a large scale . . Therefore, researchers are trying to use various cheap carbon sources, including agro - industrial byproducts . One of the initial trials was conducted by mussatto and roberto to evaluate the applicability of acidic hydrolyzate bsg as culture medium for xylitol production by candida guilliermondii, and obtained a xylitol yield of 0.78 g / g . Recently, the production of pullulan from aureobasidium pullulans was tested using csl as nutrient along with 15% (w / v) glucose as carbon source . G / l), which accounted for 18% increase compared with glucose . Also, production of polyhydroxyalkanoate (pha) by sinorhizobium meliloti using enzymatic hydrolyzate of rice bran was tested . The pha contents increased with the increase in fermentation period from 24 to 96 h, with a maximum production of 3.6 valorization options could be extended to the pharmaceutical field for the bioproduction of antibiotics . The rifamycins are a group of antibiotics that are synthesized either artificially or naturally by the bacterium amycolatopsis . Was investigated under ssf using different agro - industrial byproducts (corn husk, corn cobs, and wheat bran). Corn husk was the most suitable substrate (1.95 g / kg) with 4-fold higher production than wheat bran and corn cobs . As a result of growing environmental attentiveness and the prominence placed on an ecological society in agreement with the global environment, biosurfactants of microbial origin have recently been recommended to substitute chemically synthesized surface active agents . Low - cost media based on animal fat and csl combined with glucose, yeast extract, urea, and other inorganic nitrogen sources were evaluated for the production of biosurfactants by the yeast candida lipolytica. (82) only 2.5% of the csl remained after six days and 2.2 g / l of crude biosurfactant was produced, causing a maximum reduction in surface tension from 50 to 28 mn / m . Hydrogen gas is an eco - friendly fuel with high energy content (122 kj / g). Unlike fossil fuels emissions, only water vapor is released when hydrogen gas is used which contributes to reducing greenhouse effects significantly . Although h2 has many obvious advantages, it remains a problem with storage and transportation . Pressurised hydrogen gas takes a great deal of volume compared with other fuels like for example, gasoline that with equal energy content, needs about 30 times less volume at 100 bar gas pressure . Due to the high explositivity there are also obvious safety concerns with the use of pressurized or liquefied hydrogen in vehicles as well as additional energy use for pressurizing or liquefication . Pf by purple nonsulfur (pns) bacteria, like rhodobacter species, is among biohydrogen production methods that has attracted distinctive consideration . This is due to the fact that these photosynthetic bacteria have a number of advantages such as performing anoxygenic photosynthesis to produce hydrogen without oxygen, absence of hydrogen gas inhibition due to nitrogenase enzyme, utilization of a wide variety of substrates for hydrogen production and conferring different growth modes with respect to energy and carbon requirements . Df is more effective than photo because of the sensitiveness of pf and requirement of low concentration substrates . Biomass containing starch and cellulose, that is, agro - industrial byproducts, establish rich and consistent raw materials for biohydrogen production . An integrated biohydrogen refinery was suggested by several researchers, in which pretreated barley malt byproduct is utilized to produce biohydrogen by rhodobacter sphaeroides . Used the obtained biohydrogen (1.07 l / kg) as a fuel for the production of electricity through fuel cell, while kars and ceylan coproduced biohydrogen (0.4 l / l) and 5-aminolevulinic acid (67.4 m) using the same byproduct and microorganism . Moreover, wheat starch was investigated using fed - batch operation for biohydrogen production by combined dark and light fermentation . The optimum dark / light ratio was /2 yielding the highest cumulative hydrogen (65.2 cm / g). Also, alkaline - treated wheat milling byproduct was fermented with sewage sludge in different fermentation modes (batch, semicontinuous and continuous). The maximum hydrogen yields of 64 cm / g of wheat feed dry weight were produced in batch mode . The anaerobic bacteria caldicellulosiruptor saccharolyticus was investigated for the production of biohydrogen from the alkaline pretreated sweet sorghum bagasse (ssb), and a maximal volumetric hydrogen production rate of 10.6 mmol / lh was obtained . Bioethanol produced from low - cost biomass is considered as an attractive substitute to fossil fuels to minimize dependence on oil and reduce carbon dioxide emissions . Saccharomyces cerevisiae cells have average theoretical and practical ethanol yields of 0.5 and 0.4 g / g of glucose, respectively . Currently, the main biomass for bioethanol is starch - rich feedstock in which high yields of glucose are rapidly obtained by enzymatic hydrolysis . Substrate is one of the key costs in ethanol production . Putting this aim into focus, many studies tried to investigate the possibility of using agro - industrial byproducts as the main substrate for bioethanol production . Wheat bran is among these byproducts, in which it was used for bioethanol production by saccharomyces species after being hydrolyzed by acid or hydrothermal followed by enzymatic treatment . Under these conditions, g / l) when acidic hydrolyzed wheat stillage (main residue from the starch - to - ethanol fermentation process) was fermented by zymomonas mobiliz. (100) also, s. cerevisiae was investigated for the production of bioethanol using ssb pretreated with hydrothermal or acid treatment followed by enzymatic hydrolysis in submerged fermentation . G / l due to the usage of sweet sorghum juice mixed with the pretreated and dewatered bagasse . On the other hand, ssb without any pretreatment was used to immobilize s. cerevisiae through ssf for the production of bioethanol . The ethanol productivity of the immobilized cells was 2.24 times higher than the free cells, with an ethanol yield that reached 4.9 g / g . The ability to ferment ssb is not restricted only to bacterial cells, but it could be performed with fungal cells as well . Goshadrou et al . Studied the production of ethanol from the pretreated ssb by a zygomycetes fungus mucor hiemalis . The bagasse was treated with sodium hydroxide, prior to enzymatic hydrolysis by commercial cellulase and -glucosidase enzymes . A 24 h fermentation on the pretreated bagasse resulted in about 81% of the corresponding theoretical ethanol yield . The effect of different pretreatments and microbial strains on ssb for bioethanol production was investigated . It was found that maximum production of bioethanol was obtained (209.2 mg / g) after 24 h fermentation by issatchenkia orientalis with dry bagasse previously treated with hcl and enzymes as a substrate . Moreover, s. cerevisiae with acid / enzyme pretreated rice hull as a substrate was used for bioethanol production, and 0.11 g / g of rice hull was obtained with 84% of dissolved sugars converted to bioethanol . This yeast was also combined with candida shehatae to ferment the acidic hydrolyzed rice hull to ethanol . The pretreatment of rice hull by the wet air oxidation (wao) method was inspected by banerjee et al . The remained cellulose was around 92%, while the recovered lignin was 20%, showing oxidation of the majority of lignin . The high cellulose content and minor residual lignin in the solid fraction would greatly enable subsequent enzymatic hydrolysis, with improved ethanol yields from rice hull . Different pretreatments with various corn milling byproducts were evaluated for the production of bioethanol . Corn fiber was hydrolyzed by dilute sulfuric acid, and subsequently fermented by e. coli to produce 44 g / l of bioethanol . Coupling a pervaporation membrane unit to the hydrolyzate fed - batch fermentation maintained the ethanol production below 25 g / l, with sugar utilized completely, for 5 days, which circumvented the toxicity effect of high ethanol concentration on cells viability . Furthermore, the yeast strain pichia guilliermondii was anaerobically adapted to hydrolyzed corn cob and used for ethanol production without any detoxification or external nutrient supplementation . Studied the feasibility of substituting yeast extract by the low cost csl to produce ethanol using clostridium strain . / l) was produced after 360 h of fermentation in a 7.5 l bioreactor . Butanol is a carbon branched chain primary alcohol that could be an alternative fuel to ethanol due to its superior properties . Butanol is a metabolite of acetone / butanol / ethanol (abe) fermentation by clostridium species . Abe fermentation has major hindrances, mainly the high cost of substrates and recovery . In order to overcome this shortcoming, the objective of several studies was to evaluate the economical use of pretreated agro - industrial byproducts to produce biobutanol . Qureshi et al . Compared acidic to enzymatic pretreatment of corn fiber byproduct subsequently fermented to biobutanol by clostridium beijerinckii . G / l, when a mutant of c. beijerinckii with considerable inhibitor - tolerance was used for butanol production from nondetoxified acidic corn fiber hydrolyzate . Not only for the detoxification of the treated byproduct, but also for butanol separation from its fermentation broth . Anaerobic biomethanation can be used as an alternative potential treatment for valorization of biodegradable solid waste . Some of these organic solid wastes, that is, lignocellulosic byproducts have a limited biodegradability despite the high cod content . Therefore, several studies were conducted to enhance the biomethanation process of such byproducts . For example, barley waste was pretreated by alkaline before it was anaerobically fermented by activated sludge . This pretreatment reflected positively on the production of methane, which increased to 222 ml / g of volatile solids . Also, corn stillage was evaluated for anaerobic biogas production by biomethanator biomass after pretreatment with alkaline, and 763 ml / g of volatile solids was obtained . Regaining biochemical energy available in agricultural biomass is one approach that could be used to counterweight the world s current fossil fuel consumption . Agricultural crop byproducts, that is, cellulose and lignin, contain great amounts of biochemical energy . The potential for direct transformation of lignocellulosic biomass residues to electricity in a microbial fuel cell (mfc) was explored by several authors . Gregoire and becker developed a new single chamber solid - substrate mfc to hold the cellulose hydrolysis and fermentation processes . Untreated corn cob pellets were used as a fuel for the cell, which continuously produced electricity for more than 60 days . Bioaugmentation with geobacter metallireducens improved mfc performance to generate a maximum power density of 230 mw / m . The acidic / enzymatic hydrolyzate of corn stover was used as a substrate for a mixture of electrogenic culture in one chamber mfc to produce bioelectricity, and 1180 mw / m was obtained . Also the performance of mfcs was evaluated while treating rice mill wastewater by anaerobic sludge . Maximum cod removal efficiency of 96.5% and lignin removal of 84% were obtained . Maximum sustainable volumetric power (2.3 w / m) was achieved with 100 external resistance . Lignin is one of the key components of lignocellulosic biomass together with cellulose and hemicellulose, representing about 435% of most biomass feedstock . Lignins in cereals are guayacyl - syringyl - p - hydroxyphenyl with some structural units derived from trans - p - coumaryl alcohol . The lignin is separated by either dissolution using solvents, that is, alkali, organosolv and milled wood) or separation of insoluble lignin by acid hydrolysis of cellulose and hemicellulose, that is, klason and hydrolytic . This is clear in the klason procedure that provides quantitative separation of altered lignin . Kraft lignins (i.e., lignoboost) are accompanied by structural changes during the pulping process resulting in high content of free phenolic groups and c c linkages . This review contributes to knowledge about the future application of carbon rich agro - industrial byproducts for their value addition to biological chemicals and energy . It also offers economic and environmental benefits over traditional ways used to dispose off agro - industrial residues . Massive amounts of agricultural biomass are burnt in an open environment resulting in the release of harmful gases, which is not a sustainable method . It is clear that the utilization of agro - industrial byproducts as fermentation substrates require hydrolysis pretreatment due to the presence of complex polysaccharides in such biomass . Chemical hydrolysis uses up chemicals and produces chemically destructive effluents, whereas enzymatic hydrolysis requires optimization to achieve the best combination of enzymes for each feedstock and cannot quickly adapt to variable feedstock composition . On the contrary, hydrothermal hydrolysis is an eco - friendly method, even though it consumes energy . Although biomass may be cheap, the costs of processing technologies can be high . These technologies include not only pretreatment of biomass, but enzymatic saccharification of the pretreated biomass, and fermentation of the hexose and pentose sugars released by hydrolysis and saccharification . This can be achieved by increasing the efficiency of biorefinery in order to save resources and integrate in the existing industrial facilities, with eye on configurations that are eco - friendly . Therefore, prior to the successful application of this process on large - scale setups, consideration must be focused on the improvement of techniques for the hydrolysis of lignocellulosic biomass that result in higher sugar yields . Enzymatic hydrolysis requires further improvements especially in the terms of enzyme separation and reutilization after biomass treatment . Also, the complete characterization of the agro - industrial byproducts is required, in which it could be useful for the production of new microbial valorized products . The genetically modified microorganisms could be used to ferment c5 sugars from hemicellulose, which is not normally assimilated by the natural microbial strains.
Radiation therapy (rt) with or without concurrent chemotherapy is an established definitive treatment modality for both early and locally advanced stage nasopharyngeal cancer (npc). Although it allows favorable oncologic outcomes, there is a wide range of response to rt, highlighting the need to identify patients who might have radiation resistance and high risk of recurrence . Thus, earlier prediction of rt response and consequent long - term prognosis might be a critical issue for tailoring subsequent treatment to increase the chance of a cure in an individual patient . The tnm staging system is the most commonly accepted staging method for head and neck cancer, and it aids clinicians in determining prognosis and in selecting the most optimal therapeutic modality . The tnm staging system is considered the most important prognostic factor; however, because it is usually based on the size and/or extent of the primary tumor and/or metastatic lymph node, it sometimes does not correlate with actual tumor burden . Although this uni- or bi - dimensional measurement is an important factor, three - dimensional volumetric data have become another important factor that should be considered, particularly when using non - surgical therapies, such as rt or chemotherapy . Many studies have demonstrated the prognostic value of tumor volume in various cancers, and there is mounting evidence that pretreatment tumor volume and/or residual tumor volume have prognostic value [3 - 6]. However, few studies investigating changes in tumor volume during rt as a prognostic factor have been reported [7 - 10]. We previously reported on the prognostic impact of tumor volume reduction rate (tvrr) measured during adaptive rt for oropharyngeal cancer . In regard to the nasopharynx subsite of head and neck cancer, however, tvrr as a prognostic factor has not been reported yet, thus its prognostic role remains uncertain . The aim of this study was therefore to examine the volumetric parameters measured before and during definitive rt to determine possible prognostic implications in npc patients . To the best of our knowledge, the present study is the first to report on the value of tvrr as an independent prognostic factor in npc . We reviewed the medical records of consecutive npc patients treated with definitive rt with or without concurrent chemotherapy at samsung medical center between march 2006 and february 2013 . To be eligible for the current study, patients were required to have (1) histologically confirmed carcinoma of the nasopharynx, (2) no distant metastasis at the time of initial diagnosis, (3) completed more than 90% of the planned rt course without significant interruption, and (4) available rt plans . The exclusion criteria were (1) other non - squamous cell carcinoma histologic types such as lymphoma and salivary type carcinoma, (2) patients whose primary tumor or neck node was surgically removed or who received induction chemotherapy before rt, and (3) immediate follow - up loss after completion of rt . Complete medical history taking and physical examination, direct flexible fiberoptic endoscopic examination, computed tomography (ct) scans and magnetic resonance image of the head - and - neck region, and whole - body f - fluorodeoxyglucose positron emission tomography (pet) with ct were performed in all patients . Each patient underwent ct simulation in the supine position, immobilized by a thermoplastic mask, with a mouthpiece to keep the mouth open . Simulation ct images were obtained at 2.5- to 3.75-mm slice intervals with intravenous contrast enhancement . A second ct simulation was performed in all patients in order to generate an adaptive re - plan after delivery of the median 14 fractions (12 to 17 fractions). All sets of acquired simulation ct images were imported into the pinnacle3 treatment planning system (ver . 9.2, royal phillips electronics, miami, fl), and the gross tumor volumes (gtvs) of the primary tumor and the metastatic lymph nodes were manually contoured . The delineation of gtv was based on both clinical examination findings as well as all available diagnostic images . Rt techniques were either 3-dimensional conformal rt (3d - crt) using 4 mv and/or 10 mv photons from linear accelerator or intensity - modulated radiation therapy (imrt) using 6 mv photons from helical tomotherapy . The clinical target volume (ctv) was arbitrarily subdivided into two risk levels: high risk ctv encompassed the immediately adjacent regions to the gtvs and the lymph nodes considered at equivocal risk of metastasis based on size, shape, and pet uptake; and low risk ctv covered the apparently uninvolved lymphatics and more than one station away from the nodal gtv . The prescribed radiation dose was differently according to the rt technique . When using 3d - crt technique, the doses to the gtv, high risk ctv and low risk ctv were 70 gy in 35 fractions, 54 gy in 27 fractions, and 36 gy in 18 fractions, respectively, with 2 gy per fraction . When using imrt technique, simultaneous integrated boost (sib) was adapted, and the dose schedules changed during the study period . In earlier cases, the fraction sizes were 2.2 gy to the gtv and 2.0 gy to the ctv throughout the rt course . In later cases, the fraction sizes to the gtv and ctv were 2.2 gy and 2.0 gy during the first 18 fractions, and then 2.4 gy and 2.0 gy during the following 12 fractions, respectively . As a result, the actually delivered doses to the gtv, high risk and low risk ctvs were 66.0 or 68.4 gy in 30 fractions, 60 gy in 30 fractions, and 36 gy in 18 fractions, respectively . The schemes of the adaptive re - plans and measurements of the gtvs along the rt courses are illustrated in fig . 1 . In accordance with institutional guidelines, patients with locally advanced disease such as clinical t3 or t4 tumors and/or lymph node metastasis received concurrent chemotherapy with rt, unless they had no contraindication to addition of chemotherapy . Three weeks after completion of rt, adjuvant chemotherapy (1 to 3 cycles of cisplatin+5-fluorouracil) the primary tumor and the metastatic lymph nodes were delineated on both the pre- and mid - rt simulation ct images, and 3-dimensional tumor volumes were calculated on the rt planning system . Tvrr was defined as the percent (%) reduction of the gtv in relation to the pre - rt gtv, where tvrr=(pre - rt gtv mid - rt gtv)/pre - rt gtv . All patients were asked to visit for follow - up evaluation of disease status on a regular basis . The first evaluation was scheduled at one month of rt completion, and then every 3 months for the first 2 years, every 6 months until 5 years, and annually thereafter . On each visit, a thorough physical examination was performed together with imaging studies alternating between ct of the head - and - neck region and pet - ct . The overall survival (os) duration was measured from the date of rt initiation to the date of death or last follow - up visit, and progression - free survival (pfs) from the date of rt initiation to the date of first recurrence of disease of any type or the last clinical follow - up . The rates of os and pfs were calculated using kaplan - meier estimates with the log - rank test for univariate comparison . The relationship between the volumetric parameters and clinical outcome was assessed using mann - whitney u test . Prognostic factor analyses between tumor volume parameters were performed as both continuous and categorical variables to determine which independently affected prognosis . The optimal thresholds of the continuous variables were determined to be those producing the highest accuracy . The discriminatory performances of the volumetric data were assessed by receiver operating characteristic (roc) analysis . The log - rank test was used for validation of the determined cut - off values . Cox proportional hazard regression analysis was used to determine the implications of the potential prognosticators . The statistical tests were two - sided, and a p - value of <0.05 was considered statistically significant . Factors with p - value of <0.2 in univariate analysis were included in the multivariate analyses . Variable risk was expressed as a hazard ratio (hr) with a corresponding 95% confidence interval (95% ci). We reviewed the medical records of consecutive npc patients treated with definitive rt with or without concurrent chemotherapy at samsung medical center between march 2006 and february 2013 . To be eligible for the current study, patients were required to have (1) histologically confirmed carcinoma of the nasopharynx, (2) no distant metastasis at the time of initial diagnosis, (3) completed more than 90% of the planned rt course without significant interruption, and (4) available rt plans . The exclusion criteria were (1) other non - squamous cell carcinoma histologic types such as lymphoma and salivary type carcinoma, (2) patients whose primary tumor or neck node was surgically removed or who received induction chemotherapy before rt, and (3) immediate follow - up loss after completion of rt . Complete medical history taking and physical examination, direct flexible fiberoptic endoscopic examination, computed tomography (ct) scans and magnetic resonance image of the head - and - neck region, and whole - body f - fluorodeoxyglucose positron emission tomography (pet) with ct were performed in all patients . Each patient underwent ct simulation in the supine position, immobilized by a thermoplastic mask, with a mouthpiece to keep the mouth open . Simulation ct images were obtained at 2.5- to 3.75-mm slice intervals with intravenous contrast enhancement . A second ct simulation was performed in all patients in order to generate an adaptive re - plan after delivery of the median 14 fractions (12 to 17 fractions). All sets of acquired simulation ct images were imported into the pinnacle3 treatment planning system (ver . 9.2, royal phillips electronics, miami, fl), and the gross tumor volumes (gtvs) of the primary tumor and the metastatic lymph nodes were manually contoured . The delineation of gtv was based on both clinical examination findings as well as all available diagnostic images . Rt techniques were either 3-dimensional conformal rt (3d - crt) using 4 mv and/or 10 mv photons from linear accelerator or intensity - modulated radiation therapy (imrt) using 6 mv photons from helical tomotherapy . The clinical target volume (ctv) was arbitrarily subdivided into two risk levels: high risk ctv encompassed the immediately adjacent regions to the gtvs and the lymph nodes considered at equivocal risk of metastasis based on size, shape, and pet uptake; and low risk ctv covered the apparently uninvolved lymphatics and more than one station away from the nodal gtv . The prescribed radiation dose was differently according to the rt technique . When using 3d - crt technique, the doses to the gtv, high risk ctv and low risk ctv were 70 gy in 35 fractions, 54 gy in 27 fractions, and 36 gy in 18 fractions, respectively, with 2 gy per fraction . When using imrt technique, simultaneous integrated boost (sib) was adapted, and the dose schedules changed during the study period . In earlier cases, the fraction sizes were 2.2 gy to the gtv and 2.0 gy to the ctv throughout the rt course . In later cases, the fraction sizes to the gtv and ctv were 2.2 gy and 2.0 gy during the first 18 fractions, and then 2.4 gy and 2.0 gy during the following 12 fractions, respectively . As a result, the actually delivered doses to the gtv, high risk and low risk ctvs were 66.0 or 68.4 gy in 30 fractions, 60 gy in 30 fractions, and 36 gy in 18 fractions, respectively . The schemes of the adaptive re - plans and measurements of the gtvs along the rt courses are illustrated in fig . In accordance with institutional guidelines, patients with locally advanced disease such as clinical t3 or t4 tumors and/or lymph node metastasis received concurrent chemotherapy with rt, unless they had no contraindication to addition of chemotherapy . Three weeks after completion of rt, adjuvant chemotherapy (1 to 3 cycles of cisplatin+5-fluorouracil) was optionally administered to some npc patients . The primary tumor and the metastatic lymph nodes were delineated on both the pre- and mid - rt simulation ct images, and 3-dimensional tumor volumes were calculated on the rt planning system . Tvrr was defined as the percent (%) reduction of the gtv in relation to the pre - rt gtv, where tvrr=(pre - rt gtv mid - rt gtv)/pre - rt gtv . All patients were asked to visit for follow - up evaluation of disease status on a regular basis . The first evaluation was scheduled at one month of rt completion, and then every 3 months for the first 2 years, every 6 months until 5 years, and annually thereafter . On each visit, a thorough physical examination was performed together with imaging studies alternating between ct of the head - and - neck region and pet - ct . The overall survival (os) duration was measured from the date of rt initiation to the date of death or last follow - up visit, and progression - free survival (pfs) from the date of rt initiation to the date of first recurrence of disease of any type or the last clinical follow - up . The rates of os and pfs were calculated using kaplan - meier estimates with the log - rank test for univariate comparison . The relationship between the volumetric parameters and clinical outcome was assessed using mann - whitney u test . Prognostic factor analyses between tumor volume parameters were performed as both continuous and categorical variables to determine which independently affected prognosis . The optimal thresholds of the continuous variables were determined to be those producing the highest accuracy . The discriminatory performances of the volumetric data were assessed by receiver operating characteristic (roc) analysis . The log - rank test was used for validation of the determined cut - off values . Cox proportional hazard regression analysis was used to determine the implications of the potential prognosticators . The statistical tests were two - sided, and a p - value of <0.05 was considered statistically significant . Factors with p - value of <0.2 in univariate analysis were included in the multivariate analyses . Variable risk was expressed as a hazard ratio (hr) with a corresponding 95% confidence interval (95% ci). The median age was 51 years, ranging from 17 to 86 years, and 94.3% of the patients were male . Approximately half of the pretreatment clinical t stage was ct1 (77 patients, 44.7%), but the majority of patients (130 patients, 81.8%) had metastatic lymph nodes on initial diagnosis . The distribution of tnm stage based on the american joint committee on cancer (ajcc) seventh edition was stage i in 15 patients (9.4%), ii in 39 (34.5%), iii in 70 (44.0%), and iva / b in 35 (22.0%). The median total radiation dose was 68.4 gy (66 to 72.4 gy), and 113 patients (71.1%) received imrt . A cisplatin - based regimen was administered concurrently to all patients: cisplatin 100 mg / m every 3 weeks was administered to 111 patients (69.8%); weekly cisplatin 20 mg / m to 23 (14.5%); and cisplatin plus docetaxel 20 mg / m every 3 weeks to one (0.6%), respectively . After completion of rt, adjuvant chemotherapy with cisplatin plus 5-fluorouracil was administered to 34.6% of patients . The median follow - up duration for survivors was 41.5 months (range, 11.2 to 91.8 months). Treatment failure of any type was observed in 43 patients (27.0%), and 13 patients died during the follow - up period . As expected, patients with more advanced tnm stage had worse os and pfs: os rates at 5 years were 100% in stages i and ii, 84.7% in stage iii, and 82% in stage iv (p=0.045), respectively . Five - year pfs rates were 100% in stage i, 77% in stage ii, 64.0% in stage iii, and 58.2% in stage iv disease (p=0.022). The mean volumes of pre - rt and mid - rt gtvs were 45.9 cm (range, 1.5 to 185.3 cm) and 26.7 cm (range, 1.0 to 113.8 cm), respectively . The mean tvrr relative to pre - rt baseline was 41.9%, ranging from 87% to 78% . Tvrr did not correlate with the pre - rt gtv (spearman s correlation coefficient, 0.041; p=0.511). In correlation analyses between tnm stage and volumetric parameters, the meanstandard deviation (sd) of pre - rt gtv according to each stage was as follows: 12.66.3 cm in stage i, 29.422.5 cm in stage ii, 50.231.0 cm in stage iii, and 70.041.5 cm in stage iv (spearman correlation coefficient, 0.562; p <0.001). The meansd of mid - rt gtv according to each stage was 7.14.6 cm in stage i, 13.78.6 cm in stage ii, 29.420.0 cm in stage iii, and 44.028.3 cm in stage iv (spearman correlation coefficient, 0.583; p <0.001). Tvrr showed a marginally significant correlation with clinical stages: 46.8%25.1%, 39.1%25.9%, 30.7%24.8%, and 30.8%21.7%, respectively (spearman correlation coefficient, 0.138; p=0.082). The results of the correlation analyses of the volumetric parameters according to recurrence status are shown in table 2 . Patients without recurrence tended to show lower pre- and mid - rt gtvs and higher tvrr compared to patients with recurrence . As continuous variables, all volumetric parameters showed statistical significance in univariate analysis for pfs (pre - rt gtv [hr, 1.011; 95% ci, 1.004 to 1.018; p=0.03], mid - rt gtv [hr, 1.024; 95% ci, 1.013 to 1.035; p <0.001], and tvrr [hr, 7.514; 95% ci, 2.777 to 20.329; p <0.001]), but only tvrr displayed prognostic significance in multivariate analysis (hr, 5.949; 95% ci, 1.090 to 32.467; p=0.038). In roc analysis, a tvrr cut - off value of 35% showed the maximal discriminative power (sensitivity, 66.7%; specificity, 71.2%). When dichotomized by various tvrr cut - off values, the pfs rate for the higher tvrr group was higher than that of the lower tvrr group (fig . The pfs rate of patients with tvrr> 35% was 79.2%, while that of those with tvrr 35% was 53.2% (fig . Results of univariate and multivariate analyses of the impacts of patient- and treatment - related factors on pfs are shown in table 3 . In multivariate analysis, tvrr was identified as an independently significant prognostic factor for pfs when adjusted for clinical factors (hr, 2.877; 95% ci, 1.555 to 5.326; p=0.001). The median age was 51 years, ranging from 17 to 86 years, and 94.3% of the patients were male . Approximately half of the pretreatment clinical t stage was ct1 (77 patients, 44.7%), but the majority of patients (130 patients, 81.8%) had metastatic lymph nodes on initial diagnosis . The distribution of tnm stage based on the american joint committee on cancer (ajcc) seventh edition was stage i in 15 patients (9.4%), ii in 39 (34.5%), iii in 70 (44.0%), and iva / b in 35 (22.0%). The median total radiation dose was 68.4 gy (66 to 72.4 gy), and 113 patients (71.1%) received imrt . A cisplatin - based regimen was administered concurrently to all patients: cisplatin 100 mg / m every 3 weeks was administered to 111 patients (69.8%); weekly cisplatin 20 mg / m to 23 (14.5%); and cisplatin plus docetaxel 20 mg / m every 3 weeks to one (0.6%), respectively . After completion of rt, adjuvant chemotherapy with cisplatin plus 5-fluorouracil was administered to 34.6% of patients . The median follow - up duration for survivors was 41.5 months (range, 11.2 to 91.8 months). Treatment failure of any type was observed in 43 patients (27.0%), and 13 patients died during the follow - up period . As expected, patients with more advanced tnm stage had worse os and pfs: os rates at 5 years were 100% in stages i and ii, 84.7% in stage iii, and 82% in stage iv (p=0.045), respectively . Five - year pfs rates were 100% in stage i, 77% in stage ii, 64.0% in stage iii, and 58.2% in stage iv disease (p=0.022). The mean volumes of pre - rt and mid - rt gtvs were 45.9 cm (range, 1.5 to 185.3 cm) and 26.7 cm (range, 1.0 to 113.8 cm), respectively . The mean tvrr relative to pre - rt baseline was 41.9%, ranging from 87% to 78% . Tvrr did not correlate with the pre - rt gtv (spearman s correlation coefficient, 0.041; p=0.511). In correlation analyses between tnm stage and volumetric parameters, the meanstandard deviation (sd) of pre - rt gtv according to each stage was as follows: 12.66.3 cm in stage i, 29.422.5 cm in stage ii, 50.231.0 cm in stage iii, and 70.041.5 cm in stage iv (spearman correlation coefficient, 0.562; p <0.001). The meansd of mid - rt gtv according to each stage was 7.14.6 cm in stage i, 13.78.6 cm in stage ii, 29.420.0 cm in stage iii, and 44.028.3 cm in stage iv (spearman correlation coefficient, 0.583; p <0.001). Tvrr showed a marginally significant correlation with clinical stages: 46.8%25.1%, 39.1%25.9%, 30.7%24.8%, and 30.8%21.7%, respectively (spearman correlation coefficient, 0.138; p=0.082). The results of the correlation analyses of the volumetric parameters according to recurrence status are shown in table 2 . Patients without recurrence tended to show lower pre- and mid - rt gtvs and higher tvrr compared to patients with recurrence . As continuous variables, all volumetric parameters showed statistical significance in univariate analysis for pfs (pre - rt gtv [hr, 1.011; 95% ci, 1.004 to 1.018; p=0.03], mid - rt gtv [hr, 1.024; 95% ci, 1.013 to 1.035; p <0.001], and tvrr [hr, 7.514; 95% ci, 2.777 to 20.329; p <0.001]), but only tvrr displayed prognostic significance in multivariate analysis (hr, 5.949; 95% ci, 1.090 to 32.467; p=0.038). In roc analysis, a tvrr cut - off value of 35% showed the maximal discriminative power (sensitivity, 66.7%; specificity, 71.2%). When dichotomized by various tvrr cut - off values, the pfs rate for the higher tvrr group was higher than that of the lower tvrr group (fig . The pfs rate of patients with tvrr> 35% was 79.2%, while that of those with tvrr 35% was 53.2% (fig . Results of univariate and multivariate analyses of the impacts of patient- and treatment - related factors on pfs are shown in table 3 . In multivariate analysis, tvrr was identified as an independently significant prognostic factor for pfs when adjusted for clinical factors (hr, 2.877; 95% ci, 1.555 to 5.326; p=0.001). In this study, we investigated the usefulness of tvrr measured from simulation ct before and during adaptive rt in patients with npc . The most significant result of the present study was that tvrr was an independent prognostic factor in terms of predicting pfs in npc patients treated with definitive rt with or without concurrent chemotherapy . Tvrr during the rt course has recently emerged as a prognostic factor in various malignancies including head and neck cancers . In a previous study, we reported on the prognostic impact of tvrr measured during definitive rt on loco - regional control . With a 35% cut - off value of tvrr, the 3-year loco - regional control rate was 94.4% in patients with higher tvrr, but 72.4% in those with less tvrr . Yang et al . Also demonstrated that tvrr was a predictor for local control in patients with oro- and hypopharyngeal cancer treated with imrt . Hoeben et al ., who studied f - fluorothymidine pet - ct before and during the course of rt for head and neck squamous cell carcinoma, reported that gtv decrease above median (31%) was associated with significantly better 3-year loco - regional control and disease - free survival . Our results were consistent with those reported in these studies in that tvrr also influenced prognosis in npc . The other main finding from the present study was that pre- and mid - rt absolute tumor volumes had less impact on clinical outcomes than tvrr . Several studies have reported association of pre - treatment tumor volume with the probability of tumor control and survival in patients with head - and - neck cancer arising in the subglottic larynx, the hypopharynx, the oropharynx, and the nasopharynx . The present study also showed that pre- and mid - rt gtv were statistically significant in univariate analysis . After adjustment for other volumetric parameters, however, these factors lost their prognostic significance (table 3). Based on this result, we would speculate that tvrr has a greater prognostic value than absolute tumor volume . In addition to the prognostic role of tvrr, it could provide additional discriminative efficacy in risk group stratification during the course of definitive rt . Adaptive re - planning during the rt course is an emerging issue being routinely incorporated into clinical practice at many institutions . Variable degrees of volumetric and geometric changes are commonly observed during the rt course in treatment of head and neck cancer . Many radiation oncologists recommend repeated ct simulation and subsequent re - planning during the rt course, as the adaptive rt may provide more optimal dose delivery to the target while decreasing the normal tissue toxicities [16 - 18], and can even improve local control and quality of life . Accordingly, adaptive rt has become essential in treatment of head - and - neck cancer patients . In the current clinical setting, volumetric parameters, which could be easily acquired, tvrr may have more than just prognostic value, and it could aid clinicians in the individualization of therapeutic strategy . Most tumor responses are evaluated after completion of a definitive rt course, when it might be too late to commence an appropriate therapeutic modification . By predicting recurrence before the completion of definitive treatment an escalation of total radiation dose to a higher level than initially planned might be one example . It can be, more or less, quite easily implemented by using imrt that employs the sib technique . A second example is the indicator used in selection of candidates for intensified adjuvant chemotherapy . All of these modifications will hopefully contribute to improving clinical outcomes by early identification of patients who might not benefit from the initial treatment plan . We generated rt plans, either conventionally fractionated 3d - crt or dose - painting imrt, using the shrinking field technique . The initial rt plan was designed for 36 gy to the low risk ctv, and the subsequent plan was designed without further irradiation to this region . For this reason, the second simulation ct was performed in the third week after the initiation of rt at authors institution . One question that has arisen from the current study involves the optimal timing of the second simulation ct during the rt course . The nodal and primary tumor gtvs were reported to shrink by 1.2%-3% per treatment day on average, but their regression did not occur at a constant rate . The largest tumor volume changes occurred after the second week of the rt course and appeared to be significant after 3 - 4 weeks of rt, and could have a potential dosimetric impact when highly conformal rt techniques were used . In addition, wang et al . Reported that re - planning for npc patients before the 25th fraction during imrt helps to ensure an adequate dose to the target volumes, as well as safely limited doses to the critical normal structures . A speculative conclusion that could be drawn from these studies is that the appropriate timing of the second ct, either for effective adaptive intervention or for assessment of tumor regression, might be longer than 3 weeks after the start of rt . However, if adaptive re - planning is performed in the late phase of the rt course, this advantage might decrease, as there might remain only a few fractions of rt . In the current study, most re - planning ct scans of patients were obtained between 13 to 15 fractions according to the institutional policy . It appears that this timing is quite reasonable in achieving the benefits of adaptive re - planning . Second, epstein - barr virus (ebv) status, which is known to have prognostic importance in npc patients, was not fully addressed . Again, because of the limitations of the retrospective data, tests identifying ebv were conducted in only 26 patients (16.4%). Finally, there is a possibility of inter- and/or intra - physician variation in gtv measurements . Despite these limitations, the discriminatory performance of the volumetric parameters could be utilized as an indicator for tailoring therapy on an individual patient basis . The current study shows that tvrr measured during the rt course is a proven independent prognostic factor predicting pfs . Tvrr can be easily calculated during the process of adaptive rt by simply comparing the pre- and mid - rt simulation ct images . Tvrr can provide not only prognostic information, but can also be a useful indicator for tailoring treatment strategies before the completion of definitive rt.
Addition of glcnac to the extracellular medium activates signal transduction pathways in the yeasts c. albicans, candida lusitaniae, and yarrowia lipolytica . Glcnac induces these species to switch from growing as budding cells to forming long filamentous hyphal cells . Glcnac signaling has been best studied in the human fungal pathogen c. albicans, as the more commonly studied model yeasts saccharomyces cerevisiae and schizzosaccharomyces pombe lack the genes required to catabolize glcnac and do not appear to respond to this sugar . One pathway results in increased camp signaling, which then triggers hyphal morphogenesis and expression of virulence factors . Glcnac activation of camp signaling also induces an epigenetic switch known as white - opaque switching that results in altered morphological characteristics and gene expression in mating type homozygous forms of c. albicans . The second pathway activated by glcnac, which is independent of camp signaling, results in increased expression of the genes needed to catabolize glcnac . Initial attempts to determine whether glcnac has to be taken up by c. albicans to induce signaling led to conflicting conclusions . This was due in part to the lack of genetic approaches available at the time to study this diploid organism . However, the more recent discovery of the glcnac transporter (ngt1) in a proteomics study of c. albicans plasma membrane proteins has provided an important new tool for analysis of glcnac signaling . Ngt1 was identified as a glcnac transporter because an ngt1 deletion mutant was very strongly impaired in glcnac uptake, and heterologous expression of ngt1 in s. cerevisiae conferred the ability to take up glcnac . Additional studies showed that ngt1 was specific for glcnac and did not promote the transport of other related sugars . Although ngt1 cells were strongly defective in glcnac uptake, they could still import low levels of this sugar . Thus, it was very significant that the ngt1 mutant cells could still be stimulated to induce hyphal morphogenesis in the presence of 1,000-fold higher levels of glcnac than are required to induce the wild type . These results support the conclusion that ngt1 facilitates uptake of glcnac, and that intracellular glcnac then induces signaling in c. albicans . Interestingly, ngt1 has also turned out to be a valuable new tool for studying the roles of glcnac in other organisms, since it is the first eukaryotic glcnac transporter gene to be identified . Subsequent studies now indicate that glcnac metabolism is not necessary for its ability to induce signaling in c. albicans . This was tested in part by mutating the hxk1, nag1 and dac1 genes needed to catabolize glcnac and use it for energy . These genes encode the enzymes needed for glcnac to be phosphorylated, deacetylated and then deaminated, resulting in its conversion to fructose-6-po4 . Significantly, a triple mutant lacking all three genes could still respond to glcnac to induce both the formation of hyphae and increased expression of ngt1 . Since phosphorylation of glcnac by hxk1 is also required for cells to convert glcnac into udp - glcnac for use in anabolic pathways, the ability of the triple mutant to be stimulated indicates that glcnac metabolism is not required for signaling . The ability of cells to induce signaling in the absence of the glcnac kinase (hxk1) indicates that non - phosphorylated glcnac is capable of inducing c. albicans . This has important advantages for signaling as it allows cells to distinguish the non - phosphorylated glcnac transported into the cell from the glcnac-6-po4 that is synthesized de novo within the cell (fig . Consequently, significant amounts of non - phosphorylated glcnac are not expected to occur in the cell unless it taken up from an exogenous source . Furthermore, detecting non - phosphorylated glcnac is also expected to provide a higher degree of sensitivity for low levels of extracellular glcnac imported into the cell in the presence of endogenously synthesized glcnac-6-po4 . Cells must synthesize large amounts of glcnac-6-po4 to keep up with the demands for it in n - linked glycosylation, gpi anchor synthesis and synthesis of cell wall chitin . Not too surprisingly, signaling pathways activated by other sugars have also been reported to sense the non - phosphorylated form that is imported into the cell . Altogether, these results demonstrate that the capacity of c. albicans to sense non - phosphorylated glcnac taken up into the cell represents a novel signal transduction mechanism that may also occur in other organisms . Figure 1 . Note that (a) c. albicans and (b) bacteria such as e. coli are thought to sense forms of glcnac that are not synthesized in the cell . The response of c. albicans to glcnac raises the question of what might be the sources of glcnac that it could encounter . One possibility is that c. albicans responds to glcnac released by chitinases that remodel the cell wall during growth or released by the action of human chitinase during infection, since the fungal cell wall contains chitin, a polymer of glcnac . Similarly, glcnac is also released during growth of bacteria due to remodeling of the peptidoglycan cell wall layer, which is composed in part of glcnac . This latter source of glcnac may be important for signaling in mixed microbial environments, such as the g.i . Glcnac is also an abundant component of the cell surface on mammalian cells, and is present in polymers such as glycosaminoglycans . One example is pseudomonas aeruginosa, which was recently reported to respond to glcnac in the lung secretions (sputum) of cystic fibrosis patients . Microarray analysis revealed that growth in sputum induced expression of the glcnac catabolic genes in p. aeruginosa, consistent with the presence in sputum of glcnac - containing polymers, such as hyaluronic acid and mucins . Interestingly, glcnac also induced the production of phenazine antimicrobial compounds by p. aeruginosa . It was proposed that this antimicrobial defense system might be induced by glcnac since p. aeruginosa also lives in the soil where glcnac would be an indicator of the presence of other bacteria or fungi in the area . Similarly, glcnac also induces production of antimicrobial compounds and regulates development during sporulation of some soil bacteria, such as streptomyces coelicolor . Another bacterial pathogen, escherichia coli, responds to glcnac by reducing expression of curli fibers and type 1 fimbriae . Curli fibers are important for biofilm formation, adhesion and the internalization of e. coli by epithelial cells . Fimbriae are important for pathogenicity by promoting adhesion to mammalian cells . Decreased production of these cell surface molecules this response to glcnac may also be involved in balancing the interaction between e. coli and the host immune response, since curli and fimbriae are thought to be proinflammatory . Glcnac may also have other roles in virulence, as suggested by studies showing that the ability of e. coli to catabolize glcnac is also important for it to colonize the g.i . Glcnac also induces the genes needed for its catabolism in bacteria . In the bacteria where this has been studied, the glcnac catabolic genes are located in an operon that is regulated by a repressor, such as nagc in e. coli . Glcnac is taken up by bacteria through a phosphotransferase transporter system that results in glcnac-6-po4 entering the cell . Binding of glcnac-6-po4 to the nagc repressor causes allosteric changes that derepress the operon and promote expression of the genes needed for glcnac catabolism . Interestingly, bacteria are distinct from eukaryotic cells in that they do not synthesize glcnac-6-po4 . Intracellular glcnac-6-po4is only expected to occur in bacteria as a result of the import of exogenous glcnac (fig . Thus, bacteria have a mechanism for distinguishing exogenous glcnac from the glcnac synthesized in the cell that is analogous to the detection of non - phosphorylated glcnac in c. albicans (fig . 1). In this regard it is also interesting that glcnac-6-po4 imported into gram - positive bacteria acts as a cofactor for the ribozyme activity of the glms mrna, which results in its cleavage . Since glms encodes glucosamine-6-po4 synthetase, the first committed step in the glcnac synthesis pathway, this specialized mechanism turns off glcnac synthesis in the presence of exogenous glcnac . Thus, there are at least two mechanisms in bacteria by which glcnac-6-po4 regulates gene expression . The ability of mammalian cells and plants to respond to exogenous glcnac has not been well studied . These organisms contain the genes needed to catabolize glcnac, so they presumably have mechanisms to regulate their expression . In contrast, there is a rapidly growing body of work demonstrating that plants and mammals respond to changes in the intracellular levels of glcnac . Changes in nutrition that result in elevated glcnac synthesis lead to increased formation of udp - glcnac, which is a substrate for the enzyme o - glcnac transferase (ogt). Ogt modifies proteins by catalyzing the transfer of the glcnac moiety of udp - glcnac to ser or thr residues on proteins . This is a reversible modification in animal cells analogous to protein phosphorylation, as they also encode an enzyme that removes o - glcnac from proteins . Many of the substrates that have been identified for ogt play key roles in cellular regulation, such as the transcription factors c - myc, p53 and nf-b . This type of o - linked glcnac attachment has therefore been implicated in a range of human diseases, including cancer and diabetes . The presence of glcnac on the surface of so many different cell types makes it a good indicator of either the presence of other organisms or potentially an attack on the outer surface of a cell . Since cells in nature typically grow in mixed microbial environments, glcnac is therefore also well suited to be part of the communication that goes on between cells . An interesting example of this type of interspecies communication occurs between c. albicans and p. aeruginosa . Glcnac and other bacterial cell wall breakdown products induce c. albicans to undergo hyphal morphogenesis . Aeruginosa cells are then able to form a dense biofilm on the filamentous hyphal cells and kill them . C. albicans also responds to a quorum factor produced by p. aeruginosa (3-oxo - c12 homoserinelactone) by restricting its growth to the budding pattern, thereby protecting it from being killed since budding cells are not attacked by p. aeruginosa . Significantly, the p. aeruginosa cells are also on the receiving end of a signal from c. albicans . A quorum factor produced by c. albicans (farnesol) interferes with quorum signaling in p. aeruginosa and prevents induction of virulence factors . Thus, glcnac is part of a complex exchange of signaling molecules between c. albicans and p. aeruginosa . Given the prevalence of glcnac, it seems likely to be involved in other forms of interspecies communication.
Over the last 10 years, incidence rates of melanoma have increased for both men and women in all age groups, and, according to the united states cancer statistics report, there will be an estimated 65,647 melanoma cases in 2014 [1, 2]. Previous studies attributed the increased rates to increased awareness and detection of thin (1 mm), perhaps less aggressive, lesions [35]. However, more recent studies have found a statistically significant increase in incidence for tumors of all varying histological subtypes and thickness, including tumors> 4 mm, indicating that the incidence rates are a true increase [6, 7]. Melanoma, along with other cancers (not including basal or squamous cell carcinomas), is mandated by law to be reported by all physicians and treatment facilities to state cancer registries . However, reporting has been particularly challenging . The advent of new techniques such as mohs micrographic surgery and the high percentage of melanomas found to be thin lesions, approximately 70% of invasive melanomas reported to the seer in last decade, have led to decentralization of melanoma management from hospitals to outpatient settings [8, 9]. Also, given the relative ease of access to the skin, the cycle of care may not involve a hospital, as many dermatologists now utilize outpatient - based dermatopathologists and surgical options . Most of the data compiled by state cancer registries are obtained from hospital cancer registries . Therefore, a lack of or delay in detection of many thin melanomas is likely to occur, resulting in underreporting of early stage melanoma [2, 8, 11]. Generating inaccurate trend estimates on melanoma incidence and burden will undermine efforts to effectively allocate resources and implement public health initiatives . A survey at a national conference found that 50% of dermatologists were unaware of mandated melanoma reporting and about 56% do not report newly diagnosed cases . Tools to assist the practitioner awareness of methods to report to their state cancer registry may help narrow the knowledge and practice gaps . The purpose of this study is to generate a cross - sectional snapshot of the various reporting methods available to hospitals, outpatient dermatologists, and outpatient pathology labs for reporting diagnosed melanoma cases to their individual state cancer registries . Also, to investigate the dynamics of available reporting methods, we compared this snapshot to data that we obtained in 2012 . As this was publicly available information, irb approval was not required . In order to accurately capture various reporting methods available to hospitals, independent pathology laboratories, and nonhospital affiliated physicians initially, 50 state cancer registry websites were perused for reporting procedures . After this initial investigation, a brief query was made to every state cancer registry by either email or telephone inquiring about (1) available electronic reporting methods, (2) downloadable online reporting forms, and (3) submission of hard copy pathology reports . Hospital here encompasses hospital - based pathology labs and hospital - affiliated physicians . Pathology labs and physicians refer to independent, non - hospital - affiliated entities . From the information gathered, a table of our data was generated depicting various reporting methods available to hospitals, independent pathology labs, and non - hospital - affiliated physicians . An identical survey that was conducted in 2012 was also perused to observe whether there had been a national shift towards electronic reporting methods for melanoma compared to our current survey . Forty - seven (96%) were equipped for electronic transmission of confirmed melanoma cases from hospitals, pathology laboratories, and physicians (table 1). Maine and massachusetts were the only 2 states that had yet to implement an electronic method of melanoma reporting (table 1). Seven (14%) states accepted electronic - only transmittal methods while 40 states (82%) had options for both electronic and nonelectronic reporting methods such as submission of hardcopy pathology reports (figure 1). For states that allowed nonelectronic reporting, the alternative methods available to mandated reporters included submission of hard copy pathology reports, registries actively requesting pathology reports of diagnosed cases, and allowing reporters to request melanoma reporting forms . Forty - two (86%) state cancer registries permitted submission of hard copy pathology reports (table 1). Twenty - seven (55%) state cancer registries actively requested pathology reports from dermatology practices by sending out periodic surveys (table 1). Nine (18%) states allowed dermatologists to request melanoma reporting forms directly from them (table 1), and 22 (45%) states had reporting forms available for download on their registry websites (table 1). Comparison data exploring shifts in allowable reporting methods showed an increase in electronic reporting methods . In 2012, 4 state cancer registries had non - electronic - only methods for reporting melanoma . In comparison, also, since 2012, three additional state cancer registries have joined four others in requiring an electronic - only reporting method . The implementation of the national program of cancer registries (npcr) in 1992 signified a push to expand and augment state cancer registries and was a crucial step in developing a system that permitted up - to - date cancer surveillance . This, in turn, enhanced research efforts to shed light on cancer distribution, population heterogeneity, and environmental etiologies, all required for more efficient resource allocation for education and screening purposes [1315]. To ensure that these registries obtain accurate and comprehensive data, it is crucial to recognize gaps in data reporting and generate tools targeted at minimizing this gap as much as possible . To address clinician knowledge and practice gaps, an easy - to - reference table depicting reporting method options may facilitate better data acquisition and thus more robust estimates of melanoma incidence . We propose that these data be made available on the american academy of dermatology (aad) website . Given the mandate of electronic health records, it is natural to expect that all state registries eventually adopt electronic methods for melanoma reporting . There was a variation in the actual methods each state registry utilized for data transmissions . Some state registries had developed their own software, while, more interestingly, some had implemented the ability for providers eligible for meaningful use stage 2 (mu-2) to report melanoma by transmitting their electronic health records directly to the registries . This latter method sounds the most feasible and efficient given the ever - increasing administrative burden on physician practices . However, such a streamlined process faces several challenges given that several facets have yet to be implemented including promotion of physician reporting, on - boarding new providers, and declaration of readiness from state cancer registries . The comparison data from 2012 and 2014 demonstrated an increased shift towards electronic reporting methods by more state registries; however this shift is mostly reflective of hospitals and independent pathology labs being able to report electronically . Outpatient dermatologists still do not have the means to report melanoma cases electronically in 11 states (data not shown) but we speculate a trend towards mandated electronic reporting from clinicians . The main limitation of our study is the cross - sectional nature of data collection . While the purpose was to create a tool depicting state - specific reporting for dermatologists and independent pathology labs, to be most effective, there should be an ongoing, real - time update such that consumers (dermatologists and pathology labs) can reference current information . Until such a tool can be developed, depicting our data on the publicly accessible website of a national organization such as aad will hopefully lead to ease of reporting . At the minimum
Auditory nerve fibers diverge into different divisions of the cochlear nucleus setting the stage for multiple ascending pathways . At the thalamocortical level, parallel pathways arise from three distinct divisions of the medial geniculate body (mgb; for review see winer et al ., 2005). Thalamocortical connections include a tonotopic pathway through the mgbv (ventral division) which forms the cortical substrate for functional subregions within isofrequency contours (reviewed in read et al ., 2002). The pathway through the mgbm (medial division) is implicated in multisensory integration and learning (wepsic, 1966; edeline and weinberger, 1992; bordi and ledoux, 1994; for review see hu, 2003). The non - tonotopic pathway through the mgbd (dorsal division, raczkowski et al ., 1976; andersen et al ., 1980; morel and imig, 1987; for review see imig and morel, 1983; rouiller, 1997) may be involved in representing complex sounds (aitkin and dunlop, 1968). The hypothesis that parallel thalamocortical pathways are involved in different aspects of hearing is supported by studies in three species of bats from different families: the mustached bat (pteronotus parnellii; mormoopidae family), the horseshoe bat (rhinolophus rouxi; rhinolophidae family) and the pallid bat (antrozous pallidus; vespertilionidae family). The mustached bat auditory cortex contains a primary auditory cortex (a1) with a tonotopic map (reviewed in suga, 1989). This map, as in all other species examined, receives input from the mgbv . Some specializations for echolocation, such as over - representation of dominant harmonic frequencies of the echolocation call, are present in this pathway, but within the context of tonotopic representation . Information relevant to target distance is represented by delay - tuned combination - sensitive neurons . Injections of different tracers in different delay - tuned areas label separate regions in the mgb indicating that multiple delay - tuned areas are present in the thalamus (pearson et al ., 2007; also see wenstrup, 1999 for physiological studies with a similar conclusion) and that each thalamic delay - tuned area representation projects independently, and in parallel, to a cortical delay - tuned area (pearson et al ., 2007). Particularly, labeling was found in the rostral pole nucleus . Although it remains debated whether the rostral pole nucleus should be part of the dorsal division, the lateral part of the posterior group or an extension of the ventral division (morel and imig, 1987; wenstrup et al ., 1994; lee et al ., 2004; pearson et al ., 2007), it is clear there are parallel pathways conveying information from the mgb to the cortex . In the horseshoe bat, the cortical region termed dorsal cortex contains neurons that may be involved in target ranging . These neurons receive input from the mgbd (radtke - schuller, 2004; radtke - schuller et al ., 2004). The primary auditory cortex receives input from the mgbv . In both the mustached and horseshoe bat the suprageniculate (sg) nucleus, a part of the mgbd, projects diffusely to the entire auditory cortex, with a significant input to non - primary cortical regions involved in processing echolocation calls . Taken together, these data indicate that the thalamocortical pathways that serve fine frequency analysis and target distance calculation in the mustached and horseshoe bat are mostly segregated(figure 1a). Schematic of thalamocortical pathways in adult mustached, horseshoe and pallid bats and young pallid bat . (a) in the horseshoe bat, the mgbd projects mainly to regions dorsal to a1 that contain the combination - sensitive neurons used in target distance computation . The mgbv projects to a1 . In the mustached bat, the rostral pole nucleus projects to the delay - tuned areas . It is not clear if the rostral pole nucleus should be considered a part of the ventral or dorsal mgb . Therefore, the term mgbd / mgbv - rp is used in this schematic . In both species, the sg, considered a part of the mgbd, projects diffusely but more to the non - primary cortex than primary cortex . (b) in adult pallid bats, the hfr involved in echolocation behavior receives input from the sg and the mgbd, but not the mgbv . The lfr, involved in passive localization, receives input from the mgbv, but not the sg . Based on response selectivity in the auditory cortex, the sg hfr pathway is involved in echolocation behavior while the mgbv lfr pathway is involved in passive localization of prey - generated noise . (c) in a 2-week - old pallid bat pup, however, the pathways overlap . Studies on the pallid bat provide evidence that parallel auditory pathways may represent sounds used in two different behaviors . The pallid bat localizes terrestrial prey by listening to prey - generated noise while reserving echolocation calls for general orientation and obstacle avoidance (bell, 1982; fuzessery et al ., 1993). For echolocation, it uses a downward frequency modulated (fm) sweep (60 30 khz). For prey localization it depends on noise transients (540 khz). Its auditory cortex and inferior colliculus (ic) are organized to process fm sweeps and noise in mostly segregated regions (fuzessery, 1994; razak and fuzessery, 2002). In the auditory cortex, there is a tonotopic map with frequencies from 5 to 70 khz . Most neurons with tuning between 5 and 30 khz respond best to noise transients . Most neurons with tuning between 30 and 60 khz respond best to downward fm sweeps . The majority of inputs to the lfr and hfr in adult pallid bats arise from different divisions of the mgb (figure 2, schematized in figure 1b). Placement of retrograde tracers in the lfr labeled neurons predominantly in the mgbv with no label in the sg (razak et al ., 2007). Placement of tracers in the hfr labeled neurons in the sg, but not the mgbv . The mgbm and parts of mgbd outside of the sg send minor projections to both the lfr and the hfr . Thus parallel thalamocortical pathways represent sounds involved in two different behaviors, with the sg hfr connections putatively involved in echolocation behavior and mgbv lfr connections putatively involved with passive prey localization . The injection was made near neurons tuned 42 khz and selective for downward fm sweeps . (c f) photomicrographs of increasingly more rostral locations of the mgb show that labeled neurons were found in the sg, mgbm, and mggd, but not in the mgbv . Ii (g p) an example of injections made in the lfr . ((j n) schematics of increasingly rostral sections through the mgb showing that labeled neurons were found in the mgbv, mgbm, and mgbd, but not the sg . (o, p) photomicrographs showing fg and fr labeled cells near the rostrocaudal center of the mgb . (q s) an experiment in which fg was injected in the lfr and fr was injected in the hfr . (r) schematic of a section at the rostrocaudal center of the mgb showing that fg labeled neurons were present in mgbv, but not the sg, and that fr labeled cells were found in the sg, but not the mgbv . While the existence and functional significance of parallel thalamocortical pathways are established, very little is known about the development of these pathways . The development of these pathways between the mgb and the auditory cortex has been the subject of only one study . Gurung and fritzsch (2004) found that the mgb innervates the auditory cortex before onset of patterned sensory input . However, whether segregation of mgb - auditory cortex inputs into parallel pathways depends on sensory experience remains unknown . Studies of connections of the midbrain (friauf and kandler, 1990; gabriele et al ., 2000, 2007) and lower brainstem (leake et al ., 2002) suggest that the nucleotopic connections are mostly adult - like before hearing onset . Postnatal refinement or maintenance of connections, perhaps in an activity - dependent manner, is restricted to sharpening tonotopic organization (kim and kandler, 2003; franklin et al ., 2006; leake et al ., 2006) and fine tuning bilateral inputs within nuclei involved in sound localization (kapfer et al ., 2002). The pallid bat is suited to address the development of thalamocortical connections for several reasons . First, as mentioned above, parallel pathways can be linked to representation of different behaviorally relevant sounds . Second, the ontogeny of both echolocation behavior (brown et al ., 1978) and functional organization of auditory cortex (razak and fuzessery, 2007) has been studied . The overall functional organization of auditory cortex, including frequency representation, is adult - like early in development (razak and fuzessery, 2002, 2007). An exception is the prevalence and anatomical distribution of functionally bimodal neurons (razak et al ., 1999). These neurons appear to receive input from both pathways, and have two discrete tuning curves tuned to frequencies used in echolocation and passive sound localization . In adults, these bimodal neurons are found near the interface of the lfr and hfr . In pups, however, they are more widely distributed (razak and fuzessery, 2007), suggesting that projections from the mgbv and sg may show greater overlap in their cortical targets . We tested this hypothesis by retrograde tracing of mgb inputs from physiologically identified injection sites in the lfr and hfr of auditory cortex . Details about injection method, sizes, and definition of divisions of the mgb have been addressed in detail in the original papers (razak and fuzessery, 2007, 2009). Although it was relatively easy to distinguish the sg from rest of the mgbd and mgbv, the boundary between mgbd and mgbv was less obvious based on nissl or neutral red stains . However, luxol fast blue, neutral red stained sections show that mgbv stains weakly for myelin, whereas the mgbd contains fibers that run in a dorsomedial to ventrolateral direction . The boundary between the dorsal and the ventral divisions was drawn based on myelin staining . A comparison of adult and pup brain photomicrographs shows similar injection sizes for fluroruby and flurogold (figures 2 and 3). (a) fg was injected in the lfr near sites with tuning 15 khz . (c f) photomicrographs of increasingly rostral sections through the mgb demonstrate that both the sg and mgbv show labeled cells . (g, h) magnified view of the sg and (i, j) mgbv show strong label in both areas . It can also be noted that caudal sg sends more projections to the lfr in pups than rostral sg . (m p) schematic and (q s) photomicrographs of the mgb show that labeled cells were present in the sg and mgbd, but not the mgbv . (v y) fg and fr labeled cells were seen in the sg, while only fg labeled cells were seen in the mgbv . (z) magnified view of the sg shows fg and fr labeled cells, although no double labeled cells were seen . F; 200 m in g j . There is greater overlap in thalamocortical connections during early development (razak et al ., 2009). Injections of retrograde tracers in the lfr labeled neurons in both the mgbv and the sg (figure 3, schematic in figure 1c). The input from the sg to lfr was prominent from the caudal half of the mgb, and was at least as strong as the input from the mgbv to the lfr based on the number of labeled neurons . However, as in adults, there was no input from the pup mgbv to the hfr . An overlap of pathways was seen in p60 bats, but not in p150 bats, indicating that the refinement is occurring during this time period . Pallid bats begin to fly and use echolocation in flight around 56 weeks (brown et al ., 1978) and thus the refinement occurs a few weeks after flight onset, and more than 6 weeks after onset of adult - like hearing . These data show that the parallel thalamocortical pathways seen in adults emerge during postnatal development and may involve refinement of overlapping connections well after the bat begins to experience patterned input . We have suggested that the parallel pathways for noise and fm sweep processing observed in the ic, mgb, and cortex are an adaptation for gleaning behavior in the pallid bat (barber et al ., 2003). As the bat hunts, it receives both echoes from the flight path and prey - generated noise from the ground . The noise and fm sweeps used in prey localization and echolocation differ in spectral (low versus high frequency), temporal (noise versus sweep), and spatial (ground versus flight path) qualities . These differences should enable the bat to segregate the two sounds (bregman, 1990). In addition, the parallel processing with strong filters (noise versus fm sweep selectivity) will provide a substrate for additional segregation of the two auditory streams . During development, the onset of adult - like hearing thresholds may not be the critical experience for initiation of segregated pathways . The experience with dual processing of echoes and terrestrial prey - generated noise may be more important in initiating segregated pathways; therefore refinement occurs when the bat begins to hunt . Such experience - dependent plasticity has been described previously in the barn owl midbrain (bergan et al ., 2005). The sg appears to be a phylogenetically and ontogentically malleable region of the thalamus . In non - chiropterans the sg is dominated by visual input from the superior colliculus (cat: calford and aitkin, 1983; katoh and benedek, 1995; rat: tanaka et al ., 1985). Less than 15% of the neurons respond only to auditory stimuli, whereas 65% of the sg neurons respond to visual stimuli alone (benedek et al ., 1997). The entire rostrocaudal extent of the sg receives input from the superior colliculus (sc) (katoh and benedek, 1995). Auditory inputs to the sg may arise from the nucleus of the central acoustic tract in the brainstem and the external nucleus of the ic (henkel, 1983; katoh and benedek, 1995), both of which are considered to be a part of the extralemniscal auditory pathways . The sg projects primarily to non - primary auditory cortical areas, although sparse connections to the primary auditory cortex are found in many species (owl monkey: morel and kaas, 1992; macaque monkey: hackett et al . 1989; dog: malinowska and kosmal, 2003; rat: roger and arnault, 1989). In bats, the sg of the mustached bat, unlike non - chiropterans, receives inputs from all frequency bands of the central nucleus of the ic (wenstrup et al ., 1994). Like non - chiropterans, the sg also receives direct input from the nucleus of the central acoustic tract (casseday et al ., 1989 in addition to broad regions of the auditory cortex, the sg of the mustached bat projects to frontal cortex, which in turn projects to the sc, suggesting a role in acoustic - motor reflexes (kobler et al ., 1987). In the horseshoe bat, the sg projects to both primary and non - primary cortical fields (radtke - schuller et al ., 2004). There is overlap in projections from the sg and the mgbv to any given cortical region, but the dorsal cortical fields (non - primary) receive most sg inputs . In the pallid bat, the echolocation pathway is primarily routed through the sg . Taken together, these studies suggest that the sg can be taken over to increase thalamic representation of species - specific dominant sensory modalities . The developmental plasticity of sg connections is illustrated by cross - modal plasticity in ferrets (pallas et al ., however, an anomalous input to the primary auditory cortex arises from the dorsal thalamus, including the sg, in the cross - modal animals . This finding suggests that the sg typically receives and sends more exuberant connections during early development the data from the pallid bat thalamocortical development support the hypothesis that the sg sends exuberant projections during early development . It is possible to modify echolocation experience during development (razak et al ., 2008). Future studies will address how refinement of pathways proceeds in bats with altered experience with echolocation calls . Although several studies have focused on development of auditory pathways below the thalamus, the pallid bat is the only animal we are aware of in which postnatal development of auditory thalamocortical connections has been examined . Therefore it remains unclear if the postnatal refinement of parallel thalamocortical pathways is a general phenomenon . In animals, such as the cat, in which the adult auditory pathways have been well characterized, a key hypothesis to emerge based on physiological and anatomical studies of the pallid bat auditory system is that in gleaning bats, parallel pathways from the ic this hypothesis is supported by physiological studies in the ic of megaderma lyra, another gleaning bat from a different family (rubsamen et al ., 1988). Gleaning as a hunting strategy appears to have evolved independently in different families of bats . If parallel pathways are an adaptation for gleaning in the pallid bat, then a similar organization of the auditory system must be present in other gleaning bats . One unusual aspect of the pallid bat thalamocortical connections is that the cortical tonotopic map is composed of non - overlapping inputs from two different divisions of the mgb (razak et al ., 2007). The presence of such an organization in other gleaners will strongly suggest a modified thalamocortical plan in gleaners compared to other mammals, including obligate echolocating bats . Recent studies that compared foxp2 expression between echolocating and non - echolocating bats identified the sg as one of the nuclei in which expression was higher in the echolocators (berquist et al ., sfn abstracts, 2009). Expression of foxp2 in the mgb also changes in an activity - dependent manner (horng et al ., 2009). Foxp2 may therefore be a part of the molecular milieu of the sg that makes it developmentally and evolutionarily more labile . Future studies comparing the expression of foxp2 across various divisions of the mgb will be useful in determining the mechanisms of sg malleability and to address mechanisms underlying connectional and therefore functional plasticity in the thalamocortical connections . The authors declare that research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflict of interest.
Atrial fibrillation (af) is a common arrhythmia, and its prevalence is increasing as the population ages and more individuals survive with cardiovascular disease . Af is the major cause of ischemic stroke and is moderately associated with increased mortality from stroke, heart failure, and cardiovascular disease . The use of anticoagulants is limited, however, due to poor patient compliance and increased risks for bleeding complications . Considering these factors contributing to an insufficient success rate for stroke prevention, other treatment strategies are urgently needed to reduce the clinical and economic burden of af . Rhythm control strategies have been proposed as a potential means to decrease stroke risk in af patients . Anti - arrhythmic drug treatment is the standard approach to patients with af in the clinic, but adverse events commonly contribute to poor adherence . Mostly, the efficacy of antiarrhythmic drug therapy (adt) is limited for there are high rates of af recurrence even when adt is used ., catheter ablation (ca) is currently more successful in maintaining sinus rhythm than antiarrhythmic drugs and has been proven superior to adt in selected patients . Whether ca reduces af related stroke and death rates, however, has not been systematically reviewed . In the current study, we performed a meta - analysis of randomized controlled trials to evaluate stroke and death rates caused by af after ablation compared to drug therapy . We conducted a systematic literature search of pubmed and the cochrane central register of controlled trials covering the period from january 1990 to december 2014 by using the following search terms: randomized, ablation, atrial fibrillation, and studies were included in this meta - analysis if they met the following criteria: (1) had a random study design; (2) the intervention group included patients ongoing catheter ablation compared with patients received antiarrhythmic drug therapy; and (3) the endpoint was non - procedure related thromboembolic stroke / transient ischemic attack . As catheter ablation is an invasive procedure with attendant potential risks, including procedural stroke, we recorded the dates of the all - cause and procedure - related events . We excluded trials if catheter ablation was used in both treatment groups and if surgery for af was used . The study was performed according to the quorom statement for high - quality meta - analyses . Information was recorded as follows: last name of the first author, year of publication, length of follow - up, mean age of study participants, sample size of each treatment, and number of end points in the intervention and control arms . In addition, left atrium size, left ventricular ejection fraction (lvef), coronary artery disease (cad), hypertension, chads2 score, anticoagulation time, and number of patient crossover to the ca arm in the adt arm was also recorded . We reviewed article titles and abstracts from the initial search and excluded those that did not meet the inclusion criteria . If the same population was studied in more than one study, we included the study with the longest follow - up time . The primary end point was major thromboembolic events that were not considered to be related to the treatment (including stroke / transient ischemic attacks), and the secondary end point was all - cause mortality caused by af itself . The measure of treatment effect for the end point was reported by risk difference with 95% ci . Fixed - effect models were used to calculate the combined risk difference and in sensitivity analyses . Potential publication bias was assessed using the begg's funnel plot and egger's regression test . Meta - regression models on stroke risk between the two groups using mean age in each study and a sensitivity analysis comparing risk of stroke by per protocol was performed to assess whether these factors would have affected the results . The effect of different follow - up periods among different trials on the heterogeneity of the pooled results was assessed by repeating the analysis using the number of strokes per 1000 patient - year . Power analyses of individual studies and meta - analyses were all conducted by the software power and sample size calculation . All reported p values were two - sided, and p <0.05 was determined as statistically significant . We conducted a systematic literature search of pubmed and the cochrane central register of controlled trials covering the period from january 1990 to december 2014 by using the following search terms: randomized, ablation, atrial fibrillation, and studies were included in this meta - analysis if they met the following criteria: (1) had a random study design; (2) the intervention group included patients ongoing catheter ablation compared with patients received antiarrhythmic drug therapy; and (3) the endpoint was non - procedure related thromboembolic stroke / transient ischemic attack . As catheter ablation is an invasive procedure with attendant potential risks, including procedural stroke, we recorded the dates of the all - cause and procedure - related events . We excluded trials if catheter ablation was used in both treatment groups and if surgery for af was used . The study was performed according to the quorom statement for high - quality meta - analyses . Information was recorded as follows: last name of the first author, year of publication, length of follow - up, mean age of study participants, sample size of each treatment, and number of end points in the intervention and control arms . In addition, left atrium size, left ventricular ejection fraction (lvef), coronary artery disease (cad), hypertension, chads2 score, anticoagulation time, and number of patient crossover to the ca arm in the adt arm was also recorded . We reviewed article titles and abstracts from the initial search and excluded those that did not meet the inclusion criteria . If the same population was studied in more than one study, we included the study with the longest follow - up time . The primary end point was major thromboembolic events that were not considered to be related to the treatment (including stroke / transient ischemic attacks), and the secondary end point was all - cause mortality caused by af itself . The measure of treatment effect for the end point was reported by risk difference with 95% ci . Fixed - effect models were used to calculate the combined risk difference and in sensitivity analyses . Potential publication bias was assessed using the begg's funnel plot and egger's regression test . Meta - regression models on stroke risk between the two groups using mean age in each study and a sensitivity analysis comparing risk of stroke by per protocol was performed to assess whether these factors would have affected the results . The effect of different follow - up periods among different trials on the heterogeneity of the pooled results was assessed by repeating the analysis using the number of strokes per 1000 patient - year . Power analyses of individual studies and meta - analyses were all conducted by the software power and sample size calculation . All reported p values were two - sided, and p <0.05 was determined as statistically significant . Thirteen randomized trials with 1952 patients were included in this review. The 13 trials were published between 2003 and 2014 . Two trials were conducted at one center, whereas the rest of the trials were multicenter . One trial included patients with chronic af, while four trials included patients with paroxysmal and persistent af., all of these trials compared pulmonary vein isolation with antiarrhythmic drug therapy . Table 1 & 2 provides the baseline characteristics of the included studies . In most trials, the patients were followed for 12 months . There were no differences seen in patient characteristics . The mean age of enrolled patients was 57 9 years for ca patients and 57 11 years for adt patients . The mean left atrial diameter among patients in ca group was 41.6 5.7 mm, and in adt group was 41.9 5.9 mm . Many patients had a history of hypertension, although few had significant structural heart disease . The average value of chads2 score, mentioned in four studies, were comparable (0.6 0.8 for ca patients and 0.7 0.8 for adt patients). As the data show, mean chads2 score, lvef, left atrium size, and the percentages of coronary artery disease, diabetes were comparable between the two groups . Previous embolic events included transient ischemic events, stroke, pulmonary embolism, deep vein thrombosis and other peripheral embolism . In the antiarrhythmic therapy arm, drugs included mainly class i and class iii antiarrhythmic agents, either single or combination use, with the antiarrhythmic drug restricted to amiodarone in two of the trials ., in the catheter ablation group, no one had received antiarrhythmic drug therapy before enrollment in three trials .,, in the remaining trials, ca was used in patients after at least one antiarrhythmic drug regimen had failed . The ablation technique was pulmonary vein ablation combined with ablation of linear lesions in the left and right atria, ostia of the pulmonary veins, and cavotricuspid isthmus . The use of additional lesion ablations outside the pulmonary vein region was left to the discretion of the operator . Oral anticoagulation (international normalized ratio between 2 and 3) was required for at least three weeks before ablation in each trial, with a period ranging from 1 to 12 months after ca . In two studies, the period of anticoagulation use after ca was not noted . Adt: antiarrhythmic drug therapy; ca: catheter ablation; nr: not reported; tia: transient ischemic attack . The chads2 score is a measure of stroke risk in patients with atrial fibrillation, with scores ranging from 0 to 6 . Congestive heart failure, hypertension, an age of 75 years or older, and diabetes mellitus were each assigned 1 point, and previous stroke or tia was assigned 2 points . Adt: antiarrhythmic drug therapy; at: minimum anticoagulation time after ablation or antiarrhythmic drug therapy; ca: catheter ablation; cad: coronary artery disease; lad: left atrial diameter; lvef: left ventricular ejection fraction; htn: hypertension; nr: not reported; pee: previous embolic events; shd: structural heart disease; tia: transient ischemic attack . Before the study finished, 445 of a total 855 drug - treated patients crossed over to a ca procedure after failure or intolerance to adt . A total of 23 in the ca arm and 26 in the adt arm had previous embolic events . Ten patients were lost follow - up: four in the ablation arm and six in the aad arm . A total of seven patients in the group that underwent ca (0.64%) and two patients in the adt group (0.23%) had stroke or transient ischemic attack in the 13 trials . There was no difference in the rate of stroke or transient ischemic attack between the af ablation and adt therapy groups (figure 2). Little evidence of heterogeneity was observed (i = 0, p = 0.981), indicating the studies were very well - matched . The probability of potential publication bias existing among the 13 studies was estimated by begg's funnel plots (figure 3) and egger's regression test (p = 0.981 with 95%ci: 1.030 to 1.054). Meta - regression models showed there was no statistical evidence for heterogeneity due to mean age (p = 0.95). We performed a sensitivity analysis comparing risk of stroke by per protocol, no significant difference was shown between the two groups [risk differences (rd): 0.003, 95%ci: 0.006 to 0.012, p = 0.475]. A sensitivity analysis according to the follow - up years in the trials (trial follow up> 24 months vs. <24 months), also did not reveal any influence of follow - up years on trial results (table 3). Similarly, no change was found in the effects of ca on stroke compared with adt (4.6 vs. 1.5 per 1000 patient - years in stroke rates; rd: 0.002, 95% ci: 0.004 to 0.008) (figure 4). The power of the meta - analysis with respect to stroke was 25.1%, using the risk of the medical therapy arm reported in this study . If there is a 50% reduction risk, 20,443 ca subjects with one control per case would be needed . A total of 13 deaths were reported (five in the ablation arm and eight in the anti - arrhythmic drug arm). There was no difference in death rates between the ablation and anti - arrhythmic therapy groups (figure 5). The risk difference of deaths in all trials between patients randomized to ablation and those randomized to adt was 0.004 (95%ci: 0.014 to 0.006, p = 0.472), with no evidence for heterogeneity (i = 0, p = 0.953). Heterogeneity chi - squared = 4.15 (d.f . = 12), p = 0.981; i - squared (variation in rd attributable to heterogeneity) = 0.0; test of rd = 0, z = 0.72, p = 0.470 . Adt: antiarrhythmic drug therapy; ca: catheter ablation; rd: risk differences . The risk difference for each study was plotted against se of rd . Thirteen randomized trials with 1952 patients were included in this review. The 13 trials were published between 2003 and 2014 . Two trials were conducted at one center, whereas the rest of the trials were multicenter . One trial included patients with chronic af, while four trials included patients with paroxysmal and persistent af., all of these trials compared pulmonary vein isolation with antiarrhythmic drug therapy . Table 1 & 2 provides the baseline characteristics of the included studies . In most trials, the patients were followed for 12 months . There were no differences seen in patient characteristics . The mean age of enrolled patients was 57 9 years for ca patients and 57 11 years for adt patients . The mean left atrial diameter among patients in ca group was 41.6 5.7 mm, and in adt group was 41.9 5.9 mm . Many patients had a history of hypertension, although few had significant structural heart disease . The average value of chads2 score, mentioned in four studies, were comparable (0.6 0.8 for ca patients and 0.7 0.8 for adt patients). As the data show, mean chads2 score, lvef, left atrium size, and the percentages of coronary artery disease, diabetes were comparable between the two groups . Previous embolic events included transient ischemic events, stroke, pulmonary embolism, deep vein thrombosis and other peripheral embolism . In the antiarrhythmic therapy arm, drugs included mainly class i and class iii antiarrhythmic agents, either single or combination use, with the antiarrhythmic drug restricted to amiodarone in two of the trials ., in the catheter ablation group, no one had received antiarrhythmic drug therapy before enrollment in three trials .,, in the remaining trials, ca was used in patients after at least one antiarrhythmic drug regimen had failed . The ablation technique was pulmonary vein ablation combined with ablation of linear lesions in the left and right atria, ostia of the pulmonary veins, and cavotricuspid isthmus . The use of additional lesion ablations outside the pulmonary vein region was left to the discretion of the operator . Oral anticoagulation (international normalized ratio between 2 and 3) was required for at least three weeks before ablation in each trial, with a period ranging from 1 to 12 months after ca . In two studies, the period of anticoagulation adt: antiarrhythmic drug therapy; ca: catheter ablation; nr: not reported; tia: transient ischemic attack . The chads2 score is a measure of stroke risk in patients with atrial fibrillation, with scores ranging from 0 to 6 . Congestive heart failure, hypertension, an age of 75 years or older, and diabetes mellitus were each assigned 1 point, and previous stroke or tia was assigned 2 points . Adt: antiarrhythmic drug therapy; at: minimum anticoagulation time after ablation or antiarrhythmic drug therapy; ca: catheter ablation; cad: coronary artery disease; lad: left atrial diameter; lvef: left ventricular ejection fraction; htn: hypertension; nr: not reported; pee: previous embolic events; shd: structural heart disease; tia: transient ischemic attack . Before the study finished, 445 of a total 855 drug - treated patients crossed over to a ca procedure after failure or intolerance to adt . A total of 23 in the ca arm and 26 in the adt arm had previous embolic events . Ten patients were lost follow - up: four in the ablation arm and six in the aad arm . A total of seven patients in the group that underwent ca (0.64%) and two patients in the adt group (0.23%) had stroke or transient ischemic attack in the 13 trials . There was no difference in the rate of stroke or transient ischemic attack between the af ablation and adt therapy groups (figure 2). Little evidence of heterogeneity was observed (i = 0, p = 0.981), indicating the studies were very well - matched . The probability of potential publication bias existing among the 13 studies was estimated by begg's funnel plots (figure 3) and egger's regression test (p = 0.981 with 95%ci: 1.030 to 1.054). Meta - regression models showed there was no statistical evidence for heterogeneity due to mean age (p = 0.95). We performed a sensitivity analysis comparing risk of stroke by per protocol, no significant difference was shown between the two groups [risk differences (rd): 0.003, 95%ci: 0.006 to 0.012, p = 0.475]. A sensitivity analysis according to the follow - up years in the trials (trial follow up> 24 months vs. <24 months), also did not reveal any influence of follow - up years on trial results (table 3). Similarly, no change was found in the effects of ca on stroke compared with adt (4.6 vs. 1.5 per 1000 patient - years in stroke rates; rd: 0.002, 95% ci: 0.004 to 0.008) (figure 4). The power of the meta - analysis with respect to stroke was 25.1%, using the risk of the medical therapy arm reported in this study . If there is a 50% reduction risk, 20,443 ca subjects with one control per case would be needed . A total of 13 deaths were reported (five in the ablation arm and eight in the anti - arrhythmic drug arm). There was no difference in death rates between the ablation and anti - arrhythmic therapy groups (figure 5). The risk difference of deaths in all trials between patients randomized to ablation and those randomized to adt was 0.004 (95%ci: 0.014 to 0.006, p = 0.472), with no evidence for heterogeneity (i = 0, p = 0.953). = 12), p = 0.981; i - squared (variation in rd attributable to heterogeneity) = 0.0; test of rd = 0, z = 0.72, p = 0.470 . Adt: antiarrhythmic drug therapy; ca: catheter ablation; rd: risk differences . The risk difference for each study was plotted against se of rd . The goal of this study was to conduct a meta - analysis to determine whether af ablation reduces the long - term risk of stroke compared to anti - arrhythmic drug therapy in randomized controlled trials . To our knowledge, this is to date the first meta - analysis to compare ca to adt for the risk of stroke and mortality induced by af . Several observational research studies have suggested that af ablation strategy may associate with lower stroke and death rates compared to drug therapy. It also has been shown that the patients had long - term stroke rates similar to patients without atrial fibrillation in several studies ., one previous meta - analysis of eight randomized trials reported no significant difference between the two arms for death or stroke, although, all these studies recorded all - cause events . Clearly, there are risks of stroke associated with ablation or drugs, the dilemma whether ca could reduce stroke rate of af was still unsolved . Adt: antiarrhythmic drug therapy; ca: catheter ablation; rd: risk differences . = 12), p = 0.953; i - squared (variation in rd attributable to heterogeneity) = 0.0; test of rd = 0, z = 0.72, p = 0.472 . Adt: antiarrhythmic drug therapy; ca: catheter ablation; rd: risk differences . The potential risk of stroke in patients with af in the two groups with like age, sex, hypertension, vascular disease, and prior stroke or tia are matched well, as shown on following tables . Stroke is an uncommon complication of ablation or drugs, while still having great impact on our rare end events . To find out the direct effect of ablation on stroke risk of af, we recorded events directly induced by af itself, and excluded events that were due to the procedure or drugs used . The major finding was that the two groups had similar rates of stroke and death, indicating homogeneous responses . Our results provide insight into rhythm control treatment strategies, including ablative interventions that may not reduce the stroke rate on the basis that catheter ablation was more effective than drug therapy in maintaining sinus rhythm . In this meta - analysis, these findings indicate that there is not enough evidence on the relative effects between catheter ablation and anti - arrhythmic drugs . Because stroke and death were rare study events, the analysis included studies which differed in the types of af, different definition of procedure - related or device - related adverse events, previous embolism events, definition for procedure - related events, the use of adt in the ca arm, and the time to anticoagulation before and after ablation . The patients included in the trials were mostly younger and may not reflect that typical age seen in patients with af . Of note, there was minimal evidence of structural heart disease, the left atrial diameter was <50 mm, a mean chad2s scores <1, and there was well - preserved systolic function with normal ejection fraction (lvef> 50%) in this cohort . Thus, our results may not apply to other populations of patients, including very elderly patients or patients with underlying structural heart disease or more severe heart disease . These strengths include the absence of heterogeneity between the groups and the low probability of publication bias . Despite these strengths, there are a few limitations that should be considered . Most importantly, 445 of the total 855 drug - treated patients (52%) crossed over to a ca procedure after failure or intolerance to adt before the end of the follow - up, and the significant crossover rate in the adt arm may contribute to the results that both groups had similar stroke rates . Second, the total sample size of this meta - analysis may have been insufficient to detect significant differences between groups . More definitive evidence on the effect of ablation on af on the clinical end - points of stroke and mortality will be provided by several on - going multi - center rcts ., finally, there was a relatively short follow - up period of 12 months or less with few outcome data beyond one year . The follow - up period is a possible contributor to the lack of statistical difference observed . Indeed, in one study, the stroke / tia regression models showed statistically significant different rates of events between cohorts that emerged after the first year of follow - up . A more prolonged follow - up period in a randomized trial may reveal differences not observed in the current study . In conclusion, our analysis revealed similar rates of stroke or transient ischemic attack in patients treated by catheter ablation or anti - arrhythmic drugs for atrial fibrillation . While catheter ablation did not reduce the rates of stroke or death compared to drug therapy, a large - scale clinical research trial to confirm these findings is warranted . These strengths include the absence of heterogeneity between the groups and the low probability of publication bias . Despite these strengths, there are a few limitations that should be considered . Most importantly, 445 of the total 855 drug - treated patients (52%) crossed over to a ca procedure after failure or intolerance to adt before the end of the follow - up, and the significant crossover rate in the adt arm may contribute to the results that both groups had similar stroke rates . Second, the total sample size of this meta - analysis may have been insufficient to detect significant differences between groups . More definitive evidence on the effect of ablation on af on the clinical end - points of stroke and mortality will be provided by several on - going multi - center rcts ., finally, there was a relatively short follow - up period of 12 months or less with few outcome data beyond one year . The follow - up period is a possible contributor to the lack of statistical difference observed . Indeed, in one study, the stroke / tia regression models showed statistically significant different rates of events between cohorts that emerged after the first year of follow - up . A more prolonged follow - up period in a randomized trial in conclusion, our analysis revealed similar rates of stroke or transient ischemic attack in patients treated by catheter ablation or anti - arrhythmic drugs for atrial fibrillation . While catheter ablation did not reduce the rates of stroke or death compared to drug therapy, a large - scale clinical research trial to confirm these findings is warranted.
Complex regional pain syndrome (crps) type ii, known as causalgia, develops after a nerve injury, and tends to show the more painful and more severe symptoms of crps . The ordinary strategy in crps treatment is often multi - disciplinary, with the use of various kinds of medications and physical therapies combined with sympathetic nerve block . Even with several treatments, it is very difficult to manage the acute stage of crps type ii . And neurostimulation (spinal cord stimulator) could be surgically implanted to control the pain by directly stimulating the spinal cord for the treatment of intractable crps . A systematic review concluded that spinal cord stimulation appears to be an effective therapy in the management of patients with crps type i (level a evidence) than type ii (level d evidence)14). Moreover, there is evidence to reveal that scs is a cost - effective treatment for crps type i. we report a case of acute stage crps type ii treated with new anti - inflammatory agent [polydeoxyribonucleotide (pdrn) solution]. As far as we know, this is 1st case report showing effectiveness of pdrn for the treatment of crps type ii . A 32-year - old female patient came to the hospital complaining of left leg numbness . She had experienced a traumatic event, a fall into a waterway, one month ago . After which, she had severe low back pain and was evacuated to the nearby general hospital . Computed tomography (ct) showed an l5 left transverse process fracture and an s2 body fracture, and absolute bed rest (abr) for one month was recommended (fig . Her left leg numbness was progressively aggravated . To evaluate whether symptom aggravation during the abr period was related to the patient's history of a left - side l5s1 discectomy 2 years previously the mri did not show recurrent disc material, but it did show an annular tear at l45 and postoperative scar tissue at l5s1, and the previous doctor recommended continued conservative management . After the period of abr was complete, the patient began ambulation and was transferred to our hospital in her hometown . She complained of left leg numbness initially upon arrival, and the more she walked, the more the pain was aggravated, showing hyperalgesia and allodynia . Even as the symptoms seemed to subside somewhat, skin flushing still appeared on the left lower leg when the patient was in a standing position (fig . 2). The persistent skin discoloration seemed to worsen as time went on, despite frequent sympathetic nerve blocks using steroid . To rule out the possibility of arterial injury, we performed lower extremity ct angiography, and there was no sign of arterial injury around the fracture site . We concluded that her symptoms resulted from lumbosacral plexus injury due to the transverse process fracture . At that time, it was 2 months after the initial trauma . Because this was the acute phase, we wanted to prevent the disease from progressing to the chronic phase . Our clinics have been performing prolotherapy using pdrn solution, and we are familiar with the effects of pdrn . We injected pdrn solution at the ventral surface of the left l5 transverse process, superior and inferior, one ampoule (3 cc) each by using needles used for medial branch block (fig . Allodynia and hyperalgesia were improved one day after the procedure and the skin flushing was diminished (fig . The patient was discharged 3 days after the pdrn injection and at the 1-month follow - up, her symptoms were very much improved . Autonomic disturbance can lead to significant pain in patients with peripheral nerve injuries and is characteristic of crps . Severe burning pain, allodynia, hyperalgesia, careful attempts to protect the involved extremity from movement or contact, and evidence of autonomic hyper- or hypoactivity are features of crps . Vascular changes such as skin flushing and discoloration and temperature fluctuations are hallmarks of this deranged autonomic activity . Crps type i usually occurs after a minor injury to the extremity (e.g., ankle sprain), whereas crps type ii occurs after significant injury to a major mixed nerve (e.g., brachial plexus injury), such as in our case . Crps may change within a patient over time, particularly in the transition from " warm crps " (acute) to " cold crps " (chronic)5). Several pathophysiological concepts have been proposed to explain the complex symptoms of crps: 1) facilitated neurogenic inflammation; 2) pathological sympatho - afferent coupling; and 3) neuroplastic changes within the cns11). Management of crps remains challenging, with many patients having only partial or minimal relief with treatment . Pharmacotherapy is based on individual symptoms and includes steroids, free radical scavengers, antineuralgic agents, and finally agents interfering with bone metabolism (calcitonin, bisphosphonates). Nonsteroidal anti - inflammatory drugs (nsaids), corticosteroids, cyclooxygenase (cox)-2 inhibitors, and free radical scavengers are used in crps with intent to treat for pain as well as for inflammation . However, crps - related inflammation may be largely neurogenic, initiated by inflammatory mediators from the terminals of afferent nociceptors, and no drugs have been studied for this type of inflammation8). Sympathetic nerve block is traditionally recognized as an important procedure, both in the diagnosis and treatment of crps . The pain relief following sympathetic nerve block generally outlasts the effect of the local anesthetic13). If after numerous therapies, a patient's pain is intractable, sympathectomy or spinal cord stimulation (scs) may be recommended as surgical treatment alternatives . Recently, scs seems to be the general trend, and it is reported that scs is effective in reducing the chronic neuropathic pain of crps type i914). Specific treatment of crps type ii at the chronic stage is also reported, but to our knowledge, no specific interventional or invasive treatment for control of acute stage crps type ii has been reported6). Pdrn (placentex) is a low molecular weight dna complex extracted from trout sperm . The adenosine a2a receptor alters the cytokine system by decreasing secretion of inflammatory cytokines such as tumor necrosis factor-, macrophage inflammatory protein 1, and interleukin (il)-6 . The adenosine a2a receptor is also associated with increased circulation of il-10, anti - inflammatory cytokine4). Therefore, pdrn has an anti - inflammatory effect and could be a potential pharmacological treatment for inflammatory diseases such as rheumatoid arthritis315). This also explains how pdrn can help to control the acute phase of crps, such as in our case . However, this doesn't seem to be enough to explain the dramatic result of our case . The adenosine a2a receptor also has a tissue regeneration effect, as indicated by its stimulation of endothelial cell migration and proliferation . Activation of this receptor is also associated with increased levels of vascular endothelial growth factor, which enhances tissue function712). Promoting cell proliferation by acting as a mitogen for fibroblasts and, endothelial cells could result in extracellular matrix production and remodeling, and can be useful for burn treatment and prolotherapy210). We suppose that this regeneration mechanism has an additional effect on our case . Because we have been performing prolotherapy in our clinics, we have observed these mechanisms of pdrn, not only the regeneration effect, but also the anti - inflammatory effect . Pdrn has been found to be a safe modality, and no harmful effect has been reported1). Patients with acute stage crps type ii need appropriate treatment to prevent progression to the chronic phase . Treatment is based on a multidisciplinary approach, and pdrn could be an option for the treatment of the acute inflammatory phase of crps type ii.
Oral health is fundamental to general health and well being, significantly impacting on quality of life . Bad oral health may have a negative impact on appearance, ability to eat, communication with people, on daily tasks at work, home or school . Children with poor oral health suffer from dental pain more often, miss school and perform poorly in school . Regular tooth brushing with fluoride toothpaste can reduce dental conditions of tooth decay and periodontal disease . A survey, performed in lithuania among 373 adolescents showed that majority of them (73.7%) brush teeth two times a day or more often . The results of international survey, among 11, 13 and 15 year - old schoolchildren, regarding how often they brush their teeth, showed that only about 40% of boys (42% - 11, 40% - 13, 40% - 15 year - olds) and more than a half of the girls (52% - 11, 62% - 13, 65% - 15 year - olds) in lithuania brush their teeth more often than once a day . The study among 586 schoolchildren aged 7, 12 and 15 years in kaunas (lithuania) showed that 64.5% of participants, brushing their teeth daily, had a good oral hygiene and 19% - fair . Children s knowledge and skills of oral hygiene mostly depends on habits and approach of their parents and their attitude regarding oral hygiene of their children . Children living in child care homes do not get appropriate information about health care and oral hygiene from their parents . It becomes a responsibility of supervisors to teach children by providing them necessary knowledge and encouraging kids to take a better care of their oral hygiene . Studies has shown that orphans have fair oral hygiene because of limited education and access to services [10 - 12]. Knowledge about proper oral hygiene is better perceived through motivation, practical skills development and involvement of parents . Studies in different countries showed significant improvement, progress in oral health results after practical and motivational interventions . We hypothesized that child care home children s tooth brushing habits and oral hygiene quality can be influenced by motivation and simple individual instructions . The aim of this study was to evaluate and compare two different oral health promotion methods as well as outcome of both methods on a quality of oral hygiene . Study design and sampling the present study was conducted from september 15, 2013 to december 15, 2013 . The study was designed as a 3 month intervention study involving children and adolescents from two child care homes in kaunas, lithuania . The data included the children s clinical plaque accumulation examinations at baseline and at study end, as well as a self - administered questionnaire for the participants at baseline and at study end . Two child care homes were selected out of five in the city as the remaining three lacked participants . The number of children provided by the two child care homes was almost equal and both institutions were willing to participate . In total, 68 participants (32 of practical application group and 36 of motivation group) were enrolled into this study (figure 1). Age, gender of the participants in the motivation and practical application groups is presented in table 1 . Distribution of participants according to age, gender and intervention group type p value considered significant when p> 0.05 by chi - square test (). The protocol was approved by the ethics committee of lithuanian university of health sciences, kaunas, lithuania . Participation in the study was voluntary and the participants were informed about study details before . For both groups participants, a similar clinical plaque accumulation examination was conducted at baseline and at the study end, using a dental mirror, a sterile probe, and a portable dental equipment lamp in the health office at the care home before lunch . Four gingival areas (distal, facial, mesial and lingual) were recorded for each tooth to indicate the child s oral hygiene status . Dental plaque was recorded as 1) no plaque, plaque on gingival margins and adjacent area of the tooth; 2) clearly visible plaque on the tooth and gingival margin; 3) soft thick layer of plaque on the tooth and gingival margin with scores correspondingly 0, 1, 2, 3 . Scores for each tooth were added and divided by the number of the teeth examined . The final result was obtained by adding the total surfaces with plaque and dividing this by total number of teeth examined . The baseline clinical examination was conducted by one of the authors (g. m.). For the outcome examination, another dentist, one not involved in the present study procedures and blind to group assignment was involved . Each was examined by one of the examiners and then re - examined by the other examiner within 1 hour . This procedure resulted in an inter - examiner reliability with a kappa value of 0.76, representing an excellent agreement between examiners . Children were asked to complete a self - administered questionnaire at the baseline and at the study end . Questionnaire inquired about oral hygiene habits, self - perception, dental attendance, dietary habits . At baseline, 19 item questionnaire was used to assess the oral hygiene knowledge and practices and at the end 21 . Two questions were added in order to know, whether the programme was beneficial and if the respondents got to know something new . Approximately, one week after data collection at baseline, interventions began, and the follow - up examinations occurred 3 month later . The execution of the intervention was monitored by one of the authors (g. m.) through visits to the child care homes and discussions with staff . This intervention was applied in children care home by means of five lectures prepared by one of the authors (g. m.). Lectures covered different topics: 1) tooth structure and development (lecture included tooth brushing demonstration); 2) risk factors of oral diseases, dental caries development; 3) how to manage in the dental clinic, treatment of dental caries, prevention; 4) dental plaque, as a risk factor of gingivitis, 5) diet counselling . Lectures contained: comprehensive well - known oral health information of tooth structure, development, functioning, the aetiology of common oral diseases - dental caries, gingival diseases; preventability of oral diseases and the ways to keep the mouth healthy by recommending twice - daily tooth brushing, use of fluoride toothpaste and restricting sugary snacking . Lectures were read once every three weeks and used simple language and explained scientific issues plainly and clearly to make it easy for children to understand . Practical application group this intervention was applied by means of practical teaching children once a week . One of the authors (g. m.) every week for 3 month period was visiting children in child care home and supervised how they brushing teeth . One lecture (dental caries risk factors, prevention of tooth decay, tooth brushing demonstration) before starting intervention was delivered to children and staff of the children care home . Evaluation of the intervention after 3 month 54 children were examined clinically and scored for pli . The reasons for the 20.6% not attendance was absence of children in the children care home at the day of examination (figure 1). Changes in pli calculated as differences in score of plaque from baseline to the outcome examination . A positive value indicated improvement in plaque accumulation reducing . Statistical data analysis was conducted using the statistical package for social sciences (spss) version 20 for windows . The data were analysed using descriptive statistics and verified statistical hypotheses about average rate differences and signs of mutual interdependence . Parametric data were expressed as mean and standard deviation (m [sd]). . Chi - square test () or fishers exact test was used to compare qualitative variables based on cross tabulation . For comparing averages of quantitative variables between groups, spearman s rho correlation coefficient was used to evaluate oral hygiene status dependence on age . All children had a plaque on at least one tooth in both groups: motivation 1.14 (0.51) and practical application 1.08 (0.4) (p = 0.58). Older children had poorer oral hygiene, however no statistically significant difference between age groups determined (p = 0.13). Comparisons between different intervention and age groups are in table 2 . According to gender significant difference between motivation (63.9% boys, 36.1% girls) and practical application groups (56.3% boys, 43.8% girls) was not found (p = 0.52) (table 1). Distribution of study participants oral hygiene status according to intervention type and age groups comparisons between motivation and practical application groups . Sd = standard deviation gingival health at baseline oral hygiene status between genders was statistically significant (p = 0.007). Girls oral hygiene status 0.86 (0.36) was significantly better than boys 1.28 (0.45) (p <0.001). Significantly better oral hygiene status of the girls was found in both groups: motivation group (girls 0.82 [0.39], boys 1.33 [0.49]) (p = 0.003) (figure 2) and practical application group (girls 0.9 [0.33], boys 1.23 [0.4]) (p = 0.03) (figure 3). Oral hygiene status by gender before and after intervention in motivation group . P <0.05; student's t - test for difference between girls and boys at baseline . Ci = confidence interval . Oral hygiene status by gender before and after intervention in practical application group . P <0.05; student's t - test for difference between girls and boys at baseline . Sixty percent (n = 21) of 7 - 12 year - olds and 39.4% (n = 13) of 13 - 17 year - olds, reported that brush their teeth twice a day (table 3). There were more boys (61%) than girls (33%) (p = 0.03) who brush their teeth less than twice a day . Dental floss was used by 8.8% (n = 3) of 7 - 12 year - olds and 9.1% (n = 3) of 13 - 17 year - olds (p = 0.97). Satisfied with their oral health answered 74.3% (n = 26) of 7 - 12 year - olds, and 57.6% (n = 19) of 13 - 17 year - olds (p = 0.15). Regular tooth brushing is important in having healthy teeth stated 82.9% (n = 29) of 7 - 12 year - olds and 97% (n = 32) of 13 - 17 year - olds (p = 0.06) (table 3). Participants opinion about their oral health before and after intervention significantly more boys, 75.6% (n = 31), were satisfied with their oral health, than girls, 51.9% (n = 14) (p = 0.04). Within the last 12 months: 77.1% (n = 27) children of age 7 - 12 years and 90.9% (n = 30) children of age 13 - 17 years, visited dentist . Most of the orphans visited dentist for check up 45.2% (n = 28) while few of respondents for orthodontic treatment 4.8% (n = 3). The children were asked how often soft drinks and sweets they consumed during a week: 76.4% (n = 52) children reported that they drank soft drinks and 89.6% (n = 60) ate sweets at least once a week . Gingival health after 3 months intervention after 3 months intervention oral hygiene improved significantly in both groups 0.4 (0.35) (p <0.001). Practical application group 0.19 (0.27) showed significantly better oral hygiene than motivation group 0.55 (0.32) (p <0.001). Before the examination, the difference was not significant (p = 0.58) (table 2). Oral hygiene status in different age groups was not statistically significant after intervention (p = 0.19), however younger children group demonstrated better oral hygiene, the same tendency as at baseline (table 2). The same tendency was observed as at baseline (figure 2 - 3). Before the study, the oral hygiene status of boys (1.28 [0.45]) was significantly lower than girls (0.86 [0.36]) (p <0.001). In the motivation group: girls 0.82 (0.39), boys 1.33 (0.49) (p = 0.003); and practical application group: girls 0.90 (0.33), boys 1.23 (0.4) (p = 0.03). At the end of intervention, the difference was significant in motivation group girls 0.33 (0.35), boys 0.7 (0.0) (p = 0.018) and became insignificant in practical application group girls 0.74 (0.3), boys 0.84 (0.37) (p = 0.556) (figure 2 - 3). After the study oral hygiene improvement was higher for boys 0.75 (0.39) than girls 0.56 (0.37), but statistically insignificant (p = 0.09). Questionnaire after 3 months intervention children s tooth brushing habits did not change statistically significant between both training groups (p = 0.91) and between age groups (p = 0.85) after the study . Tooth brushing frequency statistically significantly changed among girls in comparison with the boys (p <0.001). After the study, significantly more girls (91.3%) were satisfied with their oral health than boys (67.7%) (p = 0.04). Before the study significantly more boys (75.6%) were satisfied with their oral health (51.9%) (p = 0.04). After intervention 7 - 12 year - olds were more satisfied with their oral health than 13 - 17 year - olds, but significant difference was not found (p = 0.11) (table 3). After the study, children from practical application group were more satisfied with their oral health than before study, but statistically significant difference were not found (p = 0.16). Comparing results of questionnaire surveys at baseline and after intervention on use of soft drinks, a statistically significant decrease of their use the habits of sweets use did not change significantly (p = 0.34) during the surveys . After 3 months, children were asked what they thought about the programme: 83% they have learned something new and 88.7% found that the program was useful . Results of the present study showed that oral health promotion based on motivation and practical skills development is effective in improving oral hygiene among orphan children . It has been found significant improvement of oral hygiene in both practical application and motivation groups . Most of the children at baseline demonstrated good or fair oral hygiene and after three months - excellent or good (table 2). Oral hygiene of all children at a baseline was 1.11 (0.46) and significantly better among girls than the boys (p <0.001). Our findings are in accordance with the findings of the other study performed in nigeria among orphans . Other study from india conducted by khare et al . The findings of our study showed that younger boys in both groups had better oral hygiene before intervention and after three month . It is well known that the best way to remove dental plaque is a proper regular tooth brushing including hardly reachable surfaces of the teeth . It also very important to apply a proper method since children differ in their abilities of brushing and that is dependent on their experience as well as physical and neurological development [16 - 18]. It has been proven that brushing skills get better with age and that the duration of brushing is significant for oral hygiene of children as well . Results of questionnaire survey conducted in lithuania has showed that 73.7% of teenagers brush their teeth at least twice a day or more often . Half of our study participants (50.7%) reported brushing their teeth twice a day while the rest of children doing this only once a day or less . The explanation of such low number of regular brushers is that children are not living in families . Several studies has found that children s knowledge and skills of oral hygiene mostly depends on their parents attitude regarding oral hygiene of their children . Taking into account statistical results from sweden, it is noticeable that 6 year - old children brush their teeth in a significantly shorter amount of time compared to children from other age groups (6 - 12 year - olds were measured). Brushing their teeth for less than 1 minute indicated 21% of kids . Brushing their teeth at least twice a day or results from another study showed, that only 32.8% of kids brush their teeth at least twice a day . Various studies provided similar results about oral hygiene of boys and girls, who is better or dedicate more time to take care of their dental health . Our survey showed a significant difference in oral hygiene between genders (p = 0.007). Girls oral hygiene status 0.86 (0.36) was significantly better than boys 1.28 (0.45) (p <0.001). It was also noticed that there are more boys (61%) than girls (33%) (p = 0.03) who brush their teeth less than twice a day . Considering the results of the study on dental caries prevalence and treatment needs it is also clearly that girls are more concern about their oral health than boys . So it could be recommended as an educational tool for teenagers with the emphasis on boys education . Based on the findings of the study in lithuania, it should be stated that frequency of tooth brushing does not always mean better oral hygiene . After three month period, the statistically significant increase of good oral hygiene was registered in both groups (p <0.001). Based on our findings seems that both types of education are effective . It is also known that practice without sufficient motivation can show only temporarily positive results . Despite fact that motivation group showed less improvement, probably they can have more permanent effect in the long term in comparison with practical application group . Dcruz and aradhya state that lectures may increase theoretical knowledge and practical skills to children, however lectures together with practical means shows even better results . Combination of both methods: practical and motivational teaching for the benefits to be greater and longer lasting . During a period of 3 months the authors of a study that was conducted in india came to a conclusion that short term research and educational programs, taking a period of 3 months, are effective to measure positive changes in oral hygiene . The 3 month research program performed in iran, showed increased quality of oral hygiene not only in intervention group, but control group as well . Comparing results of the two questionnaires at baseline and at the end significantly less children reported using soft drinks in both groups (p = 0.004). Another survey provides the same results . Before the survey sweet drinks actively brought to schools 22.4% of kids and 13.3% of kids the habits of confectionary use did not change significantly (p = 0.34) during the surveys . Health educational programs are as an effective tool in children motivation to perform oral hygiene were found by the different authors . It order to obtain / gain best possible results and have a long term effect, those should include and stimulate collaboration between school personal, medical staff and parents . Good results the previous programs were reached when oral hygiene was regularly performed at home as well . Teaching and explaining the importance of oral hygiene improves the state of hygiene temporally during the process of program or a short time afterwards . The present program involved not only the children but orphanage personnel as well . We hope that motivated personnel will help to continue children motivation and supervision with long term positive benefits to the program . After three month, statistically significant improvement of oral hygiene was observed, which shows that motivational, as well as practical, programs, stimulating care of oral health, are efficient in complementing oral hygiene, but before and after results of anonymous survey did not provide meaningful results . It is safe to say that kids need more than 3 months to absorb information and change attitude . It is also clear that there was a lack of motivation in practice group for kids to change their mindset . Thereof, we may guess that improvement of oral hygiene in motivation group, despite being less visible, may have longer lasting effects than those of practical application group . Teaching programs should include both practical and motivational teaching to have the best possible outcome for the longest possible time . Years spent at school are greatly influential for human being, meaning that habits of oral health, as well as other convictions and attitude, are developed for the rest of one s life and one of the main and most efficient means of teaching kids to properly take care of their oral hygiene are oral hygiene educational programs in schools . Educational programmes are effective to improve the oral hygiene, especially when they re based on practical skills training.
Human genomic dna is maintained in intimate contact with proteins that confer the structural integrity of chromosomes . Other proteins associate intermittently with dna to affect its repair, replication, and transcription . These reactions are favored under oxidative conditions, and the production of covalent dna protein cross - links (dpcs) is enhanced by exposure of cells to diverse agents including chemical oxidants, ionizing radiation (ir), ultraviolet radiation (uv), reactive aldehydes, or chemotherapeutic drugs (reviewed in ref (1)). Sequestration of proteins required for dna repair, replication, or transcription is likely to impair these important functions . In addition, dpcs are large dna adducts that block dna replication and physically impede dna - related processes . The thiopurine 6-thioguanine (6-tg) is among the therapeutic agents that promote dpc formation . 6-thioguanine nucleotides, the end product of the metabolism of 6-tg and of the anticancer immunosuppressants azathioprine and 6-mercaptopurine (6-mp), are substrates for incorporation into dna . Additionally, exposure of cultured human cells to 6-tg or 6-mp depletes their antioxidant defenses and increases steady - state levels of reactive oxygen species (ros). Patients treated with thiopurines experience skin photosensitivity and have a significantly increased risk of developing skin cancer . Dna 6-tg can act both as a type i and type ii uva photosensitizer (reviewed in ref (9)). In the type i mode, extremely reactive purine radical cations or purine thiyl radicals are generated following uva activation of dna 6-tg . As a type ii sensitizer, dna 6-tg interacts with uva in the presence of molecular oxygen to generate singlet oxygen (o2), a form of ros that is particularly damaging to proteins . These include oxidized forms of dna 6-tg (guanine sulfinate (g) and guanine sulfonate (g)) and guanine (8-oxo-7,8-dihydroguanine) as well as dna single- and double - strand breaks and dna interstrand cross - links (icls). The combination of dna 6-tg and uva also induces protein damage in the form of carbonyls and oxidized thiols . It causes oxidation - related cross - linking between the subunits of multiprotein complexes including the pcna, ku, rpa, and mcm27 dna replication / repair complexes and between dna and proteins . Importantly, protein damage induced by 6-tg+uva is associated with a significant attenuation of dna repair capacity . Ciprofloxacin is a member of the fluoroquinolone family of antibiotics that are uva photosensitizers . Protein damage by ciprofloxacin+uva also includes oxidation and cross - linking between subunits of dna replication and repair complexes and is associated with impaired dna repair . To our knowledge, the possible we previously demonstrated the formation of heat- and reducing agent - resistant dpcs between oligonucleotides containing oxidized 6-tg (g) and the amino or thiol groups of oligopeptides . The same study also presented preliminary evidence for the formation of dpcs in vivo in cultured human cells treated with 6-tg and exposed to low doses of uva radiation . Immunoblotting identified dna repair proteins among the cross - linked species from cells treated with 6-tg and uva . Specifically, the pcna dna repair / replication protein that is known to be susceptible to oxidation was identified along with msh2 and xpa, essential components of the dna mismatch repair and nucleotide excision repair pathways, respectively . The presence of these important dna repair factors in dpcs suggested that the obligatory, albeit transient association of dna repair proteins with dna might make them particularly vulnerable to inactivation by dna cross - linking . We have developed a sensitive and statistically rigorous approach to identifying cross - linked proteins . Based on stable isotope labeling with amino acids in cell culture (silac) and mass spectrometry (ms), the method is generally applicable to dna damaging treatments . Here we describe the application of this proteomics - based technique to analyze in detail dpc formation by 6-tg treatment and by the uva activation of dna 6-tg in human cells . We also report the application of the same approach to examine dpc induction by uva activation of ciprofloxacin, a representative of a family of non dna - embedded uva photosensitizers . Ccrf - cem cells were routinely grown in rpmi 1640 medium (thermo fisher) supplemented with 10% dialyzed fetal calf serum . For silac, growth medium was supplemented with a combination of either 100 mg per liter of light (n, c) or heavy (n, c) lysine and arginine (ck isotopes). Following growth for 7 d in this medium, full labeling of proteins was confirmed by ms (data not shown). The drug (3 mm) was included in the medium for 6 h prior to and during growth in 6-tg . Dna was extracted, and dna 6-tg incorporation was quantified as described previously . Cells were uva irradiated in phosphate buffered saline using a uvh 253 lamp (uv light technology limited) with maximum emission at 365 nm and a dose rate of 0.1 kj m s. neither photosensitizer treatment nor uva irradiation reduced cell viability, whereas photosensitizer+uva combinations were highly lethal . Ros were determined by facs using cm - h2dcfda (invitrogen) as previously described . Following treatment, 10 isotopically labeled control / treated cells were mixed and nuclei prepared by resuspension in 200 l of 10 mm tris - hcl ph 7.4, 2.5 mm mgcl2, 0.5% np40, 1 mm dithiothreitol (dtt) and were harvested by centrifugation . Chromatin was released from the nuclear pellet by resuspension in 25 mm na phosphate, ph 7.4, 5 mm mgcl2, 500 mm nacl, 0.5% triton, 1 mm edta, 1 mm dtt, 10% glycerol plus protease inhibitors . The chromatin pellet was washed three times by resuspension in the same buffer and then sheared by sequential passage through 19 g, 25 g, and 27 g needles (20 each). Sheared chromatin samples containing 10 g of dna were applied to a hybond - n membrane using a slot blot apparatus (ge healthcare). Dna was cross - linked to the membrane by uvc irradiation from a stratalinker (stratagene) and was then washed extensively with 8 m urea (sigma - aldrich) and water . Membrane - bound proteins were reduced with 10 mm dtt at 50 c for 30 min and alkylated by treatment with 55 mm iodoacetamide for 30 min at room temperature in the dark . The alkylation reaction was stopped by incubation with 10 mm dtt for 10 min at room temperature . Following three washes with 10 mm triethylammonium bicarbonate (teab), the proteins were digested by immersing the membrane in trypsin (12.5 ng/l) overnight at 37 c . Dtt, iodoacetamide, and trypsin were all prepared in 10 mm teab . For ms analysis of total cell lysates, whole - cell extracts were prepared with ripa buffer (50 mm tris - hcl ph 7.5, 150 mm nacl, 0.1% sds, 0.5% na deoxycholate, 1% triton, and protease inhibitors). Twenty micrograms of protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds - page) and stained with colloidal coomassie (instant blue, expedeon). Gel bands were excised and trypsin digested using a perkinelmer janus liquid handling system . Tryptic peptides were analyzed by liquid chromatography - mass spectrometry (lc ms) using an ultimate 3000 uhplc system connected to either a q - exactive or orbitrap velos pro mass spectrometer (thermo fisher scientific) and acquired in data - dependent mode . The data were searched against human uniprot (uniprot kb2012_08 taxonomy human 9606 canonical with contaminants 20 120 921) using the andromeda search engine and maxquant (version 1.3.0.5). For maxquant, a false discovery rate of 0.1% the data were uploaded into perseus version 1.4.0.11 (maxquant) for statistical analyses . Chromatin extracts (20 g of protein) were separated on 10% polyacrylamide gels (invitrogen) and transferred to hybond - n membranes . Following washing with urea and water, membranes were probed with antibodies against msh2, msh6, pcna (santa cruz), and rpa70 (abcam). Growth of ccrf - cem cells in the presence of 6-tg (0.30.9 m) resulted in the thiopurine replacing around 0.050.6% of dna guanine . When dna was prepared from a standard number of 6-tg treated cells by the wizard (promega) extraction protocol, the amount recovered declined in a 6-tg concentration - dependent manner . Irradiation of cells containing dna 6-tg with a modest dose of uva (50 kj / m) further exacerbated dna losses . The effects of 6-tg and uva were synergistic, and uva alone had no detectable effect on dna recovery . The wizard extraction protocol involves a protein precipitation step prior to dna harvesting and the inclusion of a proteinase k digestion step prior to dna precipitation restored quantitative dna yields (supplementary figure s1a). The reduced dna recovery from cells treated with 6-tg or 6-tg+uva was dependent on the presence of 6-tg in dna . The wizard dna purification protocol yielded quantitative dna recovery from 6-tg treated gm03467 lesch - nyhan cells without the inclusion of the protease digestion step (data not shown). These cells do not express hypoxanthine - guanine phosphoribosyltransferase and cannot scavenge 6-tg for incorporation into dna . In addition, quantitative dna yields were achieved without the additional protease digestion step if 6-tg incorporation into dna was prevented by treatment of ccrf - cem cells with 6-tg in the presence of hydroxyurea (supplementary figure 1a). Facs analysis (supplementary figure 1b) confirmed that exclusion of 6-tg from dna also reduced uva - induced ros levels . Their dependence on protease digestion suggested that dna yields were reduced by cross - linking of dna to protein . This possibility was investigated further using chromatin from treated cells . To selectively enrich for proteins covalently attached to dna, chromatin extracted from 6-tg+uva treated ccrf - cem cells was washed extensively with high salt (500 mm nacl) to deplete noncovalently associated proteins . Sheared, salt - washed chromatin was applied to a hybond - n membrane that was then sequentially washed with 8 m urea and water . Staining with syproruby and sybr green confirmed that extensive urea washing removed all detectable membrane - associated proteins from untreated chromatin while having no noticeable impact on the amount of bound dna . Subsequent probing of the filter with a panel of antibodies confirmed that uva induced a 6-tg dose - dependent increase in the amount of the xpa, pcna, msh2, and rpa70 (the 70 kda subunit of the rpa single strand dna binding complex) dna repair / replication proteins associated with the washed membrane (figure 1a, b). (a) sheared, salt - washed chromatin from ccrf - cem cells that were untreated or treated with 6-tg and 50 kj / m uva as indicated was applied to hybond - n membranes . Membrane - associated protein and dna was visualized by staining with sypro ruby and sybr green . . Sheared chromatin from untreated ccrf - cem cells or cells treated with uva, 6-tg or 6-tg+uva was applied to hybond - n membranes . Following extensive washing with water and 8 m urea, membranes were probed with antibodies as indicated . The observation that dna replication / repair proteins are enriched in hybond - n membrane - bound chromatin prompted us to undertake a comprehensive analysis of the proteome associated with hybond - n - bound dna to provide an unbiased screen for proteins involved in dpcs . The protocol is outlined in figure 2, panel a. outline of silac analysis . (a) ccrf - cem cells were labeled with heavy or light arginine and lysine isotopes . Half the cells were treated with 0.9 m 6-tg for 24 h and the other half left untreated . Half of each of these two cultures was then irradiated with 50 kj / m uva . Cells were mixed in 1:1 ratios for preparation of chromatin or whole cell extracts (ripa) as indicated . Chromatin extracts were applied to a hybond - n membrane that was water and 8 m urea washed prior to in situ trypsin digestion and ms analysis . Ripa extracts were subjected to short sds - page and in - gel trypsin digestion prior to ms analysis . Briefly, ccrf - cem cells labeled with heavy or light isotopes of arginine and lysine were treated with 6-tg (0.9 m). Half of each culture was then irradiated with uva (50 kj / m). Chromatin was prepared from a total of 16 1:1 mixes of heavy and light isotope labeled cells that had been treated with 6-tg, uva, 6-tg+uva, or left untreated . The compositions of these mixes are shown in figure 2, panel b. high salt - washed chromatin mixtures were loaded onto a hybond - n membrane that was extensively washed with water and 8 m urea . Remaining membrane - associated proteins a total of 2611 proteins were identified in two independent experiments (forward and reverse labeling analyses) of hybond - n membrane bound chromatin from uva, 6-tg, or 6-tg+uva treated ccrf - cem cells (supporting information). Because the analysis was carried out with chromatin prepared from mixtures of heavy and light labeled cells, changes in the log2 heavy / light (h: l) protein ratios reveal an enrichment of proteins associated with membrane - bound dna . Supplementary figure s2a presents the protein distribution for mix 1 in which the chromatin applied to the membrane was prepared from mixtures of untreated heavy- and untreated light - labeled cells . The tight symmetrical clustering of log2 h: l ratios around the zero value in the histogram confirms the expected equal representation of heavy and light labeled proteins . Because> 99.5% of log2 values for these untreated cells lie between 1 and + 1, in the subsequent analysis of treated chromatin we considered values that fall outside this range (representing> 2-fold enrichment) to be significant treatment - related changes that reflect dpc formation . Comparison of mixtures of heavy 6-tg - treated / light untreated cells and heavy untreated / light 6-tg - treated cells (mixes 3 and 9) revealed the effects of 6-tg treatment . Supplementary figure s2b, c shows that 6-tg treatment shifted the membrane - associated protein distribution in the direction of the label in the treated cells . In the scatter plot (figure 3a), 6-tg - induced asymmetry in log2 h: l ratios results in the majority of the data points occupying the lower right quadrant, a shift consistent with dpc formation . The effect was small, however, and only reached significance for approximately 10% (7 and 16% in two determinations) of the detected proteins, which indicated that 6-tg - induces a low level of dna protein cross - linking . (a) the effect of 6-tg treatment (6-tg vs untreated). Mix 3 versus mix 9 (from figure 2b). (d) the additional effect of uva on 6-tg treated cells (6-tg+uva vs 6-tg). The calculated absolute values for log2 h: l ratio shown confirm that 6-tg+uva induces a significant change . They also validate synergy between 6-tg and uva . (e) heat map of log2 h: l ratio intensities of identified proteins . Mix numbers refer to those in figure 2, panel b. mixes of h- and l - labeled cells that received the same treatment are shown in bold . The moderate uva dose we used (50 kj / m) did not induce detectable dna protein cross - linking, and the log2 h: l ratios for the comparison of mixes 2 and 5 (figure 3b) that addresses the effect of uva remain tightly clustered around the origin of the scatter plot . Analysis of mixes 4 and 13 (figure 3c) revealed that the combination of 6-tg and uva caused extensive dna protein cross - linking . By comparing mixes 12 and 15, we specifically examined the effect of uva on cells treated with 6-tg . In the absence of synergy between 6-tg and uva, the log2 h: l ratios would cluster around the origin of the scatter plot as they do for samples from cells treated with uva alone . Figure 3, panel d and supplementary figure s2d, e confirm that uva induces extensive dpc formation in cells treated with 6-tg . Dpc induction by 6-tg, uva, and combined 6-tg+uva is summarized in the heat map in figure 3, panel e. hierarchical cluster analysis reveals a family of proteins (arrowed) that appeared to be largely unaffected by any of the treatments and represent a set of false positives . Subsequent analysis of the cross - linking profiles of these proteins (see below) confirmed their absence of susceptibility to cross - linking by either 6-tg or 6-tg+uva . In summary, silac analysis demonstrates that 6-tg induces a low level of dpcs in ccrf - cem cells . It also reveals that 6-tg and uva combine synergistically to cause extensive dpc formation . The ms analysis revealed changes in log2 h: l ratios that indicate decreases in protein yield from cells treated with 6-tg and 6-tg+uva relative to that from untreated cells (the upper left quadrant of the scatter plots in figure 3). While these changes are consistent with dpc formation, we considered two alternative mechanisms that might contribute to this protein underrepresentation . If the inhibition is sufficiently severe, it could reduce overall cellular protein content . An alternative (and not exclusive) possibility is that the acknowledged insolubility of oxidized proteins might contribute to diminished protein recovery . To investigate these eventualities, we compared the hybond - n membrane - bound proteome with proteins in unfractionated extracts . Cultures of isotopically labeled ccrf - cem cells that had been treated with uva+6-tg, 6-tg alone, or uva alone were combined with an equal number of untreated reverse labeled cells, and the mixture was divided into two equal parts . Chromatin extracted from one aliquot of cells was bound to a hybond - n membrane and processed for ms as described above . The remaining cells were used to prepare a dna - free whole cell extract using a standard (ripa) extraction procedure . Trypsin digests of these whole cell extracts were compared with those of the corresponding hybond - n proteins . Figure 4, panels a and b show the effect of 6-tg treatment (mixes 3 and 9). Scatter plots of log2 silac h: l ratios comparing the effects of treatments on chromatin and ripa (whole cell) extracts . Mean values for log2 h: l ratios ripa extract mixes are presented on each panel . The vertical shifts in log2 h: l ratios from the origin of the scatter plot in the direction of the label of the chromatin from 6-tg - treated cells confirm that 6-tg induces dpcs . The small changes in mean log2 h: l ratio for the ripa extract proteins (mix 3 = 0.31; mix 9 = 0.15) indicate that yields are largely unaffected by 6-tg treatment . In contrast, ripa extracts are enriched for proteins from untreated cells relative to those from cells treated with 6-tg+uva (figure 4c, d) yielding significant changes in mean log2 h: l values (mix 4 = 1.15; mix 13 = 0.81). As expected, uva did not affect protein recovery (mean log2 h: l ratios 0.19 and 0.16 for mixes 2 and 5, respectively). Since neither 6-tg nor uva alone significantly influenced protein recovery, the changes in protein abundance in extracts from cells treated with 6-tg+uva can be ascribed to their combined effect . It follows that interference with transcription / translation during prolonged (24 h) 6-tg treatment does not have a significant impact on protein yield . We conclude that the diminished protein recovery from 6-tg+uva treated cells most likely reflects depletion due to protein dna cross - linking allied to losses resulting from precipitation of proteins oxidized by 6-tg+uva . The observation that treatment with 6-tg+uva causes a measurable reduction in protein recovery indicates that silac analysis may slightly underestimate the extent of dpc formation by this combination . To identify proteins that were most susceptible to dna cross - linking, we used r combined with ggplot2 to determine and visualize the 95th (for heavy - labeled treated cells) and fifth (for light - labeled treated cells) percentile values of log2 h: l ratio changes for each of the 16 analyses . These values are shown in figure 5, panel a along with a plot that provides a graphic representation of the predicted maximal effect of uva, 6-tg, or 6-tg+uva on dna protein cross - linking . Cross - linking profile of ccrf - cem chromatin proteins most vulnerable to dpc formation . (a) 95th (treated heavy - labeled) or 5th (treated light - labeled) percentile values for log2 h: l ratios are presented and plotted for each of the 16 comparisons . (b) the cross - linking profile of the 200 proteins that best fit the profile in panel a is shown superimposed on the profile for all 2611 detected proteins (in gray). (c) protein ontology of the best fit 200 proteins in panel b, as specified by uniprot . (d) cross - linking profiles of the 114 potential false positive proteins (figure 3e). We used the perseus program (maxquant) to identify 200 proteins that best fit this profile . In figure 5, panel b, the cross - linking profile of these proteins (red) is superimposed on that of all other identified proteins (gray). Figure 5, panels a and b confirm that both 6-tg and 6-tg+uva induce cross - linking . Of the 200 selected proteins, 192 are either predominantly nuclear or have been detected in the nucleus (uniprot / genecards). Around one - third of these proteins are involved in gene expression, and dna repair / replication comprises the next largest category (16%, figure 5c). Dna and rna binding proteins are equally represented (figure 5c) and together account for about half of the 200 selected . Since 6-tg is incorporated extensively into rna and uva cross - links protein to rna containing a photoreactive purine analog, some of these rna binding proteins may be present as rna - protein cross - links . Direct measurements (data not shown) indicated that rna accounted for <2% of the total nucleic acid in our chromatin preparations . We therefore rule out a significant contribution from rna - protein cross - links, and we conclude that these rna processing proteins are most likely present in dpcs . The cross - linking profiles for the 114 proteins identified as potential false positives by cluster analysis (figure 3e) are shown in figure 5, panel d and their categorization in figure 5, panel e. cross - linking analysis confirms that none of the treatments increased their representation in dpcs . These include particularly high abundance leading to high background and lower probability of detecting treatment - related dpc formation, a high affinity for dna, or strong direct binding to the hybond - n membrane independently of dna . Consistent with these possibilities, histones (see below) and the highly abundant intermediate filament protein vimentin (pi = 5) were among the 114 false positives . The intimate association of dna repair and replication proteins with dna is expected to increase their vulnerability to dna cross - linking . Among 179 dna repair proteins (http://sciencepark.mdanderson.org/labs/wood/dna_repair_genes.html), most matched the expected profile for 6-tg and 6-tg+uva dependent cross - linking (figure 6a). Analysis of the 30 best fits to the generic cross - link profile (supplementary table s1) is shown in figure 6, panel b. a similar analysis of whole cell (ripa) extracts of mixes 14 (figure 2b) indicated that most of these dna repair proteins were underrepresented to some degree in cells treated with 6-tg+uva (figure 6c). This observation is consistent with a significant depletion of dna repair proteins by cross - linking to dna . (a) cross - linking profiles for the 52 detected designated dna repair proteins . (b) cross - linking profiles for the 30 most vulnerable dna repair proteins . (c) log2 silac h: l ratio plots for whole cell ripa extracts for the 30 most vulnerable dna repair proteins indicating that the majority are significantly depleted . The cross - linking profiles for the 125 kda catalytic (pold1) and the 50 kda pold3 auxiliary subunits (figure 7a, c) were good fits to the generic profile of figure 5, panel a confirming their vulnerability to dna cross - linking . These profiles suggest that pold1 is susceptible to cross - linking in cells treated with either 6-tg alone or 6-tg+uva, whereas pold3-dna cross - linking appears to be predominantly photochemical and requires both 6-tg and uva . Although the 66 kda pold2 subunit was among the chromatin proteins identified by ms analysis, its profile deviated significantly from the generic plot suggesting that it is less susceptible to cross - linking . Cross - linking profiles for: (a) dna polymerase delta 125 kda, (b) 60 kda, and (c) 55 kda subunits; (d) mcm27 proteins; (e) topoisomerases 1,2a and 2b . It comprises the mcm2-mcm7 proteins that assemble on dna as a circular hexamer to initiate replication . Their cross - link profiles were essentially superimposable (figure 7d) indicating a shared vulnerability to cross - linking in cells treated with 6-tg or with 6-tg+uva . Topoisomerases relieve supercoiling by cleaving dna ahead of the transcription or replication apparatus . This essential function is performed by the major human topoisomerases top1 and top2a, 2b . They were susceptible to dna cross - linking, and like pold3, they also appeared to be particularly susceptible to photochemical cross - linking to dna containing 6-tg (figure 7e). Consistent with depletion due to dpc formation, the pold subunits, mcm proteins, and topoisomerases were all present in significantly reduced levels in whole cell extracts following 6-tg+uva treatment (supplementary figure s3). Silac - hybond - n membrane binding was used to investigate dpc formation by uva - activated ciprofloxacin . Treatment with 500 m ciprofloxacin and 50 kj / m uva induced significant dna protein cross - linking in ccrf - cem cells (figure 8b) as indicated by the clustering of most of the data for the comparison of mixes 2 and 3 in the lower right quadrant of the scatter plot . Among the 2269 cross - linked proteins we identified, the representation of dna and rna binding proteins was closely similar to that generated by 6-tg+uva (figure 8c). Forty - one dna repair proteins (http://sciencepark.mdanderson.org/labs/wood/dna_repair_genes.html) were identified in ciprofloxacin+uva treated samples . Of these, 29 fit the profile expected for dpc induction (> 2-fold change) (supplementary table s1, figure 8d). Among ciprofloxacin+uva cross - linked dna repair proteins, there was a highly significant (p <e) overlap with dna repair proteins cross - linked by 6-tg+uva treatment (figure 8e). Hierarchical cluster analysis (supplementary figure s4) confirmed ciprofloxacin+uva induced dpc formation and again revealed a group of candidate false - positive proteins . The cross - linking profiles of the 68 candidates confirmed that they were not present in treatment - related dpcs (data not shown). Histones were highly represented in this group, and there was significant overlap with false positives identified following 6-tg+uva treatment (supplementary figure s4). Heavy- or light - labeled ccrf - cem cells were treated with 500 m ciprofloxacin (cip) for 1 h and uva (50 kj / m) as indicated . Salt - washed chromatin prepared from cells mixed in 1:1 ratios as indicated was applied to a hybond - n membrane . Following washing with water and 8 m urea, (c) dna and rna binding proteins among cirofloxacin+uva (green) and 6-tg+uva (blue). (d) cross - linking profiles for 29 dna repair proteins that best fit the most vulnerable profile . (e) overlap between 29 dna repair proteins cross - linked by ciprofloxacin+uva and the 30 cross - linked by 6-tg+uva . We have devised a silac and proteomics - based method to investigate dpc formation by photosensitizer / uva combinations and describe its application to human cells treated under conditions that mimic and amplify the clinical effects of photosensitizing medications . Dpc induction by formaldehyde has been investigated by a similar approach that employs a modified chip technique . Dna cross - linking by specific subsets of proteins has also been investigated using biotinylated double - stranded oligonucleotides, specific recognition sequences inserted into genomic dna in vivo, and immunoprecipitation of cross - linked proteins or trimethylated histones . Many of these studies have employed silac or other labeling methods to quantify cross - linking . To our knowledge, selective enrichment of dpcs by hybond - n membrane binding has not previously been combined with silac - lc ms analysis . The approach identified more than 2000 cellular proteins that were cross - linked to dna by 6-tg, by 6-tg+uva, or by ciprofloxacin+uva . Among the proteins most susceptible to cross - linking, most are nuclear and are involved in control of gene expression or dna repair / replication . This distribution is consistent with incorporated dna 6-tg or dna - bound or -intercalated ciprofloxacin acting as the predominant sources of the photochemical ros that drive dpc formation . One important aspect of our study is that it addresses cross - linking targeted to dna containing a reactive center (6-tg and 6-tg+uva) as well as protein cross - linking to canonical dna constituents (ciprofloxacin+uva). Photoactivation of a site - specific dna- or rna - embedded thionucleobase, including 6-tg, has been used extensively to probe nucleic acid structure and protein nucleic acid cross - linking . Uva - induced cross - linking of oligopeptides or purified proteins to 6-tg - containing oligonucleotides has also been used to model protein dna interactions in vitro, and we have presented preliminary evidence from 2d - dige and isopycnic density gradient analysis for dpc formation in cells treated with 6-tg and uva . The present study describes an unbiased and quantitative investigation into dpc formation in human cells by uva - activated photosensitizers . A modest level of dna substitution by 6-tg (around 0.05% of dna guanine) in ccrf - cem cells was sufficient to reduce dna recovery unless the dna was treated with a protease prior to precipitation during purification . Protease - reversible dna losses during purification were exacerbated by exposure of the 6-tg - treated cells to 50 kj / m uva, a dose that had no detectable effect on dna recovery in the absence of prior 6-tg treatment . Proteomic analysis confirmed the synergistic induction of dpcs by 6-tg and 50 kj / m uva radiation . 6-tg is an atypical photosensitizer because its incorporation into dna introduces highly reactive dna thiol groups through which dpcs may form preferentially . In contrast, dpcs induced by uva - activated ciprofloxacin, which is not incorporated into dna, must involve canonical dna components . Our analysis revealed extensive dna protein cross - linking by ciprofloxacin+uva indicating that the approach is likely to be generally applicable to dpc analysis . Thiopurines like 6-tg perturb the cellular redox balance and increase the concentrations of intracellular ros including superoxide anion (o2), h2o2, and ultimately via metal - catalyzed reactions, the highly damaging hydroxyl radical (oh), which is a possible source of dpcs . We have previously shown that icl formation in cells treated with 6-tg requires both ros and incorporated dna 6-tg . Dpc induction shares these requirements, and dna - embedded 6-tg is a key participant in dna lesch - nyhan cells were invulnerable to dpc induction by either 6-tg alone or 6-tg+uva, and both treatments were without effect in ccrf - cem cells in which replication was inhibited to prevent the accumulation of dna 6-tg . On the basis of this requirement, we consider it unlikely that reactions between protein nucleophiles and oxidized dna guanine are a significant source of dpcs mediated by 6-tg or 6-tg+uva . Oligopeptide cross - linking involving g, which is a good leaving group in nucleophilic substitution reactions, and peptide sh or nh2 groups . Nucleophilic attack by proteins at dna g generated in the ros - rich environment is a possible mechanism for 6-tg - mediated dpc formation . Cross - linking between a free protein nh2 group and a dna 6-tg radical cation or a thiyl radical generated by oxidation of dna 6-tg is a possible alternative reaction . Ros generated during 6-tg treatment cause widespread protein oxidation, and dpcs may also form via the reaction of oxidized proteins with dna 6-tg . The more extensive photochemical cross - linking by 6-tg+uva most likely involves type ii photosensitization . Like dna 6-tg since proteins are susceptible to o2-mediated oxidation, reactions between oxidized proteins and canonical dna constituents are likely to be a significant factor in dpc formation by 6-tg+uva and ciprofloxacin+uva . Consistent with a common etiology, more than 75% of the dna repair proteins that were identified as highly vulnerable to cross - linking by ciprofloxacin+uva were among similar proteins identified in 6-tg+uva dpcs . Members of the group containing the large catalytic pold1 subunit of dna polymerase and the mcm proteins were efficiently cross - linked by both 6-tg and 6-tg+uva, whereas cross - linking of proteins typified by the smaller pold3 and the topoisomerases appeared to be largely photochemical and depended on 6-tg+uva . These different cross - linking behaviors may reflect different cross - linking chemistries or may simply be related to the positioning of the proteins on dna . In the case of protein multimers such as dna pol or mcm27 that can also form intersubunit protein protein cross - links, their presence in dpcs might also reflect dna cross - linking of covalent protein transient dpc formation is a feature of many dna processing enzymes, and interference with the correct reversal of topoisomerase dna complexes is the basis of the therapeutic action of drugs such as camptothecin and etoposide . These specific enzyme - related dpcs are, however, atypical, and dpcs are likely to be structurally heterogeneous . Most studies of the induction, processing, and biological effects of dpcs have used formaldehyde, a highly reactive molecule that causes protein damage, depletes cellular reduced glutathione levels, and induces icls as well as dpcs . The possible involvement of nucleotide excision repair, homologous recombination, and the proteasome in dpc reversal in human cells has been suggested but is not firmly established (see ref (42) for review). Fanconi anemia cells are extremely sensitive to formaldehyde, and evidence from mice and from chicken cells with defective aldehyde metabolism suggests a requirement for this dna repair pathway to repair aldehyde - induced dpcs . Whether sprtn, the human homologue of putative dpc - specific proteases that has been identified in yeast and xenopus laevis, participates in the same or in a separate repair pathway remains to be determined . Like formaldehyde, 6-tg+uva and ciprofloxacin+uva (and most other treatments that cause dna protein cross - linking) induce other dna lesions as well as dpcs and protein damage . This pleiotropy may have hampered attempts to define dpc repair pathways in human cells . Among the putative systems for dpc repair, we identified essential ner, homologous recombinational repair, and fanconi pathway proteins as particularly susceptible to dna cross - linking . This observation raises the possibility that dpc repair might be compromised by depletion or oxidation of the repair proteins themselves . Because the non - dpc damage induced by uva activated photosensitizers is unlikely to be the same as that caused by aldehydes, uva / photosensitizer combinations may be a useful addition to studies of dpc induction and repair in human cells.
The incidence of hepatocellular carcinoma (hcc) has tripled in the united states during the past three decades with an annual increase of 4.5% . Additionally, the mortality rates associated with this disease have continued to rise [1, 2]. The poor survival rates for hcc are in part due to the coexistence of advanced liver disease, which limits treatment options . Currently, orthotopic liver transplantation (olt) has proven to be an excellent therapeutic option for long - term survival in patients presenting with early - stage hcc . In order to facilitate equitable allocation of donor livers, the united network for organ sharing (unos) was established . Under unos guidelines, livers are appropriated based on a waiting list, in which patients are rank - ordered by their pretransplant mortality risk . Unfortunately, relatively few eligible patients with hcc patients undergo olt due to organ shortage . Each year, there are many more patients on the unos waitlist than there are organs available for transplantation . When olt was first proposed as a treatment for hcc, only patients who were unresectable due to tumor burden or other underlying liver dysfunction were selected as candidates . As a result, patients who underwent olt exhibited low survival and high recurrence rates after transplantation [6, 7]. These discouraging outcomes eventually resulted in refined selection criteria, such as those first proposed by mazzaferro et al . In 1996 . These criteria, known as the milan criteria, have significantly improved survival . However, a subsequent report by yao et al . Indicated that the milan criteria may be too restrictive . Patients transplanted under more liberal selection criteria, designated as the ucsf criteria, and had outcomes comparable to those within milan criteria . These data suggested that expansion of conventional criteria could broaden the patient pool for olt without affecting the oncologic outcomes of olt for hcc . Despite an increase in organ donation over the last two decades more than 15,000 patients were listed for olt in 2008, yet only approximately 5000 patients underwent olt that year . Appropriate selection of patients with hcc is therefore necessary to optimize the allocation of these organs . In light of this, the expansion of olt criteria for hcc needs to be carefully weighed with regards to the limited organ supply and the outcomes of patients undergoing olt with tumors outside of milan criteria should be closely examined . The unos database has yet to be queried for evaluation of liver transplantation outcomes based on selection criteria . In this paper, we have used the unos database to compare the short - term survival outcomes of patients who underwent olt for hcc within milan criteria versus those undergoing olt for hcc outside of milan criteria, but within ucsf criteria . We hypothesized that patients transplanted within milan criteria and those transplanted outside of milan criteria but within ucsf criteria had equivalent outcomes . Registered under the united states department of health and human services, unos maintains a database with detailed information about transplants performed in united states transplant centers . Via an internet - based database system known as unet, this database application provides demographic, clinical, and pathologic data for all transplanted candidates and recipients . After obtaining approval for our study from unos and the city of hope's institutional review board (irb), the unos database was queried for patients who underwent olt for hcc from 2002 through 2007 . The year 2002 was selected as the first year of our study, since it was the first year following publication of the ucsf criteria . Size criteria for milan or ucsf criteria were determined via pretransplant diagnostic imaging, which is reported to unos by a given transplant center prior to olt as part of ongoing patient registration . From the unos registry, a total of 3,434 patients underwent olt for hcc during the study period . After excluding patients with missing tumor size or number, patients exceeding ucsf criteria, and patients <18 years of age these patients underwent olt for hcc within milan criteria (n = 1, 913) or outside of milan criteria but within ucsf criteria (n = 59). Milan criteria were defined as 1 tumor 5 cm; or 3 tumors with each tumor 3 cm . Ucsf criteria were defined as 1 tumor 6.5 cm or 3 tumors with the largest tumor diameter 4.5 cm and total tumor diameter 8 cm . Comorbidities of the study cohorts included diabetes mellitus (dm), body mass index (bmi), hepatitis b virus (hbv), and hepatitis c virus (hcv). The causes of death included malignancy (i.e., graft failure from recurrent disease or malignancy not otherwise specified) and other causes (i.e., cardiovascular, graft failure, multisystem organ failure, hemorrhage, infectious, and other). Liver - directed therapy (ldt) included transarterial chemoembolization (tace), radiofrequency ablation (rfa), and cryoablation . The primary prognostic factor of interest was transplant criteria (i.e., milan versus ucsf) and the association of this variable with survival . After patients were stratified by transplant criteria, the overall survival was calculated from the date of transplant to the date of death using the kaplan - meier method . The log - rank test was used to compare survival curves . Among the demographic and clinical data that were compared, age, bmi, and total tumor diameter were coded as continuous variables, whereas gender, dm, tumor number, ldt, hbv and hcv status, and cause of death were coded as categorical variables . Student's t - test and -test were used to calculate the differences in the continuous and categorical factors, respectively . Univariate cox regression analysis was performed to determine the association of each clinicopathologic factor with survival . Multivariate cox regression analysis was applied to assess the association of multiple covariate factors with survival in the two transplant criteria cohorts, while controlling for the factors found to be significant on univariate analysis . Results were presented as hazard ratios (hr) and reported with 95% confidence intervals (ci) and two - sided p values . The entire study cohort consisted of 1,972 hcc patients who were transplanted within milan or ucsf criteria from 2002 to 2007 (table 1). The majority of patients (n = 1913; 97%) were within milan criteria at the time of olt based on imaging, while only 59 patients, or 3% of the total, underwent olt with tumors that were beyond milan criteria but within ucsf criteria . The majority of patients (79%) was male and had 1 tumor (67%). Only a small percentage of patients (24%) had dm, and most patients (69%) were nonobese . Local tumor control or downsizing with ldt was performed in 36% of patients . Causes of death included malignancy (31%) and all other causes (56%). Patients who underwent olt for hcc within milan criteria and ucsf criteria were compared, as shown in table 2 . The majority of the milan criteria cohort had 1 tumor (69%), whereas the majority of the ucsf criteria cohort had 1 tumor (39%) or 2 tumors (44%) (p <0.001). Ldt was also more frequently performed in the ucsf cohort (61% versus 35%, respectively, p <0.001). Age, gender, dm, bmi, hbv status, hcv status, and cause of death were similar between the two cohorts . Kaplan - meier curves were constructed to determine survival for the milan and ucsf criteria cohorts (figure 1). Survival between the milan and ucsf criteria cohorts was similar (1-, 2-, 3- and 4-year survival rates: 89%, 81%, 76%, and 72% versus 91%, 80%, 68, and 51%, respectively, p = 0.21). By univariate and multivariate analysis, neither milan nor ucsf criteria were independently predictive of improved survival (table 3). Liver transplantation remains a preferred treatment for patients with early - stage hcc in the setting of cirrhosis . Based on the accumulation of data showing excellent disease - free survival in patients with early - stage hcc treated by transplantation, the current united states allocation system for liver transplantation has given priority to patients with hcc within milan criteria . With the incidence of hcc continuing to rise despite a relatively static organ supply, the allocation system requires continuing refinement so that patient benefit and outcome from this scarce resource are optimized . The current accepted milan criteria for transplantation originally demonstrated 4-year survival and recurrence - free rates of 75% and 83%, respectively . These results have been validated by numerous subsequent studies showing equivalent or superior survival advantages [1013]. In 2001, yao et al . Referred to as the ucsf criteria, these expanded hcc transplantation criteria resulted in a modest increase in the total number of eligible patients of approximately 510% . Using this single institution criteria, yao et al . Demonstrated a 5-year survival of 75% after olt for hcc as compared to a 50% 1-year survival in patients who exceeded these criteria . These encouraging results were corroborated in a larger single institutional series that included 185 patients transplanted for hcc who also met ucsf criteria and exceeded milan criteria . In that series, duffy et al . Reported a 5-year survival of 64% in patients beyond milan but within ucsf criteria compared to a 79% 5-year survival for patients transplanted within milan criteria (p = 0.061). Given the equivalent survival in patients transplanted within these expanded criteria, interest in revising the current organ allocation system has grown albeit with considerable caution and controversy . Opponents of selection criteria expansion have suggested that the ucsf criteria are applicable to only a small subset of patients and cannot be applied in the pretransplant setting [5, 13]. Decaens et al . Attempted to evaluate the ucsf criteria in a multiinstitutional setting . Pooling data from 14 french transplant centers, they identified 39 out of 461 patients who were transplanted beyond milan criteria but within ucsf criteria, and compared these patients to 184 patients within milan criteria . While survival was equivalent between the two groups when the criteria were applied to explant pathologic evaluation (64% versus 70%, respectively, p = 0.33), the 5-year survival in patients outside milan but meeting ucsf criteria when applied to pretransplant diagnostic imaging was only 46% . Despite no statistical difference in survival between the two criteria groups, the authors suggested that the application of these criteria may be imprecise when used in pretreatment patient selection . These concerns have been outlined by other series as well [15, 16]. Subsequently, yao et al . Reported another series of 38 patients exceeding milan criteria but within ucsf criteria based on pretreatment diagnostic imaging and compared them to patients within pretreatment milan criteria . There were no differences in survival, nor were there any differences in other risk factors for posttransplant recurrence, such as vascular invasion or poorly differentiated pathology between groups . Similarly, duffy et al . Also did not identify a survival difference when milan and ucsf criteria were compared according to pretransplant diagnostic imaging . Overall these studies suggest that in a few select centers, pretransplant imaging is a reasonably accurate predictor of explant pathologic status, and that patients undergoing olt for hcc have similar outcomes whether pretransplant imaging is within milan or ucsf criteria . Since the majority of evidence supporting the adoption of ucsf criteria comes from single institution series, we sought to evaluate these criteria within a multiinstitutional database . By using the unos database, we identified 59 patients transplanted for hcc within ucsf criteria, as compared to 1,913 patients transplanted within milan criteria . We were unable to identify a survival difference between selection groups nor was ucsf criteria an independent predictor of worse survival on multivariate analysis . Importantly, patients within ucsf criteria were more likely to receive ldt when compared to patients within milan criteria . Indeed, other single institution series have shown the benefit of downstaging via ldt in order to meet criteria, or to control disease while awaiting transplantation [18, 19]. Our results are not able to separately address the outcomes of patients who are initially outside of ucsf criteria on pretransplant imaging and subsequently undergo downstaging procedures to allow olt . Additionally, due to the limitations of the database, information concerning the time interval from diagnosis to transplant and drop - out from the waiting list could not be ascertained . The results of our study need to be carefully considered in light of our small patient cohort . Similar to other reported series, the number of patients in our study transplanted within ucsf criteria was small . However, the number of patients in our series beyond milan criteria but within ucsf criteria (n = 59) compares favorably with yao's two initial reports first establishing the ucsf criteria . Yao's initial series based on explant staging identified 18 patients meeting criteria and his subsequent analysis based on pretransplant imaging was based on 38 patients meeting ucsf criteria [9, 17]. However, given that the unos database covers nationwide transplant center reporting, we believe our findings may hold more weight in comparison to single institution series . Moreover, this is the largest multiinstitutional series comparing these two selection criteria within the united states patient population in the reported literature and the first time that the unos database has provided a comparison of transplantation outcomes for hcc based on selection criteria . Before adopting an expanded size based criteria into the current unos allocation scheme, first, the ability to adequately stage patients with hcc remains a challenge . With current imaging modalities, understaging can be expected in 2030% of patients, while overstaging can be seen in up to 15% of patients . Given survival in patients with hcc beyond ucsf criteria is clearly inferior to survival within either milan or ucsf criteria, further refinement of pretransplant staging is necessary to ensure size criteria validation . Furthermore, while size - based criteria may be comparable and reproducible in pretreatment staging, they may not predict the biologic aggressiveness of the underlying hcc . Recent evaluation into expanded criteria both from the university of pittsburgh and a european multicenter series suggests that pathologic characteristics, in particular tumor grade and microvascular invasion, may be more important determinants of recurrence - free survival than size criteria [11, 21, 22]. Additionally, biopsy samples provide the opportunity for genetic profiling and molecular analysis, which may shed further insight into the tumor's biology . However, obtaining adequate pretransplant tissue for pathologic evaluation in patients with active hepatitis or cirrhosis is not without inherent risk and thus limits the utility of such staging systems . Yao et al . Tested the ucsf criteria to predict the pittsburgh modified tnm staging system, and the results were favorable without the need for a pretransplant biopsy . Our preliminary results suggest that pretransplant imaging, while imperfect, may not negatively affect outcomes when used to stratify patients by ucsf or milan criteria . Nonetheless, we acknowledge that our conclusions are limited by the constraints of the unos dataset . Lack of information on biopsy results and other pretransplant features, as well as the possibility of unknown confounding factors, may have influenced our results and affects the generalization of our findings [23, 24]. Perhaps the most difficult question to address is how the addition of candidates for transplantation will affect the overall survival of hcc patients listed for transplantation based on an intention - to - treat analysis . Current population based analyses and single institution series suggest drop - out rates of 1218% secondary to tumor progression, while the intention to treat overall survival of all listed patients is approximately 50% [22, 25]. Patients exceeding milan criteria do not receive prioritization in the current unos allocation system, thus limiting the ability to compare these two selection criteria objectively . Given that population prediction models anticipate a 20% increase in candidates for transplantation with adoption of the ucsf criteria, the adoption of expanded criteria must be weighed heavily against the limited organ supply . However, when one considers that the drop - off rate increases to 3040% when milan criteria are used as absolute criteria, further refinement seems warranted [22, 25]. In this largest, multiinstitutional series comparing ucsf to milan criteria, we identified no difference in survival between patients transplanted by either selection criteria . Given the superior results of transplantation for appropriate patients with hcc and advanced liver disease, long - term prospective comparison of these two selection criteria appears warranted.
Pgs membranes (~250 m thick) on silicon wafers16 were situated on the x - y stage (accuracy 1 m) of a rapid x 1000 system (resonetics, nashua, nh) with a 248 nm krypton fluoride lpx200 excimer laser (coherent - lambda physik, santa clara, ca). Pores were patterned via g - code programs . For highly cross - linked pgs (16 h/160c), a power of 350 mj, burst frequency of 500/sec and burst count of 1000 were used . For partially cross - linked pgs (e.g., 7.5h/160c), scaffolds were loosened from wafers in deionized water (24 h) and 70% ethanol (24 h). Bilaminar scaffolds (~400 m) with 3-d pore networks were fabricated by microablating a lateral array of troughs in one pgs lamina (burst spacing of 0.01 sec; table speed of 0.5 mm / sec), overlaying a second pgs lamina, microablating top - to - bottom pores through both lamina (burst frequency of 500/sec; burst count of 2000), and then stabilizing the resultant scaffold by thermally cross - linking17 via autoclaving (121c for 30 min). Cells were obtained from 2-day old neonatal rat hearts via an institute - approved protocol as described8 (see supplementary information, methods). In studies of pgs scaffolds with varying effective stiffness and prototype bilaminar scaffolds (5 mm 5 mm for imaging; 10 mm 5 mm for mechanics), scaffolds were autoclave - sterilized and seeded with freshly harvested, unseparated neonatal rat heart cells at ~36 10 cells / cm . In brief, 10 ml of cell suspension were added to a vent - capped 50 ml tube (product #91253; tpp, trasadingen, switzerland) containing one scaffold, and tubes were placed in a 37c/5% co2 humidified incubator and gently mixed (8 rpm) using a rotisserie (labquake, barnstead - thermolyne, dubuque, ia). After 4 days, culture media were replaced and culture was continued statically for 3 more days . In studies of 16h/160c pgs scaffolds with varying pore structures, scaffolds (5 mm 5 mm) were autoclave - sterilized and sequentially seeded30 first with cardiac fibroblasts at ~1 10 cells / cm, followed by a heart cell population enriched for cardiomyocytes at ~36 10 cells / cm . In brief, 10 ml of cardiac fibroblast cell suspension was added to a 50 ml tissue culture tube containing a single scaffold and cultured with gentle mixing as described above . After 5 days, cardiomyocyte - enriched heart cells were added, and after an additional 3 days, grafts were transferred to petri dishes (costar ultra low attachment; corning, corning, ny) (one per 35 mm well) and cultured statically for 4 more days . Culture media (6 ml / well) were replaced every 2 days . Specimens were assessed using our previous method, wherein an electrical pulse generator applied biphasic square waveforms at 1, 2, 3, and 4 hz to a specimen positioned between two carbon rod electrodes within an environmentally - controlled, perfused test chamber34, 35 . Excitation threshold (et) was defined as the minimum voltage observed to initiate synchronous contractions . Video recordings made at twice the et used a 30 frame / s video camera (sony xcd - x710) and nikon diaphot microscope at 10x magnification . Cell orientation was evaluated using confocal laser microscopy with staining of filamentous actin (f - actin)39 and fast fourier transform (fft)-based image analysis40, 51 . In brief, specimens were rinsed, fixed in 10% neutral buffered formalin (sigma) for 2 h, rinsed, extracted in 0.2% (v / v) triton x-100 (sigma) in pbs for 2 h, rinsed, pre - incubated in 1% (w / v) bovine serum albumin (sigma) in pbs (bsa buffer) for 2 h, incubated in alexa fluor 488-phalloidin (2:150 (v / v) dilution; molecular probes) for 3 h, rinsed, and then incubated in draq5(25 m in pbs; biostatus limited, leicestershire, uk) for 30 min, all at room temperature . Specimens were then rinsed, bulk mounted on glass slides in vectashield medium (vector laboratories, burlingame, ca), and cover - slipped . Specimens were imaged with 25x and 100x oil immersion objectives on a zeiss lsm 510 laser scanning confocal microscope, with f - actin and nuclei pseudo - colored green and blue, respectively . Micrographs of f - actin filaments (i.e., green channel) were analyzed for cell orientation using a matlab program . Fft image analysis was similar to our previous study40, and adapted from ng et al.51 . See descriptions of fft in quantifying 2-d fiber network orientation52 and comparisons of fft to morphometry53 . Micrographs taken at ~30 m from the graft and rv epicardial surfaces were analyzed (n=6 pores; one representative sample per group). Orientation index (oi) was defined as the angular increment centered about the distribution mean encompassing 50% of the population41 . For random cell orientations oi approaches 90 degrees; for highly aligned cells oi approaches zero . Comparisons were made by one - factor (anisotropy ratio) or two - factor (other parameters) anova with tukey s post - hoc test (statistica v7, statsoft, tulsa, ok). Pgs membranes (~250 m thick) on silicon wafers16 were situated on the x - y stage (accuracy 1 m) of a rapid x 1000 system (resonetics, nashua, nh) with a 248 nm krypton fluoride lpx200 excimer laser (coherent - lambda physik, santa clara, ca). Pores were patterned via g - code programs . For highly cross - linked pgs (16 h/160c), a power of 350 mj, burst frequency of 500/sec and burst count of 1000 were used . For partially cross - linked pgs (e.g., 7.5h/160c), scaffolds were loosened from wafers in deionized water (24 h) and 70% ethanol (24 h). Bilaminar scaffolds (~400 m) with 3-d pore networks were fabricated by microablating a lateral array of troughs in one pgs lamina (burst spacing of 0.01 sec; table speed of 0.5 mm / sec), overlaying a second pgs lamina, microablating top - to - bottom pores through both lamina (burst frequency of 500/sec; burst count of 2000), and then stabilizing the resultant scaffold by thermally cross - linking17 via autoclaving (121c for 30 min). Cells were obtained from 2-day old neonatal rat hearts via an institute - approved protocol as described8 (see supplementary information, methods). In studies of pgs scaffolds with varying effective stiffness and prototype bilaminar scaffolds (5 mm 5 mm for imaging; 10 mm 5 mm for mechanics), scaffolds were autoclave - sterilized and seeded with freshly harvested, unseparated neonatal rat heart cells at ~36 10 cells / cm . In brief, 10 ml of cell suspension were added to a vent - capped 50 ml tube (product #91253; tpp, trasadingen, switzerland) containing one scaffold, and tubes were placed in a 37c/5% co2 humidified incubator and gently mixed (8 rpm) using a rotisserie (labquake, barnstead - thermolyne, dubuque, ia). After 4 days, culture media were replaced and culture was continued statically for 3 more days . In studies of 16h/160c pgs scaffolds with varying pore structures, scaffolds (5 mm 5 mm) were autoclave - sterilized and sequentially seeded30 first with cardiac fibroblasts at ~1 10 cells / cm, followed by a heart cell population enriched for cardiomyocytes at ~36 10 cells / cm . In brief, 10 ml of cardiac fibroblast cell suspension was added to a 50 ml tissue culture tube containing a single scaffold and cultured with gentle mixing as described above . After 5 days, cardiomyocyte - enriched heart cells were added, and after an additional 3 days, grafts were transferred to petri dishes (costar ultra low attachment; corning, corning, ny) (one per 35 mm well) and cultured statically for 4 more days . Culture media (6 ml / well) were replaced every 2 days . Specimens were assessed using our previous method, wherein an electrical pulse generator applied biphasic square waveforms at 1, 2, 3, and 4 hz to a specimen positioned between two carbon rod electrodes within an environmentally - controlled, perfused test chamber34, 35 . Excitation threshold (et) video recordings made at twice the et used a 30 frame / s video camera (sony xcd - x710) and nikon diaphot microscope at 10x magnification . Cell orientation was evaluated using confocal laser microscopy with staining of filamentous actin (f - actin)39 and fast fourier transform (fft)-based image analysis40, 51 . In brief, specimens were rinsed, fixed in 10% neutral buffered formalin (sigma) for 2 h, rinsed, extracted in 0.2% (v / v) triton x-100 (sigma) in pbs for 2 h, rinsed, pre - incubated in 1% (w / v) bovine serum albumin (sigma) in pbs (bsa buffer) for 2 h, incubated in alexa fluor 488-phalloidin (2:150 (v / v) dilution; molecular probes) for 3 h, rinsed, and then incubated in draq5(25 m in pbs; biostatus limited, leicestershire, uk) for 30 min, all at room temperature . Specimens were then rinsed, bulk mounted on glass slides in vectashield medium (vector laboratories, burlingame, ca), and cover - slipped . Specimens were imaged with 25x and 100x oil immersion objectives on a zeiss lsm 510 laser scanning confocal microscope, with f - actin and nuclei pseudo - colored green and blue, respectively . Micrographs of f - actin filaments (i.e., green channel) were analyzed for cell orientation using a matlab program . Fft image analysis was similar to our previous study40, and adapted from ng et al.51 . See descriptions of fft in quantifying 2-d fiber network orientation52 and comparisons of fft to morphometry53 . Micrographs taken at ~30 m from the graft and rv epicardial surfaces were analyzed (n=6 pores; one representative sample per group). Orientation index (oi) was defined as the angular increment centered about the distribution mean encompassing 50% of the population41 . For random cell orientations oi approaches 90 degrees; for highly aligned cells oi approaches zero . Comparisons were made by one - factor (anisotropy ratio) or two - factor (other parameters) anova with tukey s post - hoc test (statistica v7, statsoft, tulsa, ok).
To describe the course and outcome of treatment of advanced acanthamoeba sclerokeratitis with intravenous pentamidine . A case of advanced acanthamoeba sclerokeratitis was resistant to conventional therapy and was treated with intravenous pentamidine . The eye showed acanthamoeba organisms within the cornea and evidence of acute and chronic inflammation throughout the remainder of the eye . Acanthamoebal sclerokeratitis is an uncommon and severe complication of advanced acanthamoeba keratitis (ak).13 despite intensive medical and surgical treatment, enucleation may be required to manage this disease . Pentamidine has shown effectiveness against acanthamoeba with synergistic effects when used in combination with polyhexamethylene biguanide (phmb).410 in vitro testing may not always be associated with in vivo effectiveness.11 a 59-year - old female presented for a second opinion with previously diagnosed ak of her right eye . The contact lens type and solution were unknown, but she did wash her contact lens case out with tap water on a regular basis . She first noted pain and decreased vision in the right eye approximately 2 years earlier and saw an ophthalmologist . A ring stromal opacity was seen and she was treated for herpetic keratitis . Several months later she was diagnosed with acute retinal necrosis and was hospitalized and treated with foscarnet . The patient underwent penetrating keratoplasty in the right eye 9 months after initial treatment for presumed corneal opacity secondary to herpes simplex keratitis . Treatment was continued for 1 year when the patient presented at our institution for a second opinion . Her ocular medications on presentation were: topical phmb every 23 hours, topical propamidine (brolene) three times per day, topical moxifloxacin (vigamox) three times per day, prednisolone actetate 1% every 23 hours, and dorzolamide - timolol (cosopt) three times per day . She had apparently had multiple (1012) subtenons injections of triancinolone (kenalog) over the past year . On examination, visual acuity in the right eye was hand motion at 1 foot with light projection in all quadrants . The sclera showed moderate injection, and there was a white plaque consistent with triancinolone present beneath tenons capsule superiorly . The retina of the right eye could not be visualized, but ultrasound b scan showed a clear vitreous cavity and intact retina . Confocal microscopy (confoscan 3 nidek) of the corneal graft showed cystic structures consistent with acanthamoeba throughout the depth of the cornea (figure 2). Famcyclovir was discontinued, and topical propamidine, clotrimazole 1% and chlorhexidine 0.02% were added every hour for the first week and then decreased to every 2 hours . Prednisolone actetate 1% was reduced to four times per day, and dorzolamide - timolol was reduced to twice per day . The patient was admitted to the hospital under the infectious disease service for a course of intravenous pentamidine . The treatment was associated with nausea, but no episodes of hypotension or hypoglycemia occurred . Eleven days into this course of treatment a combined penetrating keratoplasty, cataract removal with lens implant was performed . Orbital and brain computed tomography (ct) scan and magnetic resonance imaging (mri) were performed during the hospitalization and did not demonstrate any orbital or intracranial extension . One month following her eye surgery her cornea was found to be clear with a quiet anterior chamber and posterior chamber lens implant . Her topical medications were moxifloxacin, neosporin, phmb, clotrimazole 1%, chlorhexidine 0.02%, and prednisolone actetate 1%, all four times per day . She was also using atropine 1% and dorzolamide - timolol, each twice per day . The eye remained stable, and the patient continued her medications for 6 months when she presented with a 1 week history of pain . Examination noted a 2 mm cream - colored loculated mass in the superior anterior chamber (figure 4). The mass was removed in the operating room and sent for bacterial, fungal, and acanthamoebal cultures . Cultures grew one colony of coagulase negative staphylococcus, which was felt to be a contaminant . The intraocular inflammation progressed over the next 3 weeks and a trans pars plana vitrectomy was performed with intravitreal injection of vancomycin 1.0 mg and ceftazidine 2.25 mg . At the time of this surgery the patient was again admitted and given a 4-day course of intravenous pentamidine 200 mg per day . The postoperative medications were phmb, clotrimazole, chlorhexidine, neosporin, propamidine, and prednisolone actetate, all every hour for the first week and then tapered to 4 times per day . The eye remained inflamed and painful with vision of light perception but no accurate projection . Confocal microscopy demonstrated structures within the corneal stroma consistent with acanthamoeba (figure 5). The pathology of the enucleated globe demonstrated acanthamoebal cysts within the cornea (figure 6) and evidence of acute and chronic inflammation throughout the globe but no evidence of active organisms (figure 7). The treatment of advanced acanthamoeba sclerokeratitis is a challenge.13 it is diagnostically difficult to distinguish between secondary inflammatory reaction and actual acanthamoebal infection of the sclera.3 topical treatment of the cornea may not achieve therapeutic levels in the sclera.12 systemic treatment with ketaconazole and fluconazole has been used in the treatment of acanthamoeba keratitis and sclerokeratitis.12 pentamidine has been shown to have effectiveness against acanthamoeba and has synergism with the biguanide compounds.410 the presence of chorioretinal inflammation in this eye does not necessarily indicate presence of acanthamoeba in the posterior segment . Chorioretinal inflammation has been seen with the presence of acanthamoeba13 and also as a sterile inflammation.14 the sudden development of intense ocular inflammation in this patient after apparent successful treatment of the infection led to the concern that active infection had recurred . The failure of a prolonged therapeutic response to intravenous pentamidine could indicate that the treatment was of insufficient duration, that therapeutic levels were not attained and/or maintained, or that the acanthamoeba was resistant to pentamidine . If the scleritis was an immune - mediated response,3 then aggressive systemic immunosuppression may have changed the course of this case . This was not used in this case because of the concern that the acanthamoeba was active . It is clear that the use of systemic pentamidine in this case did not effectively alter the progression of the disease and eventual loss of the eye . Perhaps earlier intervention or combined systemic antiamebial therapy and immunosuppression would achieve a better outcome . Miltefosine and voriconazole have shown strong inhibitory effects on acanthamoeba trophozoites in vitro and might be candidates for in vivo testing.15
Case subjects included 851 unrelated african americans with type 2 diabetes associated end - stage renal disease (esrd) from dialysis centers in winston - salem, greensboro, and hickory, nc . All diabetic nephropathy case subjects had esrd and were on dialysis at the time of recruitment . Diabetes was considered to be the primary cause of nephropathy if subjects developed diabetes after the age of 35 years and diabetes was present> 5 years before initiation of renal replacement therapy and/or in the presence of diabetic retinopathy or proteinuria exceeding 500 mg/24 h. an additional 317 unrelated african americans with type 2 diabetes and no renal disease, and 871 nondiabetic unrelated control subjects were recruited from medical clinics, churches, and health fairs in north carolina . Diabetic subjects lacking nephropathy had diabetes for> 10 years with a spot urine albumin - to - creatinine ratio <30 mg / g and serum creatinine concentration <1.5 mg / dl in men or <1.3 mg / dl in women . Nondiabetic control subjects were self - reported african americans born in the southeast, age 18 years, who denied a personal history of diabetes or a personal or family history of kidney disease in first - degree relatives . Dna was isolated using an autopure ls automated dna extraction robot (gentra systems, minneapolis, mn). Recruitment and sample collection procedures were approved by the institutional review board at wake forest university, and all subjects provided written informed consent . A total of 24 snps were genotyped in 1,168 african american case subjects (851 with diabetic nephropathy and 317 with type 2 diabetes lacking nephropathy) and 871 nondiabetic control subjects . Snps were chosen based on their ability to capture genetic information for african (yoruban) and european (ceu) populations in hapmap (www.hapmap.org) using tagger (haploview). Tagged snps had a minor allele frequency> 0.05 and captured an inter - snp r value> 0.8 for known polymorphisms in the region . Three snps, rs17300539, rs4632532, and rs266729, were included based on previous association with type 1 diabetic nephropathy or insulin resistance syndrome phenotypes (13,17). A total of 34 snps were initially chosen for analysis . Two snps failed design, six snps could not be incorporated into a multiplex, and two snps were eliminated because of low genotyping efficiency . The genotyped snps captured at least 64% of the variation in the yoruban hapmap sample . Snps were genotyped using the massarray genotyping system (sequenom, san diego, ca). All case and control subjects were genotyped at 70 admixture informative markers (aims) to estimate the percentage of african ancestry for each individual (18,19). The associated snp rs182052 was sequenced in 175 african americans with diabetic nephropathy to verify the accuracy of the genotype calls . The region was pcr amplified and products were purified and directly sequenced using big dye ready reaction mix on an abi3730xl sequencer (applied biosystems, foster city, ca). Sequence data were visualized using sequencher software version 4.6 (genecodes corporation, ann arbor, mi). Biometric data were compared using a kruskal - wallis one - way anova on ranks (sigmastat; systat software, san jose, ca) with a dunn's method multiple comparison test . Age at onset of type 2 diabetes in the diabetic nephropathy and type 2 diabetic case subjects was compared using a mann - whitney rank - sum test (sigmastat). Each snp was tested for departures from the hardy - weinberg equilibrium using the goodness - of - fit test in the statistical analysis program snpgwa (www.phs.wfubmc.edu) (20). Tests for genotypic association were performed on each snp individually using snpgwa - admix, a component of the snpgwa program that includes the capability to perform association calculations adjusting for covariates . If significant, then the individual genetic models (dominant, additive, and recessive) were examined for context . Percentage of african ancestry was computed from 70 admixture informative markers using the program frappe (18,19). The influence of other possible covariates (age, bmi, and sex) on evidence of association was tested using snpgwa - admix . We computed a series of cox proportional hazards models and the corresponding likelihood ratio statistics to test for associations between adiponectin polymorphisms and age at type 2 diabetes onset for 1) diabetic nephropathy case subjects, 2) type 2 diabetic (no nephropathy) case subjects, and 3) type 2 diabetic and diabetic nephropathy case subjects combined . Here, age at type 2 diabetes onset was computed for the above three conditions and was contrasted with the age of nondiabetic control subjects at time of enrollment; specifically, control subjects had age at onset of type 2 diabetes censored at age of enrollment . The hazards ratio (hr) and corresponding 95% ci was computed as exp() and exp [1.96 se ()], respectively . Tests for association and the estimates for the hazards ratio were computed without covariate adjustment and adjusting for sex, bmi, and admixture estimates . As discussed above, a total of 24 snps were genotyped in 1,168 african american case subjects (851 with diabetic nephropathy and 317 with type 2 diabetes lacking nephropathy) and 871 nondiabetic control subjects . Snps were chosen based on their ability to capture genetic information for african (yoruban) and european (ceu) populations in hapmap (www.hapmap.org) using tagger (haploview). Tagged snps had a minor allele frequency> 0.05 and captured an inter - snp r value> 0.8 for known polymorphisms in the region . Three snps, rs17300539, rs4632532, and rs266729, were included based on previous association with type 1 diabetic nephropathy or insulin resistance syndrome phenotypes (13,17). A total of 34 snps were initially chosen for analysis . Two snps failed design, six snps could not be incorporated into a multiplex, and two snps were eliminated because of low genotyping efficiency . The genotyped snps captured at least 64% of the variation in the yoruban hapmap sample . Snps were genotyped using the massarray genotyping system (sequenom, san diego, ca). All case and control subjects were genotyped at 70 admixture informative markers (aims) to estimate the percentage of african ancestry for each individual (18,19). The associated snp rs182052 was sequenced in 175 african americans with diabetic nephropathy to verify the accuracy of the genotype calls . The region was pcr amplified and products were purified and directly sequenced using big dye ready reaction mix on an abi3730xl sequencer (applied biosystems, foster city, ca). Sequence data were visualized using sequencher software version 4.6 (genecodes corporation, ann arbor, mi). Biometric data were compared using a kruskal - wallis one - way anova on ranks (sigmastat; systat software, san jose, ca) with a dunn's method multiple comparison test . Age at onset of type 2 diabetes in the diabetic nephropathy and type 2 diabetic case subjects was compared using a mann - whitney rank - sum test (sigmastat). Each snp was tested for departures from the hardy - weinberg equilibrium using the goodness - of - fit test in the statistical analysis program snpgwa (www.phs.wfubmc.edu) (20). Tests for genotypic association were performed on each snp individually using snpgwa - admix, a component of the snpgwa program that includes the capability to perform association calculations adjusting for covariates . If significant, then the individual genetic models (dominant, additive, and recessive) were examined for context . Percentage of african ancestry was computed from 70 admixture informative markers using the program frappe (18,19). The influence of other possible covariates (age, bmi, and sex) on evidence of association was tested using snpgwa - admix . We computed a series of cox proportional hazards models and the corresponding likelihood ratio statistics to test for associations between adiponectin polymorphisms and age at type 2 diabetes onset for 1) diabetic nephropathy case subjects, 2) type 2 diabetic (no nephropathy) case subjects, and 3) type 2 diabetic and diabetic nephropathy case subjects combined . Here, age at type 2 diabetes onset was computed for the above three conditions and was contrasted with the age of nondiabetic control subjects at time of enrollment; specifically, control subjects had age at onset of type 2 diabetes the hazards ratio (hr) and corresponding 95% ci was computed as exp() and exp [1.96 se ()], respectively . Tests for association and the estimates for the hazards ratio were computed without covariate adjustment and adjusting for sex, bmi, and admixture estimates . As discussed above, diabetic nephropathy and type 2 diabetic case subjects were older and more were female compared with the nondiabetic control subjects . Type 2 diabetic case subjects had increased bmi compared with diabetic nephropathy case subjects and nondiabetic control subjects . The average age at type 2 diabetes onset for case subjects with diabetic nephropathy was less than that of case subjects with type 2 diabetes without nephropathy . A total of 24 snps in the adiponectin gene were successfully genotyped in diabetic nephropathy case subjects and nondiabetic control subjects . None of the snps departed from the hardy - weinberg equilibrium after correction for multiple comparisons . Two snps, rs182052 and rs3821799, showed evidence of association with diabetic nephropathy (supplemental table 1, found in an online appendix at http://dx.doi.org/10.2337/db08-0598). Test (p = 0.002) and under the dominant model (p = 0.002, odds ratio [or] 1.37; 95% ci 1.131.67) (table 2). Snp genotype calls for rs182052 were verified by direct dna sequence analysis in 175 case subjects with diabetic nephropathy to ensure that the slight departure from hardy - weinberg equilibrium in the case subjects was not the result of erroneous genotyping . Genotypes observed from dna sequencing were 100% concordant with genotypes generated from the sequenom massarray system . The snp rs3821799 was also associated (p = 0.039) but did not retain statistical significance after adjusting for multiple comparisons . Snp rs182052 was associated with bmi and waist measures in hispanic americans in the insulin resistance and atherosclerosis (iras) family study (22). To ascertain whether the association of rs182052 with diabetic nephropathy reflected an association with bmi or other phenotypes known to be associated with increased risk of type 2 diabetes or diabetic nephropathy, genotypic association was evaluated with individual adjustments for bmi, sex, and age at exam (table 2) and showed little effect on the evidence of association . We also tested association after simultaneously adjusting for admixture, bmi, sex, and age . Evidence of association between rs182052 and diabetic nephropathy remained significant (p = 0.0019, or 1.46, ci 1.151.86) under the dominant model . To discern whether rs182052 was associated with nephropathy or diabetes in our african american sample, this snp was tested for association in 317 african americans with longstanding type 2 diabetes without nephropathy versus the 871 nondiabetic control subjects . This snp was associated with type 2 diabetes under the dominant model (p = 0.0054; or 1.46, ci 1.121.91) after adjustment for african ancestry proportions, and the association remained significant with adjustment for all four covariates (p = 0.0056, or 1.54, ci 1.132.09) (supplemental table 2). When evaluating all 1,168 african american diabetic individuals (851 with diabetic nephropathy and 317 with type 2 diabetes without nephropathy), rs182052 remained associated with type 2 diabetes under the dominant model (p = 0.0003, or 1.40, ci 1.171.67) with adjustment for proportion of african ancestry (table 2). Individual and combined adjustment for african ancestry, age, bmi, and sex did not significantly alter this result . To clarify whether the association was with type 2 diabetes only or also with nephropathy, we tested rs182052 for association between the two groups of case individuals: diabetic nephropathy and type 2 diabetes lacking nephropathy . There was no association detected (p = 0.87, or 0.98, ci 0.731.30), indicating that the rs182052 is associated with type 2 diabetes status and not nephropathy . The linkage disequilibrium structure for the adiponectin gene in african american nondiabetic control subjects is shown in supplemental fig . 1 . African americans have one large block at the 3 end of the gene and four smaller blocks surrounding it . The associated snp, rs182052, is located in a small linkage disequilibrium block containing one other known snp, rs266729, and covering the promoter . A cox proportional hazards model was used to determine the relative risk (rr) for early age at onset of type 2 diabetes for genotypes at the associated snp, rs182052 (table 3). Significant risk for earlier onset of type 2 diabetes was detected under the dominant model (p = 0.00310.0002; rr 1.261.41) for the diabetic nephropathy and type 2 diabetic case groups individually and when the two groups were combined (diabetic nephropathy + type 2 diabetes). Significant risk was also found with age at onset of esrd under the dominant model (p = 0.0113; rr 1.20). Rr increased when the data were adjusted for sex, bmi, and admixture (p = 0.0084; rr 1.21). Pearson's correlation coefficient (r = 0.56; p <0.001) indicated a positive correlation between the age at onset of type 2 diabetes and the age at onset of esrd, suggesting that early age of onset of esrd may depend on an early age of onset of type 2 diabetes . We confirmed this by calculating the rr of age at onset of esrd after adjustment for age at onset of type 2 diabetes and saw no significant risk (table 3). When the survival distribution function is plotted versus the age at onset of type 2 diabetes (fig . 1), individuals with the minor allele a (a / a or a / g genotypes) have an earlier age at onset of type 2 diabetes compared with individuals who are homozygous for the g allele (g / g genotype). We have evaluated association between adiponectin gene polymorphisms and type 2 diabetes and diabetic nephropathy in a large sample of african americans . We observed evidence of association between the rs182052 polymorphism in intron 1 in african americans with type 2 diabetes . This evidence of association remained significant after adjustment for african ancestry, age, bmi, and sex . In the fully adjusted models, the p value for association of rs182052 with type 2 diabetes was 0.0005, a level of significance that survives even stringent bonferroni adjustment . Survival analysis revealed association between the minor allele and earlier age at onset of type 2 diabetes . Previous studies of association between adiponectin polymorphisms and type 2 diabetes or diabetic nephropathy have evaluated european - derived or asian populations, with only one study in non - european south africans (14) and one study with a small number of african americans (15). To our knowledge, this is the first study that has addressed adiponectin gene polymorphism associations with type 2 diabetes and diabetic nephropathy in african americans . In our case samples, this may be the result of differing allele frequencies in the european and african ancestral populations . In hapmap (www.hapmap.org), the yoruban population has a minor allele frequency of 0.395 at this snp, whereas the european population has a minor allele frequency of 0.353 . This difference underscores the importance of including adjustment for african ancestry in analyses of african american populations . In our case subjects and control subjects, the percentage of african ancestry was similar between the two groups of case subjects and the control subjects (80 10 to 82 10). This study was well powered to detect associations consistent with complex genetic traits with over 1,168 (combined) type 2 diabetes and diabetic nephropathy case subjects and more than 871 nondiabetic control subjects . With this large sample size, we had> 80% power to detect an odds ratio of 1.31.5 under the dominant model of association within a range of minor allele frequencies (0.10.3). While a number of the genotyped snps had low minor allele frequencies (<0.05) and would contribute to a reduction in power, the associated snp was relatively common (minor allele frequency = 0.348 in control subjects) and the low p value contributes a high level of confidence in this association . Previous studies have reported association between adiponectin polymorphisms and type 2 diabetes (14,15,2326) and diabetic nephropathy (13). These studies have primarily detected association with snps in the promoter region (rs17300539 and rs266729) or in exons (rs22517766 and rs1501299). With our african american sample, we have the ability to detect association with both type 2 diabetes and diabetic nephropathy . We tested three of these snps (rs17300539, rs266729, and rs1501299) and found no significant evidence for association . That the association in our african american collection is at a different snp is not unexpected . This may reflect ethnic differences in adiponectin gene structure, as discussed by ukkola et al . The lack of two distinct linkage disequilibrium blocks in the adiponectin gene in our african american sample, compared with that previously demonstrated in european - derived samples (27), also supports this conclusion . (28) identified nine snps in the adiponectin gene that had significantly different minor allele frequencies (p <0.001) between african americans and european americans . These genetic data are further supported by evidence that african americans have reduced plasma adiponectin concentrations than other ethnic groups (europeans, japanese, and pima indians) (29). The potential for ethnic differences in the adiponectin gene emphasizes the need to study genetic associations in multiple populations, particularly african americans . Whereas association with type 2 diabetes has not been previously reported with rs182052, this snp has been associated with bmi and waist measures in hispanic americans in the insulin resistance and atherosclerosis family study (22). To ascertain whether the association we observed was the result of type 2 diabetes and not increased bmi, we adjusted for bmi in the association analysis . The evidence of association remained significant after bmi adjustment, indicating that the difference in adiposity between case subjects and control subjects is not driving the association and that the mechanism is via some other pathway . In addition, rs182052 is in the same haplotype block as rs266729 (supplementary fig . 1) in african americans, an snp with prior reports of association with type 2 diabetes (11). Haplotype analysis of these two snps revealed no association with type 2 diabetes or diabetic nephropathy (supplementary table 3). (27) have demonstrated association between the minor allele of rs182052 (a) and reduced levels of plasma adiponectin in european - derived american adolescents and healthy europeans, respectively . In our study, individuals with the minor allele (a / a or a / g genotypes) had earlier onset of type 2 diabetes . If the minor allele is contributing to type 2 diabetes in our samples by reduced levels of plasma adiponectin, this would be consistent with previous studies that have demonstrated an association between hypoadiponectinemia and increased risk of type 2 diabetes (24,30). We do not have serum adiponectin concentrations for the subjects in our study; however, it would be interesting to determine whether rs182052 contributes to plasma adiponectin concentrations in our collection of african americans . (28) detected no association between rs182052 and plasma adiponectin levels in a comparably sized sample of african american adolescents . A primary issue with the observations we have made is whether the observed association reflects an association between rs182052 and type 2 diabetes, diabetic nephropathy, or both . We have sampled a relatively large number of african american subjects with diabetic nephropathy and a smaller collection with type 2 diabetes and no nephropathy to attempt to ascertain whether this gene is associated with nephropathy or diabetes . As the snp was associated in subjects with diabetic nephropathy and subjects with type 2 diabetes lacking nephropathy, we believe that the data are most consistent with the rs182052 variant contributing to type 2 diabetes susceptibility in african americans . This is supported by the cox model's rr, which indicated increased risk for earlier age at onset of type 2 diabetes . Additionally, we compared allele frequencies of rs182052 between the diabetic nephropathy case subjects (n = 851) and the type 2 diabetic (no nephropathy) case subjects (n = 317) and observed no evidence of association . Although this analysis has less power than the initial case - control analysis, it does support our conclusion that rs182052 is associated with type 2 diabetes in african americans . We observed association between a variant in the adiponectin gene and type 2 diabetes in african americans . This snp, rs182052, has not previously been associated with diabetes or diabetic nephropathy in other populations, although it has been associated with plasma adiponectin levels . That other variants previously associated with diabetes or diabetic nephropathy in europeans were not associated in our african american population emphasizes the potential that there are likely ethnic differences in the linkage disequilibrium structure of the adiponectin gene.
Smoking is known for its negative impact on health being tightly connected to early ageing, carcinogenicity, high risk of lung and cardiovascular diseases, and early mortality . Extensive research in recent years indicates a tight connection between cigarette smoking and rheumatoid arthritis (ra). The prevalence of cigarette smoking appears to be higher in patients with ra . At the preclinical and early stages of the disease smoking has been suggested to trigger the immune reactions controlled by hla - drb1 genes, which initiates joint inflammation and production of rheumatoid factor (rf) and antibodies to citrullinated peptides, classical serologic biomarkers of ra [4, 5]. In the established ra, cigarette smoking is related to the progressive joint damage, persistent disease activity, and development of rheumatic noduli [6, 7]. These reports are met by controversy from other studies, which reported a lower radiographic disease progression and no significant effects on disease activity in the smoking patients with ra [810]. In addition, nicotine exposure through smokeless tobacco was not associated with risk of ra . Thus, the molecular events connecting cigarette smoking to severe joint damage and low efficacy of antirheumatic drugs indicates the role of combined biological mechanisms triggered by smoking during arthritis . Smoking is inversely associated with circulating levels of igf1 indicating a direct inhibitory effect [12, 13]. Igf1 is an important mediator in developmental processes including proliferation, growth, differentiation, and survival [14, 15]. Systemic and paracrine igf1 deficiency is recognized by enhanced skeletal metabolism and progressive osteoporosis, development of glucose intolerance, premature atherosclerosis, and cardiovascular mortality . Modern understanding of molecular mechanisms of igf1 suppression by nicotine is connected with dysfunction of the hypothalamus - pituitary axis, where growth hormone, neuropeptide y, and adipokine leptin have central role . In human and in experimental ra, the alterations of the igf1 system encompass the reduction of igf1 levels [19, 20] and high expression of igf1 binding proteins, which reduces bioavailability of igf1 [2022] despite the increased density of igf1 receptors . The impairment of igf1 system is viewed as a result of cytokine - driven chronic inflammation . Clinically, ra patients with low levels of igf1 have higher disease activity and are prone to cachexia . However, the alleviation of inflammation with the antirheumatic treatment including corticosteroids and tnf inhibitors seldom restores igf1 system in ra patients . We hypothesise that the competitive binding and activation of the insulin / igf1 receptor (igf1r) by several members of the adipokine family including leptin, resistin, and visfatin [20, 24, 25] disturb normal function of igf1 system in ra preventing positive feedback . In ra, adipokines have been connected to the progressive joint destruction . The levels of leptin showed a negative and positive association with the radiographic damage . Additionally, the baseline levels of adipokines may predict the effect of antirheumatic treatment [31, 32]. In the present study we asked if smoking was associated with the changes in igf1 and could mediate the adipokine dependent mechanisms of inflammation in ra . The evaluation of two independent ra cohorts convincingly demonstrates that smoking is an essential factor contributing to low levels of igf1 and leptin . This novel association between smoking, igf1 and hypoleptinemia in ra may be of importance for long - term prognosis of the disease and for prediction of comorbidities . 570 patients from 2 independent ra cohorts (gothenburg, n = 367, and leiden, n = 203) were included in this cross - sectional observational study . Twenty - seven patients (4.7%, 17 patients of gothenburg and 10 patients of the leiden cohort) were excluded because of unknown smoking status or use of snuff resulting in 543 patients in total . Written informed consent was obtained from the participants . The gteborg cohort comprised the patients with rheumatoid arthritis who attended the rheumatology clinics at the sahlgrenska university hospital, gothenburg, and the rheumatology unit of the uddevalla hospital and were current users of methotrexate . The patients of leiden cohort were randomly selected from the leiden early arthritis clinic cohort . The study was approved by the ethics committee of sahlgrenska university hospital and the leiden university hospital . Patients were asked to report their smoking habits through a structured telephone interview or by filling in a questionnaire . Smoking was considered if the patients smoked or had smoked for the past year, based on self - report . Patients were divided by their smoking habits defined as current smokers, former smokers, or never smokers . Subjects who smoked previously and stopped smoking longer than 1 year before the study were considered former smokers . Blood samples were obtained from the cubital vein and centrifuged at 800 g for 15 min, aliquoted, and stored frozen at 70c until use . Biological markers were analysed by sandwich enzyme - linked immunosorbent assays (elisas) using matched pairs of specific antibodies and recombinant standards . Assays for human adiponectin (dy1065, 62 pg / ml), human leptin (dy398, 31 pg / ml), human resistin (dy1359, 10 pg / ml), and human free bioactive igf1 (dy291, 4 pg / ml) were all purchased from rnd systems (minneapolis, mn, usa). Assays specific for human visfatin (ag-45a-0006tp - ki01; 125 pg / ml) were purchased from adipogen, inc . Elisas were read with a spectramax 340 from molecular devices (sunnyvale, ca, usa). The levels of rheumatoid factor (rf) antibodies were measured at the clinical immunology laboratory of the sahlgrenska university hospital or leiden clinical immunology laboratory . Descriptive data are presented as the median, the interquartile range, the number, and the percentage . The material was stratified by smoking history into the patients who never smoked (nsm, n = 240), current smokers (sm, n = 126), and former smokers (fsm, n = 177). Bivariate correlation between variables was examined by spearman's correlation test . The differences between groups were assessed by the mann - whitney u test or kruskal wallis test followed by dunn's multiple comparisons test . The sensitivity and specificity of calculations were performed using 2 2 table analysis and chi - square tests . To compensate for the absence of established cut - off points and to retain adequate statistic power, the material was categorized by tertiles within each cohort . For the binary logistic regression predictive value of smoking and adipokines for low levels of igf1 and leptin was calculated by composing binary logistic regression models where low igf1 or low leptin were chosen as the dependent variable and adipokines in tertiles, smoking habits, gender, and age as independent variables (n = 516). A second binary logistic regression set was performed where the clinical parameters disease duration, rf positivity, das28, vas - pain, and bmi together with gender, age, and smoking habits, were added as independent variables (n = 246). The leiden cohort comprised patients with early ra and had lower disease duration (p <0.001), higher disease activity (p <0.001), and lower prevalence of rf - positive patients compared to the gothenburg cohort of patients with established ra . The two cohorts were similar in gender composition and in bmi . Within the smoking groups, the leiden cohort was sharply stratified into current smokers (sm) and never smokers (nsm) (56%), while the gothenburg cohort had a substantial proportion of former smokers (fsm) (43%). In both cohorts, sm were more often men (leiden, 46% men versus 22% women; gothenburg, 23% men and 18% women), while fsm were older (p = 0.006), had longer disease duration (p <0.001), and lower das28 (p = 0.05), and were more often rf - positive (p = 0.04) than nsm . The highest serum levels of igf1 were measured in nsm and they gradually declined with smoking . Additionally, sm and fsm had higher probability to have igf1 levels within lowest tertile compared to nsm (sm: or 2.24 [1.383.65], p = 0.0012; fsm: or 1.85 [1.182.89], p = 0.0071). Bmi, a surrogate measure of adipose tissue volume, showed no correlation with the igf1 levels (rho = 0.053) and was similar between the smoking groups (figure 1(f)). Correlations between bmi and adipokine levels are shown in supplementary table s1 (see supplementary material available online at http://dx.doi.org/10.1155/2016/3082820). The levels of leptin and adiponectin were lower in sm compared to nsm (figures 1(b) and 1(c)), while the serum levels of resistin and visfatin were similar between the smoking groups . Bivariate correlation analyses revealed that nsm presented no or only a weak association between igf1 and adipokine levels . In sm, igf1 was associated with leptin (rho = 0.233, p = 0.009) and resistin (rho = 0.210, p = 0.018). Nsm had direct association between serum leptin and das28 (rho = 0.216), while in sm this association became inverse (rho = 0.198). The difference in associations of leptin and das between sm and nsm was significant (p = 0.024) (figure 2(a)). Similar smoking dependent difference was seen in associations between leptin and vas - pain (p = 0.056) (figure 2(b)). The positive association between leptin and resistin was present in all smoking groups suggesting concordant effect of smoking on these parameters (figure 2(c)). To identify independent variables predicting low serum levels of igf1, a binary logistic regression with backwards elimination was performed . In the first step of the model, the adipokine levels in tertiles, gender, age, and smoking groups were introduced . The importance of each variable was verified using wald statistics and the variables of no importance (p> 0.1) were eliminated . The last step of the elimination analysis indicated that current smoking (or 0.54 [95% ci 0.340.86], p = 0.009) and low levels of leptin (or 0.73 [95% ci 0.570.93], p = 0.010) were predictive of low levels of igf1 . Adiponectin was eliminated from the regression model and showed no association with low igf1 levels . The complete table of elimination steps is available in supplementary table s2 . In the second model, the clinical variables (dd, rf - positivity, das28, vas - pain, and bmi) and the smoking groups were added to the analysis . The last step of the elimination demonstrated that present smoking (or 0.39 [95% ci 0.180.84], p = 0.016) and high vas - pain (or 0.986 [95% ci 0.9731.000], p = 0.044) were the independent variables to predict low igf1 in ra patients (supplementary table s3). Since low serum levels of leptin appeared to be an important predictor of low igf1, a different binary logistic regression model was constructed to understand which clinical parameters were associated with the low levels of leptin . The disease duration, das28, age, smoking groups, rf - positivity, bmi, vas - pain, and gender were included in the regression model . The final step of the backward elimination showed that high das28 (or 0.82 [95% ci 0.671.01], p = 0.060), high bmi (or 0.74 [95% ci 0.670.83], p <0.001), and female gender (or 14.12 [95% ci 5.9133.73], p <0.001) increased the probability of low leptin levels (supplementary table s4). The results of our study indicate that cigarette smoking may be a major cause of acquired igf1 and leptin deficiency in ra patients (figure 3). The multivariate analysis revealed the existing association between these parameters where hypoleptinemia was also shown to be a consequence of higher disease activity . These observations are supported by the reports of an increase in the circulating levels of leptin with a decline of inflammation in ra [34, 35] and with successful antirheumatic treatment . After adjustment to inflammation leptin still showed a negative association with the joint damage . In preclinical setting, both the leptin deficient and leptin receptor deficient mice exhibited a delayed resolution of arthritis, and leptin supplementation was reported to ameliorate arthritis . Similar to ra, the importance of leptin has been demonstrated in cardiovascular disease [3840], malnutrition, and sepsis, where low serum levels of leptin are unfavourable for survival . These observations indicate that leptin is not a simple reflection of inflammation being rather engaged in the network of molecular processes in ra pathogenesis . The physiological role of leptin is currently associated with regulation of the neuroendocrine functions including hypothalamic - growth factor axis and insulin sensitivity with consequences for the energy balance . Stimulation of the growth factor - igf1 mediated effects makes leptin essential for bone formation acting directly on the bone remodelling cells . In women with hypothalamic amenorrhea, hypoleptinemia is considered the major molecular mechanism of negative bone metabolism in women [42, 43], and leptin supplementation enhanced bone formation . Leptin has been shown to be essential for development of nave t - cells and immature b - cells supporting intracellular activation of the mechanistic target of rapamycin complex 1 (mtor) mediated processes . This mechanism explains the controls of leptin over t - cell receptor - dependent functions and the balance between the regulatory t - cells and th17-cells . Leptin - induced mtor activation defines a specific molecular and transcriptional signature controlling cd4 + effector t - cell responses . Thus, smoking - induced low levels of igf1 and leptin may support the aberrant t - cell formation at the preclinical stage of arthritis, in the poor bone remodelling and progressive joint damage in the overt arthritis, and for the early cardiovascular mortality in ra . There are opposing reports on the local intra - articular effects of leptin and its role in cartilage remodelling . In several experimental studies, leptin is shown to be pr - inflammatory and associated with cartilage damage in antigen - induced arthritis [4850]. Other studies reported potential protective effect of leptin for the joint cartilage especially in the setting of acute inflammation . Clinical studies followed similar lines; three studies showing high levels of leptin were associated with joint destructive disease [22, 51, 52]. Interestingly, intra - articular levels of leptin were reported to be low compared to serum levels favouring the hypothesis on the cartilage - protective nature of leptin inside the joints . The prospective case - control study showed a negative association between serum leptin and the cartilage changes measured by larsen score and adjusted for inflammation; a different study reported that high leptin was associated with radiographic progress of joint damage . Three other studies found no association between serum leptin and progress in radiographic changes using longitudinal analysis [26, 27, 53, 54]. The present study suggests a connection between leptin and neuroendocrine processes regulated by igf-1 in ra since the levels of leptin together with smoking appeared to be the major denominator of igf1 levels . The observational design of this study reveals association between leptin and igf1 without studying their mutual dependence . A bidirectional activation of leptin - igf1 signalling has been demonstrated experimentally, where leptin competes with igf1 for binding to igf1r, and igf1 in turn activates (phosphorylates) the leptin receptor . In the clinical setting, acquired and congenital hypoleptinemia is followed by a decrease in igf1 levels and leptin treatment efficiently restores igf1 levels . The effects of leptin on the levels of igf1 occur independently of growth hormone, since the increase of igf1 during growth hormone supplementation was associated with a decrease of leptin [5557]. Stratification of smoking groups is done on the basis of self - reported smoking habits, which may present a source of bias in our study . Accuracy of self - reported smoking varies between studies and is shown to be lower in younger individuals and in socially deprived areas . None of these parameters are applicable in the studied cohorts and enhances confidence in our observations . The use of smokeless tobacco, a common substitute for cigarette smoking in sweden, is another potential source of bias . The two independent patient cohorts included in the analysis differed with respect to smoking, where the prevalence of former smokers was higher in the gothenburg cohort . The trichotomization of the whole material permitted the independent analysis of current and former smoking on the low igf1 levels and showed concordant results . Selection bias may be expected in this study since the gothenburg cohort has been collected among the methotrexate users where patients with less severe course of ra are underrepresented . However, the cohorts of this study are representative with respect to rf, which permits an assumption of sufficient balance in disease severity . Importantly, the adjustment for rf - positivity did not affect the observed associations between smoking, igf1 and leptin and makes extrapolation of the results possible on both rf - positive and rf - negative ra populations . The combination of two independent clinical materials adds certain strength and weakness to the study . On one side, it introduces heterogeneity with respect to disease duration and disease activity, also with respect to experience of different treatment modalities . On the other side, in conclusion, this cross - sectional study identified smoking as an important predictor of low igf1 and low leptin in ra patients . Importantly, this association existed independently of age, gender, and bmi and may be of importance for the long - term prognosis in ra and development of comorbidities.
Evaluation of her current complaint revealed a mobile urethra, stable cystometrogram, and complete bladder emptying . She had a positive cough stress test with a valsalva leak point pressure of 119 cm h2o with 150 ml of fluid in the bladder . One hour after surgery, she complained of lower abdominal pain, a feeling of lower abdominal distension, and nausea . Her hemoglobin changed from 12.7 g / dl preoperatively to 8.3 g / dl about 2 hours postoperatively . Computed tomography (ct) revealed a retropubic hyperdense hematoma sized up to 10 cm9 cm8 cm and active contrast leakage from the left internal pudendal artery (fig . The arteriography also confirmed left internal pudendal arterial injury, which was successfully treated by selective embolization with a gelatin sponge (gelfoam, pfizer inc ., new york, ny, usa)) with 25% n - butyl - cyanoacrylate (fig . The patient's postoperative course was uneventful requiring neither blood transfusion nor surgical removal of the hematoma . Two years after surgery, her stress incontinence was resolved, and she complained of mild urge incontinence . Since the tvt procedure was introduced, it has become the gold standard for the treatment of female stress urinary incontinence . Although this method is minimally invasive, various complications have been reported even though the complication rate is low . New minitapes, such as tvt - secur, may be a way to reduce such perioperative complications . The tvt - secur system was developed with the intention of further reducing the invasiveness of the surgical procedures by avoiding the passage of the needle carriers through the retropubic or obturator regions . The advantages of this new technique are thus related to the short passage of the needles, which minimizes the risk of vascular, nerve, or visceral injury . The tvt - secur system was designed to overcome two of the perioperative complications reported with the use of the tvt - obturator: thigh pain and bladder outlet obstruction . These complications were addressed by tailoring the tape to a length of only 8 cm and anchoring the tape edges into the internal obturator muscle, rather than passing it through the obturator foramen, muscle, and membrane . However, a possible reason for hemorrhagic complications of the tvt - secur procedure may be the scalpel - shaped tip of the inserter, which can cut the vessels . Minor intraoperative bleeding from the initial vaginal dissection can usually be controlled with direct pressure on the paraurethral and retropubic areas, held for 5 to 10 minutes and followed by vaginal packing . Greatly increased bleeding that results in a retropubic hematoma usually arises from a venous injury during trocar passage, and up to 2.8% of these cases may require transfusion . Depending on the size of the hematoma, signs and symptoms may include pain, urgency and frequency from bladder compression, a suprapubic mass, hypotension, tachycardia, lightheadedness, vaginal and abdominal tenderness, and a decrease in hematocrit . Interestingly, specified bleeding from an internal pudendal artery has not been reported previously with the tvt or tvt - obturator technique . Reported in research carried out on 141 cases of tvt - secur that no injuries to the vessels and no hematoma were observed . . Demonstrated that no bleeding or hematoma occurred during their 91 cases of the tvt - secur procedure . However, severe bleeding from the internal obturator muscle and injury of the corona mortis after tvt - secur have been reported . If abnormal abdominal pain or abdominal distension appears after the tvt - secur procedure, a ct scan should be performed . The patient should also be carefully examined if other complications occur, such as buttock or groin pain, signs of shock, vaginal bleeding, or urinary retention . To decrease the risk of vascular injury, the pelvic surgeon should pay attention to proper technique during the approach and have a high clinical suspicion of complications under the appropriate circumstances.
Mycosis fungoides palmaris et plantaris (mfpp) is an uncommon variant of mycosis fungoides (mf) limited to the palms and soles, first described by resnik et al . In 1995 . Since only 10 english papers about mfpp have been previously published, little is known about its pathogenesis . In this report, we describe a case of mfpp showing a dense deposition of periostin (postn) in the cancer stroma, which might modulate the tumor microenvi ronment through tumor - associated macrophages (tams). Our present case might suggest one of the possible reasons for the good prognosis of mfpp . An 85-year - old japanese man visited our outpatient clinic with a 2-year history of pruritic erythema on his scalp . His condition had been treated in a private clinic as pustulosis palmaris et plantaris with topical steroid and maxacalcitol, but they were ineffective . On his initial visit, physical examination revealed erosive, atrophic, well - demarcated scaly erythema on his right palm and right sole (fig . A biopsy specimen showed atypical large lymphocytes densely infiltrated mainly in the upper dermis with involvement of the overlying epidermis (fig . Immunohistochemical (ihc) staining revealed that these atypical lymphocytes, which were distributed from the upper layer of the stratum spinosum to the dermis, were positive for cd3, cd4 and cd5, and negative for cd7 and cd8 . A full blood count and biochemical profile revealed eosinophilia (19.7%), and a significantly high level of serum il-2 receptor (925 u / ml). We screened for possible metastatic lesions with positron emission tomography and computed tomography but found no evidence of metastasis . From the above findings, we diagnosed this patient as having mfpp . We administered radiation therapy 40 gy in 20 fractions to the right palm and sole, and the erosive, atrophic erythema then disappeared . To further investigate the possible immunological background of the mfpp, we employed ihc staining for postn, cd163 and cd206 . Ihc staining revealed the dense deposition of postn within the superficial to deep dermis (fig . Mfpp is a rare variant of mf limited to the palms and soles [2, 4, 5]. Since only a small number of cases have been reported in english, the pathogenesis and biological behaviors of mfpp are still incompletely understood . Among the published reports, nakai et al . Reviewed 24 cases of mfpp, which suggested that patients with mfpp present a relatively good prognosis, and that radiotherapy is one of the best therapies for mpff . Excimer laser therapy (308 nm) and bexarotene are also effective [4, 5] and, to our knowledge, no patients have died from disease progression . Postn is an extracellular matrix protein that modulates the production of a series of chemokines from macrophages and fibroblasts [3, 6]. As we previously reported, the deposition of postn is prominent in early stage (patch to plaque stage) of conventional mf . In addition, previous reports also suggested that in vitro stimulation of monocyte - derived macrophages by postn induces the production of chemokines such as cxcl5 and cxcl10, which correlates with the tumor - formation of mf in the early stage . In another report, zhou et al . Reported that postn secreted from glioblastoma stem cells increases the m2 tams in the tumor site, resulting in the growth of glioblastoma multiformes . These reports suggest that the postn / tam axis might contribute to the progression of the early stage of mf . In our present case, unlike conventional mf in the tumor stage, the dense infiltration of atypical lymphoid cells as well as dense deposition of postn were observed throughout the dermis . In addition, the main population of tams was cd163cd206 macrophages, which suggested that tams were not polarized to m2 macrophages in mfpp . As a previous reports suggested, m2 macrophages produce ccl17 and ccl22 [9, 10, 11], which recruit regulatory t cells (tregs) in the tumor microenvironment . Since a large population of tregs is associated with a poor prognosis in various types of human cancer, and the depletion of m2-like tams delays the cutaneous t - cell lymphoma development in vivo, the inhibition of m2 polarization in tams might be connected with the progression of mf . Concerning our present case, the phenotypes of tams were not polarized to the m2 phenotype, which might be responsible for the good prognosis in mfpp . Since this report presented a single case of mfpp, and since we did not directly prove the relationships between immunomodulatory effects of postn and prognosis of mfpp, further analysis of the mechanisms underlying this phenomenon may provide fundamental insights into the mechanisms of postn with mfpp.
Joint contracture tends to increase progressively in severity as the joint is kept immobile for a prolonged period of time under the influence of joint fixation or illness . One type of joint contracture is acquired contracture, which develops after birth in the presence of some factors . Depending on the factors responsible, acquired joint contracture has been subdivided into cutaneous contracture, connective tissue contracture, myogenic contracture, neurogenic contracture, and arthrogenic contracture (contracture of joint origin)1 . Of these types of joint contractures, cutaneous contracture is usually considered to develop from skin burns, injury, scarring, etc2 . Stretching of the affected skin in cases of burns has been used as a means of treating scar contracture . This therapy has been shown to be effective at improving the extensibility of scarred tissue3 and expanding the area of the scarred tissue4 . These previous reports indicate the efficacy of skin stretching on cutaneous contracture arising from scarring . It has also been shown that the skin is involved in disuse joint contracture arising from joint fixation or immobility5,6,7 . Our previous study demonstrated that 2 weeks of joint fixation reduced the extensibility of the overlying skin8 . Joint movements are normally accompanied by stretching of tissues such as the skin and muscles; however, even in cases when a joint is impossible to move, the skin retains its capability to stretch without moving the joint . Maintaining the extensibility of the skin may potentially minimize the progression of joint contractures, even when the joints cannot be moved . However, no previous study has elucidated whether skin extensibility can be maintained by stretching the skin without performing any joint movement . The present study was designed to examine the possibility of maintaining the skin extensibility through stretching of the involved skin in cases of disuse joint contracture . The animals used in this experiment were 8-week - old female wistar rats (n=18). The animals were housed in cages and given free access to food and water . The environment of the animal care room was maintained at a constant room temperature of 23 c with an air conditioner, and day and night were artificially regulated by setting lights - on for 12 h and lights - off for 12 h each day . This animal experiment was conducted in accordance with the national institute of health (nih) guide for the care and use of laboratory animals, and the experiment was carried out with the approval of the research ethics committee of the prefectural university of hiroshima . Six rats were allocated to the control group, which received no intervention for the right ankle joint . Another 6 rats were allocated to the fixation group, in which the right ankle joint was fixed in maximum plantar - flexion with a cast for one week . The remaining 6 rats were allocated to the stretching group, which received fixation of the right ankle joint in maximum plantar - flexion with a cast, and stretching of the dorsal skin over the ankle joint once daily for 30 minutes with the cast removed during the skin stretching, which was performed without joint movement . Pentobarbital (40 mg / kg) was injected intraperitoneally (i.p . ), and under anesthesia the ankle joint was held in full plantar flexion and immobilized with a plaster cast from the thigh to the foot area9 . In order to prevent the cast from breaking or falling off, it was covered with a stainless steel net . During the fixation period, close attention was paid to the onset of edema and cast loosening, and the cast was renewed as needed . In the stretching group, skin stretching was performed after removal of the net and the cast, under anesthesia induced by intraperitoneal injection of pentobarbital sodium (40 mg / kg, i.p . ). During the stretching, the rats were placed in the lateral decubitus position, with the right ankle joint placed upwards and kept in maximum plantar - flexion to avoid ankle joint movement . Stretching was carried out using an unstretchable segment of tape which could be attached to the skin, so that only the skin could be stretched, without any motion of the joint . One tape was attached to each of the proximal and distal parts of the rat s right achilles tendon . The skin was stretched along the major axis of the leg in both the proximal and distal directions . A stretching force equal to 0.3 n was applied with the use of a spring scale, following the method of a previous study10 . Skin extensibility was evaluated using a tensile strength testing machine (autograph tensile tester model ag-50kng, shimadzu corporation) on the last day of the experiment8 . The skin sample for the test was obtained from over the achilles tendon . During maximum plantar - flexion of the ankle joint, the skin was marked at 2 points: point a, 3 mm distal to the heel; point b, 10 mm proximal to point a. the length of the skin sample between the distal end (5 mm distal to point a) and the proximal end (5 mm proximal to point b) was 20 mm and its width was 4 mm . Holes were made at points a and b of the skin sample, and an unstretchable stainless steel wire was inserted through the holes, followed by fixation of both ends of the wire on the tensile testing machine with the use of a clamp . The test start point of the sample was adjusted with calipers, so that the distance between the two points of wire insertion was 10 mm (the distance between the two marked points). The tensile strength test was conducted once on each skin sample, with the starting distance of the stretch set at 0 mm and the starting tension at 0 n. the extent to which the skin was stretched by the force of 0.3 n from the start point was measured as an indicator of the skin extensibility . The non - parametric kruskal - wallis test was used to compare skin extensibility . A significant difference was concluded to exist at a probability value of less than 5% in all of the statistical tests . The results of the skin extensibility test are shown in table 1table 1.the changes in skin extensibilitygroupdistension distance (mm)control5.1 0.7fixation2.8 0.4 * stretching3.7 0.4 * values are mean sd . * significant decrease compared to the control group (p<0.05) significant increase compared to the fixation group (p<0.05). Statistical analysis of the data revealed significant differences in the skin extensibility among the three groups . The skin extensibility was significantly lower in the fixation and stretching groups than in the control group . Significant decrease compared to the control group (p<0.05) significant increase compared to the fixation group (p<0.05) this study examined whether or not skin stretching, without any joint motion, would allow skin extensibility to be maintained . Stretching is usually performed to cause the soft tissues to be stretched while moving the joint . The effects of stretching have been evaluated in previous experimental studies11, 12 . In previous experiments using animals, the ankle joints of rats were fixed in maximum plantar - flexion with the use of a cast and the range of motion of the ankle joint was measured after repetition of once daily stretching of the plantar muscles originating in the ankle joint with the cast removed during stretching . In these experiments, continuous stretching (30 min / day) resulted in suppression of the onset of joint contracture13 . In human experiments measurement of the passive torque after repeated stretching of the ankle joint in the direction of dorsiflexion (30 min / sess) showed there was a decrease in the passive torque in this direction14 . In these past experiments, the efficacy of stretching while the joint was moved was manifested by improvement of the muscle extensibility, and the target of the stretching was the muscles . However, it has been reported that the tissues responsible for joint contracture are not confined to skeletal muscles, but include the skin as well5,6,7 . As is the case with skeletal muscle, the skin can also be stretched by the motion of stretching while moving the joint . In cases where the joint is forced to be fixed with a cast, stretching of the skin without the joint moving at the same time is possible if a window is opened in a part of the cast . Therefore, unlike in previous studies, it was determined that in this study, the effects of stretching exercises targeting exclusively the skin would need to be examined by carrying out those exercises without joint movements to prevent them from exerting effects on the muscles . The results of the present study indicate that skin stretching without joint motion can also maintain skin extensibility . Tissue expansion was previously reported as a method of stretching the skin without moving the joint . In the technique of tissue expansion, a capsule is inserted under the skin and gradually inflated to cause stretching of the skin and soft tissues15 . Used a tissue expander in male patients with scarring alopecia caused the skin of the hair - covered scalp to stretch and replace the skin of the area of hair loss16 . Used a tissue expander in patients with nevus to stretch the skin of the inguinal and hypogastric regions, followed by use of the stretched skin to cover the nevus - affected area17 . The results of these studies indicate that tissue expansion can improve skin elasticity through stretching of the skin . However, because tissue expansion involves surgical stress, it is never used to suppress the onset of disuse joint contracture . The present study is significant in that it revealed the efficacy of skin stretching on the body surface without movement of the joint . The reported changes in skin area stretched by the tissue expansion technique include epidermal cell proliferation18, epidermal thickening19, dermal thinning20, etc . The improvement in the skin extensibility observed in the present study, following stretching without simultaneous joint movement, seems to be explained by changes in the skin similar to those reported by previous studies . However, although morphological changes and biochemical changes should have been examined, these were beyond the scope of our study . As a result morphological and biochemical changes in the skin will need to be investigated after performing stretching exercises without joint movements.
Paired blood and urine samples from 39 patients who were consecutively referred to the outpatient clinic of the regional reference center for multiple sclerosis of the careggi university hospital, florence, italy, were prospectively collected . The patients were recruited according to the following inclusion criteria: definite diagnosis of ms; relapsing - remitting course; fulfillment of the european medicines agency indications for natalizumab administration in ms; and written informed consent to participate in a program for pml risk stratification established in collaboration with the marketing authorization holder (mah) of natalizumab and based on a centralized anti - jcpyv ab assay in sera and on a locally organized jcpyv dna analysis in blood and urine . The results included in the present study were generated by analyzing the diagnostic data reported in the clinical records . The study was approved by the local ethic committee (comitato etico azienda ospedaliero - universitaria careggi, largo brambilla 3, firenze) according to the italian law requirements (n. oss 14.073). The authors obtained preventive unrestricted permission for analyzing the data provided by the mah of the anti - jcpyv ab assay . The jcpyv dna was extracted from peripheral blood mononuclear cells (pbmc) obtained from enriched buffy coats after density gradient separation of 8 ml of whole blood, 200 l of plasma, and 200 l of urine, and was quantified by jcpyv - specific real - time pcr targeting the large t gene performed in triplicate using 100 ng of dna (forward primer 5-gcagcttagtgattttcttagg-3, reverse primer 5-gaacacaggtgtttccacctgg-3, and taqman mgb probe fam-5-ggcactgaatattcattcatgg3). Each assay was carried out with negative controls (no template) and dna 10-fold dilutions (1010 copies) of a plasmid carrying the amplicon cloned (standard curve plot was 1010 copies vs 36 - 21 cycle threshold). The lower limit of quantification of the jcpyv dna was 100 copies per ml of plasma and urine or per g of pbmc dna . Anti - jcpyv abs were detected in serum by a 2-step virus - like particle - based elisa assay (stratify, focus diagnostics, provided by biogen idec). Patients who tested positive for jcpyv dna in blood or urine but who had undetectable serum antibodies had their serum samples retested for anti - jcpyv abs . Paired blood and urine samples from 39 patients who were consecutively referred to the outpatient clinic of the regional reference center for multiple sclerosis of the careggi university hospital, florence, italy, were prospectively collected . The patients were recruited according to the following inclusion criteria: definite diagnosis of ms; relapsing - remitting course; fulfillment of the european medicines agency indications for natalizumab administration in ms; and written informed consent to participate in a program for pml risk stratification established in collaboration with the marketing authorization holder (mah) of natalizumab and based on a centralized anti - jcpyv ab assay in sera and on a locally organized jcpyv dna analysis in blood and urine . The results included in the present study were generated by analyzing the diagnostic data reported in the clinical records . The study was approved by the local ethic committee (comitato etico azienda ospedaliero - universitaria careggi, largo brambilla 3, firenze) according to the italian law requirements (n. oss 14.073). The authors obtained preventive unrestricted permission for analyzing the data provided by the mah of the anti - jcpyv ab assay . The jcpyv dna was extracted from peripheral blood mononuclear cells (pbmc) obtained from enriched buffy coats after density gradient separation of 8 ml of whole blood, 200 l of plasma, and 200 l of urine, and was quantified by jcpyv - specific real - time pcr targeting the large t gene performed in triplicate using 100 ng of dna (forward primer 5-gcagcttagtgattttcttagg-3, reverse primer 5-gaacacaggtgtttccacctgg-3, and taqman mgb probe fam-5-ggcactgaatattcattcatgg3). Each assay was carried out with negative controls (no template) and dna 10-fold dilutions (1010 copies) of a plasmid carrying the amplicon cloned (standard curve plot was 1010 copies vs 36 - 21 cycle threshold). The lower limit of quantification of the jcpyv dna was 100 copies per ml of plasma and urine or per g of pbmc dna . Anti - jcpyv abs were detected in serum by a 2-step virus - like particle - based elisa assay (stratify, focus diagnostics, provided by biogen idec). Patients who tested positive for jcpyv dna in blood or urine but who had undetectable serum antibodies had their serum samples retested for anti - jcpyv abs . Demographic and clinical characteristics and jcpyv infection status of the 39 patients with rrms are reported in table 1 . The blood and urine samples were collected biannually at baseline and during the therapy, from july 2008 to september 2012 . We included in the study the first positive isolation of jcpyv dna and the anti - jcpyv ab assay evaluated in the serum sample of the same patient collected concurrently or after the jcpyv dna isolation . Eleven out of 39 samples included were collected at baseline, and the other 28 were collected during natalizumab administration . No obvious associations between patient demographic and clinical characteristics (age, sex, disease duration, previous treatments) and jcpyv dna status were observed . Overall, jcpyv dna was detected in at least one body fluid in 23 of 39 patients (59%; table 2 and table e-1 at neurology.org/nn): 17 patients (44%) had jcpyv dna in pbmc, 4 patients (10%) in plasma, and 13 patients (33%) in urine (table 2). Of note, the assay could detect jcpyv dna in 1 of 20 (5%) blood (pbmc or plasma) samples and in 11 of 20 (55%) urine samples of healthy donors . The range of viral load was similar in the different compartments (table 2). Baseline demographic and clinical characteristics of the patients according to jcpyv status jcpyv dna and anti - jcpyv ab status in the patients with rrms included in the study anti - jcpyv abs were present in 25 of 39 patients included (64%) and in 16 of the 23 jcpyv - infected patients (70%; table 2 and table e-1), indicating an assay sensitivity of 0.7 . In addition, jcpyv dna was present in 7 of the 14 anti - jcpyv antibody negative patients (50%; table 2), indicating that the negative predictive value of the assay was 0.5 . However, in this context, the viral load observed in seronegative patients did not differ from that observed in the seropositive patients . Of note, only 1 of the 7 patients who was anti - jcpyv ab negative despite the presence of jcpyv dna in pbmc showed seroconversion in a sample taken within 6 months . In the present study, the frequency of jcpyv dna (59%) in patients with ms treated with natalizumab was higher than that previously reported when jcpyv infection was evaluated only in urine (24%) and was consistent with that reported when the infection was evaluated in pbmc (60%). The high frequency of jcpyv in pbmc, a cell compartment well - known for its ability to harbor jcpyv - dna, was not unexpected, particularly in patients treated with natalizumab, considering the effects of this drug on lymphoid precursor cells . The frequency of anti - jcpyv abs observed in serum (64%) was consistent with previous observations in similar populations . However, in our study, anti - jcpyv abs were present in only 70% of the 23 patients with jcpyv infection . This result puts the sensitivity of the stratify test at 70%, whereas in previous studies that used the same assay to detect anti - jcpyv abs sensitivity was reported to be 97% . This difference should probably be attributed to the method of identifying the presence of the jcpyv dna, as the serologic data reported here and in previous studies have been obtained with the same methods (the stratify test). The previous data have been obtained by testing the virus dna only in urine, whereas more recent data and the present study indicate that when peripheral blood is also tested the frequency of jcpyv dna is higher than previously described, particularly during natalizumab treatment . In addition, the present study data, which put the negative predictive value of the ab assay at 50%, are consistent with other recent observations reporting that when peripheral blood was included in the analysis the false - negative rate of jcpyv serology was 37% and 35% . The present results indicate that jcpyv dna evaluation in blood and urine could detect jc virus infection earlier than anti - jcpyv ab serology . Thus, anti - jcpyv ab serology may underestimate jcpyv infection frequency, so it may be useful but not always sufficient to predict pml risk . This possibility points out the need to reevaluate the pml risk thus far reported in anti - jcpyv seronegative patients through additional virologic investigations . However, the limited number of patients included does not allow us to conclusively quantify the real frequency of anti - jcpyv ab this should be reevaluated in a larger set of patients identified by assessing jcpyv dna in paired blood and urine samples . V. clausi contributed to data collection, data analysis, and interpretation of the data . S. giannecchini contributed to data analysis, interpretation of the data, drafting of the manuscript, and revision of the manuscript . E. magnani contributed to patient recruitment and follow - up and interpretation of the data . A. repice contributed to patient recruitment and follow - up and collection, analysis, and interpretation of the data . C. mechi contributed to patient recruitment and follow - up and collection, analysis, and interpretation of the data . L. massacesi contributed to the design, interpretation of the data, and revision and supervision of the manuscript . The work was supported by grants from the fondazione istituto di ricerca virologica oretta bartolomei corsi florence, italy . V. clausi, s. giannecchini, e. magnani, a. repice, c. mechi, f. martelli, and a. azzi report no disclosures . L. massacesi received reimbursements for meeting participation or educational grants from biogen idec, merck - serono, sanofi - aventis, and novartis . L. massacesi is a member of the scientific advisory group (sag) neurology of the european medicines agency (ema) and of the italian medicines agency (agenzia italiana del farmaco, aifa), but the opinions included in this article may not represent the positions of these agencies . Go to neurology.org/nn for full disclosures.
Effective health initiatives in the nineteenth century and also most of the infrastructural measures in public health are signs of the recognition of such association between health and social status . In fact, most of the substantial reductions in mortality rates of infectious diseases, e.g. Tuberculosis, happened before introduction of modern and effective medical treatments . To be exact, such declines emanated from improvements in people s living conditions and food baskets (1). It is shown that from different determinants of health, social determinants account for approximately 50% of health differences (2). Constitution of world health organization (who) whose draft was written in 1946 also shows that along with taking challenges of medical care and effective treatments into account, who founders were also to tackle social roots of health problems . They believed that health is a complete physical, mental and social wellbeing and not only the absence of disease and infirmity (3). Although social health has been one of three staples of every definition of health, its concept bears no resemblance to physical and mental dimensions of health . Its unique feature is that it can be thought of a society and an individual characteristic simultaneously . As russell put it, on one hand a society is healthy when everyone has equal opportunities and access to basic goods and services, something which leads to high performance of citizens (4). In fact, law abiding, equality in wealth, public participation in decision making and social capital can be indicators of a healthy society (4). On the other hand, individualistic social health points to wellbeing of an individual which is, in turn, related to the quality of living and interacting with other people and also quality of reactions to social institutions and conventions (4). For the first time, belloc and breslow (1971) took a scientific and practical approach to study social health . They defined it as degree of members function in a society and then made up a social health index accordingly . They asked some questions about social, mental and physical dimensions of health to figure out members function (5). Couple of years later, cathy donald and colleagues (1978) proposed a new concept again . Their reasoning was that, health is something beyond just reporting disease symptoms, rates and individual functional abilities . They believed that wellbeing is a matter apart from physical and mental health . According to their perception, social health is both a health basis and also a subject of that (6). Considering different definitions and approaches, it seems that social health can be defined in three ways: social health as a social dimension of an individual s health, along with physical and mental health, which is concerned with being in a relationship with society . A healthy society as pro - health social conditions and finally as a better social status generally that according to each society s situations, can have different objective meanings and examples . Indices help us determine the degree of changes (7). From different indices which are used to measure social health, the following can be mentioned; fordham social health index, which measures some different variables in different age groups and also some variables regardless of age . They are child poverty, child abuse and infant mortality in childhood; suicide, drug abuse, high school drop - out and teenage pregnancy in adolescence; unemployment, income and health care coverage in adulthood; poverty and life expectancy at 65 in old age and finally violent crime, alcohol - induced mortality, affordable house and income inequality in all ages (8). Ontario healthy communities coalition index that attributes these traits to a healthy population: clean and safe environment, peace, equity and social justice, adequate access to food, water, shelter, income, security, work and recreation, adequate access to health care, opportunity for learning and developing skills, strong and supportive relations and networks, supportive work environments for family and individual wellbeing, extensive participation of dwellers in decision making, cultural and spiritual heritage, varied and dynamic economy, protection of natural environment and responsible consumption of resources that assures their sustainability (9). North carolina population health workgroup concept that believes a healthy population should enjoy security, affordable house; accessible transportation system, job security, healthy and safe environment, sustainable ecosystem and accessible and prevention - focused health care (10). And finally, healthy village concept of who eastern mediterranean office that includes clean and safe material environment, social coordination, openness to experiences, interactions and various relationships, protection and promotion of cultural and historical heritage, appropriate and accessible health care, economic variation and originality and sustainable utilization of available resources (11). In iran, rafiey and colleagues for the first time conducted a delphi survey to construct a national concept for social health and then an acceptable index for it (12). Since rafiey and colleagues concept has no empiric backing, we aimed to put that in an empiric trial . Present study is an ecologic and correlational study in which iran s provinces (30 provinces in 2007) were the study units . In rafiey and colleagues study (delphi method), there were five rounds of comment and feedback between researchers and iranian social health experts . At first, 63 experts were chosen as delphi members . Out of these initial participants, there were two criteria to choose participants: 1) scientific scholarship and reliability in health and social issues and 2) experience in health and social issues . The participants expertise ranged from economics, development, management and epidemiology to social medicine, psychiatry, social and clinical psychology and social work . The first category contained those traits on which most delphi participants agreed . According to these traits, a healthy society is a society in which 1) there is no one under poverty line, 2) there is no violence and 3) population growth is under control . However, whenever any difference could not be found in the first category, the second category can be added to concept of a healthy society; thus, a healthy society is a society in which, in addition to existing aforementioned traits in first category, 4) there is no gender discrimination, 5) all of citizens are the same in the eyes of law (in enforcing and being supported by law), 6) human rights treaty and other related treaties are established, 7) education is free and compulsory until secondary school, and is free thereafter, 8) there is universal access to health care, 9) security is assured, 10) liberty of conscience is granted, and 11) people are satisfied with their lives . The third category expresses further traits of an ideal healthy society; a healthy society is a society in which, as well as the existence of previous traits, 12) there is universal insurance coverage, 13) there is equitable distribution of income (everybody is given his / her rights and there is equal opportunity in income earning), 14) there is no unemployment, 15) there is no racial, ethnic and regional discrimination, 16) government is legitimate, 17) democracy is the only way of electing governors, and finally 18) governors are supervised democratically . In the present study, in order to provide an empirical confirmation for social health concept, 6 indicators were chosen from above 18 indicators . There were three criteria to choose these indicators; inclusion of first category traits, availability and accessibility of required and reliable data in the country (for all provinces), and convertibility to quantitative data . From this, in our study a healthy society is a society in which; 1) nobody is under poverty line, 2) there is no violence, 3) population growth is under control, 4) education is free and compulsory until secondary school, and is free thereafter, 5) there is universal insurance coverage, and finally 6) there is no unemployment . In this study, required data for population growth was extracted from 2006 national census and also 2007 population estimates . Violence data (willful murder, assault & battery) was drawn from iranian police force statistics . Data for poverty (proportion of food expenditure to non - food expenditure) was taken from a national study on household budget in 2006, published by iranian central bank . To better expression, more elaboration on the concept and definition of each indicator is provided . Absolute poverty means not having access to standards of life i.e. Adequate food, house and clothing . Relative poverty means that a person is not able to reach a particular level of living standards which is being known as necessary and desired in the society . In the present study, proportion of food expenditure to non - food expenditure was used as a measure of poverty where the higher the share of food expenditure to non - food expenditure, the higher the degree of poverty (13). 2) violence; although it seems easy to define violence but like other social issues there is no any consensus on it . As corporal punishment is the simplest type of violence, early definitions were on corporal punishment; violence is the expression of physical or verbal force against self or others, forceful action against one s will bearing hurt (14). Who has defined it as the intentional use of physical force or power, threatened or actual, against oneself, another person, or against a group or community which either leads to injury or has a high likelihood of injury, death, psychological harm, maldevelopment, or deprivation (15). In the present study, by violence we mean willful murder, assault and battery which is defined as; willful murder; killing by will and authority so that a person deliberately, consciously and premeditatedly takes the lives of others whether the deed is typically deadly or not (16). Assault & battery; causing maim and mayhem whether it is intentional or unintentional . If trauma does not result in bleeding even with inner fracture it would be assault (16). 3) population growth; population means a group of individuals who dwell in a specified area (village, city, county, province or a country) continually and normally in households which share a unique political stand and national and ethnic features (17). Natural growth rate (ngr) demonstrates increase or decrease in population number, exclusive of immigration, which can be computed only by raw mortality and morbidity rates (formula 1) and represents that how many people will be added to population annually per 1000 person . Formula: ngr = mortality - birthmean population1000 4) literacy: a literate person is somebody who can read and write a simple text in persian or in other languages, whether he / she has an official credential or not . In this study, literacy rate has been used to measure literacy status in provinces (formula 2). Formula: literacy rate = number of literate personsnumber of6 and over six years old persons1000 5) insurance coverage: in the present study insurance coverage is the number of people who were under the coverage of social security organization (including public, private, arbitrary, occupational drivers and special insurance) at the end of 2006 . 6) unemployment: in our study an unemployed is somebody who does not have any paid job or salaried job and is not self - employed, is ready to work and is a job seeker (has tried to find a paid job or salaried job or put up a self - employed job). Unemployment rate is the proportion of the unemployed population to the active population (18). This should be noted that due to some reasons we could not include gini index and assault & battery rates in our study . Assault & battery rate for the reason that there was a reverse relationship between it and willful murder unexpectedly (r= 0.248, p= 0.09). This is maybe because reports of assault & battery cases were not precise in some provinces . The reason for exclusion of gini index was that again contrary to expectations this index had no correlation with other indices (for example with poverty; r= 0.007, p= 0.48 and with unemployment r=0.005 and p= 0.48). Range restriction was our interpretation of this issue i.e. Whenever a variable has a narrow variance this variable has to be called a constant rather a variable . In order to prove the construct validity and obtain a social health index, an exploratory factor analysis was conducted on six indicators of population growth, willful murder, poverty, unemployment, insurance coverage and literacy . Factor analysis investigates that whether a number of observed correlated variables are related to a smaller number of unobserved variables called factors . Factors are latent constructs that influence observed variables and factor analysis discovers the nature of these constructs . As indicators had different units of measurement and in order to equalize the units, z scores was measured for each indicator: formula: z = x where x is the mean of sample, is the mean of population and is the standard deviation of population . And finally, to calculate the value of social health index in each province the following formula was used; formula: ishie = zlit+zins.cov(zngr+zpov+zmurd+zunemp) where ishie stands for empirical iranian social health index and z, lit, ins.cov, ngr, pov, murd and unemp denote the standardized rate, literacy, insurance coverage, natural growth rate, poverty and unemployment respectively . In our study, all of analyses were done in spss software (version 17). As table 1 shows tehran had the best status in the index of proportion of food expenditure to non - food expenditure (poverty). It indicates that 80 percent of tehran s households income is spent on non - food expenditure and they already crossed basic needs level . Also, as it was expected sistan & baluchistan had the highest rates of violence and poverty and lowest rates of literacy and insurance coverage . Plus, hormozgan had the lowest rate of population natural growth . Iran s provinces are ranked based on their social health index value in table 2 . As this table illustrates, tehran, semnan, esfahan, boushehr and mazandaran had the highest social health index values whereas sistan & baluchistan, lorestan, ilam, kohkiloyeh and kermanshah had the lowest social health index values . Table 3 shows some information on capability of variables (indicators) in construction of factors . Kaiser- meyer- olkin (kmo) measure of sampling adequacy tests the amount of variance in data which can be explained by factors bartlett s test of sphericity is used to test the hypothesis that the variables (indicators) in correlation matrix are correlated . In this test a p - value lower than 0.05 indicates that there are significant relationships between variables and it is possible to find out latent structures in data . Considering values of kmo (0.66) and bartlett s test (p - value<0.0001) in table (4), our data had this capability to make up factors and reveal a latent construct that we call it social health . Following the factor analysis, factors were extracted . As figure 1 illustrates, eigenvalue of two factors were more than 1, something which means that these two factors could explain the highest amount of variance in data . As it can be seen, variables of natural growth rate, proportion of food to non - food expenditure, insurance coverage and literacy rate were loaded on the first factor, and variables of willful murder and unemployment rate were loaded on second factor . Totally, these two factors explained 68.25 percent of social health variance (48.65 percent by the first factor and 19.60 percent by second one). The researchers named the first and second factors as diathesis and problem respectively . In the present study, we tried for the first time to investigate empirically the concept of social health in iran . According to factor analysis in our study, an iranian healthy society can be defined as a society that is stout with less deviance and problem, i.e. With more and more literate and insured people, controlled growth rate and less and less willful murder, poverty and unemployment . There are some differences and similarities between this concept and concepts of other studies . As it can be seen in following sentences, although the measures are somehow different but most of the indices of social health, used in other parts of the world, use indicators similar to our study: literacy and education: availability of learning opportunities (ontario index) (9); high school drop - out (fordham index) (8); insurance / health: adequate access to health care (ontario index) (9); prevention - focused and accessible health care (north carolina index) (10); accessibility and appropriateness of health care / access to basic health care (eastern mediterranean index) (11); infant mortality rate and health care coverage in adults (fordham index) (8). Poverty: adequate access to food, water, shelter, income, security, job and recreation by public (ontario index) (9); poverty in children and elderly (fordham index) (8); murder: security (ontario, north carolina, eastern mediterranean) (9, 10, 11); violent crime (fordham index) (8). Unemployment: adequate access to job by public (ontario index) (9); job for seekers (north carolina) (10); adult unemployment (fordham index) (8). Following substantial reduction in mortality rates in 1950s, iran had experienced a booming population growth . The number of its population had tripled in 30 years and mounted to 50 million in 1980s . Now iran has one of the youngest populations in the world and is experiencing a transient fertility phase (19). However, the importance of population growth in iran s social health index can have some policy implications . Its fertility rate fell from 7.0 births per woman in 1979 to 1.9 births per woman in 2006 (21). According to our index of social health, iran has to stick firmly with its current family planning programs to make a healthy society and any change in population policies should be made carefully . Due to its young population, the employment has been one of the most challenging matters for iran in recent years . As our study also shows, the unemployment is a matter of focus for health experts too . We have to give more weight to employment if we are to improve social health . As statistics says 12.3% of iranian people are unemployed and this matter is worse in marginalized parts and groups of the country (22). Although the government has put its stamina on this matter, but the problem somehow remains . Home business and easy start - up business are initiatives that is hoped to work in some ways . However, there are more investments needed . There is a strong relationship between poverty and sustainable development and social welfare in a country . There is no doubt that in a country with higher rates of poverty, it would be really hard to make improvements in other determinants of social health and consequently in social health . Unemployment, illiteracy, uncontrolled population growth and crime are the inevitable companions of poverty . Iran has had some plans to decrease its poverty rate and release more people off poverty trap . According to statistics, targeted subsidies plan that is being performed in iran is capable of making remarkable changes in peoples lives and improve poverty rates in iran . According to a well - known model (ecologic model) of violence, biological, social, cultural, economic and political factors all however, it is believed that social factors are the most important determinants of violence (25). As it was revealed in our study violence has a strong association with health in its various dimensions (physical, mental and social) (26). Due to some cultural issues however, as violence is one of the iran s social health components, in the first place it might be better to devise some initiatives to get access to better violence data . There is no doubt that the more people of a country are literate, the better they are in obtaining and protecting health . Program is referred as one of the effective ways to improve literacy worldwide . However, the literacy rate is still low in some regions and among some groups of iranian people, especially women . It might be the reason why experts chose the literacy as a component of social health in iran . According to data, 85% of iranian people are literate (23). Enrichment of home schooling and literacy movement initiatives in marginalized parts of the country, like rural regions, are of ways that can improve literacy rate in iran . About 10 percent of people in iran are under no insurance coverage (27). It has been performed in rural settings from 2004 and has some fruitful and promising achievements (28). It is now planned to extend to urban areas and cover all of iranian population . Improvement of social health can be one of its results . From the above - stated matters, it can be drawn that social health concept is a dynamic and fluid concept . Hence, in different societies depending on their achievements and situations and in different times there would be different concepts of social health . So, it can be suggested that there should be a redefinition of concept every 5 or 10 years in every country . Indeed, we intended to include more indicators of social health on which experts agreed in rafiey and colleagues study . It seems that by entering more indicators into factor analysis, stronger empirical evidence will be provided . Considering importance of poverty, violence, literacy, insurance coverage, population growth and unemployment in formation of a health society, these factors should be consistent parts of iran s social health policy agenda . Above that, as most of these factors are beyond the health sector, a strong intersectoral collaboration is needed to achieve that healthy society . Ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc) have been completely observed by the authors.
It accounts for approximately 3% of all adult neoplasms and in about 30% of patients it has a tendency to metastasize, usually to the lung (70%), lymph - nodes (55%), bone (42%), liver (41%), adrenal gland (15%) and brain (11%). Breast metastases from extra - mammary tumors are much less common with respecto to primary breast tumors (0.52% of breast cancer), and are usually due to melanoma, lymphoma and leukemia; metastases to the breast from rcc are a rare occurrence and take place in 3% of all metastatic rcc . Overall, 25 such cases have been reported in the literature: eleven cases presented with metastasis as the initial sign of the disease and fourteen, among which 2 were bilateral, occurred as metachronous lesions after a former nephrectomy . We report a case of rcc metastasis to the breast occurring 9 years after primary surgery for rcc . The difficulties in obtaining a correct diagnosis, which is crucial to avoid unnecessary mastectomy, and the management of axillary ipsilateral axillary lymph nodes are discussed . A 70-year - old woman was referred to our institution on april 2013 due to a quickly enlarging lump located in the upper inner quadrant of her right breast . In her medical history, she reported having undergone a right radical nephrectomy in 2004 for rcc . At the time of the operation the tumor was limited to the kidney compressing the renal capsule but with no sign of infiltration and there was no vascular invasion as well; surgical margins were free and the disease was therefore staged as t3n0m0 (g2). The patient received no further treatment and was followed up with annual whole - body computed tomography (ct) scan and mri . In april 2011 she underwent a pylorus - preserving pancreatic head resection because of a solitary, hypervascular and homogeneous mass located in close proximity to the head of the pancreas, measuring 16 mm . The patient was thereafter asymptomatic until december 2011, when she noted a quickly enlarging lump in the right breast . On admission, clinical examination revealed a palpable and mobile mass in the upper inner quadrant of the right breast with an enlarged ipsilateral axillary lymph node; no skin or nipple retraction were noticed . A mammogram showed a dense well circumscribed solid mass, not spiculate, measuring 3 cm in diameter, with no adjacent parenchymal distortion and no sign of microcalcification; there were no signs of infiltration of the skin . Sonography showed a hypoechoic homogeneous solid nodule with no posterior attenuation of the acoustic wave but with prominent peripheral vascularity and penetrating vessels detected at color and power doppler . The adjacent parenchyma was just displaced without obvious distortion, again with no skin thickening or duct ectasia . Multiple subcentimetric axillary lymphnodes were present, with a single one measuring 2 cm in diameter, with a preserved architecture . A contrast - enhanced whole body ct showed an enhancing, homogeneous right breast mass (fig . 1), and fine needle aspiration citology (fnac) was unable to show malignancy . A tru - cut biopsy was then performed and revealed a tumor composed of nests and cords of cells with clear cytoplasm separated by a prominent sinusoidal vascular network (fig . 2), which showed, at immunohistochemistry, positive vimentin and cd10 staining while ck7 and ck20 were negative . Such pattern was consistent with renal cell carcinoma of clear cell type (fig . 3). After a multidisciplinary discussion, the patient underwent metastasectomy and exeresis of the largest palpable axillary lymph node, that resulted unaffected at frozen section . The breast is a rare site of metastatic deposits, as emerges from the small number of cases reported in the literature . However, it is possible that the incidence of such finding will become more and more frequent because of the increasing number of patients who live longer bearing malignant disease . In facts, the 5-year and 10-year survival rates for the patients with rcc treated with nephrectomy are 95% and 91% for pt1; 80% and 70% for pt2; 66% and 53% for pt3a; 52% and 43% for pt3b; and 43 and 42% for pt3c, respectively . Metastases can take place either in a synchronous or metachronous fashion with respect to the primary tumor: out of 25 cases reported in the literature, 14 actually occurred after nephrectomy for rcc . A former history of rcc should therefore arouse suspicion of metastatic escape to the breast despite the fact that primary carcinoma of the breast is much more common . Reported a majority of metachronous presentation, that was claimed to carry a better prognosis than the synchronous metastases; however, to the best of our knowledge, no strong evidence of a significant prognostic difference between metachronous and synchronous presentations can be argued from the data available in the literature . Clinically, metastatic lesions in the breast present as painless and mobile discrete masses with rapid growth . The findings of the traditional diagnostic workup most likely mimic a benign breast mass, due to the absence of desmoplastic reaction; in particular the deposits of malignant cells do not show spiculation or microcalcification on the mammogram and the sonography does not reveal posterior attenuation of the ultrasounds . The only finding that can be suggestive of malignancy is the prominent peripheral and penetrating vascular network, well evident to color and power doppler, that is common to all cases reported in the literature . Awareness of a former treatment for rcc should lead to stress the preoperative diagnosis because the treatment of breast secondary lesions differs from that of primary breast tumors, either as far as the lumpectomy margins width and the management of axillary lymph nodes are concerned . In facts, the malignant extension to the breast can be explained by the spread of neoplastic cells from the renal vein into the inferior vena cava and then through the pulmonary circulation, eventually reaching the arterial circulation, diffusing throughout the whole body and thereby reaching the breast: therefore, the high rate of multiple lesions that constitutes the rationale of the efforts aimed at obtaining wide tumor - free margins is pointless in secondary disease . Besides, the mechanism of metastatic diffusion renders the the biopsy of a sentinel lymph node void of sense in such cases; since malignant cells reach the breast (via ematogenous spread), sampling the efferent lymphatics that constitute a way of diffusion from the breast is pointless . Moreover, the staging role of slnb obviously only applies to primary tumors, while sampling breast loco - regional lympnodes for tumors originating elsewhere in the body makes no sense . In cases of metastatic rcc, citology may show atypical cells which differ from those of a primary breast tumor but which, on the other hand, are frequently reported as benign . A diagnosis based on histopathological investigation is therefore crucial in order to avoid an unnecessary mastectomy with sentinel lymphnode biopsy (or primary axillary dissection). Moreover, we suggest to avoid fnac for the additional reason that the vascular network that characterizes metastases from rcc may lead to a high false - negative rates . In conclusion, we suggest to consider a micro - histological biopsy of any new breast lesion appearing in a patient with a history of treatment for rcc as mandatory . A whole body ct scan may be included in the staging work - up . In the case of an isolated lesion, metastasectomy is the treatment of choice; axillary lymph nodes should be removed only in cases of overt metastatic deposists . So far, unfortunately, no effective adjuvant therapeutic option is available for disseminated rcc because of the great resistance to chemotherapy and radiotherapy of this kind of metastases . A written and signed consent to publish the case report was obtained from the patient . Sanna paola angela was the radiologist; dubini allessandra was the pathologist; and folli secondo was the supervisor.
Asthma accounts for 13.9 million outpatient visits, 2 million emergency department (ed) visits, 500,000 hospitalizations and 5,000 deaths annually.1 the environment has increased the risk for the development of asthma . Pollution from industrial chemicals, exhaust from vehicles, crowded living conditions, infection and bioterrorism are all environmental factors that have contributed to this sensitization . Despite all these risk factors in the urban environment, the research articles reviewed showed mixed results in the prevalence of asthma in urban versus rural areas . However, the overall consensus is that environmental factors and socioeconomic issues predispose people to asthma . The hygiene hypothesis states that early exposure to infection for children may actually confer an advantage by regulating the immune system to protect against allergies.2 there were different outcomes on the prevalence of asthma in the literature reviewed . In mississippi there were higher asthma hospitalizations in the rural delta region compared to the urban jackson, mississippi metropolitan area.3 however, another study in maryland showed highest rates of ed visits for asthma in urban areas; baltimore city had 2.4 times the national rate of asthma hospitalizations for 514 year olds in 1990.4 there was no difference in asthma prevalence in rural and urban areas in arkansas.5 in mississippi the lower socioeconomic group resides in the rural region . Many of the urban living patterns were seen in the rural delta region of mississippi such as the limited access to health care, pollution, and other environmental factors . In arkansas, the use of health services was the same between the rural and urban group, but asthma morbidity was greater in the rural group; 85 percent of rural and 67 percent of urban children were black and 78 percent of rural and 37 percent of urban children had insurance from the state.5 the rural children in arkansas were more commonly diagnosed with chronic bronchitis and likely to report moderate to severe asthma symptoms as compared to urban children.5 the lower socioeconomic group in arkansas resided in the rural areas and there may be some cultural and health plan barriers in the medical care that may account for the discrepancy in morbidity . Minorities make up the majority of the government health care plans that may limit preventive care or referral to specialists to curtail costs; this has resulted in higher mortality due to increased hospitalization rates and poor health care.6 internationally, the prevalence of asthma related symptoms were higher among those adolescents living in the urban centers in comparison to the rural ones in brazil.7 furthermore, rural chinese children had significantly lower prevalence of asthma and atopic sensitization than urban children, using the validated isaac (international study of asthma and allergies in childhood) questionnaire and objective skin - prick tests.8 in tamil nadu, india, there was a higher prevalence of breathing difficulty and nocturnal cough among urban children in the age group of 612 years.9 a large study in a pediatric medicaid population found that the rural children had increased asthma prevalence and similar asthma morbidity compared with urban children.10 the prevalence of asthma in adults living in the rural areas of the kirikkale region in central turkey was significantly higher than that in the urban population.11 in this study of 12,270 from turkey, the prevalence of asthma was more common in rural areas than in urban areas (10.8 percent vs. 6.2 percent). Furthermore, a recent european study from cyprus showed that the prevalence of current wheeze nearly doubled between 2000 and 2008 in rural areas (5.4 percent vs. 9.7 percent) while no significant change was observed in urban areas (7.5 percent vs. 8.4 percent).12 a study from egypt showed the rural school children who had higher exposure to farm animals and who were fed on farm milk, had less allergic symptoms.13 rural students showed higher fev1, fev1 percentages and significant fvc versus urban students.13 these results support the hygiene theory, ie, endotoxin exposure could be protective to asthma and atopy in school children.13 on the other hand, a study in mexico city found that an exposure to particulate matter at the level of 2.5 resulted in acute airway inflammation and decrease in lung function in both asthmatic and non - asthmatic children.14 additionally, a korean study that looked at 1819 elementary school students, and air pollution in metropolitan (seongbuk), and semirural (andong) areas found no significant difference in the prevalence of self - reported asthma.15 the data from various studies on the prevalence of asthma has been summarized in table 1 . Studies have also shown that minorities may receive less inhaled corticosteroids or controller medications than caucasians . Physicians need to practice evidence based medicine and avoid cultural stereotypes during clinical assessment and treatment . Baltimore, maryland, the prototype of the urban living environment, had the typical city living conditions, lower socioeconomic class and pollution . However, the suburban howard county, maryland also had high asthma ed visits . This affluent county is between two metropolitan centers and has a major interstate that increases exposure to exhaust from automobiles and other industrial chemicals.4 this county also has a growing population, but the specific factors that led to increased suburban ed visits for asthma was not further investigated by this study . In fact, the article states that an urban environment alone cannot explain increased ed visits for asthma.4 a recent study found that the frequent use of the ed reflected the urban social issues of poverty, homelessness, chronic illness and alcohol abuse.16 in addition, evidence suggests that the higher number of hospital admissions and ed visits may partially be due to the problems faced by single parents with limited resources in properly managing their child s asthma condition.17 a study in texas showed that the majority of asthmatics who presented to the texas ed asthma surveillance project s participating eds were sub - classified as having mild - acute severity and mild - intermittent chronic disease . The majority of these patients (almost up to one third) did not have health insurance.18 in baltimore, wealthy suburban counties were found to have the higher risk of an asthma ed visit.19 children from rural counties had fewer ed asthma related visits than children from urban and suburban counties.19 after reviewing the literature, it appears that places that share similar environmental and socioeconomic risk factors have a higher prevalence of asthma regardless of whether it is in a rural or urban location . In other words, some rural areas may have similar living conditions and socioeconomic situation that are comparable to the prototype urban environment (pollution, higher population, etc). However, in most places urban areas tend to have the environmental risk factors that predispose populations to asthma . Chinatown, new york experienced high asthma rates post 9/11.20 evidence suggests that the environment may affect an individual s genetic susceptibility for asthma and hay fever development at different ages inducing changes in the prevalence of atopic diseases in populations in a time- and age - dependent way.21 there are multiple factors that play a role in the development of asthma . Certain occupations may expose people to chemicals or other irritants that can cause permanent lung damage . The decreased incidence of asthma and allergy in european farming children has to do with a genetic variation of toll like receptor 2.2 there is also the protective farming effect that explains a lower risk of asthma due to close contact with large animals; there is th-2 thymocyte suppression and th-1 stimulation that reduces ige production (prevents allergies) secondary to bacterial endotoxin exposure in the air from animals.2 however, this type of farming environment is not common now in the united states due to increased industrialization and growing populations . Currently, the increased risk of asthma is supported by an urban living effect.22 increased ed visits may also be due to lack of outpatient follow - up with primary care doctors . Some people who may benefit from referral to a specialist for asthma care may not be able to do so under their health care plan . The pollution in urban environments can decrease lung growth and function by narrowing the airways; the pathogenesis of the effect of pollution on the airways may be caused by enzymes from dust mites that harm the lining of the airway and cause narrowing.2 children may remain asymptomatic despite these changes . Decreased fef50 is a more accurate indicator of small airway disease in urban residents exposed to pollution; children exposed to more pollution have lower fvc and decreased fev1 by the time they become adults.2 the longer the duration of exposure, the more the lung damage . In a study in greece, eight to 10 year old children were followed into adolescence and watched for the development of asthma; there was no difference in asthma prevalence in the rural versus urban areas.22 the urban children may have underlying air disease secondary to the pollution even though they may not have any clinical manifestation of disease . A limitation in the research was loss of follow - up on a number of students who were not present after graduation . There may be a higher prevalence of asthma in urban adult residents as symptoms become evident with longer duration of exposure . Forty one million americans were uninsured in 2009 and asthma costs reached 37 billion dollars in 2007.23 state children s health insurance plan (schip), community health centers and quality improvement programs have already made some difference in management of asthma . Asthma mortality appears to be highest in the african american and hispanic population than in caucasians.24 asthma prevalence is 39 percent higher in african americans than in caucasians in general and 2025 percent higher in the ed setting.1 there are increased recurrent ed visits from asthmatics that are non - white and have a low social economic status.25 racial disparities in asthma status and management practices exist in managed medicaid populations . There is a greater asthma prevalence in the urban setting (7.1 percent) than in the rural setting (5.7 percent) and in a low social economic status (9.8 percent) with incomes lower than $15 000 compared to 5.9 percent prevalence in incomes greater than $75 000.26 a number of states have decreased medicaid eligibility and lowered prescription drug benefits to save money; federal health plans like medicare may refer less to specialists and preventive care in order to control budgets.6 disparities can affect parameters such as adherence to medication compliance which can be a problem among low - income and minority patients . Inhaled corticosteroid use in african - americans is sometimes less than half compared to caucasians due to lack of compliance.27 changes in the areas of recycling, fuel efficient vehicles, carpooling and affordable public transportation will help . Carbon monoxide and nitrogen dioxide are high in cities secondary to vehicle emission . Formaldehyde, toluene and chloroform found in newer buildings are also common in cities, which can lead to asthma.2 children should be educated on proper dietary habits early on . Fruits, vegetables, and omega-3 fatty acids have been shown to help lower the risk of allergies and asthma.2 families with smokers should be encouraged to stop smoking due to the effect of second hand smoke . Increased security or surveillance in cities with high crime rates and counselors in schools a recent study examined 2,746 employees in 18 norwegian smelters and found that dust exposure was associated with an increased incidence of work - related asthma - like symptoms.28 a study of 79,888 school - aged children found that a traditional farm (ie, with cows and cultivation) was protective against asthma, hay fever, and atopy.29 schip has improved health care access for children who could not afford insurance and are unable to qualify for medicaid . Community health choice (chc) are federal nonprofit programs that also provide coverage to uninsured . Programs such as quality improvement (qi) projects increase awareness of cultural and health disparities in different patients and this will provide more effective medical care . The efforts of community health care workers help educate people on how to manage their asthma . It should be mentioned that even though most studies show a difference in prevalence of asthma in rural and urban centers, the management of asthma in the rural settings was comparable to that of urban setting in alberta, canada.30 higher copayments make it difficult to afford medications and lead to asthma exacerbation . Less than half of the children with medicaid used controller medications recommended by the national institutes of health (nih) and only had a beta agonist inhaler for symptomatic relief.23 children with asthma account for three times the health care expenses compared to children without asthma.17 a recent study showed that children under 2 years of age or with persistent asthma or lower asthma quality - of - life were at greater risk for ed revisits after management of their acute asthma symptoms in the ed.31 it examined the issues related to significantly higher rates of asthma hospitalization and mortality among poor urban children and black americans compared with white americans.32,33 the ncicas was the largest funded us - based initiative and emphasized the particular social, environmental, and medical care issues associated with urban pediatric asthma.3437 the icai attempted to implement the ncicas guidelines in community settings.38 of the 4,174 children enrolled, 1,035 (25 percent) completed the entire 12-month icai protocol, while 1,355 children (32 percent) completed at least the core activities of the intervention . The icai study had lower compliance (57 percent) compared with the original ncicas compliance (80 percent) because ncicas protocols involved careful screening and provided monetary and childcare incentives . A study by wood et al38 in the inner city population focused on factors that predicted retention and provision of allergy testing . In terms of morbidity, warman et al39 found that a little more than half of all the families reported high risks of asthma morbidity . The icai study showed that implementation of the protocol was possible but retention of participants was challenging . Master s degree level social workers matched well with health care professionals to address various issues involved in caring for children with asthma and their families . The sustainability of the interventions was possible beyond external funding if local funding was sought and outcome measures were considered.39 health care providers should follow national asthma education and prevention program (naepp) guidelines for all patients . Education on proper use of inhalers and patient reiteration of the treatment plan, in order to reinforce their comprehension, is important . Physicians need to practice evidence based medicine and overcome language and cultural barriers . At the same time ultimately, a collaborative effort is needed to protect children from health risks like asthma that may be preventable.
Determination of a person's identity is a difficult job in cases of traffic accidents, acts of terrorism, and mass disasters . Visual identification, finger printing, and dna comparison are the most common techniques employed in forensic identification for fast and reliable identification . However, dna testing is very costly and cannot be conducted for everyone . Hence, analysis by dental arches, cheiloscopy, antemortem periapical radiographs, pulp and gingival morphology, missing teeth, restorative material, palatal rugae, etc . Can be considered to be sources of comparative material because mouth allows for a myriad of possibilities . Identification of an individual is possible which can legitimize the identification process, even in highly difficult circumstances . The palatal rugae pattern will not be modified even in cases of severe illness, chemical injury, or trauma . Palatal rugae also called plicae palatinae transversae and rugae palatine are asymmetrical and irregular elevations of the mucosa located in the anterior third of the palate, made from the lateral membrane of the incisive papilla and arranged in transverse direction from palatine raphe located in the mid sagittal plane . It is a well - established fact that the rugae pattern is as unique to a human as his / her fingerprints and it retains its shape throughout life . Even in twins, the anatomical position of the rugae inside the mouth surrounded by the cheeks, lips, tongue, buccal pad of fat, teeth, and bone keeps them well - protected from trauma and high temperatures . Palatal rugae pattern of an individual may be considered to be a useful tool for sex determination and identification, which is also supported by a study conducted in 2012 by manjunath et al . The palatine rugae could be used as a reference landmark during forensic identification of an individual . Many victims of natural disasters such as fires and floods can be identified by dental means . The advantage of palatal rugae is its internal position, which leads to stability and perenity . It is a cost - effective, noninvasive, and easily available mode for human identification . The aim of the present study is to analyze the morphology of palatal rugae patterns in central indian population by studying the cast . The present clinical study was planned and designed in the department of oral medicine and radiology, hitkarini dental college and hospital, jabalpur, madhya pradesh, india . Ethical clearance was obtained by the college ethical committee . All individuals who participated in the study were recruited from patients attending the outpatient department of hitkarini dental college and hospital from 2013 to 2015 . A detailed case history was recorded in a specially designed proforma [annexure 1] for the study . Thorough clinical examination was performed, patients were informed about the study, and a written consent was obtained . Click here for additional data file . On the basis of result obtained from pilot study using n - master software 2,0 version the sample size came out to be 497 which was then rounded off to 500 (95% confidence interval and 5% allowable error and 80% power of the study). Five hundred healthy adult participants were then equally divided into 250 males and 250 females according to the below inclusion and exclusion criteria . Patients in the age group 1725 years were considered for the study, and 250 healthy males and 250 healthy females were included in each group . Participants with palatal abnormalities such as cleft palate, soft tissue protuberances, trauma of palate, and patients with braces, were excluded from the study . The rugae seen as elevated impression were marked on these casts using a black permanent marker pen [figure 1] under adequate light, which enhanced the clarity of the pattern on the cast . A divider with an adjustable screw and measuring scale the palatal rugae were then analyzed on these casts on the basis of primary rugae, number of rugae, direction, unification and pattern, using the classification by thomas and kotze [figures 2 and 3]. Marking of palatal rugae shape of pattern a: curved pattern, b: straight pattern, c: wavy pattern shape of pattern circular rugae pattern the collected data were sorted, tabulated, and subjected to statistical analysis . The data obtained was transferred to microsoft excel then to the statistical package of social sciences (spss) version 19 for analysis . The mann whitney u test was used to assess the significant difference of the total number of each type of palatal rugae between males and females . Descriptive statistical analysis was applied using spss to obtain the means and standard deviation from the data of each category . All individuals who participated in the study were recruited from patients attending the outpatient department of hitkarini dental college and hospital from 2013 to 2015 . A detailed case history was recorded in a specially designed proforma [annexure 1] for the study . Thorough clinical examination was performed, patients were informed about the study, and a written consent was obtained . Click here for additional data file . On the basis of result obtained from pilot study using n - master software 2,0 version the sample size came out to be 497 which was then rounded off to 500 (95% confidence interval and 5% allowable error and 80% power of the study). Five hundred healthy adult participants were then equally divided into 250 males and 250 females according to the below inclusion and exclusion criteria . Patients in the age group 1725 years were considered for the study, and 250 healthy males and 250 healthy females were included in each group . Participants with palatal abnormalities such as cleft palate, soft tissue protuberances, trauma of palate, and patients with braces, were excluded from the study . The rugae seen as elevated impression were marked on these casts using a black permanent marker pen [figure 1] under adequate light, which enhanced the clarity of the pattern on the cast . A divider with an adjustable screw and measuring scale the palatal rugae were then analyzed on these casts on the basis of primary rugae, number of rugae, direction, unification and pattern, using the classification by thomas and kotze [figures 2 and 3]. Marking of palatal rugae shape of pattern a: curved pattern, b: straight pattern, c: wavy pattern shape of pattern circular rugae pattern the data obtained was transferred to microsoft excel then to the statistical package of social sciences (spss) version 19 for analysis . The mann whitney u test was used to assess the significant difference of the total number of each type of palatal rugae between males and females . Descriptive statistical analysis was applied using spss to obtain the means and standard deviation from the data of each category . Table 1 and graph 1 show statistically significant difference [p = 0.000 (<0.001)] with total number of rugae . Males exhibited higher number of rugae with a mean and standard deviation of 10.52 2.64 among study population . Females had a less number of rugae with a mean and standard deviation of 9.60 2.08 . Table 2 and graph 2 show statistically significant difference [p = 0.000 (<0.001)] between shapes of rugae pattern . Males showed more wavy rugae pattern with 129 (51.6%) followed by straight rugae pattern 80 (32.0%), curved rugae pattern 35 (14.0%), and circular rugae pattern 06 (2.4%). Females presented more straight rugae pattern with 138 (55.2%) followed by wavy rugae pattern 85 (34.0%), curved rugae pattern 20 (8.0%), and circular rugae pattern 07 (2.8%). Overall, it was observed that straight rugae pattern was more in number 218 (43.6%). Graph 4 shows significant difference [p = 0.000 (<0.001)] between direction of rugae between the genders among study population . Number of rugae among study population number of rugae among study population comparison of shape of rugae among study population comparison of shape of rugae among study population comparison of unification of rugae among study population comparison of direction of rugae among gender palatoscopy or palatal rugoscopy is the study of palatal rugae in order to establish a person's identity . Transverse palatine folds or palatal rugae are asymmetrical and irregular elevations of the mucosa located in the anterior third of the palate, made from the lateral membrane of the incisive papilla and arranged in transverse direction from palatine raphe located in the mid - sagittal plane . Palatoscopy is very useful in identification of decomposed or burnt bodies when fingerprint data are missing . Palatoscopy is a valuable technique in aeronautical accidents, pilots antemortem data is used for identification purpose . It is the most valuable technique in a areonautical accidents in order to ensure identification of pilots making use of ante - mortem data . In our study, males had more number of rugae when compared to females 10.52 2.64 with p <0.001 which was statistically significant . This result was in agreement with a study conducted by indira et al . Where number of rugae were slightly higher in males . . Also found in his study that the number of rugae were higher in males . Also found higher number of rugae in males; whereas verma et al . And manjunath et al . Found that there was more rugae in females . In our study, males were having more wavy rugae with 129 (51.6%)., saraf et al ., kamala et al ., nagalaxmi et al ., and bing et al . Also found wavy pattern to be higher in males . In contrast, babu et al . Found wavy pattern to be common in both the genders where males were having 4.82 1.91 and females were having 4.74 1.91 . Our study observed that males with 40 (16.0%) diverging type of rugae, 139 (55.6%) converging type of rugae and females with 49 (19.6%) diverging type of rugae, 144 (57.6%) converging type of rugae . Therefore, there was no significant difference in unification between males and females because p value was> 0.05 . Manjunath et al ., narang et al ., sharma et al ., azab et al ., and rajan et al . Studies conducted by chandra et al ., fahmi et al ., and saraf et al . Our study conducted in a central indian population observed that males were having more backwardly directed rugae with 64 (25.6%). Females were having more forward - directed rugae with 156 (62.4%) (p <0.001). Reddy et al . Found forward - directed rugae more commonly in north and south indian population . Saxena et al . Did not find any significant difference with direction of rugae between males and females . Dental identification has always played a role in natural and manmade disaster situations and in mass casualties normally associated with aviation disaster . Because of the lack of comprehensive fingerprint database, dental identification continues to be crucial . The central dogma of dental identification is that postmortem dental remains can be compared with antemortem dental records, including written notes, study casts, and radiographs to confirm identity . The palate not only represents a suitable repository for such unique and identifying features they also survive most postmortem events that can disrupt or change other body tissues . Thus, further studies are needed to investigate the possibility that there is a distinct ethnic difference in the palatal rugae morphology in human identification.
Along with colon cancer, rectal cancer is the second leading cause of cancer - associated deaths in the u.s . With about 40,000 newly diagnosed cases in 2014 . Despite the high percentage (approximately 25%) of metastatic cases at diagnosis, the 5-year survival rate has improved in recent years, mainly due to the development of systemic therapeutic strategies such as neoadjuvant chemoradiotherapy (crt). Neoadjuvant crt has been developed as a standard strategy, especially for the management of locally advanced rectal cancer (larc). With the advent of neoadjuvant crt, the survival rates of patients with larc has significantly improved . However, patients who do not reach a pathologically complete response (pcr) from neoadjuvant crt could more easily suffer local or distant recurrence at any time with poorer outcomes . The 5-year disease - free survival (dfs) hence, it is still necessary to find specific biomarkers to improve the selection of patients who will most benefit from neoadjuvant crt and therefore achieve a better outcome . This could help to prevent unnecessary radiation therapy and surgery delays . Increasing evidence suggests that cancer - associated inflammation is associated with poorer outcomes of cancers, including rectal cancer . Immune cells produce cytokines, chemokines, and inflammatory mediators to cause an inflammatory response leading to changes in circulating white blood cell levels . Various markers have been studied in solid tumors, including the neutrophil - to - lymphocyte ratio (nlr). Meta - analyses have indicated that nlr is a useful predictive factor in several cancers, such as esophageal cancer, gastric cancer, hepatocellular carcinoma, and non - small cell lung cancer . Hence, our study further explored the associations between the baseline nlr and prognosis following neoadjuvant crt for larc . The role of nlr as a predictor for pcr after neoadjuvant crt in larc was also assessed . In total, 202 patients diagnosed with larc (ct34 and/or cn12) and receiving neoadjuvant crt followed by radical surgery at zhejiang cancer hospital from february 2002 to december 2012 were included in this retrospective study . All patients had histologically confirmed rectal adenocarcinoma within 15 cm from the anal verge, and patients with distant metastasis were excluded from this study . All patients signed informed consent, and underwent pelvic radiation (range 4555 gy) with concurrent 5-fluorouracil - based chemotherapy (folfiri or folfox) before surgery . The pretreatment assessment included a detailed medical history and physical examination, digital rectal examination, endoscopy, biopsy, endorectal ultrasonography, abdominal and pelvic computed tomography (ct), chest x - ray / ct, and pelvic magnetic resonance imaging (mri). Laboratory examination included whole blood cell count, as well as liver and kidney function tests . The degree of primary tumor regression was determined by the amount of viable malignancy versus the amount of fibrosis as described by dworak et al . . Tumor regression grade 0 (trg0) was defined as no regression; trg1 as minor regression (dominant tumor with fibrosis in 25% of the tumor mass); trg2 as moderate regression (dominant tumor with fibrosis in 2650% of the tumor mass); trg3 as good regression (> 50% tumor regression); and trg4 as total regression (no viable tumor cells). Statistical analysis was performed using spss statistics software, version 20.0 (ibm, armonk, ny, usa). Continuous variables are expressed as the mean standard deviation and/or median (range). Receiver operating characteristic (roc) analysis was used to determine the cut - off value of the nlr . In this analysis, we established the cut - off value of the nlr with maximum sensitivity and specificity in predicting 3-year overall survival (os) and 3-year dfs . The correlation analysis between the nlr and the clinicopathological characteristics was performed using chi - squared tests . Survival analysis was performed using the kaplan - meier method with the log - rank test and the cox s proportional hazard regression test . A multivariate analysis was performed for the variables with p value less than 0.10 by univariate analysis . A 2-sided p value less than 0.05 was considered to be statistically significant . In total, 202 patients diagnosed with larc (ct34 and/or cn12) and receiving neoadjuvant crt followed by radical surgery at zhejiang cancer hospital from february 2002 to december 2012 were included in this retrospective study . All patients had histologically confirmed rectal adenocarcinoma within 15 cm from the anal verge, and patients with distant metastasis were excluded from this study . All patients signed informed consent, and underwent pelvic radiation (range 4555 gy) with concurrent 5-fluorouracil - based chemotherapy (folfiri or folfox) before surgery . The pretreatment assessment included a detailed medical history and physical examination, digital rectal examination, endoscopy, biopsy, endorectal ultrasonography, abdominal and pelvic computed tomography (ct), chest x - ray / ct, and pelvic magnetic resonance imaging (mri). Laboratory examination included whole blood cell count, as well as liver and kidney function tests . The degree of primary tumor regression was determined by the amount of viable malignancy versus the amount of fibrosis as described by dworak et al . . Tumor regression grade 0 (trg0) was defined as no regression; trg1 as minor regression (dominant tumor with fibrosis in 25% of the tumor mass); trg2 as moderate regression (dominant tumor with fibrosis in 2650% of the tumor mass); trg3 as good regression (> 50% tumor regression); and trg4 as total regression (no viable tumor cells). Statistical analysis was performed using spss statistics software, version 20.0 (ibm, armonk, ny, usa). Continuous variables are expressed as the mean standard deviation and/or median (range). Receiver operating characteristic (roc) analysis was used to determine the cut - off value of the nlr . In this analysis, we established the cut - off value of the nlr with maximum sensitivity and specificity in predicting 3-year overall survival (os) and 3-year dfs . The correlation analysis between the nlr and the clinicopathological characteristics was performed using chi - squared tests . Survival analysis was performed using the kaplan - meier method with the log - rank test and the cox s proportional hazard regression test . A multivariate analysis was performed for the variables with p value less than 0.10 by univariate analysis . A 2-sided p value less than 0.05 was considered to be statistically significant . The median age was 51 years (range, 2376 years) and 70.8% of the patients were male . Of these patients, 190 (94.1%) had r0 resection, and 103 (51.0%) received sphincter - preserving operations . Pathological results showed that the pcr rate was 18.8%, and the t - downstaging percentage was 61.9% . The lymph node ratio (lnr) of 52 (25.7%) patients was 0.1 . Poorly differentiated adenocarcinoma, mucinous adenocarcinoma, and signet - ring cell carcinoma were totally present in 73 (36.1%) patients . Thirty - six (17.8%) patients presented with perineural invasion (pni) and/or lymphovascular invasion (lni). Of the patients, 108 (53.5%) received adjuvant chemotherapy . The median follow - up was 45 months, with a range of 3162 months . At the last follow - up, 116 (57.4%) patients were alive, 82 (40.6%) had disease progression, 18 (8.9%) had local recurrence, 76 (37.6%) had distant metastasis, and 9 patients (4.5%) were lost to follow - up . The average nlr was 2.71.5 (median, 2.4; range, 0.612.8). In the roc analysis, we failed to identify an appropriate cut - off value because the area under the curve for nlr was 0.555 [95% confidence interval (ci), 0.4670.642; p=0.223] for the 3-year os and 0.558 (95% ci, 0.4770.638; p=0.159) for the 3-year dfs . Finally, an nlr 3.0 was considered as elevated nlr on the basis of previous studies . There were 63 (31.2%) patients with nlr 3.0 and 139 (68.8%) patients with nlr <3.0 . We compared the difference in clinicopathological characteristics between the nlr 3.0 group and the nlr <3.0 group (table 1). The results showed that elevated nlr were more common in patients ages <60 years (p=0.017). It is worth noting that there were no differences in the pcr rate or the t - downstaging rate between the 2 nlr groups . The correlations between os and various clinicopathological characteristics are shown in table 2 . Surgical margin status [hazard ratio (hr), 8.329; 95% ci, 4.16816.643; p=0.000], pcr (hr, 5.025; 95% ci, 2.03312.500; p=0.000), t - downstaging (hr, 2.049; 95% ci, 1.3443.115; p=0.001), pathological response (hr, 1.555; 95% ci, 1.0112.392; p=0.041), lnr (hr, 2.800; 95% ci, 1.8084.335; p=0.000), and perineural and/or lymphovascular invasion (hr, 2.614; 95% ci, 1.6194.222; p=0.000) were significantly associated with os on univariate analysis . In the multivariate analysis, r1/r2 resection (hr, 6.769; 95% ci, 3.12714.653; p=0.000), lnr 0.1 (hr, 2.467; 95% ci, 1.4484.203; p=0.001), and perineural and/or lymphovascular invasion (hr, 1.876; 95% ci, 1.0763.271; p=0.027) age (hr, 0.504; 95% ci, 0.2730.931; p=0.025), clinical n stage before crt (hr, 1.548; 95% ci, 0.9952.409; p=0.049), surgical margin status (hr, 6.389; 95% ci, 3.01213.551; p=0.000), pcr (hr, 2.801; 95% ci, 1.3485.814; p=0.004), t - downstaging (hr, 1.742; 95% ci, 1.1332.681; p=0.036), lnr (hr, 2.621; 95% ci, 1.6804.088; p=0.000), and perineural and/or lymphovascular invasion (hr, 2.661; 95% ci, 1.6344.334; p=0.000) were significantly associated with dfs on univariate analysis . In the multivariate analysis, r1/r2 resection (hr, 5.708; 95% ci, 2.54812.790; p=0.000), lnr 0.1 (hr, 2.376; 95% ci, 1.4193.978; p=0.001), and perineural and/or lymphovascular invasion (hr, 1.762; 95% ci, 1.0542.943; p=0.031) we did not find an association of nlr with os (hr, 1.066; 95% ci, 0.6811.668; p=0.779) or dfs (hr, 0.863; 95% ci, 0.5361.390; p=0.542) using 3.0 as a cut - off value (figures 1, 2). Furthermore, we repeated survival analyses using different cut - off values (2.0 and 4.0) and again did not find an association between nlr and survival outcomes . The median age was 51 years (range, 2376 years) and 70.8% of the patients were male . Of these patients, 190 (94.1%) had r0 resection, and 103 (51.0%) received sphincter - preserving operations . Pathological results showed that the pcr rate was 18.8%, and the t - downstaging percentage was 61.9% . The lymph node ratio (lnr) of 52 (25.7%) patients was 0.1 . Poorly differentiated adenocarcinoma, mucinous adenocarcinoma, and signet - ring cell carcinoma were totally present in 73 (36.1%) patients . Thirty - six (17.8%) patients presented with perineural invasion (pni) and/or lymphovascular invasion (lni). Of the patients, 108 (53.5%) received adjuvant chemotherapy . The median follow - up was 45 months, with a range of 3162 months . At the last follow - up, 116 (57.4%) patients were alive, 82 (40.6%) had disease progression, 18 (8.9%) had local recurrence, 76 (37.6%) had distant metastasis, and 9 patients (4.5%) were lost to follow - up . The average nlr was 2.71.5 (median, 2.4; range, 0.612.8). In the roc analysis, we failed to identify an appropriate cut - off value because the area under the curve for nlr was 0.555 [95% confidence interval (ci), 0.4670.642; p=0.223] for the 3-year os and 0.558 (95% ci, 0.4770.638; p=0.159) for the 3-year dfs . Finally, an nlr 3.0 was considered as elevated nlr on the basis of previous studies . There were 63 (31.2%) patients with nlr 3.0 and 139 (68.8%) patients with nlr <3.0 . We compared the difference in clinicopathological characteristics between the nlr 3.0 group and the nlr <3.0 group (table 1). The results showed that elevated nlr were more common in patients ages <60 years (p=0.017). It is worth noting that there were no differences in the pcr rate or the t - downstaging rate between the 2 nlr groups . The correlations between os and various clinicopathological characteristics are shown in table 2 . Surgical margin status [hazard ratio (hr), 8.329; 95% ci, 4.16816.643; p=0.000], pcr (hr, 5.025; 95% ci, 2.03312.500; p=0.000), t - downstaging (hr, 2.049; 95% ci, 1.3443.115; p=0.001), pathological response (hr, 1.555; 95% ci, 1.0112.392; p=0.041), lnr (hr, 2.800; 95% ci, 1.8084.335; p=0.000), and perineural and/or lymphovascular invasion (hr, 2.614; 95% ci, 1.6194.222; p=0.000) were significantly associated with os on univariate analysis . In the multivariate analysis, r1/r2 resection (hr, 6.769; 95% ci, 3.12714.653; p=0.000), lnr 0.1 (hr, 2.467; 95% ci, 1.4484.203; p=0.001), and perineural and/or lymphovascular invasion (hr, 1.876; 95% ci, 1.0763.271; p=0.027) age (hr, 0.504; 95% ci, 0.2730.931; p=0.025), clinical n stage before crt (hr, 1.548; 95% ci, 0.9952.409; p=0.049), surgical margin status (hr, 6.389; 95% ci, 3.01213.551; p=0.000), pcr (hr, 2.801; 95% ci, 1.3485.814; p=0.004), t - downstaging (hr, 1.742; 95% ci, 1.1332.681; p=0.036), lnr (hr, 2.621; 95% ci, 1.6804.088; p=0.000), and perineural and/or lymphovascular invasion (hr, 2.661; 95% ci, 1.6344.334; p=0.000) were significantly associated with dfs on univariate analysis . In the multivariate analysis, r1/r2 resection (hr, 5.708; 95% ci, 2.54812.790; p=0.000), lnr 0.1 (hr, 2.376; 95% ci, 1.4193.978; p=0.001), and perineural and/or lymphovascular invasion (hr, 1.762; 95% ci, 1.0542.943; p=0.031) were independently associated with worse dfs . We did not find an association of nlr with os (hr, 1.066; 95% ci, 0.6811.668; p=0.779) or dfs (hr, 0.863; 95% ci, 0.5361.390; p=0.542) using 3.0 as a cut - off value (figures 1, 2). Furthermore, we repeated survival analyses using different cut - off values (2.0 and 4.0) and again did not find an association between nlr and survival outcomes . The tumor node metastasis (tnm) staging and dukes staging system are the basis for subdividing colorectal cancer, but even patients with the same stage may turn out to have different clinical characteristics and outcomes . Hence, supplementary methods, such as the laboratory index, added to the tnm staging and dukes staging system, may have an important role in the determination of personalized treatment strategy . Data also showed that patients who used regular non - steroidal anti - inflammatory drugs had decreased risk of colorectal cancer . Clinical studies and pool analyses have shown that several markers of systematic inflammatory reactions, such as c - reactive protein, modified glasgow score, and nlr, show prognostic value in patients with cancer . Among the laboratory markers, nlr has some advantages, such as low cost and wide availability . A meta - analysis of 7 studies involving 959 patients suggested that elevated nlr was related to worse os, dfs, and recurrent - free survival . Interestingly, all the studies included in the meta - analysis obtained positive results . Leonardo et al . Reviewed a cohort of 175 patients, and analyzed the association between nlr and survival outcomes using different cut - off values (2.0, 2.5, 3.0, 4.0, and 5.0). The results did not show differences in disease - specific survival, recurrent - free survival, or pcr . In our present study, we also found no association of the nlr with either os or dfs . One explanation for these contradictory results could be the clinical heterogeneity of the patients enrolled in the different studies . In our cohort, a higher proportion (59.4%) of patients had clinical stage t4, compared with other clinical studies of rectal cancer . In our previous study, we found that tumor burden, which is regarded as an important prognostic factor, was the most pivotal factor influencing systemic inflammation before treatment in colorectal cancer patients . We speculate that nlr, as a type of systemic inflammatory marker, may predict the prognosis by distinguishing patients with different tumor burdens . If a group of patients had a wide spectrum of tumor burdens, nlr would be an effective predictor of outcomes . In contrast, if patients enrolled in a certain study had a narrow spectrum of tumor burdens, the predictive power of the nlr would be weakened . In addition, when interpreting the contradictory results in different studies, other possible reasons need to be taken into account, such as random error caused by small sample and variability of treatment in different centers . In addition to finding no association between the nlr and survival outcomes, we also found no association between the nlr and short - term effects indicators of crt (e.g., pcr, downstaging, and pathological response), for which previous studies have yielded conflicting results . However, there is no convincing explanation of the mechanism by which systemic inflammatory factors affect the short - term actions of crt . In the present study, we found that younger patients were associated with higher nlr, while previous studies have not found this correlation . The most probable reason for this disagreement is the randomly caused uneven distribution of baseline characteristics in different age groups . First, this was a retrospective study, and some comorbidity that may affect the level of nlr had not been included in our analysis . Second, nearly 3/5 of the patients in our cohort were ct4, and the percentage was higher when compared with that of other studies . Finally, the heterogeneity of the tumor burden in different studies may affect the predictive power of the nlr . In our cohort, the nlr did not correlate with survival outcomes in larc patients undergoing neoadjuvant crt . The prognostic value of nlr should be validated in future large - scale prospective studies.
It promotes calcium and phosphate absorption in the intestine and maintains an adequate concentration of these minerals in circulation to allow for normal bone mineralization . Furthermore, vitamin d deficiency can lead to rickets in children and osteomalacia in adults . Recently, many nonskeletal actions of vitamin d on cellular proliferation and differentiation, muscle function, and immunity have also been suggested . Several clinical studies have shown that vitamin d deficiency or insufficiency is associated with an increased risk of nonskeletal health conditions, including cardiovascular diseases, diabetes mellitus, cancers, infections, and autoimmune diseases [2 - 15]. Although vitamin d can be obtained from dietary sources such as fatty fish and meat, the vitamin d in the human body is mainly acquired by cutaneous synthesis in response to sunlight . Once vitamin d is formed in the skin surface, it undergoes two sequential hydroxylations in the liver and the kidney in order to become biologically active . As people increasingly live in cities and spend the majority of their time indoors, it has become more difficult to get sufficient sun exposure for adequate cutaneous production of vitamin d. furthermore, even when outdoors, many people use sunscreen and avoid direct exposure to sunlight due to concerns about skin cancer and aging . Therefore, there is a very high prevalence of vitamin d insufficiency worldwide . In particular, as korea has experienced rapid industrialization and economic development over the past few decades, vitamin d insufficiency has become a public health problem not only in elderly people, but also in younger generations due to changes in lifestyle and occupations . It has been well established that vitamin d status is best assessed using the serum concentration of 25-hydroxyvitamin d [25(oh)d], which is produced in the liver and reflects vitamin d obtained from both cutaneous synthesis and dietary sources . Although previous studies have investigated what constitutes a sufficient level of 25(oh)d using several criteria, including serum parathyroid hormone suppression, calcium absorption, bone mineral density, and fall or fracture rates, there is no clear consensus on what level of serum 25(oh)d is sufficient . According to the world health organization, serum 25(oh)d levels below 25 nmol / l however, there have been many proposals to increase the lower threshold of sufficient 25(oh)d level to upwards of 75 nmol / l . Suggested that the most advantageous serum 25(oh)d levels begin at 75 nmol / l, with the optimal level ranging from between 90 and 100 nmol / l . Most recently, the institute of medicine in the united states suggested that a serum 25(oh)d level of 50 nmol / l is sufficient to ensure bone health after an extensive literature review of vitamin d . Therefore, it is still controversial whether sufficient vitamin d status should be defined as a serum 25(oh)d level of above 50 or 75 nmol / l . Vitamin d status in the korean population has been previously investigated based on the korea national health and nutrition examination surveys iv conducted in 2008 (table 1). The mean serum 25(oh)d level measured by radioimmunoassay (ria) (diasorin, stillwater, mn, usa) was 52.9 the prevalence of vitamin d insufficiency, defined as a serum 25(oh)d level below 50 nmol / l, was 47.3% in males and 64.5% in females . With a serum 25(oh)d level of 75 nmol / l as the threshold, the prevalence of vitamin d insufficiency rose to 86.8% in men and 93.3% in women . Interestingly, the study showed that serum 25(oh)d level increases with age from 20 - 29 to 60 - 69 years in both sexes . Furthermore, contrary to the common belief that vitamin d insufficiency is more prevalent in elderly people, vitamin d insufficiency was most prevalent in the 20 to 29 age group (65.0% in males and 79.9% in females, with 50 nmol / l as the threshold of vitamin d insufficiency). In the same study, vitamin d insufficiency was more prevalent among those who work indoor occupations such as sales, service, administration, clerical work, or specialists compared with those who have outdoor occupations such as agriculture, forestry, or fishing . Although vitamin d status has been studied in many countries worldwide, relatively few studies have assessed the vitamin d status of a population based on large representative samples . In the united states and canada, the vitamin d status of the population has been investigated in nationwide surveys (table 1). In the united states, vitamin d status was investigated in the national health and nutrition examination survey (nhanes) (table 1). Serum 25(oh)d levels were measured using ria (diasorin) in the nhanes 1988 to 1994 and the nhanes 2001 to 2006 . Nmol / l in 1988 to 1994 and 55.2 nmol / l in 2001 to 2006 . The prevalence of vitamin d insufficiency, defined as a serum 25(oh)d level of less than 50 nmol / l, was 29% in the nhanes 1988 to 1994 and 32% in the nhanes 2001 to 2006 . With a serum 25(oh)d level of 75 nmol / l as the threshold, the prevalence of vitamin d insufficiency was 69% in the nhanes 1988 to 1994 and 76% in the nhanes 2001 to 2006 . Overall, there was a trend toward an increased prevalence of vitamin d insufficiency in 2001 to 2006 compared to 1988 to 1994 . In canada, vitamin d status was investigated in the canadian health measures survey from 2007 to 2009 (table 1). Serum 25(oh)d levels were measured by a chemiluminescence assay using the liaison 25-hydroxyvitamin d total assay (diasorin). For both sexes, the serum level of 25(oh)d was highest among children and seniors, and lowest in those aged 20 to 39 years . Nmol / l was 67.0% in males and 62.2% in females and highest in those aged 20 to 39 years . An individual's vitamin d status is determined by several factors related to cutaneous synthesis or dietary intake of vitamin d. accordingly, traditional risk factors for poor vitamin d status include limited sun exposure, higher latitude, winter season, darker skin pigmentation, sunscreen use, skin - covering clothing, and a diet low in fish and dairy products . Older age is commonly suggested to be a risk factor for vitamin d insufficiency because the cutaneous synthesis of vitamin d declines with age . Aging can decrease the capacity of the skin to produce previtamin d in response to ultraviolet radiation by greater than 2-fold . However, in korea and canada, vitamin d insufficiency was more prevalent among young adults than in the older people . Although the cause of this finding is not clear, it may be due to behavioral factors such as an indoor lifestyle, sunscreen use, outdoor activity, or dietary habits . Previously, the national diet and nutrition survey 1992 to 2001 of the united kingdom also showed that vitamin d insufficiency was most prevalent among young adults aged 19 to 24 years as well as in elderly people over 85 years of age . Despite growing awareness of the multiple health benefits of vitamin d, vitamin d insufficiency remains a major health problem worldwide . It is not only a health concern of elderly people, but has been shown to affect all age groups . In korea, compared with the united states and canada, korea has a lower mean 25(oh)d level and a higher prevalence of vitamin d insufficiency, although it is important to exercise caution when comparing results from surveys conducted in different laboratories or using different methods (table 1). Therefore, it should be a critical public health issue to improve the vitamin d status of the korean population . Due to the heavily indoor lifestyle of modern society, it is difficult to get adequate sunlight exposure for sufficient vitamin d production . Furthermore, it is also challenging to change the dietary habits of a population in order to increase the intake of foods rich in vitamin d such as fish . More realistic ways of improving vitamin d status include food fortification and supplementation with vitamin d. an aggressive national policy on food fortification for the general population should be adopted . Likewise, the promotion of vitamin d supplementation is necessary, especially for specific groups at risk for vitamin d insufficiency.
Exogenous agents such as ionising radiation (ir) and ultraviolet light or endogenous agents such as free radicals produced within cells can damage the dna of eukaryotic organisms . Diverse mechanisms have evolved to detect and repair dna damage that threatens the integrity of the genome . Here, we study two dna damaging agents used in the treatment of cancer, ir and the epipodophyllotoxin drug etoposide that acts on topoisomerase ii . Cellular dna damage responses to ir exposure have been extensively investigated and several pathways exist within the cell to respond to double strand breaks (dsbs) induced by ionising radiation (ir). Histone h2ax is rapidly phosphorylated following ir, with foci observed within the first minute following exposure [13]. The phosphorylation of h2ax occurs over megabase regions of chromatin extending away from the site of dna damage and initiates assembly of several proteins involved in the dna damage response . Phosphorylation has been shown to be essential for correct amplification of the dna damage response [57]. At sublethal levels of dna damage, phosphorylated h2ax (termed h2ax) forms distinct foci within the cell nuclei . At less than 150 dsbs per nucleus, there exists a 1: 1 relationship of h2ax foci: dsbs . At these levels of dna damage, h2ax can be used as an accurate and sensitive surrogate reporter of dna dsb levels . Can also be phosphorylated in response to topoisomerase ii - targeting agents [912]. Topoisomerase ii is an enzyme that alters the topological state of dna via a transient covalent enzyme - bridged double strand break in the dna, through which a second dna helix can pass . These protein associated breaks can be stabilised by drugs such as etoposide [13, 14]. Two isoforms of topoisomerase ii exist, termed and, these are both targeted by etoposide [1618]. The genotoxic effects of etoposide are generally considered to be mediated through conversion of stabilised protein - dna complexes to protein free frank dsbs [19, 20], possibly via collisions between the drug - stabilised topoisomerase - dna complex and rna polymerase during transcription or with dna replication forks, analogous to the situation seen with topoisomerase i [21, 22]. Frank dsbs may also be generated by proteolytic degradation of the topoisomerase ii moiety [2326] and topoisomerase ii is thought to be preferentially degraded over the isoform . A reduction in etoposide induced dsb levels was reported in cells cotreated with the proteasome inhibitor mg-132, suggesting that the proteasome has a role in converting etoposide - stabilised protein - dna complexes into frank dsbs . Additionally a 5 tyrosyl dna phosphodiesterase (ttrap) has recently been identified that may play a role in generating frank dsbs ready for repair . Topoisomerase ii has been implicated in the cellular response to dna dsbs . Down regulating topoisomerase ii by sirna altered the response to radiation whilst topoisomerase ii has been reported to play a role in promoting dsb repair following peroxide damage . The damage sensor topbp1 was first identified as a topoisomerase ii interacting protein, and wstf (williams syndrome transcription factor) which regulates the h2ax dna damage response interacts with winac, a topoisomerase ii containing multi protein complex . Thus topoisomerase ii may be directly involved in damage detection and signalling following ir via protein - protein interactions . Alternatively, topoisomerase ii may be required for proper regulation of genes involved in the damage responses . For example, cells downregulated for topoisomerase ii have been reported to express reduced peroxiredoxin 2 . To investigate whether topoisomerase ii affects the cellular response to ir or etoposide induced dna damage, we used wt and top2 mefs, we used h2ax assays and in parallel the trapped in agarose dna immunostaining (tardis) assay to examine the kinetics of formation and removal of topoisomerase ii - dna complexes in response to ir or etoposide treatment . Wild - type mtop2-4 (containing both topoisomerase ii and topoisomerase ii) [wt] and mtop2-5 [top2] immortalized mouse embryonic fibroblasts (mefs) have been described previously and were maintained as monolayers in dulbecco's modified eagle's medium (dmem) supplemented with 10% foetal bovine serum (fbs) and penicillin (50 g / ml)/streptomycin (50 g / ml). All chemicals were obtained from sigma - aldrich (poole, uk) and vwr international (lutterworth, uk). Ir exposure was carried out using a gammacell 1000 irradiator with a [cs] source (nordion international, inc .) And was delivered at a rate of approximately 3.08 gy / min . The h2ax focus assay was performed as described previously in detail by watters and colleagues . Wt and top2 mefs were seeded at 3 10 cells / well into 6-well tissue culture plates and grown to approximately 80% confluency . Cells were exposed to the appropriate dna damaging agent (ir or etoposide) and harvested by trypsinisation at specific time - points as detailed in the text . Trypsinised cells were resuspended in 1 ml of ice - cold pbs, centrifuged at 1000 rpm for 3 minutes and then resuspended in 50 l of ice - cold pbs . Slides were probed with an antibody that detects topoisomerase ii and . Quantification of complex levels has been described previously . Statistical analyses were carried out using graphpad prism 4 software (cherwell scientific, oxford, uk . ). Two - tailed, paired, and unpaired students t - tests were generally used to compare data sets and data sets between cell lines; analysis of variance (anova) was used for multiple comparisons where appropriate, as detailed in the text . All statistical analyses were calculated using a 95% confidence interval (p <.05). H2ax formation was used as a surrogate marker for dsbs in parallel with topoisomerase ii - dna complex measurement in both wild - type and topoisomerase ii null murine embryo fibroblast cell lines (mefs). The response to three doses of ir (0.5, 1 and 2 gy) was determined in mefs wild type (wt) or null for topoisomerase ii (top2). In response to 1 gy, h2ax focus numbers were similar between the two cell lines, with ~35 h2ax foci per nucleus at 20 minutes (figure 1(a)). H2ax foci were most abundant at the 20 minute time - point after irradiation and decreased at each subsequent time - point investigated and returned to background levels at 24 hours (figure 1(a)). Following 2 gy (figure 1(b)), focus numbers appeared higher in the wt cell line than the top2 at 20 minutes, 2 hours, and 5 hours following exposure . The lower foci number in the top2 cells suggests the initial dna damage responses following ir are altered in these cells, however, the differences were not statistically significant by two way anova or students t - test . Nor was there any significant difference between the levels of apoptosis following ir between the two cell lines . The trapped in agarose dna immunostaining (tardis) assay was used to detect topoisomerase ii protein - dna complex levels in these cell lines following ir . Topoisomerase ii complex formation did not increase in response to ir in wt or top2 cells when compared to the untreated cells, as previously reported for cem cells (data not shown), thus ir had no effect upon topoisomerase ii - dna complex levels in the 24 hours following exposure to 2 gy . In an etoposide dose finding experiment topoisomerase ii wild - type and null mef cell lines were exposed to 0, 0.1, 1, 10, or 100 m etoposide for 2 hours, after which time cells were placed in drug - free media for 20 minutes before being assayed for h2ax foci . Foci numbers per nucleus increased in both cell lines with increasing dose of drug . At 100 m to ensure foci were within a countable range, subsequent experiments were performed using doses of 1 m and 10 m only . Wt and top2 cells were exposed to 1.0 m etoposide for 2 hours, cells were then resuspended in drug - free media and assayed for h2ax at different time points after drug removal, 20 minutes, 2 hours, 5 hours, and 24 hours following removal of drug (figure 3). The kinetics of focus formation and removal were comparable between the wild - type and top2 cell lines, and by 24 hours postdrug removal foci numbers were similar to untreated controls . No statistically significant difference was found in focus numbers between the two cell lines, indicating that topoisomerase ii status did not affect the kinetics of disappearance of h2ax phosphorylation following a 2 hour exposure to 1 m etoposide, this is consistent with the evidence that the cytotoxic effect of etoposide is mainly mediated via topoisomerase ii in these cells, since the ic50 for etoposide did not differ significantly between the two cell lines . The topoisomerase ii - dna adducts levels measured by tardis after a 2 hour exposure to etoposide has previously been reported . Wild - type and top2 cells were also exposed continuously to 1.0 m etoposide over the course of 24 hours, and samples removed to quantify h2ax foci formation at various time points (figure 4). After two hours of etoposide exposure the foci numbers were comparable to that seen after the two - hour short term exposure, numbers then increased further with continued exposure to etoposide . The maximal foci numbers were seen after 8 hours of etoposide, and they then decreased by 24 hours even though etoposide was still present . The focus numbers were statistically different between the two cell lines only at the 24 hours point (p <.05), when the wt cell line had approximately double the number of foci compared to the top2 cell line . Topoisomerase ii - dna complex levels in wt and top2 cells were determined using the tardis assay at time - points over a 24 hour continuous exposure to etoposide at 1 m (figure 5(a)) or 10 m (figure 5(b)). At both drug doses and in both cell lines, treatment induced a time - dependent increase in topoisomerase ii dna adduct levels (fitc immunofluorescence) up to the 8 hour time - point followed by a decrease at the 24 hour time - point (figures 5(a) and 5(b)). With 1 m etoposide, the increase was significant at 8 hours in wt cells and at both 5 hours (p <.05) and 8 hours (p <.01) in top2 cells . Although levels decreased at 24 hours, immunofluorescence was still significantly greater than background levels in both wild - type and top2 cells (p <.05). When cells were exposed to 10 m etoposide, immunofluorescence levels in wt cells became statistically significant at 2 hours after drug addition (p <.05) and remained elevated at all other time - points (p <.01). In top2 cells, levels were significant at 1 hour (p <.01), 5 hours (p <.05), and 24 hours (p <.001). Notably immunofluorescence levels were greater in the top2 cells than the wild - type cells (figure 5) at both drug doses and all time - points (all p - values <.05). The higher complex levels seen in cells lacking topoisomerase ii may result from the longer half life of topoisomerase ii complexes or be due to a role of topoisomerase ii in sensing and/or promoting repair or alternatively to downregulation of peroxiredoxins in top2 cells [32, 33]. To investigate whether the decrease in topoisomerase ii - dna complexes between the 8 hour and 24 hour time - points was due to proteasomal degradation of topoisomerase ii, wt and top2 cells were incubated in the presence of the proteasome inhibitor, mg-132, for 30 minutes prior to and during 24 hour exposures to 1 m and 10 m etoposide . As shown in figure 6(e), when treated with mg-132 alone, topoisomerase ii - dna complex levels were only significantly elevated above background levels at the 24 hour time - point . Immunofluorescence levels were comparable in the wt and top2 cells at all time - points considered . In cells cotreated with etoposide and mg-132, topoisomerase ii - dna complex levels did not decrease between the 8 hour and 24 hour time - points, as seen in cells treated with etoposide alone . Figure 6(a)6(d), demonstrate that cotreatment led to increased immunofluorescence at the 24 hour time - point, in both cell lines and at both drug doses . This increase was most dramatic in wt cells, where cotreatment led to a 6-fold increase in immunofluorescence in cells treated with 1 m etoposide and a 15-fold increase in cells treated with 10 m etoposide . In the top2 cells, this indicates that topoisomerase ii removal at 24 hours is mediated via the proteasome, and that the effect is greatest on topoisomerase ii, in agreement with previous studies [23, 25, 26]. In response to ir phosphorylation of histone h2ax was triggered immediately, and slightly less phosphorylation was seen in top2 cells, which may indicate altered dna damage sensing . As previously reported, ir did not produce topoisomerase ii - dna adducts . In response to etoposide both h2ax foci and topoisomerase ii - dna adducts were formed, with foci appearing 1 hour after treatment in wt cells and after 2 hours in top2 cells . This was slower than following treatment with ir, and presumably reflects the need to remove the topoisomerase ii adduct to produce a frank dsb to trigger phosphorylation of histone h2ax . In both instances, levels become maximal at the 8 hour time - point and subsequently decrease at 24 hours . Topoisomerase ii complexes in wt cells but not top2 cells increased significantly at 24 hours when the proteasome was inhibited, suggesting that topoisomerase ii adducts are removed by the proteasome.
Neurofibromatosis type 1 (nf1) is an autosomal dominant genetic disorder occurring in approximately 1 in 3000 live births . Mutation of the nf1 gene (chromosome 17q11.2), neurofibromin 1, results in activation of the ras signaling pathway which has far - reaching effects in many cellular and neurodevelopmental processes . As a tumor suppressor, reduced production of neurofibromin results in the characteristic tumors that define nf1 . Common physical characteristics include caf au lait spots, cutaneous or subcutaneous neurofibromas, axiliary freckling, and lisch nodules, with diagnosis made based on physical characteristics . About half of cases are inherited from a parent and about half are sporadic mutations . Children with nf1 are at increased risk for cognitive, attention, and learning problems [36]. Features identified in individuals with nf1 include poor performance in the areas of language expression and comprehension, written language, reading accuracy, mathematics, and fine motor skills [710]. However, there has been little comprehensive examination of the functional impact of these difficulties in everyday life . The goal of this study is to evaluate the pattern of adaptive functioning in young children with nf1 as an indication of the functional impact of the difficulties seen in nf1 . The american association on mental retardation (aamr) defines adaptive behavior as the collection of conceptual, social, and practical skills that have been learned by people in order to function in their everyday lives (p. 73). Sattler and hoge further elaborate, indicating that conceptual skills are seen in receptive as well as expressive language, reading, writing, and handling money, that social skills involve establishing friendships, social reasoning, interpretation, and comprehension, and examples of practical skills include actions such as bathing, preparing food, washing dishes, basic housekeeping, and taking medicine . Hence, adaptive behavior relates to practical skills and abilities needed to function independently in home, social, school, work, and community settings . Most measures of adaptive behavior in the preschool and early school - age years include measures of social, communication, daily living, and community living skills and also include motor skills especially in young children, most frequently as described by parents . Studies of relations between adaptive behavior and cognitive functioning suggest that these are closely coupled . There have been few investigations of broad adaptive behavior in children with nf1 . As part of a much larger descriptive study n = 20) were less functionally independent than were unaffected siblings (n = 20) in the areas of broad independence, motor skills, social and communication skills, and community living skills, but not in personal living skills, with the largest group difference in motor skill development . Using the adaptive scales of the achenbach teacher report form, with 79 children ages 8 through 16, barton and north found that children with nf1 had significantly worse adaptive functioning than children without nf1, as well as poorer adaptive functioning when compared to normative data . The adaptive scales of the achenbach use an open - ended format to ask parents to list activities, hobbies, and chores and are not considered an adequate comprehensive measure of adaptive functioning [12, 18], rather serving as a screening tool . In sum, the study of adaptive behavior in nf1 is sparse, and there have been no studies of adaptive behavior in younger children . There is also an accumulation of prior research suggestive of difficulties with the separate conceptual, social, and motor skills components of adaptive behavior in children with nf1, using structured standardized assessments . Related to conceptual skills, a downward shift in intellectual functioning and increased risk of learning difficulties is consistently described based on direct measurement of these abilities with standardized assessment methods . The vast majority of children with nf1 show overall cognitive functioning in the average range, although specific areas of difficulty are generally noted for close to half of people with nf1 [19, 20]. Language and communication difficulties are also commonly described, with some beginning evidence that these difficulties are discernible on standardized individually administered language assessments even in the preschool years [2123]. Related to social skills, a number of studies have demonstrated social skills difficulties, based on parent and teacher report as well as peer ratings [15, 17, 24, 25] in comparison to same - aged peers or normative data, even for children as young as kindergarteners . In the area of motor skills, as mentioned, dilts and colleagues pointed specifically to the prominence of everyday difficulties in motor skills . Several other investigations have similarly revealed that motor difficulties are common for children with nf1 based on parental report and lab - based measures [2730], although these difficulties typically do not reach the level of clear impairment . Hence, difficulties with many of the components and contributors to overall adaptive functioning have been described in the literature about school - aged children with nf1 . However, few have been overarching studies of all of these domains together, focusing on the day - to - day functional impact of these difficulties from parents' perspectives . Moreover, the literature about adaptive functioning in young children with nf1 is sparse; there have been no published studies of adaptive behavior in young children with nf1 using one of the commonly used comprehensive measures of adaptive behavior . In the current study, adaptive functioning of children 3 through 8 years is examined in comparison to unaffected children using a broad parent interview measure covering social, communication, daily living, community, and motor skills domains . Given that a downward shift in intellectual functioning is seen in nf1 and adaptive functioning is closely coupled with intellectual functioning, it is hypothesized that the children with nf1 will show weaker adaptive skills in comparison to same - aged peers . In other words, it is expected that the cognitive difficulties in children with nf1 will translate into difficulties in everyday functioning . Based on prior research, it is expected that motor skills and social / communication skills will be particularly affected . Participants were 61 children with nf1 (36 male, 25 female; 24 familial cases, 37 sporadic) and 55 unaffected children (34 male, 21 female) consisting of 30 siblings of children with nf1 and 25 community participants between the ages of 3 and 8 years . All children with nf1 and siblings were included in the sample regardless of intellectual functioning . Among the unaffected children recruited from the community (n = 36), there was an overrepresentation of children with above - average intellectual functioning, so those with a general cognitive ability score over 115 were excluded from the study (n = 11). The sibling and community participants were compared on a number of relevant variables prior to combining them into one group . The groups did not differ in age, cognitive functioning, gender distribution, or adaptive behavior at the overall or domain level . Therefore, the two groups were combined to maximize power rather than using a three - group analytic approach . Participants with nf1 and their siblings were recruited from neurofibromatosis specialty clinics in the midwest designed for the medical management and anticipatory guidance regarding nf1 . Participants were approached by study personnel at their yearly medical check - in; participants attending clinic for initial diagnostic assessment were not approached . It was emphasized to families that the purpose of the study was to describe cognitive and psychosocial functioning of young children with nf1 and that it was important that participants with a range of functioning be included both children experiencing developmental difficulties and children for whom no difficulties were currently noted . Hence, the assessments conducted were not conducted due to clinical concerns about neurodevelopmental functioning . The response rate was high; only two of the families approached declined to participate . Inclusion criteria included the following: (1) clinical diagnosis of nf1; (2) primary language english . Three participants had identified optic glioma for which chemotherapy treatment was provided together with shunting for hydrocephalus, one had hydrocephalus alone, and four had asymptomatic optic glioma that were untreated . A physician completed severity report, based on a medical severity measure, was available for 50 of the 61 participants indicating minimal severity for the vast majority of the participants (n = 30), mild severity for 11 participants, moderate severity for 8 participants, and high severity for 1 participant . Analyses were conducted both including and excluding those with a history of optic glioma and/or hydrocephalus, with no substantial differences in findings . Community participants were recruited by posting fliers in areas frequented by families with young children (coffee shops, ymca, and libraries). Participants were ineligible for participation if they had an identified neurodevelopmental disorder or received special education services at school . The scales of independent behavior revised (sib - r;) is a widely used comprehensive measure of adaptive behavior . The assessment is arranged into 4 clusters which in turn are comprised of multiple subscales (table 4 contains a listing of the domains and subdomains). The broad independence (bi) index of the measure provides an overall assessment of adaptive behavior, the domain scores provide a sense of functioning in specific areas, and subdomain scores provide a sharper view of specific abilities . Standard scores (m = 100, sd = 15) are available at the domain level, and qualitative descriptors are available at the subdomain level . The sib - r was normed on approximately 1,700 individuals who range in age from infants less than three months old to mature adults . Parents were interviewed and asked to rate their children's behavior using a four point rating scale which ranges from 0 (never or rarely (does)even if asked) to 3 (does very well always or almost always without being asked). In this study, standard scores in broad independence and at the domain level were examined, and qualitative descriptors were examined at the domain and subdomain levels . The qualitative descriptors indicate how difficult age - level tasks in the given domain are expected to be for the child, based on a relative mastery index . The number of children for whom age - level tasks are expected to be difficult was examined . The differential ability scales second edition (das - ii;) is a comprehensive individually administered assessment of cognitive abilities that are important to learning including verbal reasoning, nonverbal reasoning, and spatial abilities . It yields a general cognitive ability (gca) standard score, which reflects overall conceptual / reasoning ability, and is similar to an intelligence quotient . This measure is a validated measure of cognitive functioning for use with children ages 2 years, 6 months through 17 years, 11 months . This study used the early years form for children three to eight years of age . The sib - r was individually administered to parents as a structured face - to - face interview, as standardized . Children were individually administered the das - ii, as the first part of a larger battery that included age - appropriate lab - based experimental measures of temperament and emerging executive functioning, with specific measures appropriate to chronological age . Parents also completed additional questionnaires assessing psychosocial functioning that are not included here as the versions of the measures varied across the age range of the current study . The data were analyzed using spss . Findings at p <.05 were generally considered significant . When multiple comparisons were conducted (i.e., more than 4), skewness and kurtosis were acceptable for all variables for both groups of participants . For t - test, levene's test for equality of variances occasionally indicated a violation of this assumption, but pooled variance t - test showed identical findings to traditional t - test . Bivariate pearson correlations between age and scaled scores on the sib - r were examined (see table 3). For the nf1 group, there were no significant correlations with age . For the unaffected participants, a significant correlation between age and personal living standard score was observed, with older children showing stronger personal living skills relative to their peers . No group differences in gender were found at the domain level or scale level . T - test indicated that a significant group difference in gca was observed (t = 6.17, p <.001). Bivariate pearson correlations with iq were also examined (see table 3). For both the nf1 group and the unaffected group, there were significant correlations between iq and all adaptive behavior standard scores, with the exception of motor skills (r(61) = .318, p = .013 for the nf1 group, r(61) = .310, p = .021) for the unaffected participants . Based on t - tests, significant group differences were found in overall adaptive functioning, motor skills, and community living skills, with a trend towards a significant difference in social / communication (see table 2). To examine whether group differences in adaptive functioning were largely driven by group differences in intellectual functioning, this analysis showed no significant group differences in overall adaptive functioning or any specific domains once intellectual functioning was taken into account, although there was a trend for group differences in motor skills (f = 3.30, p = .072). To further probe adaptive functioning problems related to nf1 and minimize the potential role of decrements in intellectual functioning through experimental design rather than statistical control, the manova was rerun including only participants in both groups with intellectual functioning in the average range or stronger (gca> 84). This subgroup was comprised of 45 children with nf1 (27 male, 18 female) and 54 unaffected children (33 male, 21 female). The two subgroups continued to show significant differences in gca (t(97) = 4.48, p <.001) but did not differ on ses (t(93) = .36, p = .72) or age (t(97) = .94, p = .35). The children with nf1 exhibited significantly lower functioning in motor skills (t(97) = 2.73, p = .008). The proportion of the children in each group showing difficulties in adaptive functioning was also examined and compared across the groups (see table 4). Difficulties were identified based on relative mastery index scores classified by the sib - r as reflecting difficulty . At the domain level, group differences in motor functioning and in broad independence were noted . At the subdomain level, group differences were present for gross and fine motor skills, language expression, and time and punctuality . In this examination of everyday adaptive behavior of young children with nf1, children with nf1 showed weaker adaptive behavior than same - aged peers at the overall level and in motor and community living skills, suggesting that the well - documented general intellectual difficulties seen in children with nf1 translate into difficulties in real - world functioning . Analogous to findings regarding intellectual functioning in nf1, a slight downward shift in average adaptive functioning was observed . While children with nf1 do not show stark areas of impairment in adaptive functioning, these results nevertheless provide evidence that nf1 leads to a dampening of adaptive functioning and that, like in other populations, decrements in adaptive functioning appear to be closely intertwined with general intellectual difficulties . There is controversy regarding the appropriateness of controlling for intellectual functioning when examining the behavioral phenotype associated with neurodevelopmental disorders and for nf1 specifically, with the argument that iq is a global functioning variable and that controlling for this general outcome variable can lead to meaningless findings, given that iq would be expected to be related to many other variables, including adaptive behavior . Therefore, some discussion of group differences in adaptive functioning, before considering patterns of findings taking into account the role of intellectual functioning, is warranted . First, group differences in adaptive behavior, across the majority of the domains, were noted . While mean scores indicated functioning in the average range for the nf1 group, examinations of the frequency of difficulties in each of the domains and subdomains revealed that more children with nf1 than children in the contrast group show adaptive difficulties in all areas . At the subdomain level, difficulties with both fine and gross motor skills and with language expression were most notable . Practitioners may find that administration of measures of adaptive behavior to parents is useful to identify children with nf1 with motor or language difficulties, who would benefit from more comprehensive assessment and developmental support . In comparison to the only other study broadly examining adaptive functioning in children with nf1, the level of adaptive functioning in this sample was considerably higher, especially in the motor skills domain . More specifically, mean adaptive behavior in the dilts study was 88, while in the current study it was 96 . At the domain level, the greatest difference in findings between the studies is in the motor domain; dilts and colleagues found that mean motor functioning was in the borderline range, while in the current study it was in the average range . Findings in the other domains are largely analogous . While no age effects were seen in the current study, it remains possible that motor difficulties might be seen as becoming more pronounced over time if a larger age span was considered . Alternatively, this difference may relate to differences in ascertainment approaches or to improvements in early identification of children with nf1 without significant symptomatology . It is possible that nf1 is now being diagnosed at younger ages by physicians and that children with physical signs of nf1 but no developmental difficulties may be more likely to be identified now than at the time of dilts and colleagues' study . While there is indeed controversy about statistically controlling for intellectual functioning, as discussed above, inclusion of analysis statistically controlling for intellectual functioning is nonetheless informative as it demonstrates a potentially strong role for intellectual functioning in adaptive skills . These abilities are closely intertwined for children with nf1, as they are in the general population . Based on both the statistical and experimental design approaches to controlling for intellectual functioning, there is suggestive evidence that nf1 has an impact on everyday motor functioning that is separate from its impact on intellectual functioning . Motor functioning is indeed a very commonly noted area affected by nf1 [27, 28, 36]. Even for those children who have average or above - average cognitive functioning, there may still be additional difficulties with motor skills in comparison to same - aged peers . These motor difficulties may translate into greater frustration with school - based tasks such as learning handwriting and may also potentially affect recreational skills such as participation in group - based sports activities . Further research about the effects of motor functioning difficulties on the lives of children with nf1 is warranted . In sum, this is one of the first studies to comprehensively examine adaptive behavior in young children with nf1 in comparison to same - aged peers . Mild decrements in adaptive behavior were observed across the range of adaptive behavior domains, with relative difficulty with motor skills remaining even after intellectual functioning was taken into account . One limitation of this study is the reliance on parental report alone to characterize adaptive behavior . Studies that include measurement of real - world behavior and examination of relations between parental report and lab - based measurement of the abilities examined are important next steps . Additionally, potential relations with other cognitive skills such as emerging executive functioning and attention should be explored . Finally, further research about the developmental trajectory of early motor difficulties in particular is also needed.
Who discovered lepra bacilli? Asked my professor as we gathered around a patient afflicted with leprosy, in our first clinical session on the disease in a leprosy ward . Pleased with the answer, the professor insisted on hearing a little more than just the name from his students . But as much as we struggled to connect a few disjointed words like norway, leprosy and bacilli to this name, none of us really knew the history well enough for a narration . The rest of the lecture focused mainly on clinical skills and basics of leprosy knowledge . But the untold story of hansen that day, kept lurking at the back of my mind and the large portrait on the wall was a like a bookmark that urged me to turn the pages of history, every time i walked past dr . As i sought to know more, i found a fascinating story of a brave heart scientist who was very justifiably famous as the discoverer of the leprosy bacilli, and a man who dedicated a lifetime of scientific research towards transforming one of the great scourges of mankind to an understandable and treatable disease . The story dates back to 1841 in bergen, a small town in the west of norway . In the summer that year, was born gerhard henrik armauer hansen, 8 of fifteen children to mrs . Claus hansen, on the july 29 . After spending a mischievous childhood moderated by his strict father in a middle class household, this destiny's child went to the university of christiania to study medicine in 1859 . This exceptional research talent acquired his degree with honours in 1866 . After completing his internship, he served as a community doctor in lofoten, a small norwegian fishing village in northern norway, for a year . But as destiny had it planned, hansen returned to his people in bergen in the year 1868 . Norway, which was already fighting its battle against leprosy, by the middle of nineteenth century, was probably the worst hit by leprosy in europe and it was bergen where the leprosy research centre was established with three other lepra hospitals . It was estimated that in the 1850s, roughly two individuals per thousand in norway were afflicted with leprosy, but the prevalence in bergen was as much as 25 per thousand . On his return to his hometown, hansen met dr . Carl wilhelm boeck, the two celebrated stalwarts of ancient leprosy research, who had the very famous (on leprosy) publication to their credit, along with a great deal of researched knowledge . Danielssen was especially considered as the foremost authority in leprosy scientific research and the guiding force behind all public health efforts to combat leprosy, not only in norway but entire europe . He however was a staunch believer of the concept of hereditary affliction of leprosy, supported by the rest of the medical fraternity of his time . An infectious etiology was not obvious because of the long incubation period that obscured the trail of contact . But as we all know today, history was yet to be written, and written differently . However absurd the theory of hereditary transmission of leprosy sounds today, one cannot deny dr . It was to his credit that bergen was made the epicenter of leprosy research . While working under dr . Danielssen, a young and inquisitive hansen travelled with him all across norway to study the disease, collect pathological samples from the lepers and research relentlessly to finally come to a revolutionary conclusion that challenged dr . He believed an organism carried the disease from person to person and emphasized the theory of contagion, ruling out hereditary etiology for this condition . It was a daring speculation at a time when the concept of contagion was still poorly understood . By declaring this theory, hansen ran into a professional conflict with his superior but continued to believe firmly in his observation . Despite all the negative criticism and resistance against such a challenging proposal, he continued to research further to prove his theory . In 1869, in his but his technique of staining bacteria was exceedingly primitive and furthermore his poor equipment complicated his work and it would be unfair to expect him to have delivered more adequate description of the lepra bacilli than he did . It became clear to hansen that he needed improved skills in pathologic anatomy and particularly in microscopy . In 1870, a grant allowed dr . Hansen to travel to vienna to for advanced training in staining and histopathology, which enabled him to strengthen his research technique and a more determined hansen launched his search for that infectious substance, a culprit yet to be captured and produced in front of the world . He would sit unceasingly for days on end, focusing through the microscope and looking at stained tissue slides of leprous tissue . Finally in the year 1873, when he was merely 32 years old, he concluded his successful attempt at identifying the infectious substance in leprous material and published his historic work . In 1875, he was made physician - in - chief for all the lepers in norway . In tissues taken from nodules of leprosy patients he described rod shaped bodies, resembling bacteria and claimed that these bacteria were indeed the causative agent for leprosy and leprosy was neither a hereditary disease nor a curse given by god that the lepers must suffer for their sins . Hansen was in fact the first investigator to suggest that microorganisms can cause human diseases . The rest of the medical fraternity worldwide still laughed at this revelation and his daring proposal, and thought of it as a figment of hansen's imagination . Some of the investigators though were convinced about the existence of such a substance in the experimented material, but rejected its role in causing leprosy . However; his attempts at inoculation of the organism into other animals made it more difficult for him to prove his stand . He was unable to satisfy koch's postulates, which required any putative pathogen to be first isolated and then shown to cause the same disease when re - introduced into another animal . A desperate hansen even tried to inoculate the eye of a woman with material drawn from leprosy patient without her consent . It was undoubtedly a reckless attempt at human experimentation and to nobody's surprise this unethical attempt landed him in a medico legal quagmire, which later proved to be a major setback in his medical career . He also suffered a stroke at the young age of 36 in 1877, but continued his untiring work unfettered and uninhibited . In 1879, a young german bacteriologist caller albert neisser (who later gained fame for discovering the causative organism of gonorrheoa), a pupil of robert koch, on his research trip to norway to study leprosy, had the opportunity to meet dr . He received preparations made from lepra nodes . On his return to germany, neisser made all attempts to stain them better to yield more convincing results and equipped with his advanced staining methods and by virtue of being a bacteriologist himself, he succeeded . A very excited dr . Neisser went ahead to publish his scientific find in 1880, without giving the due credit to dr . Soon the term neisser's bacterium was in use while hansen was busy in bergen fighting his court case for violating medical ethics for human experimentation . The court found him guilty for failing to obtain consent from the subject and he was removed from his post as resident physician of the bergen leprosy hospital in 1880 . But once the news of albert's claim reached bergen, the entire medical fraternity of norway defended hansen's stand and it was none other than dr . Fortunately the truth was unveiled and the conflict was officially addressed in a lepra congress held in berlin where hansen [figure 1] was recognized as the true discoverer of the lepra bacilli . This also marks the world's awakening and acceptance of the contagion theory and changed the way leprosy as a disease was approached . It was the fruit of his untiring work that the amended act of 1885 was passed, which ordered health authorities to allow lepers to live in precautionary isolation, away from the unaffected section of the community, which led to quick and steady decline in the leprosy disease burden in norway . Henrik armauer hansen: the discoverer of m. leprae if one looks at his personal life, he did have his share of ups and downs . In his seventies coming from a humble middle class background, pursuing medical education itself was a challenge which he took upon himself, serving as a school teacher in a girls school and later as a prosector in anatomy, in order to meet the expenses of medical school . Hansen was also an accomplished biologist and an ardent follower of darwin's theory of evolution and propagated it for wider acceptance . He is described as a radical in his time who doubted the benefit of visiting the church, which earned him a lot of criticism and displeasure from the clergy . Hansen sometimes was considered unflinching in his views and was not known to be amenable to debate . He was critical of women's freedom and even doubted women's credential to become doctors . But hansen was well respected by his colleagues who described him as a humble, hardworking, warm - hearted and entertaining person with a great sense of humour . Danielssen's daughter stephanie marie in 1873, but lost her to tuberculosis within a year of their marriage . He named his son daniel cornelius armauer hansen, after his previous father in law dr . He encouraged his son to pursue medical education, which rightfully carried his legacy forward and later went on to become the chief of the tuberculosis hospital in bergen . After an episode of a brain stroke at a very early age, hansen started experiencing symptoms of heart disease in his fifties, which nearly confined him to bed . In february 12, 1912 in flor, a little town on the western coast, he breathed his last . He was given a funeral at state expense; he had been the president of the bergen museum and the ceremony took place from its hall . It was much later that the cause of his illness was revealed and history tells us that he actually suffered from syphilis and probably his stroke and subsequent heart disease was in fact due to syphilis . It is also rumored that he might have contracted the disease from a seamstress in christiana during his student years in 1860s . The real significance of hansen's contribution to medicine in general, and to bacteriology in particular, can be fully grasped only when one recalls the position investigative medicine had reached in 1873, when the leprae bacillum was discovered . He shared the fate of other great minds that labored in advance of their time and was met with opposition, scathing criticism and a lack of recognition . It was his misfortune to have fought a battle against a bacterium which even today defies most attempts at artificial cultivation . But his numerous contributions to leprosy with his discovery of the causative organism will always be held in reverent esteem in the history of medicine . This article encapsulates the unyielding determination and astute scientific acumen that gerhard - henrik armauer hansen exhibited in his lifetime and gives us a glimpse into the life of a legend in the field of leprology.
The exponential demand for energy is evidenced by dwindling fossil fuel supplies and record - high oil and gas prices due to global population growth and economic development (fig . This energy shortage has significant implications to the future of our society for example, in order for 10 billion people to sustain their current lifestyle with their current energy consumption, we need a minimum of ten additional terawatts (tws), an equivalent of 150 millions of barrels of oil per day (150 m boe / day), until the year 2050 . The energy crisis is further exacerbated by major concerns about global warming from greenhouse gas emissions due to increasing fossil fuel consumption [6 - 8].) At this large scale, solar energy seems to be the most viable choice to meet our clean energy demand . The sun continuously delivers to the earth 120,000 tw of energy, which dramatically exceeds our current rate of energy needs (13 tw). This implies that covering only 0.1% of the earth s surface with solar cells of 10% efficiency would satisfy our current energy needs; however, the energy currently produced from sunlight remains less than 0.1% of the global energy demand (fig . The major barrier for the large - scale use of solar energy is the high cost and inadequate efficiencies of existing solar cells . The best commercial solar cells based on single - crystal silicon are about 18% efficient . These conventional p - n junction cells, so - called first - generation devices, suffer from the high cost of manufacturing and installation . The second - generation devices consisting of cuingase2 (cigs) polycrystalline semiconductor thin films can reduce the price significantly, but it does not reduce the challenge to make their efficiencies more practical . Now the third - generation solar cells, such as dye - sensitized solar cells (dsscs), bulk heterojunction cells [17 - 19], and organic cells, are promising for inexpensive and large - scale solar energy conversion (table 1); however, laboratory dsscs based on cheap dye sensitization of oxide semiconductors are typically less than 10% efficient, and those based on even cheaper organic materials are 25% efficient . Nanostructured semiconductors, organic - inorganic hybrid assemblies, and molecular assemblies present new opportunities to design such third - generation light energy conversion devices . Considerable efforts have been devoted to the development of more efficient photoanode materials, such as ordered mesostructured materials and one - dimensional (1-d) nanostructures (nanowires, nanotubes, and nanorods) [22 - 24]. Examples of 1-d nanostructures include highly ordered tio2 nanotube arrays synthesized with anodization of ti foils, zno nanowire arrays synthesized with aqueous solutions, and carbon nanotube (cnt) and si nanowire arrays synthesized with chemical vapor deposition (cvd) [23 - 34]. Bandgap - tunable semiconductor zero - dimensional (0-d) nanomaterials, such as cds [35 - 37], pbs, bi2s3, cdse, and inp quantum dots (qds), have demonstrated extraordinary optical and electronic properties that open up possibilities for revolutionary advances in photovoltaic (pv) devices . The combination of 1-d with 0-d nanostructures is attracting more interests from the solar cell community . Assembly methods for these hybrid nanostructures include wet - chemistry processes through chemical functionalization [43 - 47] and dry routes through the electrostatic - force - directed assembly . In particular, 1-d nanostructures are promising for photovoltaic devices due to several performance and processing benefits, such as a direct path for charge transport and large surface areas for light harvest offered by the geometry of such nanostructures . For example, the mobility of electrons in 1-d nanostructures is typically several orders of magnitude higher than that in semiconductor nanoparticle films commonly used in dsscs (table 2, data from [23,50 - 54]). This review addresses some of the current research issues in the field of new solar cells based on 1-d semiconductor nanostructures, and hybrids of 1-d nanomaterials and dye molecules or qds . These solar cells include dsscs with tio2 nanotube or zno nanowire arrays as photoanodes in lieu of nanoparticle networks, solar cells with tunable - bandgap qds supported by wide - bandgap semiconductor nanotube / nanowire or cnt arrays, and solar cells with coaxial p - n junctions in vertically - aligned 1-d nanostructures or p - n junctions between the 1-d nanostructure and the substrate . We summarize recent advances and discuss the performance and properties of these innovative solar energy harvesting and conversion devices . Comparison of electron mobilities (cm v s) of 1-d nanomaterials and nanoparticles field - effect and; intrinsic mobility of a 300 nm long and 3.9 nm diameter single - walled carbon nanotube (swcnt); 830 nm wide si nanowires; 20 nm wide ge nanowires; 1617 m long and 130200 nm wide zno nanowire; for particle size around 4 nm; for 48 nm anatase tio2 nanocrystals; it is 15 cm v s in single crystal tio2 a dye - sensitized solar cell (dssc) is a type of photoelectrochemical (pec) solar cell which has been studied extensively [55 - 57]. In a dssc, dye molecules are used to sensitize wide - bandgap semiconductors, such as tio2 and zno, which assist in separating electrons from photo - excited dye molecules . The visible light absorbed by dye molecules is more intensive than the uv light absorbed by wide - bandgap semiconductors for solar radiation, even if the energy of visible light is lower than that of uv light . The sensitization of wide - bandgap semiconductors by adsorbed monolayers of dye molecules began in the late 1960s with the work of gerishcer and memming . A conceptual and practical breakthrough occurred in the late 1980s when grtzel and coworkers started using high - surface - area semiconductors for dsscs [16,60 - 63]. A schematic representation of a dssc is shown in fig . Its working principle is: (1) the incident photon is absorbed by the dye molecule adsorbed on the surface of nanocrystalline tio2 particles and an electron from the molecular ground state s is excited to an excited state s *; (2) the excited electron of the dye is injected into the conduction band of the tio2 particles, leaving the dye molecule to an oxidized state s; (3) the injected electron percolates through the porous nanocrystalline structure to the transparent conducting oxide (tco) layer of the glass substrate (negative electrode or anode) and finally through an external load to the counter electrode (positive electrode or cathode); (4) at the counter electrode, the electron is transferred to the triiodide () in electrolyte to yield iodide (i); (5) the cycle is closed through reducing the oxidized dye by the iodide in the electrolyte . However, there is no net chemistry in terms of chemicals created or destroyed in the device, so the cell is regenerative . The working principle of a dye - sensitized nanostructure solar cell (adapted from ref .) The anodes of dsscs are typically constructed with a thin film (~10 m) of wide bandgap semiconductor nanoparticles involving sno2, zno, or tio2[68 - 74]. The nanoparticle film provides a large surface (~1000 times higher than the geometrical area of the electrode) for absorption of light - harvesting molecules (usually ruthenium - based dyes); however, electrons are usually trapped by isolated nanoparticles, surface states, or defect states . The so - called trap effects slow down the electron transport through diffusion and limit the device efficiency, which has been proved by time - resolved photocurrent and photovoltage measurements and modeling studies . Under full sunlight, the average injected electron may experience a million trapping events before either being collected by the electrode or recombining with an oxidizing species . For example, electron transport in crystalline wires is expected to be several orders of magnitude faster than percolation through a random polycrystalline network (table 2). The zno nanowire array of high surface area was synthesized using a simple two - step process in aqueous solutions . Briefly, a 1015-nm - thick film of zno quantum dots was deposited onto fluorine - doped tin oxide (fto) conductive glass substrates by dip coating, and wires were then grown from these nuclei through thermal decomposition of a zinc complex . Individual zno nanowire resistivity varied from 0.3 to 2.0 cm, with an electron concentration of 15 10 cm and mobility () of 15 cm v s. using the einstein relation, d = kbt/e, an electron diffusivity dn = 0.050.5 cm s for a single nanowire can be estimated . Compared with zno nanoparticles (dn 10 cm s), the nanowire array anode can collect charge carriers much more effectively by introducing highly ordered architectures . Law et al . Showed that the injection process in nanowires is complete after ~5 ps, but continues for ~100 ps in the nanoparticles . The new cell exhibited a short - circuit current density jsc = 5.35.85 ma / cm, an open - circuit voltage voc = 0.610.71 v, a fill factor ff = 0.360.38, and an efficiency = 1.21.5% under am1.5 sun illumination (100 3 mw / cm). The external quantum efficiency of these cells peaks at 4043% near the absorption maximum of the dye and is limited primarily by the relatively low dye loading of the nanowire film . Analogous to zno nanowire arrays, well - aligned, self - organized tio2 nanotubes have been fabricated with the goal to improve electron transport pathways for solar energy conversion devices [24,29,30,81 - 83]. Even though the efficiencies of these devices are not as high as cells fabricated with standard tio2 nanoparticles, respectable performance has been demonstrated . Tio2 nanotube arrays allow direct charge transport along the length of the nanotube toward the electrode; however, this assumes that the charge transport in mesoporous tio2 is limited by interparticle traps . The advantage of nanorod or nanotube arrays will not be apparent if the surface trapping limits the charge transport . Comparison between these 1-d nanostructures and standard films fabricated from sintered nanoparticles may very well assist in elucidating the mechanism for electron transport in these materials . Recently, highly ordered tio2 nanotube arrays were synthesized by anodic oxidation of titanium and have generated considerable scientific interest [24 - 32]. To fabricate nanotube devices, these nanopores initially have an amorphous structure, which can be transformed to anatase tio2 upon annealing to over 450 c . The porous film forms on the titanium foil and a compact titanium dioxide layer forms between the unoxidized titanium and the nanotubes during the heating process . Reported high open - circuit voltages of up to 860 mv for this cell structure . Their best cells reached efficiencies of over 4% under am1.5 sun (iodide / triiodide - based cells). The nanotube devices display inhibited recombination characteristics with longer electron lifetimes, indicating fewer recombination centers in the nanotube film compared with a nanoparticle film . A disadvantage of using tio2 nanotube arrays for anode fabrication is that the device requires illumination from the back side (through the pt cathode) because the counter electrode fabricated from ti is opaque . This is not the optimal configuration for dsscs because the platinum counter electrode partially reflects light and the iodine in the electrolyte absorbs photons at lower wavelengths . Therefore, the challenge is to achieve highly ordered tio2 nanotube arrays on fto substrates, especially nanotubes with increased film thickness . Taking advantage of extremely high electron mobilities of single - walled cnts (swcnts), brown et al . Deposited tio2 nanoparticles on an swcnt network . When modified with a sensitizer such as ru(ii)(bpy)(dcbpy), the swcnt / tio2 film provided an unnoticeable influence on the charge injection from dye molecules into tio2 nanoparticles, but improved charge separation according to transient absorption and emission measurements . The rate of the back electron transfer between the oxidized sensitizer (ru(iii)) and tio2 was slower in the presence of the swcnt scaffold . The incident photon to charge carrier efficiency (ipce) at all wavelengths was enhanced by a factor of ~1.4 as a result of introducing a swcnt scaffold in the mesoscopic tio2 film . This is due to the suppressed back electron transfer and the improved electron transport within the nanostructured tio2 film . However, the improvement in photocurrent generation was neutralized by a lower photovoltage, as the apparent fermi level of the tio2 and swcnt composite became more positive than that of pristine tio2 . The dye - sensitized swcnt / tio2 cell had = 0.13%, voc = 0.26 v, and jsc = 1.8 ma / cm . It is not surprising that the semiconductor nanotube / nanowire arrays are not always highly ordered, however . For instance, clumps of nanotubes and crack - like features in the films prepared by electrochemically anodizing titanium metal have been observed . The formation of clusters of bundled nanotubes in nominally oriented arrays could adversely affect the transport and recombination dynamics in tio2 films . Clusters of bundled nanotubes could be produced during the anodization process or during the cleaning and evaporative drying process of the as - grown films through capillary forces of the liquid acting between the nanotubes . Such capillary forces have the potential to not only bundle nanotubes but also to crack the film . Removing liquids from the mesopores of the arrays by the supercritical co2 (scco2) drying technique can yield bundle - free and crack - free nanotube films . Compared with h2o / air - dried tio2 nanotube array film, zhu et al . Found that the ethanol / scco2-dried films could prevent morphological disorders induced by capillary stress and enhance the total surface area of a film accessible to dye molecules by 23% . The electron transport was about twice as fast in the ethanol / scco2-dried film than in the h2o / air - dried film . The photoresponse of the dssc with ordered films exhibited jsc = 5.7 ma / cmvoc = 0.58 v, and ff = 0.56 to offer a solar conversion efficiency = 1.9% . In contrast, the dssc with less - ordered films showed a jsc of 4.9 ma / cmvoc of 0.60 v, and an ff of 0.53 to yield = 1.6% . In general, dsscs are relatively well developed in recent years with the following possible improvements . The growth of tio2 nanotube arrays on a transparent anode will be beneficial for the light absorption . A compact tio2 particle film between the fto anode and the electrolyte was recently proved to reduce charge recombination losses, which might be a valuable hint to design tio2 nanotube array for dsscs . Use of bundle - free and crack - free 1-d nanostructure arrays is another principle for solar cell assembly . High aspect ratio nanotube / nanowire arrays are expected to load more dye molecules, but the dilemma is that tubes / wires longer than the diffusion length of electrons will degrade the efficiency of electron collection . Recent work also includes the molecular engineering of suitable ruthenium compounds, which are known for their excellent stability . The use of solvent - free electrolytes such as ionic liquids has made striking advances during the past few years . These nonvolatile redox melts show great promise for use in outdoor photovoltaic systems and have been discussed in detail elsewhere . The combination of two or more nanostructure architectures provides another option to modulate the performance of light - harvesting devices [94 - 97]. As presented in the previous section, the electron transport across particles is susceptible to recombination loss at the grain boundaries and charge trapping in nanostructured semiconductor films prepared from particles . The use of nanotube / nanowire support to anchor light - harvesting assemblies (e.g., semiconductor particles and dye molecules) provides a convenient way to capture photogenerated charges and transport them to electrodes . Quantum - dot - sensitized solar cells (qdsscs) provide additional opportunities that are not available with dye - sensitized solar cells (fig . First, the use of quantum dots in lieu of the dye molecules provides the ability to tune the optical absorption in the solar cell through selection of semiconductor material and particle size . Second, qdsscs can potentially exploit the recently observed multiple electron - hole pair generation per photon to achieve higher efficiencies than that predicted by shockley and queisser . An array of zno nanowires, grown vertically from an fto / glass substrate and decorated with cdse quantum dots, serves as the photoanode . A second fto / glass substrate, coated with a 100 layer of pt, is the photocathode . The space between the two electrodes is filled with a liquid electrolyte and the cell is illuminated from the bottom (reprinted with permission from ref . . A swcnt is an ideal channel for collecting and transporting charges across light - harvesting assemblies . Significant progress has been achieved in synthesizing semiconductor - cnt composite films in recent years [102 - 109]. These earlier studies have mainly focused on establishing synthetic strategies and characterizing the composite systems, including cnts with tio2, sno2, cdse, and cds nanocrystals . Most of the wet - chemistry strategies involve chemical functionalization of the cnt surface followed by the assembly of nanocrystals onto the cnts via covalent, noncovalent, or electrostatic interactions . Swcnts were dispersed in tetrahydrofuran (thf) with the aid of tetraoctylammonium bromide (toab). The adsorption of cd ions on the swcnt surface followed by reaction with s provides a simple and convenient method of preparing swcnt cds composites . The cds - swcnt composite is capable of generating a photocurrent from visible light with unusually high efficiencies, in which the luminescence of cds is quenched by swcnt . Transient absorption experiments have confirmed the quick deactivation of excited cds on the swcnt surfaces, as the transient bleaching recovers in about 200 ps . The ability of the cds - swcnt nanocomposite system to undergo photoinduced charge separation opens up new ways to design light - harvesting assemblies . Although the wet - chemistry methods work well with randomly dispersed cnts, they are not suitable for vertically aligned cnts . The aligned structure will be damaged in the wet processing because the upper ends of the neighboring nanotubes have been seen to bundle together and cause some nanotubes to lay down . Have recently developed a material - independent dry route based on the electrostatic - force - directed assembly (esfda) to assemble aerosol nanocrystals onto cnts . In principle, the esfda technique works for both random cnts and aligned cnts without the need for chemical functionalization or other pretreatments of the cnts . In esfda, charged and nonagglomerated aerosol nanocrystals were produced from a mini - arc plasma source and then delivered to the electrically biased cnts in an inert carrier gas . The electric field near the cnt surface was enhanced significantly and the aerosol nanocrystals were attracted to the external surface of the cnts . With this technique, chen et al . Have demonstrated the successful assembly of various nanocrystals, including single - component nanocrystals (au, ag, and sno2) and multicomponent nanocrystals (sno2 and ag), onto randomly dispersed multiwalled cnts (mwcnts), swcnts, and vertically aligned mwcnts . The highly ordered charge transport channel is not limited to only cnts, but also to other semiconductor (zno or tio2) 1-d nanostructures . Quantum - dot - sensitized nanowire solar cell based on photosensitization of zno nanowires with cdse quantum dots has been demonstrated . A zno nanowire array can be grown directly onto transparent and conducting fto substrates from an aqueous solution of zn(no3)2 and methenamine between 80 and 95 c . Cdse qds were assembled on the zno nanowires by using bifunctional molecules of the type x - r - y, where x and y are groups that bind to cdse (x = sh) and zno (y = cooh), respectively . The photocurrent is generated from visible light by the excitation of electron - hole pairs in the cdse qds . The electrons are injected across the qd - nanowire interface into the zno, a process that is facilitated by the overlap between the electronic states in the qd and the zno conduction band . The morphology of the nanowires provides the photoinjected electrons with a direct electrical pathway to the photoanode . The zno nanowire - based qdsscs exhibited = 0.4%, voc = 0.50.6 v, jsc = 12 ma / cm, and a fill factor ff ~0.3 . Tio2 is another important wide - bandgap semiconductor that is widely used in both dsscs and qdsscs . Sun et al . Reported an investigation on the cds qds sensitized tio2 nanotube array photoelectrodes and their performance in photoelectrochemical solar cells . The highly ordered tio2 nanotube films were synthesized by anodic oxidation in a nh4f organic electrolyte . The cds qds were deposited into the crystalline tio2 nanotubes by the sequential chemical bath deposition method . The cds - tio2 cells exhibited impressive = 4.15%, voc = 1.27 v, jsc = 7.82 ma / cm, and ff = 0.578 under am1.5 illuminations . These results clearly demonstrated that significant improvement on the pec cell efficiency can be obtained via incorporating inorganic semiconductor qds into the tio2 nanotube array films . A number of active research areas could significantly contribute to the advancement of qdsscs . Maximizing the overlap between the solar spectrum and the solar cell absorption spectrum through judiciously selecting tunable - bandgap qds the multiple exciton generation with uv photons demonstrated in qds is yet to be proved in solar cells . The challenge is to find ways to effectively collect the resulting excitons before they recombine since recombination occurs at a femtosecond time scale . Assembling qds onto nanotubes / nanowires with wet - chemistry methods will likely damage the ordered arrays, and molecule linkers between qds and 1-d nanostructures are likely to be potential barriers for charge transfer . Dry methods such as esfda could potentially achieve better electronic transfer between qds and 1-d nanostructures . Use of fe or ni as catalysts in cvd growth of cnts constrains the material types of substrates and makes it difficult to achieve ohmic contacts between cnts and substrates . More understanding on the electronic transfer at the qds - nanotubes / nanowires heterojunction interface and cnts - substrate interface is imperative to further improve the performance of qdsscs . Many researchers have been eager to explore carbon nanostructures such as swcnt assemblies for energy conversion devices because of their unique electrical and electronic properties, wide electrochemical stability window, and high surface area [118 - 120]. Fullerenes, for example, exhibit rich photochemistry and act as an electron shuttle in photochemical solar cells . They also play an important role in improving the performance of organic photovoltaic cells . On the other hand, the exciton annihilation and charge separation processes have been characterized by transient absorption and emission measurements . For example, the photoresponse of cnt filaments was realized in early years from the elastic response of the aligned bundles between two metal electrodes . Hot carrier luminescence from ambipolar cnt field - effect transistors (fets) has been monitored by avouris and coworkers . The relaxation of electrons and holes to the fundamental band edge occurs within 100 fs after photoexcitation . These early studies confirmed the ability of cnts to possess a band structure that can undergo electron - hole charge separation with visible light excitation . However, spatially confined charge carriers in the nanotube are bound by coulombic interactions with the bound pair referred to as an exciton [125 - 127]. A small fraction of the excitons are able to dissociate and form unbounded electron - hole (e - h) pairs . A key question is whether the photoinduced charge carriers generated in swcnts can be collected suitably for photocurrent generation, similar to the photovoltaic application of other semiconductors . Double - walled carbon nanotubes (dwcnts) were also directly configured as energy conversion materials to fabricate thin film solar cells, with nanotubes serving as both photogeneration sites and a charge carriers collecting / transport layer . The solar cells consisted of a semi - transparent thin film of nanotubes conformally coated on an n - type crystalline silicon substrate to create high - density p - n heterojunctions between nanotubes and n - si to favor charge separation and extract electrons (through n - si) and holes (through nanotubes). The p - type dwcnts were first formed as an ultrathin film on the water surface with the aid of ethanol, and then transferred to an n - si substrate . Experiments have shown = 1.38%, voc = 0.5 v, jsc = 13.8 ma / cm, and ff = 19% under am1.5 illumination, proving that dwcnts - on - si is a potentially suitable configuration for solar cells . Two key constraints for this type of solar cell are (1) the material must be sufficiently thick and pure to absorb most of the solar photons with energies above the material s bandgap; and (2) the material must have a high minority carrier diffusion length to effectively collect the photogenerated charge carriers . One attractive method to improve the light absorption and charge carrier collection involves high aspect ratio cylindrical absorbers, such as 1-d nanowires . A preferred implementation includes the use of wires that are sufficiently long to absorb most of the incident light and have sufficiently small diameters to facilitate efficient radial collection of carriers, even for relatively impure absorber materials (fig . Methods are required (1) to prepare large area arrays of vertically aligned core - shell nanowires; (2) to make electrical junctions to such wire arrays; and (3) to make electrical contacts to the backsides of these devices . These challenges have been investigated by various means, including chemical vapor deposition (cvd) growth of wire arrays, etching of flat substrates to produce wire arrays, and creating conductive polymer electrical junctions with wires . Schematic cross section of a radial p - n junction core - shell nanowire solar cell . The light gray area is n type; the dark gray area is p type (reused with permission from ref . . Copyright 2005, american institute of physics .) In an earlier theoretical work by kayes et al ., a device physics model has been developed for radial p - n junction nanowire / nanorod solar cells, in which densely packed nanorods, each having a p - n junction in the radial direction, are oriented with the rod axis parallel to the incident light direction . The radial p - n junction nanorod geometry produces significant improvements in the efficiencies of cells made from materials that have diffusion lengths at least two orders of magnitude less than their optical thickness and low recombination in the depletion region (for example, exciton lifetime> optimal cells have a radius approximately equal to the minority - electron diffusion length in the p - type core, and their doping levels must be high enough that a rod of such radius is not fully depleted . In silicon with very low diffusion lengths (ln= 100 nm), extremely large efficiency gains (from 1.5% to 11%) are possible by exploiting the radial p - n junction nanorod geometry, provided that the trap density in the depletion region remains fixed at a relatively low level (<~310 cm). . Experimentally showed that optimal efficiencies can be obtained when the si wires have a diameter comparable to the minority carrier diffusion length . Smaller diameters increase the surface area, thereby increasing the surface and junction recombination with few accompanying improvements in carrier collection . Based on the study of kayes et al ., tsakalakos et al . Also estimated an efficiency of 1518% for si nanowire solar cells . According to the calculation, the lateral diffusion of minority carriers to the p - n junction, which is at most 50500 nm away, was proposed rather than many microns away as in bulk si solar cells . An array of si nanowires (diameter = 189 30 nm, length ~16 m) was fabricated with cvd . A current density of ~1.6 ma / cm for 1.8 cm cells was obtained, and a broad external quantum efficiency was measured with a maximum value of~12% at 690 nm . The optical reflectance of the silicon nanowire solar cells was reduced by one to two orders of magnitude compared with planar cells . The junction area of a cnts / si cell is no more than the surface area of the anode (si substrate). Therefore, rough architectures rather than planar ones are expected to improve the junction areas . Semiconductor nanowire / nanorods with radial p - n junctions make it possible to orthogonalize the direction of light absorption and carrier collection, and thus can enable efficient carrier collection in optically thick nanowire arrays even when minority carrier diffusion lengths are shorter than the optical absorption length . Although coaxial si nanowire / nanorod p - n junction solar cells have high theoretical efficiencies, the cost of cvd growth of si nanowires on si substrates is high . Au as a catalyst used in the cvd growth of si nanowire arrays also reduces the lifetime of carriers in silicon due to the presence of au in the nanowire . Identifying inexpensive catalysts such as the sun provides enormous potential in helping to resolve the growing demand for energy worldwide; however, the high costs of implementing solar energy is a significant barrier compared with traditional energy sources, such as fossil fuels . The cost of a photovoltaic system is directly related to the low conversion efficiency, diluted energy density of solar radiation, and costly materials and fabrication process . During the past decade, the development of nanoscience and nanotechnology has launched new ways to design efficient solar cells . Strategies have been developed to design nanostructure architectures of semiconductors, metals, and polymers for solar cells . Theoretical and modeling studies have also helped to understand the optical and electrical processes of the photovoltaic conversion . The examples discussed in this review summarized how 1-d nanostructures, hybrids of 1-d nanomaterials / molecules, and qds could aid in dsscs, qdsscs, and conventional p - n junction solar cells . While the new generation of photovoltaic cells offers many opportunities, it also presents challenges such as in the following directions (1) ordered assemblies of two or more nanocomponents on electrode surfaces; (2) new sensitizers or semiconductor systems that can harvest infrared photons; and (3) multiple exciton generation in semiconductor qds . Worldwide, solar power is the star attraction for venture capitalists due to its booming laboratory research and commercialization . Although other forms of renewable energy can make significant contributions to current markets, sunlight is most available in the amount required to substitute completely for the energy quantities currently derived from hydrocarbons . This work was financially supported by the national science foundation through an ner grant (cmmi-0609059).
Verrucous carcinoma (vc) is a slow - growing cancer type which has been reported in the esophagus, oropharynx, larynx, glans penis, scrotum, vulva, vagina, cervix, endometrium, urinary bladder, anus, and in the sole of the feet . The first 5 cases of esophageal vc ever described were reported by minielly at al . In 1967 . In total, vc is known to be associated with several medical conditions, such as chronic esophagitis, esophageal diverticular disease, gastroesophageal reflux disease, hiatal hernia, achalasia, and caustic injury . Known risk factors include smoking and alcohol abuse [1, 3, 6, 7, 8]. Vc occurs more frequently in males and has been reported at all ages from 36 to 76 years . The diagnosis is rare and often delayed since initial superficial biopsies usually show nonspecific chronic inflammation and candida infection but only rarely malignancy . The majority of cases follow a similar symptom pattern and have similar endoscopic and histological characteristics . The endoscopic appearance is often described as a shaggy, papillary mucosa with white plaques ., we diagnosed 2 male patients at the department of gastrointestinal surgery and the department of pathology, rigshospitalet, copenhagen . The 2 cases will be presented in the following to highlight the characteristics of this rare type of carcinoma . Our first patient was a 67-year - old male with a medical history of diabetes mellitus type ii, hypertension, and benign polyps in the colon (three polypectomies). The patient had a history of tobacco use during the last 50 years; at the time of reference, he smoked 10 cigarettes daily . When referred, the patient had suffered from dysphagia for 2 months, and he only consumed fluids . Besides a weight loss of 8 kg (weight at referral 83 kg, weight loss corresponding to 9% of initial body weight), the first physical examination was unremarkable, as were the initial blood tests . The primary endoscopy revealed a mucosa covered with white plaques spreading from 30 to 40 cm from the incisors . In the distal part of the affected area, the mucosa protruded and covered almost 2/3 of the lumen and involved the entire circumference (fig . Biopsies showed severe acute inflammation with reactive changes but without dysplasia or malignancy . A computed tomography (ct) scan showed a thickened wall in the distal part of the esophagus . Since no malignancy was found, four months later, the patient was referred again due to a further massive weight loss of 25 kg; at this time he weighed only 58 kg (weight loss corresponding to 36% of initial body weight). Gastroscopy and blood test were performed again, and human immunodeficiency virus, herpes, and hpv infection were ruled out . To rule out achalasia of the esophagus and gastroesophageal junction, a high - resolution impedance manometry examination was performed followed by a positron emission tomography (pet)-ct scan, but neither could explain the condition the patient suffered from . During this period of approximately 4 months six months after the first visit, another gastroscopy was performed showing ulceration and a process protruding into the lumen . The biopsy revealed a squamous cell papilloma . In view of the severe weight loss and the poor condition of the patient, he was a heavy smoker (1015 cigarettes daily since 18 years of age) and had a history of previous alcohol abuse of> 168 g pure alcohol per week during a period of 10 years . In addition, he had a hiatal hernia and barrett's esophagus for which he was treated with a proton pump inhibitor (pantoprazole 40 mg daily). The initial symptom was dysphagia which he had suffered from intermittently during the last one and a half years . An area of a 10-cm, white, irregular mucosa with a 1-cm large polyp - like process was observed at gastroscopy (fig . 3). Biopsies revealed acute and chronic inflammation as well as infection with nonhemolytic streptococcus, escherichia coli, and a few fungal hyphae . The patient underwent a series of gastroscopies without malignant findings, just as the initial ct scan was without suspicion of malignant disease . Three months later, a biopsy from the esophagus showed a squamous cell papilloma . The following pet - ct scan performed 6 months after the initial visit found tumor growth and a suspicion of malignancy, stage t2n0m0 . An endoscopic ultrasonography - guided fine - needle aspiration found no malignant cells . During this period, the patient suffered from dysphagia but had no weight loss, nor was he hospitalized . Despite the lack of malignant findings, the patient was offered an esophagectomy and underwent surgery . Nine months after the first visit, the pathology report of the resected specimen showed a hyperplastic mucosa with a low - grade vc, a mostly exophytic papillary tumor . The tumor was highly parakeratinized with no dissemination into the lymph nodes or other organs . After a minor pneumonia, for which he received antibiotic treatment, in our 2 cases, as in almost all cases previously described, there has been a considerable delay of diagnosis . For our patients this seems to be a consistent pattern for this group of patients, including the presence of dysphagia, occasionally a massive weight loss, and a typical endoscopic picture . The mucosa in our 2 cases was white and covered with plaques and wart - like polyps . Furthermore, the mucosa is often described as exophytic, and the description a multilobular polypoid mass has also been used . The only strategy to identify and, moreover, to diagnose and treat this limited group of patients is to be familiar with this typical white, wart - like mucosa and to initiate aggressive treatment already after a few endoscopic examinations . Even if all biopsies are without malignancy, a suspicion of vc should be present just based on the macroscopic appearance . A german study including 15 patients with vc in the esophagus from 1999 to 2011 and an american study including 11 patients from 1995 to 2010 have been published . How many underdiagnosed cases of vc exist remains unknown, but an awareness of this type of carcinoma is certainly desirable in a worldwide context . The resected specimen often shows a relatively low - grade tumor and rarely dissemination into the lymph nodes or other organs, which indicates a generally good prognosis . Unfortunately, the patients often suffer from a long period of undiagnosed illness and may, as a consequence, be in such a poor condition that curative treatment is no longer an option . The etiology of this carcinoma is still unknown, but different causes, such as chronic inflammatory conditions, have been proposed . However, neither of our patients had an hpv infection in the mucosa . In the literature, the carcinoma is often localized in the distal part of the esophagus, which was also the case in our patients . The endoscopic appearance was very similar to the typical findings described earlier a white, irregular, exophytic mucosa covered with plaques and wart - like polyps, and all biopsies taken from the area were without malignancy . Due to the exophytic nature of the vc and the bland appearance of the epithelial cells, it is extremely difficult to obtain biopsies that are sufficiently deep to visualize the invasive part of the tumor . Therefore, biopsies are often false - negative and only show nonspecific inflammatory changes or changes compatible with squamous cell papilloma . In conclusion, vc has a very characteristic pattern of symptoms but also a typical endoscopic appearance . Thus, when clinical suspicion is raised and disseminated disease has been ruled out, the patient should be considered a candidate for potential curative treatment despite negative biopsies . Furthermore, the diagnosis and treatment require a close collaboration between specialists including pathologists, radiologists, and surgeons . As corresponding author i certify that all authors have participated sufficiently in the planning of the study, intellectual content, and the writing of the work to take public responsibility for it.
Medial ankle sprains are somewhat rare, accounting for approximately 1015% of all ankle sprains in athletes, and can result in long - term disability3 . Most of the pain and tenderness caused by a medial ankle sprain is localized to the medial aspect of the ankle, especially around the deltoid ligament and the medial malleolus ., we report the effects of ankle inversion taping (ait) using kinesiology tape in a patient with a medial ankle sprain . A 28-year - old amateur soccer player complained of severe pain on the medial aspect of his left ankle after sustaining a grade 2 medial ankle sprain during a soccer match one month prior to his visit . He presented with a history of left ankle injury 3 years ago, following which, his ankle was in a cast for 3 weeks . He experienced painful eversion and dorsiflexion of the left ankle and severe pain during sport - related activities such as jumping and turning . In particular, full weight bearing on the left leg was difficult because of severe pain . Prior to participation in this study, the patient demonstrated an understanding of the study purpose and provided written informed consent . This protocol was in accordance with the ethical standards of the declaration of helsinki . In the initial assessment, the tenderness on the medial aspect of the ankle, at a pressure of 3 kg, was measured using an algometer (pain test - model fpk; wagner instruments, greenwich, ct, usa) on a 010 scale, with 0 indicating no pain and 10 indicating the worst pain5 . The patient s self - reported functional ankle deficits using the cumberland ankle instability tool (cait) questionnaire, which is comprised of 9 items scored on a 30-point scale6, were investigated . Dynamic balance was assessed using the star excursion balance test (sebt)7 . The sebt is a valid and reliable tool for dynamic postural control assessment of subjects with ankle instability8 . The maximal reach distances of the opposite leg in the anterior, posteromedial, and posterolateral directions when standing on the sprained leg were 57 cm, 60.5 cm, and 65.5 cm, respectively . Ankle flexibility was assessed using weight - bearing ankle dorsiflexion (the distance from big toe to wall in maximal ankle dorsiflexion with knee flexion without lifting the heel) was assessed9 . At the initial ankle active range of motion assessment, the eversion, inversion, plantarflexion, and dorsiflexion ranges of the left ankle were 6/20, 25/45, 46/60, and 14/30, respectively . Seoul, korea) was applied to the sprained ankle every day for 2 months . The first i - shaped tape was applied from the talus to the calcaneus in a minimal dorsiflexion position for assisting posterior talar glide (fig . The second i - shaped tape was applied from 12 cm above the medial malleolus, over the lateral calcaneus, to the outside of the instep of the foot in a maximal inversion position for assisting ankle inversion and preventing painful eversion (fig . The third i - shaped tape was reapplied in a similar manner as the second step for reinforcement (fig . The fourth i - shaped tape was reapplied in a similar manner as the first step to reinforce the posterior talar glide (fig . We reapplied ait to the sprained ankle every day after removal of the previously applied ait even if the patient did not report any itching and instructed the patient to remove the tape immediately if itching was experienced . In addition, to prevent skin problems, no stretch was applied to the start and end points of approximately 45 cm when the i - shaped tape was applied10 . The patient did not undergo any other interventions for treatment of the medial ankle sprain . At the final assessment, the tenderness at 3 kg of pressure decreased from 8 to 1, and the cait score increased from 11/30 to 27/30 . The maximal reach distances in the anterior, posteromedial, and posterolateral directions increased from 57 cm to 72 cm, from 60.5 cm to 86 cm, and from 65.5 cm to 87 cm, respectively . The maximal distance of weight - bearing ankle dorsiflexion increased from 2.6 cm to 7.0 cm . The eversion, inversion, dorsiflexion, and plantarflexion range of motion increased from 6/20 to 20/20, from 25/45 to 42/45, from 14/30 to 28/30, and from 46/60 to 57/60, respectively . Following this treatment, the patient did not experience any medial ankle pain during sport - related activities such as jumping, turning, and was able to perform full weight - bearing on the left leg . This study showed that the ait application for 2 months decreased medial ankle pain and the self - reported functional ankle deficits, and it improved the ankle range of motion and dynamic balance . The first treatment strategy with ait application is protection of the sprained ankle from further injury and avoidance of eversion that can cause pain due to the mechanical effects of inversion . Painful eversion and dorsiflexion of the sprained ankle were avoided through a more inverted ankle with the application of the kinesiology tape . Although no research on the ankle joint has been reported, previous studies have reported on the application of kinesiology tape to support joint structure11 and modified joint structure such as mechanical effect12 . We assume that the recurrence of ankle sprain and motions that cause medial ankle pain were avoided . The kinesiology tape used in this case study stimulates cutaneous mechanoreceptors13 and improves the joint position sense14 . The elasticity of the kinesiology tape applied to the skin around the ankle may activate the proprioceptors, while the skin is stretched and shortened during ankle movement . Therefore, maintaining the correct ankle position increased ankle stability, which resulted in an increase in the distances of the sebt and weight - bearing ankle dorsiflexion . Future mechanical studies on ait using kinesiology tape in patients with medial ankle sprain are needed.
Additional causes of nontraumatic ich include hemorrhagic infarction, septic embolism, brain tumors, bleeding disorders, anticoagulants, vasculopathy (including moyamoya disease and arteriovenous malformations), and drugs . Among these, hemorrhage rates in patients with intracranial neoplasms range from 2% to 5%2). However, apoplectic onset of hemorrhage from a silent brain tumor is even more uncommon ., we report a rare case of anaplastic astrocytoma with tumor bleeding mimicking a spontaneous hypertensive intracerebral hemorrhage . A 69-year - old woman visited our hospital presenting with nausea, vomiting, dysarthria, and right hemiparesis . Brain computed tomography (ct) scans with contrast enhancement and ct angiograms demonstrated an acute intracerebral hematoma in the left basal ganglia, including the caudate nucleus, accompanied by a small amount of intraventricular hemorrhage, without any underlying enhanced lesion or vascular pathologies (fig . The patient was treated conservatively with anti - hypertensive drugs under a diagnosis of hypertensive basal ganglia ich . Additional physical rehabilitation was performed, and the patient was discharged with improved motor function and mental status 6 weeks after admission . During follow - up as an outpatient, the patient was re - admitted for physical malaise and deteriorated mental status 6 weeks after discharge . Her mental status was temporarily improved and the ventricle size was decreased following a ventriculoperitoneal (vp) shunt operation . Three months after vp shunt placement, the patient's mental status deteriorated further and quadriparesis was noted . Brain magnetic resonance imaging (mri) with gadolinium enhancement revealed an irregular, enhanced, round mass with multiple necrotic foci in the left basal ganglia, where the previous hemorrhage had occurred (fig . Navigation - assisted biopsy was performed, revealing an anaplastic astrocytoma (who grade iii). The patient underwent radiotherapy, and repeat mr imaging revealed non - progression of the tumor (data not shown). However, she has been bed - ridden during 6 months of follow - up . Intratumoral hemorrhage is thought to originate from abnormal newborn vessels that traverse necrotic areas9) or from tumoral invasion of large vessels12), leading to thinning and rupture of the vessels walls . Another potential mechanism would be relatively weak tumor vessels, which are not well invested with a glial meshwork; this may contribute to reduced resistance to the shearing forces of the brain1). Endothelial proliferation with subsequent obliteration of the lumen or presence of intratumoral arteriovenous fistulae are alternate explanations for intratumoral bleeding7). Thus, hemostasis often cannot be easily achieved at hematoma removal; this finding indicates the possibility of brain tumors as a cause of bleeding10). Hemorrhage more often develops in malignant tumors such as glioblastoma and metastatic brain tumors4,9,11). The incidence of tumor bleeding in malignant astrocytoma in one study was 6%4) while that in glioblastoma and metastatic brain tumors were 6.5 - 8% and 7 - 9%, respectively4,9). Among benign neuroepithelial tumors, the incidence of hemorrhage from mixed glioma and oligodendroglioma was much higher than the other tumors4,9). On the other hand, pituitary adenoma and meningiomas have the high risk for developing intratumoral hemorrhage among benign non - neuroepithelial tumors11). The location of bleeding depends on the different site of brain tumor even though intratumoral hemorrhage usually develops in the atypical location of hypertensive intracerebral hemorrhage and the patients often have no history of hypertension11,12). Radiological studies with contrast material usually distinguish tumors from hemorrhage, as the border between the tumors and hemorrhage is usually clear6). In contrast, if the tumors are compressed by a large hemorrhage, or the border between the tumors and hemorrhage is unclear, intratumoral hemorrhage may be indistinguishable from spontaneous ich, even though contrast material is used3). Thus, a ct with contrast cannot exclude underlying pathologies that may cause ich, especially if the patient has a history of hypertension, and the location is typical for hypertensive ich . Mri with gadolinium in the early follow - up period would likely have lead to earlier detection of the tumors in the present case . However, inamasu et al.3) suggested that in terms of cost effectiveness, it is controversial to have every patient presenting with typical hypertensive ich undergo mri with gadolinium to rule out intratumoral bleeding . Histopathological examination of hematoma specimens with brain parenchyma may have detected the brain tumor, if hematoma evacuation or aspiration surgery had been performed in the current case . However, the relatively small volume of hematoma and patient's age led us to choose conservative treatments, rather than surgical options . It is controversial to undergo surgical treatments for histological diagnosis in typical hypertensive small ich, though potential surgical modalities include stereotactic hematoma aspiration or endoscopic hematoma evacuation . However, if surgical treatment is indicated, a considerable hematoma specimen with adjacent brain parenchyma would be preferred to rule out underlying pathologies through histological examination . Interestingly, normal initial neuroimaging including mri is not uncommon among patients with malignant primary brain tumors . Thaler et al.8) reported that among 193 patients with malignant primary brain tumors, initial imaging preceding diagnosis with a short interval was normal in nine patients and abnormal but non - diagnositic in an additional eight patients . Thus, in our case, it cannot be excluded that basal ganglia hemorrhage was a hypertensive hemorrhage which was not related with brain tumor, and then anaplastic astrocytoma rapidly developed in the site of hemorrhage . In the present case, there was an opportunity to detect the underlying high - grade glioma during outpatient follow - up . The patient suffered from physical malaise and a deteriorated mental status 6 weeks after discharge . However, only a ct was performed as the etiology of the ich was presumed to be hypertension . A follow - up ct revealed asymmetric ventriculomegaly and periventricular edema, with shunt operation performed without additional radiological studies . As a result, the final diagnosis of the tumor was delayed . However, in retrospect, asymmetric ventriculomegaly suggests an underlying pathology in the region of the pre - existing hemorrhage . Thus, follow - up mri in the chronic stage of ich seems to be necessary for detecting underlying pathologies masquerading as hemorrhage if the patient undergoes subtle neurological deterioration during follow - up in order to avoid diagnostic delay, even in cases typical for hypertensive hemorrhage . The present case indicates that some brain tumors, masquerading as a hypertensive ich, may be difficult to diagnosis in the acute phase . A diagnostic delay for highly malignant tumors leads to progression without proper treatment and resultant poor outcomes . Thus, although the morphology and location of the hematoma and patient medical history are typical for a hypertensive ich, brain tumors should be suspected as a cause of spontaneous hemorrhage . If needed, aggressive histological investigation at hematoma evacuation and mri follow - up should be considered to avoid hidden, underlying pathologies.
Giant cell tumor (gct) of a phalanx of a finger is extremely rare . The metaphyseal region of the metacarpals and phalanges has been found to be the common site of gcts in most of the reported cases [24]. Though gct is not a sarcoma, its relatively high recurrence rate coupled with local aggressiveness after simple curettage often requires extensive en bloc excision . Local recurrence following curettage and bone grafting has been reported to be as high as 90% [1, 4, 6, 7]. Wide resection and reconstruction with structural bone grafting is also reported to have a high local recurrence rate . Multiple procedures like excision (local or wide), ray amputation, and amputation are used to eradicate the disease completely . Even with single- or double - ray resection for primary or recurrent tumors, local tumor control may not be absolute . Here, we report two cases of gct on phalanges of the finger, noting the rarity of a lesion at this site . A 50 year old man presented in june 2001 with an ulcerated swelling of two months duration over the amputated left ring finger . Careful history taking and a review of his records led to a diagnosis of gct of the proximal phalanx of the left ring finger, where amputation of the proximal phalanx through its base had been performed elsewhere one year earlier . He gave an associated history of traumatic amputation of the pulp of the left index finger in his childhood . A review of previous radiographs showed an expansile lytic lesion at the proximal phalanx involving only the proximal metaphysis and diaphysis, sparing the distal end with an intact articular surface . 1) showed sheets of mononuclear stromal cells with numerous osteoclastic giant cells and osteoid tissue formation in certain areas . He was asymptomatic for one year after the surgery until he noticed a progressive swelling on the amputated stump . The surgical scar was subsequently broken after a month, leading to ulcer formation, and exposing the underlying tumor mass.fig . 1histopathological picture showing typical features of the giant cell tumor (40 magnification) histopathological picture showing typical features of the giant cell tumor (40 magnification) on examination, an ulcerative swelling about 2 cm 3 cm in size involving all of the amputated stump was seen (fig . The margin of the ulcer was not everted and was fixed to the tumor mass . Regional lymph nodes were not palpable.fig . 2a amputation stump with ulcer at the tip . B radiograph showing partial amputation of the proximal phalanx with expansile lesion involving the articular surface . C ct scan showing the irregular expansile lytic lesion with cortical erosion a amputation stump with ulcer at the tip . B radiograph showing partial amputation of the proximal phalanx with expansile lesion involving the articular surface . C ct scan showing the irregular expansile lytic lesion with cortical erosion a radiograph revealed partial amputation of the proximal phalanx through the proximal one - third, with an expansile lesion in the remaining part involving the articular surface that was associated with soft tissue swelling (fig . Ct scan clearly demonstrated an irregular expansile lytic lesion with cortical erosion (fig . 2c). 3) was found to involve the entire base of the proximal phalanx, including the articular cartilage, with areas of new bone formation . 3resected tumor mass extending up to the joint resected tumor mass extending up to the joint at his most recent follow - up (80 months), neither clinical nor radiological evidence of local recurrence was seen . There was no limitation on the strength or motion of the uninvolved digits or wrist . A 26 year old female presented with a painless swelling of the left little finger of six months duration without any history of trauma or fever . Examination revealed a fusiform swelling in the middle phalanx of the little finger (fig . Radiographs demonstrated an expansile lytic lesion involving the entire phalanx with a cortical break (fig . 5a radiograph (ap, lateral) reveals an expansile lytic lesion with cortical break . C postoperative radiograph (ap, oblique) at 14 months showing incorporation of the cancellous graft clinical photograph showing fusiform swelling of the little finger a radiograph (ap, lateral) reveals an expansile lytic lesion with cortical break . B ct scan of the middle phalanx showing marked osteolysis . C postoperative radiograph (ap, oblique) at 14 months showing incorporation of the cancellous graft under general anesthesia, curettage of the lesion was performed with cancellous bone grafting from the iliac crest . Histology confirmed the diagnosis of a gct, demonstrating osteoclastic giant cells admixed with stromal cells . The patient is undergoing regular check - ups for 24 months and has not shown any evidence of local recurrence clinically or radiologically (fig . A 50 year old man presented in june 2001 with an ulcerated swelling of two months duration over the amputated left ring finger . Careful history taking and a review of his records led to a diagnosis of gct of the proximal phalanx of the left ring finger, where amputation of the proximal phalanx through its base had been performed elsewhere one year earlier . He gave an associated history of traumatic amputation of the pulp of the left index finger in his childhood . A review of previous radiographs showed an expansile lytic lesion at the proximal phalanx involving only the proximal metaphysis and diaphysis, sparing the distal end with an intact articular surface . 1) showed sheets of mononuclear stromal cells with numerous osteoclastic giant cells and osteoid tissue formation in certain areas . He was asymptomatic for one year after the surgery until he noticed a progressive swelling on the amputated stump . The surgical scar was subsequently broken after a month, leading to ulcer formation, and exposing the underlying tumor mass.fig . 1histopathological picture showing typical features of the giant cell tumor (40 magnification) histopathological picture showing typical features of the giant cell tumor (40 magnification) on examination, an ulcerative swelling about 2 cm 3 cm in size involving all of the amputated stump was seen (fig . The margin of the ulcer was not everted and was fixed to the tumor mass . Regional lymph nodes were not palpable.fig . 2a amputation stump with ulcer at the tip . B radiograph showing partial amputation of the proximal phalanx with expansile lesion involving the articular surface . C ct scan showing the irregular expansile lytic lesion with cortical erosion a amputation stump with ulcer at the tip . B radiograph showing partial amputation of the proximal phalanx with expansile lesion involving the articular surface . C ct scan showing the irregular expansile lytic lesion with cortical erosion a radiograph revealed partial amputation of the proximal phalanx through the proximal one - third, with an expansile lesion in the remaining part involving the articular surface that was associated with soft tissue swelling (fig . Ct scan clearly demonstrated an irregular expansile lytic lesion with cortical erosion (fig . 2c). 3) was found to involve the entire base of the proximal phalanx, including the articular cartilage, with areas of new bone formation . 3resected tumor mass extending up to the joint resected tumor mass extending up to the joint at his most recent follow - up (80 months), neither clinical nor radiological evidence of local recurrence was seen . There was no limitation on the strength or motion of the uninvolved digits or wrist . A 26 year old female presented with a painless swelling of the left little finger of six months duration without any history of trauma or fever . Examination revealed a fusiform swelling in the middle phalanx of the little finger (fig . Radiographs demonstrated an expansile lytic lesion involving the entire phalanx with a cortical break (fig . 5a radiograph (ap, lateral) reveals an expansile lytic lesion with cortical break . B ct scan of the middle phalanx showing marked osteolysis . C postoperative radiograph (ap, oblique) at 14 months showing incorporation of the cancellous graft clinical photograph showing fusiform swelling of the little finger a radiograph (ap, lateral) reveals an expansile lytic lesion with cortical break . B ct scan of the middle phalanx showing marked osteolysis . C postoperative radiograph (ap, oblique) at 14 months showing incorporation of the cancellous graft under general anesthesia, curettage of the lesion was performed with cancellous bone grafting from the iliac crest . The recovery was uneventful . Histology confirmed the diagnosis of a gct, demonstrating osteoclastic giant cells admixed with stromal cells . The patient is undergoing regular check - ups for 24 months and has not shown any evidence of local recurrence clinically or radiologically (fig . Gct of the hand is rare and seems to be different from conventional gct, which occurs at other sites in the skeleton . It is even rarer to encounter a gct arising from the phalanges . Of the more than 2,400 skeletal gcts reported in the literature, less than 50 were found to involve the phalanges of the hand [7, 8]. . Reported only two cases of gct arising from the phalanges in their series of 108 cases . Goldenberg et al ., in their analysis of 218 cases of gct, reported six cases involving the phalanges . In another two large series of 568 and 327 cases of gct, authors found only four and one cases of phalangeal involvement, respectively [11, 12]. Reported a multicentric giant cell tumor of the hand involving a finger and the wrist . Benign metastazing giant cell tumor of the hand has also been reported . In our series of 124 skeletal gcts giant cell tumors of the hand have been treated with curettage and cancellous bone grafting, wide resection, and structural bone grafting or ray amputation [1, 4, 6, 7, 15]. High local recurrence rates have been reported with these treatment modalities [1, 4, 6, 7]. Reported a gct of the middle phalanx treated with curettage and bone grafting, which recurred at 9 months and was successfully treated by excision and allograft replacement . Resection iliac graft and double arthrodesis for gct of the proximal phalanx of the thumb has also been reported . Our patient (case no . 2) with curettage and iliac bone grafting recovered uneventfully . To avoid stiffness of fingers and tendon adhesion, we started active finger rehabilitation measures as early as the second week . The patient was still disease free at the most recent follow - up, and was performing normal activities with a good grip strength . Most local recurrences of gct cases of the hand are reported to occur within one year of primary surgery [1, 6]. Patel et al . Treated three cases of gct of the hand with curettage and bone grafting, two of which had local recurrence and required ray resection . 1) presented with local recurrence one year after primary surgery elsewhere, but has been disease free for more than 80 months following ray amputation of the amputated ring finger . . Reported one of the largest series of gct of the hand, consisting of 28 lesions (14 in the phalanges) in 21 patients . Two of the six cases of finger phalangeal involvement reported in goldenberg s series developed local recurrence . Table 1 shows the reported local recurrence rates of gct of the hand following curettage, resection and amputation.table 1reported local recurrence after curettage, resection and amputation for gct of the handsl no.authorsno . Of casescurettage bone graftresection or ray amputationnos.local recurrencenos.local recurrence1.goldenberg 611512.averill et al . 1117.this study2116448 (75%)389 (24%) reported local recurrence after curettage, resection and amputation for gct of the hand most of the gct cases with recurrent tumors require ray amputation to prevent recurrence . There are reports of success with ray resection or amputation at the cost of the loss of a functional finger [7, 15]. We chose ray resection of the amputated ring finger with the aim of preventing recurrence . The recurrent tumor in our patient expanded eccentrically, leading to breakage of the surgical scar and ulcer formation . Contrary to malignant ulcers, the margin of the ulcer was not everted and did not bleed on touch . Kumar and tuli and dahlin et al . Reported similar histological findings in their series . Following ray resection of the ring finger the patient was satisfied with a cosmetically improved hand . In view of the comparative rarity of a tumor arising from the phalanges of the finger,
Numerous studies have shown a worldwide increase in the incidence of thyroid cancer (tc) in recent years due to the increased frequency of papillary thyroid carcinoma (ptc). The reason for this increase is not well understood, although some authors argue that excessive medicalization, sometimes called thus, a greater number of ultrasound scans (us) and other imaging techniques, along with an increase in the number of thyroidectomies for benign conditions and better histological examination of surgical specimens, have led to the diagnosis of ptc cases with low clinical impact . This argument is corroborated by epidemiological data which suggest that the increase in the diagnosis of ptc is at the expense of smaller tumors, especially microcarcinomas (ptc <1 cm). Other authors, however, point to a real increase in the incidence of ptc, which could be related to the effect of ionizing radiation or iodine supplementation in the general population . Other reasons, such as hormonal factors, the increased incidence of obesity, environmental toxins such as polybrominated diphenyl ethers, or changing histological criteria for ptc have also been postulated . The aim of this study was to retrospectively analyze a series of thyroidectomies performed at our center over the course of a decade (2001 - 2010) and to determine the incidence of tc in these cases . Between january 2001 and december 2010, a total of 1,323 subjects underwent thyroid surgery . All included patients had a solitary nodule or a multinodular goiter detected by clinical examination, us, or both . A fine - needle aspiration biopsy (fnab) was performed for thyroid nodules> 1 cm and for nodules <1 cm with suspicious us features . Thyroidectomy was prescribed for patients with malignant, suspicious, or repetitive indeterminate nodules according to fnab results . Moreover, surgery was indicated for benign disease when local symptoms were present or for esthetic reasons . Total cohorts were separated into two groups: group 1 included patients operated on between 2001 and 2005, whereas group 2 included patients operated on between 2006 and 2010 . In both groups, demographic characteristics, type of surgery (total thyroidectomy vs. non - total thyroidectomy), preoperative serum levels of thyrotropin (tsh), presence of thyroid antibodies, and incidence of tc according to histological types were compared . Histological variants were classified as differentiated tc (dtc), including ptc and follicular thyroid carcinoma (ftc), and non - dtc, which included all other types of malignancies (medullary carcinoma, undifferentiated carcinoma, metastatic thyroid, and lymphoma). Finally, we compared the characteristics of ptc detected in both periods in relation to the following variables: age, preoperative tsh value, tumor size, percentage of microcarcinomas, mutlifocality, lymph node metastases, associated thyroiditis, and mode of diagnosis (incidental vs. non - incidental). We defined an incidental diagnosis as ptc diagnosed at final histology in a patient whose reason for intervention was benign thyroid disease . Quantitative variables were expressed as mean and standard deviation, and categorical variables were expressed as frequencies and proportions . Statistical analysis was performed using the test for categorical variables and the student t test for quantitative variables . A total of 1,263 cases, 1,023 women and 240 men (average age of 51.9 years), were included retrospectively . Thyroidectomy was prescribed for malignancy or suspicious of malignancy in 221 patients (17.5%). Final histological examination revealed the presence of tc in 253 cases (20%), and of these, 216 patients had dtc (195 ptc cases, 21 ftc cases) (fig . The mean size of the ptc was 15.4 (12.6) mm, and no significant differences in size were observed over the 10 years (fig . Group 1 included 593 cases (487 women and 106 men) with a mean age of 51.8 (14.2) years . In group 2, there were 670 subjects (536 females and 134 males) with a mean age of 51.9 (16.3) years . However, there was a significant increase in the number of malignancies in the second period: 90 tc cases (15.2% of all interventions) in group 1 compared to 163 cases (24.3%) in group 2 (p <0.001). Differences were due to a significant increase in the number of dtc cases: 73 (12.1%) in the first half of the decade versus 143 (21.3%) in the second half (p <0.001). Within dtc cases, there was no significant variation in the number of ftc cases: 7 (1.2%) in group 1 compared to 14 (2%) in group 2 (p = 0.29). Thus, the observed increase in the incidence of tc was due solely to an increase in ptc: 66 cases between 2001 and 2005, accounting for 11.13% of all thyroidectomies performed, compared to 129 cases between 2006 and 2010, which accounted for 19.25% of all surgical interventions . Non - dtc thyroid malignancies included 13 and 11 medullary tcs, 5 and 3 anaplastic tcs, and 1 and 6 other tumors (secondary metastasis and primary thyroid lymphoma) in groups 1 and 2, respectively . The preoperative tsh value was 1.8 (5.9) mu / dl in group 1 patients and 1.5 (1.9) mu / dl in group 2 patients . Although not statistically significant, there was an increase in the number of total thyroidectomies in the second half of the decade: 409 cases (69%) between 2001 and 2005 compared to 494 cases (73.7%) between 2006 and 2010 (p = 0.06). In both groups, it can be seen that there were no differences in any of the observed variables, which included age, preoperative tsh value, tumor size, percentage of microcarcinomas, multifocality, metastatic disease, thyroiditis, and mode of diagnosis . Thus, there were a total of 84 incidental cancers, 31 in the first period (47%) and 53 in the second period (41%) (p = 0.43). These numbers represented 7.6% of the patient cohort with total thyroidectomies in group 1 and 10.7% of the patient cohort with total thyroidectomies in group 2 (p = 0.1). In addition, only 6 cases (9%) in the first period and 12 cases (9%) in the second period were non - incidental microcarcinomas . This study supports, in our environment, the trend of increased incidence of tc, due to an increase in ptc, during the first decade of the century in the cohort of patients who underwent thyroid surgery . The percentage of ptc increased by almost 10% between 2006 and 2010 compared to 2001 and 2005 . One presumed reason for this increase in the incidence of ptc is the screening effect, namely the higher and better performance of various tests in the general population, such as us and fnabs of thyroid nodules, which has led to increased diagnosis of ptc . This argument is corroborated by epidemiological data suggesting an increase in ptc diagnosis at the expense of smaller ptcs, especially microcarcinomas . It has been hypothesized that if the entire pool of occult ptc were identified antemorten, the resulting ascertainment bias could result in a 50-fold increase in the apparent incidence of ptc . In our study, we did not observe an increase in microcarcinomas, and the average size of ptc did not change over the decade . Furthermore, although it is postulated as being more frequent, not all reports found that the increase in ptc was only at the expense of smaller ptcs . Davies and welch found, based on data from the national cancer institute's surveillance, epidemiology and end results (seer) program, that between 1988 and 2002, the largest percent increases in ptc were in tumors <2 cm . Hughes et al . Also analyzed the seer registry and found that the greatest increase in ptc occurred in patients older than 45 years with microcarcinomas; the authors attributed these findings to overdiagnosis . However, using the same database, zhu et al . Found that the rate of the age - adjusted incidence showed a significant increase in ptc for all tumor sizes between 1988 and 2003 . In a detailed analysis conducted by enewold, the authors found that ptcs> 5 cm increased 222% (similar to the 248% in tumors <1 cm) in american white women . Similarly, yu et al . Compared the incidence of ptc in two different periods (1992 - 1996 and 2000 - 2004) and found a significant increase in all tumor sizes . Also, in an italian study that compared the most recent periods (1998 - 2003 and 2004 - 2009), the authors did not find differences in the percentage of microcarcinomas . Finally, morris and myssiorek reported a similar increase in the percentage of cases with extrathyroidal extension, a factor that does not support the screening effect as the cause of the observed changes . Similar to other studies on the incidence of neoplasms, data are recorded in relation to the reference population (usually per 100,000 population). Since a high percentage of ptc cases are incidentally diagnosed after thyroidectomies for benign disease, the population incidence of thyroid interventions and the extent of the thyroidectomy may influence this kind of malignancy . In a recent report, sassolas et al . Found that 64% of microcarcinomas are diagnosed incidentally; in a study by alevizaki et al . The frequency of tc was not related to the general population, but to cases of thyroidectomies performed . We observed an increment in the percentage of interventions that were positive for malignancy, reaching 24.3% in the second period . Using a similar design, sassolas et al . Found that 22% of all thyroid interventions for any cause were related to tc . It has been proposed that the increase in ptc may be due to an improvement in the selection of patients referred for surgery . However, the fact that incidental and non - incidental cases increased in parallel does not support this hypothesis . In the present study, the combination of lack of specific increasing trends of smaller tumors and stability in the percentage of cases incidentally diagnosed seems to favor a true increase in the incidence of ptc . Moreover, in most recent years, there have been no substantial changes in the diagnosis and therapeutic management of patients with nodular thyroid disease . Thus, it appears that the increase in ptc is due to both improvements in diagnostic techniques and their high use (overdiagnosis), and a real increase in the incidence of ptc . The study population is not representative of the general population nor is it a population with thyroid disease . Therefore, we cannot rule out bias caused by changes in social or demographic parameters in the reference population . Moreover, the comparison periods were two successive 5-year periods, a very short time interval for the identification of significant changes in incidence trends . However, the relatively short study period also reduced the likelihood of changes in socio - demographic status, referral criteria, surgical indications, and diagnostic (clinical and histological) and therapeutic protocols . Therefore, this variability could be higher in studies that compare longer periods and thus produce greater bias . In conclusion, this study suggested an increase in the number of tc cases in the first decade of the century in patients undergoing thyroid surgery.
Over the last decade, intestinal transplantation has become an established treatment modality in the management of intestinal failure . The preoperative status of patients has been found to influence the outcome of surgery, and scores developed to semiquantify this [2, 3] have been used in routine practice to facilitate preoperative risk assessment . Much of this improvement has arisen from advances in immunosuppressive regimes and better preoperative preparation and postoperative care of patients . Although survival after transplantation remains inferior to that on parenteral nutrition (pn), the gap is closing, and as a consequence patients are being considered for transplantation at an earlier stage . Potential improvement in quality of life is also now being factored in to the decision regarding a patient's suitability for transplantation . It has therefore become very important to accurately assess patient's individual survival chance . In our routine clinical practice we have semiquantified comorbidity for each patient and compared this to postoperative survival to determine the relationship . Preoperative comorbidity was transformed into a numerical score to allow service analysis to take place . Comorbidity factors included were loss of venous access and impairment of organs or systems not corrected by transplantation . A score of 3 indicated a functional abnormality not fully corrected by treatment of a severity approaching a contraindication for transplantation . A score of 1 describes a characteristic which does not present an immediate risk but which may develop into a risk factor in the future . A score of 2 indicates an abnormality of function which is corrected by ongoing treatment and conveys moderate risk (table 1). Following the loss of 2 large venous access sites suitable for parenteral nutrition and high flow infusions (i.e., internal jugular, subclavian, and femoral vein), each subsequent loss was given a score of 1 . Patients were selected for transplantation according to the conventional indications as previously published [5, 6]. In summary, these were irreversible intestinal failure and inability to continue parenteral nutrition, due to such conditions as loss of venous access and parenteral nutrition - induced liver disease . Patients received an intestinal graft either in isolation or as part of a cluster of abdominal organs including liver (multivisceral) or excluding liver (modified multivisceral transplant). All patients received induction therapy with either alemtuzumab (campath 1h), daclizumab (zenapax), or a few with glucocorticosteroids . The data were collected prospectively during routine preoperative evaluation at the transplant centres at the university of cambridge, united kingdom, and university of miami, united states of america, from 1997 to 2003 . Patient data were consecutively entered into a database as a routine at each institution from which data were acquired for the analysis . Where comorbidity data were incomplete, patients were excluded from the analysis; this was undertaken without knowledge of their subsequent survival . The primary outcome measured was median recipient survival with 95% confidence interval (ci) and was calculated by kaplan - meier (km) approach with comparison using logrank test . The accuracy of the comorbidity score to predict postoperative survival was tested by using harrell's c statistic which estimates the proportion of accurate predictions . A harrell's c index varies from 0.5 to 1 reflecting no discrimination to perfect discrimination, respectively . The donor and patient demographics are detailed in (tables 2 and 3, resp . ). A total of 72 adults (m: f patients received either an isolated intestinal graft (n = 27) or intestine in combination with other organs including liver (multivisceral) (n = 27) or excluding liver (modified multivisceral) (n = 18). In 31 patients, the large intestine was also transplanted, and 6 received a kidney . The cause of intestinal failure in recipients was most commonly short gut syndrome (33%), and the most frequent procedure in this cohort was multivisceral transplantation (intestine, liver, and additional organs) in 38%, followed by isolated intestine (37%), and modified multivisceral (intestine and other organs excluding liver) 25% (table 3). Most patients received alemtuzumab (campath 1h) induction immunosuppression, and the mean (sd) number of rejection episodes per patient was 1.22 1.34 (table 4). Individual patient comorbidity scores and their postoperative survivals the kaplan - meier (km) curve analysis of the cumulative recipient survival revealed a significant inverse association between patient survival and comorbidity score as evaluated by the logrank test for trend (p <0.0001) (figure 1). For the purposes of comparing survival chance, patients were grouped into comorbidity scores of 0 and 1, 2 and 3, 4 and 5, 6, and above . The hazard ratio (95% ci) for death (compared to group 0 + 1) was found to increase as the comorbidity score increased: 1.945 (0.76225.816), 5.075 (3.31436.17), and 13.77 (463.3 - 120100), respectively; this became significantly greater than group 0 + 1 at group 4 + 5 (p <0.0001) (figure 1). The survival of patients receiving isolated intestinal grafts was compared to those who underwent multivisceral transplantation . Kaplan - meier analysis revealed no difference in survival between patients receiving isolated and multivisceral transplantation: logrank (mantel - cox) test: p = 0.43; hazard ratio: 0.8129 (0.4599 to 1.366). Median survival for isolated transplants (2007 days) was similar to that for multivisceral grafts (3107 days). The accuracy of the score to predict postoperative survival was evaluated using the receiver - operator characteristic analysis . A receiver - operator curve (roc curve) was constructed for survival prediction at 1, 3, 5, and 10 years . The harrell's c statistic was calculated for each curve and represents the overall accuracy when assessed in terms of specificity and sensitivity at all possible points . The c statistic indicated very good or excellent accuracy of the comorbidity score at all times frames (figure 2, table 5). We recognise that the interpretation of these data should be undertaken in the context that there will be differences in the protocols of transplant centres which might have an influence on the relative importance of comorbidity on survival . For instance, the majority of patients in our cohort received alemtuzumab as induction therapy which may have made the impact of comorbidity more significant . There will also be additional influences on survival, particularly in the long term, that are not included in this analysis . Nevertheless, in this cohort of patients, our results indicate that the degree of preoperative comorbidity appears to predict posttransplant survival following small intestinal and multivisceral transplantation in adults . The predictive value of the comorbidity score might allow better patient selection, focus on pretransplant optimization, and facilitate informed consent for the procedure . Patients who have a low comorbidity score might be considered for earlier transplantation or even preemptive transplantation, where transplantation is offered at the time of developing intestinal failure . This would be particularly appropriate for those with high risk circumstances, where they are likely to have a reduced life expectancy on pn, such as pseudoobstruction and systemic sclerosis and possibly ultrashort bowel syndrome (<30 cm of jejunum), rather than waiting for them to develop the traditional indications through complications of their pn . In better performing centres, it might be reasonable to offer patients preemptive transplantation on the basis of improving a very poor quality of life (qol). Patients in comorbidity groups 0 + 1 have a 10-year survival similar or better than unselected patients on pn . However, before this becomes common practice, it will be important to develop methods of determining which patients will benefit in terms of qol from transplantation, as it is clear that certain aspects of patient's lives do not improve with transplantation . Furthermore, the timing of transplantation might be influenced by the desire to limit comorbidity to keep the comorbidity score low . For instance, a patient with early comorbidity in several body systems might benefit from early transplantation before these deteriorate; conversely, patients with active comorbidity might have a better survival chance if transplantation is delayed until they receive treatment to stabilise concurrent illness . As postoperative survival improves poor quality of life, it is likely to become a more frequent indication for transplantation . Under these circumstances, it will be crucial to predict postoperative survival for individual patients to better evaluate the risk / benefit ratio of transplantation and facilitate informed consent, and the comorbidity score might also be of considerable use for this purpose . As the role of other factors, such as the presence of hla antibodies, becomes clearer,, the numeric score (comorbidity score) developed to allow us to semiquantify preoperative comorbidity appears to be an accurate and convenient method of assessing risk using preoperative risk factors . A future multicentre international prospective study to evaluate this survival risk factor is important to confirm our initial observation and may lead to a useful clinical tool in the selection and timing of patients for transplantation.
The fusion between the sperm and the egg marks the beginning of a new individual and ensuing development of either man or beast depends upon a number of additional cell fusion events (reviewed by ogle et al . Thus, mononuclear cytotrophoblasts fuse to form the syncytiotrophoblast layer of the placenta in man and certain other mammals . Syncytiotrophoblasts control the exchange of gases, nutrients and waste products between the fetus and the mother, protect the fetus against the maternal immune system and are also responsible for the production of hormones, which like chorionic gonadotropin, regulate the continuation of pregnancy . In addition, in the developing individual, myoblasts fuse to form multinucleated skeletal muscle fibers, while cells of monocytic origin fuse to form osteoclasts, which participate in bone sculpturing and remodeling as well as in the regulation of serum calcium concentrations . It is well - known that skeletal muscle is regenerated through fusion of muscle fibers with satellite cells (bischoff 1994) and that macrophages may fuse to form multinucleated giant cells with enhanced phagocytic capabilities in response to injury and antigenic challenges (vignery 2005). Recent data indicate that additional cell fusions may contribute to tissue repair in the adult (vassilopoulos et al . 2003; wang et al . 2003). Bone - marrow - derived (bmd) cells have been shown to fuse with hepatic cells, nerve cells and gastrointestinal cells and the theory has been put forward that such fusions may serve to repair damaged or corrupted cells (alvarez - dolado et al . However, it is still debated whether such fusions are important to tissue repair and whether they engage bmd stem cells or more differentiated fusogenic cells like monocytes / macrophages (reviewed by vignery 2005). Intriguingly, the accumulation of macrophages in injured organs may reflect not only the need to remove debris but also to repair compromised cells through heterotypic fusions (vignery 2005). Moreover, in recent transplant experiments, irradiated mice showed evidence of fusions between crypt stem cells and donor bmd cells (rizvi et al . 2006). In thus transplanted mice, representatives of all cell lineages derived from intestinal crypt stem cells (absorptive columnar, enteroendocrine, goblet and paneth cells) showed expression of chromosomal markers characterizing donor bmd cells . This phenomenon appeared to depend upon tissue injury since expression of donor cell markers never was detected in non - irradiated mice (rizvi et al . (1999), who have demonstrated that cells phagocytosing apoptotic cells may acquire functional dna . Moreover, a mechanism of cell cell invasion (entosis) was recently described and could potentially transfer dna between cells (overholtzer et al . Thus, viral as well as normal sequences from apoptotic cells have been found to be active also in phagocytosing cells (holmgren et al . This observation challenges our understanding of apoptosis as an efficient way of clearing corrupted or alien dna . The viral genome encodes envelope (env) proteins, which bind to cell surface receptors and assist the virus in entering the cell . The infected cell commences to synthesize env proteins, which, upon insertion in the plasmalemma, engages receptor proteins in neighboring cells and initiate fusions . Interestingly, modifications of the cytoplasmic tail of several env proteins appear to modify fusions . Thus, proteolytic cleavage of the cytoplasmic tail regulates fusions and also cellular signaling events, such as tyrosine kinase activity, may be involved in regulation of virally induced cell fusions (kubo et al . Thus, a cell infected by a specific virus becomes resistant to infections with other viruses that bind to the same receptor . This phenomenon, which probably reflects receptor saturation, has led to the grouping of retroviruses into different subtypes, which bind to the same receptor . Other types of viruses encode fusogenic proteins (like influenza hemagglutinin), which do not bind to surface receptors, but which liberate a fusion peptide in the acidic milieu of endosomes and thus initiate viral host cell fusion following endocytic uptake of viral particles . Both these proteins and the env proteins are referred to as class i viral fusion proteins and contain hydrophobic fusion peptides buried within their sequences . The cleaved peptides undergo conformational changes, resulting in hairpin - like, alpha - helical bundles, which bring the viral and cell membranes into close apposition and thereby facilitates fusion (reviewed by chen and olson 2005). In 1911 aichel suggested that cancer cells might spontaneously fuse with host cells to produce hybrids with supernumerary chromosomes that could evolve into cells of increased malignancy . Subsequent studies have confirmed this prediction by demonstrating that cells with mixed phenotypes spontaneously appear in co - cultures of normal and malignant cells (barski and cornefert 1962; busund et al . 1994) (figs . 1, 2, 3). Additionally, transplanted tumor cells of animal (busund et al . 1974a, b) or human (goldenberg et al . 1974; goldenberg and pavia 1982; mortensen et al . 2004) origin may fuse with and acquire phenotypic characteristics of normal host cells (fig ., data suggested that the putative fusion partner was of macrophage or endothelial origin but, in most cases, definite identification was not achieved . In a few cases, it has also been demonstrated that the fused cells expressed genetic markers of both parental cell types (goldenberg et al . 1974; mortensen et al . (2004) demonstrated that, initially after fusion, bi- or multinucleated cells formed and that such cells had the parental genomes seggregated in diffferent nuclei (heterokaryons) (fig . Subsequently, mitotic figures appeared showing an admixture of the parental chromosomes (fig . Eventually, cells with the parental genomes mixed in a single nucleus (synkaryons) were detected (fig . 2k o). In agreement with the notion that synkaryons appear after mitotic divison of heterokaryons, 3). A few recent studies present compelling evidence that cells with genetic characteristics of hybrid cells also may appear in human tumors (andersen et al . 2007; chakraborty et al . Thus, in two cases of renal carcinomas, arising in bone marrow transplant recipients, some tumor cells showed genetic markers characterizing the healthy donor (chakraborty et al . Moreover, in patients with multiple myelomas, osteoclasts have been shown to contain an admixture of nuclei, of which some possess tumor - specific chromosomal translocations while others are devoid of translocations (andersen et al . Finally, lymphoma - specific genetic abnormalities were described in endothelial cells in b cell lymphomas (streubel et al . Host cell hybrids did form in these patients, the combined results from studies in vitro and in vivo do present compelling evidence that cell fusions do occur in tumors (reviewed by pawelek 2005). Co - culture of human breast cancer (mcf-7) cells and human umbilical vein endothelial cells (huvec). Cells were stained for the cancer cell marker cytokeratin (red, c), the endothelial cell marker vimentin (green, b) and dna (bisbenzimide, blue, d). Note in the merged image (a) one fused, multinucleated cell reacting for both cytokeratin and vimentin (orange - yellow)fig . The cultures were subjected to fluorescent in situ hybridization (fish) with probes recognizing all bovine chromosomes (green; b, g, l) and all human chromosomes (red; c, h, m) and counter - stained for dna with dapi (blue; d, i, n) and observed in differential interference contrast (dic; e, j, o). Merged red - green images are shown in a, f, k. note that initially, binuclear cells (heterokaryons) form, having one bovine and one human nucleus (a e). Occasionally, tri- or multinuclear cells with different admixtures of bovine and human nuclei are also detected . At longer times after mixing, mitotic figures, containing an admixture of bovine and human chromosomes appear (f j) and, eventually, cells with a single nucleus, containing an admixture of the two genomes in mixture (synkaryons) are detected (k o)fig . Mixed culture of human breast cancer cells and bovine endothelial cells submitted to double fish as in fig . 2 (a double fish; b combined dapi and dic). Low power micrograph showing a pair of synkaryons with the bovine and human genomes admixed in single nuclei (arrows). In addition, nuclei hybridizing only for the bovine (green) or only for the human (red) genome occurfig . A, b section from lung of a nude mice injected with human breast cancer (mda - mb-231) cells in the tail vein (mortensen et al . The section underwent fish for the mouse genome (red) and the human genome (green) (a) and dna was counterstained with dapi (b; blue). Note one nucleus in which the human and mouse genome co - localize (arrow). C: similar section, stained with an antibody detecting human (but not mouse) p53 (red; p53 is mutated and overexpressed by the breast cancer cells), an antibody to beta - catenin (to mark cell membranes) and for dna with bisbenzimide (blue). Note a micrometastasis of human breast cancer cells having violet (red + blue) fluorescent nuclei . D section stained for human p53 (red) and the endothelial marker von willebrand factor (green) and dna (blue). Note a human cancer cell with a violet (red + blue) nucleus showing membrane - staining for von willebrand factor . Since von willebrand factor is not normally expressed by the breast cancer cells, this image is suggestive of a fusion between a human breast cancer cell and a mouse endothelial cell . Similar results were obtained using double fish for the human and mouse genome and imunofluorescent staining for von willebrand factor (described by mortensen et al . Co - culture of human breast cancer (mcf-7) cells and human umbilical vein endothelial cells (huvec). Cells were stained for the cancer cell marker cytokeratin (red, c), the endothelial cell marker vimentin (green, b) and dna (bisbenzimide, blue, d). Note in the merged image (a) one fused, multinucleated cell reacting for both cytokeratin and vimentin (orange - yellow) chromosomal markers and cell fusions . The cultures were subjected to fluorescent in situ hybridization (fish) with probes recognizing all bovine chromosomes (green; b, g, l) and all human chromosomes (red; c, h, m) and counter - stained for dna with dapi (blue; d, i, n) and observed in differential interference contrast (dic; e, j, o). Merged red - green images are shown in a, f, k. note that initially, binuclear cells (heterokaryons) form, having one bovine and one human nucleus (a e). Occasionally, tri- or multinuclear cells with different admixtures of bovine and human nuclei are also detected . At longer times after mixing, mitotic figures, containing an admixture of bovine and human chromosomes appear (f j) and, eventually, cells with a single nucleus, containing an admixture of the two genomes in mixture (synkaryons) are detected (k o) chromosomal markers and cell fusions . Mixed culture of human breast cancer cells and bovine endothelial cells submitted to double fish as in fig . 2 (a double fish; b combined dapi and dic). Low power micrograph showing a pair of synkaryons with the bovine and human genomes admixed in single nuclei (arrows). In addition, nuclei hybridizing only for the bovine (green) or only for the human (red) genome occur cancer host cell fusion in vivo . A, b section from lung of a nude mice injected with human breast cancer (mda - mb-231) cells in the tail vein (mortensen et al . 2004). The section underwent fish for the mouse genome (red) and the human genome (green) (a) and dna was counterstained with dapi (b; blue). Note one nucleus in which the human and mouse genome co - localize (arrow). C: similar section, stained with an antibody detecting human (but not mouse) p53 (red; p53 is mutated and overexpressed by the breast cancer cells), an antibody to beta - catenin (to mark cell membranes) and for dna with bisbenzimide (blue). Note a micrometastasis of human breast cancer cells having violet (red + blue) fluorescent nuclei . D section stained for human p53 (red) and the endothelial marker von willebrand factor (green) and dna (blue). Note a human cancer cell with a violet (red + blue) nucleus showing membrane - staining for von willebrand factor . Since von willebrand factor is not normally expressed by the breast cancer cells, this image is suggestive of a fusion between a human breast cancer cell and a mouse endothelial cell . Similar results were obtained using double fish for the human and mouse genome and imunofluorescent staining for von willebrand factor (described by mortensen et al . Host cell fusions is, of course, if they are relevant to the patient . In fact, there are two opposing views . The first is based on early experiments on fusions induced to occur between cancer cells and normal cells in culture . With few exceptions these studies were, in fact, seminal to the discovery of tumor suppressor genes (reviewed by anderson and stanbridge 1993). Since tumor suppressor genes, like p53 and rb, frequently are inactivated in cancer cells, fusions would present cancer cells with unperturbed tumor suppressors from the normal fusion partner and consequently initiate cell cycle arrest or apoptosis . Although, this certainly applied for the cell types studied in the contributions cited above, it may not be a general rule . Thus, production of monoclonal antibodies depends upon the fact that it is possible to fuse antibody - producing spleen cells with myeloma cells to obtain hybridomas that retain the unlimited proliferative ability of the tumor cell partner and the antibody production of the normal cell (kohler and milstein 1975). In fact, several studies documented that some fusions may lead to cells of increased malignancy (barski and cornefert 1962; busund et al . 2003; chakraborty et al . Possibly, the genetic make - up of tumors may dictate the outcome of individual cancer host cell fusions . Moreover, fusions lead to aneuploidy and chromosomal instability, which characterizes most cancers and may, by itself, stimulate carcinogenesis (duelli et al . 2007). Cell fusion events must be extremely well controlled . Due to their major importance to fertilization, placentation, muscle development, bone structure, calcium homeostasis and the immune defense system, additionally, the potential role of cell fusions as a repair mechanism and the role of cell fusions in cancer development and progression have further stimulated research in this field . In spite of this, much less is known about cell cell fusion mechanisms than is known about how intracellular membranes fuse through v- and t - snares . Interestingly, engineered flipping of the v- and t - snare machinery has been shown to promote cell cell fusions (weber et al . However, it is evident that this mechanism is not a physiological mediator of cell cell fusions . Interestingly, v- and t - snares act similar to class i viral fusion proteins in that they form bundles of alpha - helices, which result in membrane apposition and fusion (blumenthal et al . 2003; jahn et al . 2003). Of the many proteins, which to date have been shown (or proposed) to be involved in cell fusions in mammals, only the syncytin family appears to use a similar alpha - helical mechanism . That they do is not surprising in view of the fact that syncytins represent conserved endogenous retroviral env sequences . The founding family member, syncytin-1, was discovered as a protein capable of mediating fusions between cytotrophoblasts into syncytiotrophoblasts (blond et al . This capability may have contributed to a high degree of evolutionary conservation of the syncytin-1 sequence . Syncytin-1 represents the env protein of the human endogenous retroviral (herv) w sequence, which entered the primate genome 2540 million years ago . In contrast, most other retroviral sequences inserted in our genome have been subject to inactivating changes and probably represent garbage sequences . Molecular studies have shown that syncytin-1 (env w) shares a structure similar to class i viral fusion proteins, especially in the region of the n- and c - terminal heptad repeats (nhr and chr), and shares a common fusion mechanism with these proteins (chang et al . A synthetic peptide derived from the chr is also capable of inhibiting syncytin-1-mediated fusions by perturbing this mechanism (chang et al . Syncytin-1 binds to the d - type retroviral receptor asct-2 (blond et al . 2000) and may use another neutral amino acid transporter (asct-1) as an auxiliary receptor (lavillette et al . Syncytin antibodies, syncytin-1 downregulation through antisense oligonucleotides and the syncytin-1 chr peptide have been shown to inhibit fusions between trophoblast - derived cells (blond et al . Agents increasing cellular levels of camp or camp analogues have been shown to promote cytotrophoblast fusions in vitro (keryer et al . 1998) and such agents are also known to elevate protein and mrna levels of syncytin-1 in isolated cytotrophoblasts (frendo et al . Also estradiol may regulate syncytin-1 expression (carino et al . 2003) and the placenta - specific transcription factor gcma interacts with two upstream sites in the herv - w 5-long terminal repeat and stimulates syncytin-1 transcription (yu et al . Additionally, experimentally induced truncations in the cytoplasmic tail of syncytin-1 increases its fusogenicity (drewlo et al . 2006), similar to what has been observed for some virally derived env proteins (reviewed by kubo et al . However, if modifications in the cytoplasmic tail of syncytin-1 are of physiological importance for regulating fusogenicity has yet to be demonstrated . A second syncytin - family member, syncytin-2, syncytin-2 also represents a highly conserved endogenous retroviral envelope gene and is derived from the herv frd sequence (blaise et al . Immunocytochemical studies have localized syncytin-1 primarily to syncytiotrophoblasts as well as to cytotrophoblasts (fig . 2004), which may reflect differences in antibodies, fixation procedures and controls . A major point of concern is also the degree of cross - reactivity to the related sequences in syncytin-2, which recently was localized to a subpopulation of cytotrophoblasts (malassin et al . The syncytin-1 receptor protein asct-2 was recently localized to cytotrophoblasts (hayward et al . 2007). More studies on the exact distribution of syncytin expression in the placenta using syncytin-1 and -2 specific antibodies seem warranted in order to exactly localize where fusions are likely to occur . Nuclei are lightly counterstained with haematoxylin human term placenta immunocytochemically stained with a syncytin-1 peptide antiserum . Nuclei are lightly counterstained with haematoxylin trophoblast cell fusions are, however, not unique to the primate placenta . Thus, also in the mouse placenta, trophoblasts fuse to form syncytiotrophoblasts but mice do, of course, not express herv sequences . Amazingly, an in - silico search of the mouse genome unraveled the existence of two murine endogenous retroviral (merv) env genes, labeled syncytin - a and -b (dupresssoir et al . These genes were also fusogenic and orthologous genes were present in additional species of muridae (rats, gerbils, voles and hamsters) and appear to have entered the rodent lineage some 20 million years ago (dupresssoir et al . 2005). They were expressed in the placenta and at least syncytin - a has been shown to be involved in the formation of syncytiotrophoblasts in mice (gong et al . This represents an amazing example of parallel acquisition of retroviral genes of importance to reproduction in primates and rodents and poses the question whether also other species may have acquired similar viral genes of importance to cell fusions . The ability of syncytins to induce cell fusions may not be their only physiologic role . Thus, recent studies have shown that while syncytin-2 and syncytin - b also possess immunosuppressive activity, syncytin-1 and syncytin - a do not (mangeney et al . 2000) but subsequent studies have revealed the presence of syncytin-1 also in the brain (antony et al . 2004) and in breast, colon and endometrial cancers (bjerregaard et al . 2007, larsen, talts, andersen, bjerregaard and larsson: work in progress). In fact, in all systems studied so far, from mating yeast to man, a bewildering array of mechanisms have been identified (see chen and olson 2005; chen et al . Molecules potentially involved in mammalian cell fusions include adam (a disintegrin and metalloproteinase domain) 12 (meltrin alpha), which has been associated with myoblast and osteoclast cell fusions (abe et al . 1999; in addition, adams 1 and 2 (fertilins) may be involved in sperm oocyte fusions, but do not seem indispensable for this function and, in man, the fertilin alpha gene is dysfunctional (chen and olson 2005; cho et al . Cd9 is also expressed by bewo trophoblast tumor (choriocarcinoma) cells and has been linked both to bewo invasiveness and to invasion during mouse embryo implantation (hirano et al . 1999; liu et al . Cd9 has, together with another tetraspanin family member, cd81, also been linked to myoblast fusion and myotube maintenance (tachibana and hemler 1999). Moreover, antibodies to either cd9 or cd81 have been shown to block fusions induced by mason pfizer monkey virus a d - type retrovirus (duelli et al . 2005). Tetraspanins are known to organize other proteins into membrane microdomains and may link to the actin cytoskeleton via ewi (glu - trp - ile) and erm (ezrin radixin moesin) proteins (hemler 2003; sala - valds et al . Possibly, their role as organizers of other proteins into microdomains may play a role in their involvement with cell fusions (zivyat et al . They do not express characteristics of fusogenic proteins like the syncytins, snares and class i viral envelope proteins, but are coexpressed with syncytin-1, at least in bewo cells . A macrophage fusion receptor (mfr, sirpalpha), resembling cd4the cell surface receptor for hiv has been identified in macrophages (saginario et al . Mfr, which belongs to the immunoglobulin superfamily, binds another member of this family, cd47 . Cd47 is also structurally related to proteins expressed by vaccinia and variola viruses (chen et al . Whereas expression of mfr is restricted to myeloid cells and neurons, cd47 is ubiquitously expressed . Mfr is transiently induced in macrophages at the onset of fusion while cd47 expression is constant . It has been hypothesized that cd47 initially binds to a long form of mfr to secure recognition and then switches to bind a shorter form to bring the plasma membranes closer (510 nm) for fusion (vignery 2005). Additionally, or alternatively, cd47 may promote calcium entry by forming a membrane pore (reviewed in chen et al . Also the hyaluronan receptor, cd44, is induced transiently when macrophages start to fuse . Mice lacking dc - stamp are osteopetrotic and lack multinucleated osteoclasts and giant cells (yagi et al . 2005). Dc - stamp is a seven - transmembrane receptor, somewhat similar to the hiv co - receptor cxcr4, but a ligand has yet to be identified . These and many more molecules, including integrins, vacuolar atpase and receptors and their ligands, as well as different signaling intermediates have been associated with cell fusions . With the exception of the syncytins, most of the mammalian molecules so far studied do not fulfill strict criteria for fusogens and several are dispensable for fusions (reviewed by chen and olson 2005; chen et al . However, this may reflect molecular redundancy and does not definitely exclude that these molecules may participate in fusions . It is noteworthy that, in both man and mouse, two different syncytins are expressed and are both fusogenic . It is possible that, in the mammalian system, fusion requires a flotilla of molecules organized into membrane microdomains by proteins like cd9, encompassing receptors / ligands, signaling entities, proteases and fusogens of retroviral origin that are capable of forming alpha - helical bundles, which bring membranes closer . Syncytins and related retroviral envelope sequences may, in this connection, function both as fusogens and receptor ligands . However, it seems unlikely that as irreversible an event as a cell cell fusion should depend upon a single receptor ligand interaction . Thus, both facilitatory and inhibitory factors are expected to be part of the flotilla . (2004) documented that human breast cancer cells fused with endothelial cells in culture . Stimulated by studies showing that syncytin-1 was involved in cytotrophoblast cell fusions (blond et al . 2000; mi et al . 2000), we examined whether a similar mechanism could account for cancer endothelial cell fusions . Expression of syncytin-1 was documented in the breast cancer cell lines examined (mcf-7 and mda - mb-231 cells; bjerregaard et al . 2006, sk - br-3 cells: talts, bjerregaard and larsson: unpublished data). Moreover, we found that both tumor cells and endothelial cells expressed the syncytin-1 receptor asct-2 . Use of phosphorthioate - protected syncytin-1 antisense oligonucleotides downmodulated syncytin-1 expression as measured by either quantitative rt - pcr or western blotting and inhibited breast cancer endothelial cell fusions, whereas a scrambled oligonucleotide control was without effect . Additionally, the syncytin-1 chr peptide, referred to above, also inhibited the fusions whereas a control peptide was without effect (bjerregaard et al . However, neither the antisense nor the chr peptide experiments effected a total inhibition of cancer - endothelial cell fusions . There may be several reasons to this . Thus, the antisense oligonucleotide did not totally downmodulate syncytin-1 levels and could therefore not be expected to decrease cell fusions to zero and the lack of total inhibition by the chr peptide could potentially be ascribed to proteolytic degradation . However, we also detected that the breast cancer cells produced syncytin-2 and cannot exclude that also this molecule contributed to the cancer further experiments using shrna - directed downmodulation of both syncytin-1 and -2 as well as of additional putative fusogenic retroviral sequence are now underway to test this . We next examined two series of human breast cancer patients for tumoral expression of syncytin-1 using a polyclonal antiserum raised to a synthetic nonapeptide derived from the syncytin-1 sequence . In addition, tumors were also screened for expression of asct-2 using a peptide antiserum . Preabsorption of the antisera with the corresponding peptides, but not with irrelevant peptides, abolished staining (larsson et al . The results showed that 38% of all breast cancer samples showed detectable staining for syncytin and that endothelial cells expressed asct-2 (fig . 6). Moreover, significant expression of asct-2 was detected also in many tumor cells (fig . The degree of syncytin immunostaining was visually graded using coded specimens and statistical analysis showed that it correlated positively with disease - free survival of the patients (larsson et al . 2007b). Multivariate analysis included age dichotomized at 40 years, tumor size dichotomized at 20 mm, grade and adjuvant therapy and identified syncytin expression as an independent prognostic indicator of increased disease - free survival also when used as a continuous variable, syncytin expression emerged as a significant prognostic indicator for disease - free survival in the cox model (p = 0.02) (larsson et al . . 6human breast cancers immunocytochemically stained for syncytin-1 (a) or with antigen - preabsorbed antiserum (b) and for asct-2 (c, e) or with antigen - preabsorbed antiserum (d). Note presence of syncytin immunoreactivity in tumor cells and of asct-2 immunoreactivity in endothelial cells (arrows) and in tumor cells human breast cancers immunocytochemically stained for syncytin-1 (a) or with antigen - preabsorbed antiserum (b) and for asct-2 (c, e) or with antigen - preabsorbed antiserum (d). Note presence of syncytin immunoreactivity in tumor cells and of asct-2 immunoreactivity in endothelial cells (arrows) and in tumor cells the involvement of syncytin-1 in tumor cell fusion events was subsequently confirmed by strick et al . (2007), working with endometrial carcinomas . In their study, downmodulation of synctytin-1 expression also inhibited fusions between endometrial tumor cells . In agreement with findings on placental cells this apparent discrepancy may possibly be ascribed to the fact that estrogen treatment upregulated tgfbeta, which interfered with syncytin - induced fusions . Thus, estrogen did, in fact, stimulate cell fusions if tgfbeta was immunoneutralized . Conversely, additions of tgfbeta 1 or 3 reduced fusions induced by camp - elevating agents . This effect was observed both in endometrial carcinoma cells and in trophoblast - derived cells . These results show that both camp and estrogens positively may regulate syncytin-1 expression in tumor cells and that the tgfbeta family may negatively regulate the fusogenic effects of syncytin-1 in both trophoblasts and cancer cells (strick et al . 2007). The role of syncytin-1 and cell fusions in cancer needs further study . Thus, our data show that syncytin-1 is not the only fusogenic protein expressed by breast cancer cells and the results presented by strick et al . (2007) show that additional regulators, such as tgfbeta isoforms may be important modulators of cell fusions . So far, our breast cancer data indicate that syncytin-1 expression constitutes a positive prognostic factor . This is not the same as to say that cell fusions may be universally beneficial to cancers . First, syncytins may have additional effects within the tumor environment (larsson et al . Secondly, it seems likely that several factors within the cancer and its stroma (inactivated tumor suppressor genes, activated oncogenes, expression of fusogens and of cd9 and cd81 as well as tgfbeta) may act together to bring about a tumor profile that may be as diverse as the one demonstrated by the cell fusion experiments referred to above . Interestingly, however, expression of cd9 has been analyzed in a number of tumors and seems, like syncytin expression, to predict a good prognosis (funakoshi et al . We propose that syncytins may be fusogens of importance to both trophoblast and cancer cell fusions and that they possibly also may mediate additional cell fusion events, acting in concert with other molecules with both enhancing and inhibitory regulatory effects . Cell fusion events must be extremely well controlled . Due to their major importance to fertilization, placentation, muscle development, bone structure, calcium homeostasis and the immune defense system, additionally, the potential role of cell fusions as a repair mechanism and the role of cell fusions in cancer development and progression have further stimulated research in this field . In spite of this, much less is known about cell cell fusion mechanisms than is known about how intracellular membranes fuse through v- and t - snares . Interestingly, engineered flipping of the v- and t - snare machinery has been shown to promote cell cell fusions (weber et al . However, it is evident that this mechanism is not a physiological mediator of cell cell fusions . Interestingly, v- and t - snares act similar to class i viral fusion proteins in that they form bundles of alpha - helices, which result in membrane apposition and fusion (blumenthal et al . 2003; jahn et al . 2003). Of the many proteins, which to date have been shown (or proposed) to be involved in cell fusions in mammals, only the syncytin family appears to use a similar alpha - helical mechanism . That they do is not surprising in view of the fact that syncytins represent conserved endogenous retroviral env sequences . The founding family member, syncytin-1, was discovered as a protein capable of mediating fusions between cytotrophoblasts into syncytiotrophoblasts (blond et al . This capability may have contributed to a high degree of evolutionary conservation of the syncytin-1 sequence . Syncytin-1 represents the env protein of the human endogenous retroviral (herv) w sequence, which entered the primate genome 2540 million years ago . In contrast, most other retroviral sequences inserted in our genome have been subject to inactivating changes and probably represent garbage sequences . Molecular studies have shown that syncytin-1 (env w) shares a structure similar to class i viral fusion proteins, especially in the region of the n- and c - terminal heptad repeats (nhr and chr), and shares a common fusion mechanism with these proteins (chang et al . A synthetic peptide derived from the chr is also capable of inhibiting syncytin-1-mediated fusions by perturbing this mechanism (chang et al . Syncytin-1 binds to the d - type retroviral receptor asct-2 (blond et al . 2000) and may use another neutral amino acid transporter (asct-1) as an auxiliary receptor (lavillette et al . Syncytin antibodies, syncytin-1 downregulation through antisense oligonucleotides and the syncytin-1 chr peptide have been shown to inhibit fusions between trophoblast - derived cells (blond et al . Agents increasing cellular levels of camp or camp analogues have been shown to promote cytotrophoblast fusions in vitro (keryer et al . 1998) and such agents are also known to elevate protein and mrna levels of syncytin-1 in isolated cytotrophoblasts (frendo et al . Also estradiol may regulate syncytin-1 expression (carino et al . 2003) and the placenta - specific transcription factor gcma interacts with two upstream sites in the herv - w 5-long terminal repeat and stimulates syncytin-1 transcription (yu et al . Additionally, experimentally induced truncations in the cytoplasmic tail of syncytin-1 increases its fusogenicity (drewlo et al . 2006), similar to what has been observed for some virally derived env proteins (reviewed by kubo et al . However, if modifications in the cytoplasmic tail of syncytin-1 are of physiological importance for regulating fusogenicity has yet to be demonstrated . A second syncytin - family member, syncytin-2, syncytin-2 also represents a highly conserved endogenous retroviral envelope gene and is derived from the herv frd sequence (blaise et al . Immunocytochemical studies have localized syncytin-1 primarily to syncytiotrophoblasts as well as to cytotrophoblasts (fig . 2004), which may reflect differences in antibodies, fixation procedures and controls . A major point of concern is also the degree of cross - reactivity to the related sequences in syncytin-2, which recently was localized to a subpopulation of cytotrophoblasts (malassin et al . The syncytin-1 receptor protein asct-2 was recently localized to cytotrophoblasts (hayward et al . 2007). More studies on the exact distribution of syncytin expression in the placenta using syncytin-1 and -2 specific antibodies seem warranted in order to exactly localize where fusions are likely to occur . Nuclei are lightly counterstained with haematoxylin human term placenta immunocytochemically stained with a syncytin-1 peptide antiserum . Nuclei are lightly counterstained with haematoxylin trophoblast cell fusions are, however, not unique to the primate placenta . Thus, also in the mouse placenta, trophoblasts fuse to form syncytiotrophoblasts but mice do, of course, not express herv sequences . Amazingly, an in - silico search of the mouse genome unraveled the existence of two murine endogenous retroviral (merv) env genes, labeled syncytin - a and -b (dupresssoir et al . These genes were also fusogenic and orthologous genes were present in additional species of muridae (rats, gerbils, voles and hamsters) and appear to have entered the rodent lineage some 20 million years ago (dupresssoir et al . 2005). They were expressed in the placenta and at least syncytin - a has been shown to be involved in the formation of syncytiotrophoblasts in mice (gong et al . This represents an amazing example of parallel acquisition of retroviral genes of importance to reproduction in primates and rodents and poses the question whether also other species may have acquired similar viral genes of importance to cell fusions . The ability of syncytins to induce cell fusions may not be their only physiologic role . Thus, recent studies have shown that while syncytin-2 and syncytin - b also possess immunosuppressive activity, syncytin-1 and syncytin - a do not (mangeney et al . 2000) but subsequent studies have revealed the presence of syncytin-1 also in the brain (antony et al . 2004) and in breast, colon and endometrial cancers (bjerregaard et al . 2007, larsen, talts, andersen, bjerregaard and larsson: work in progress). In fact, in all systems studied so far, from mating yeast to man, a bewildering array of mechanisms have been identified (see chen and olson 2005; chen et al . Molecules potentially involved in mammalian cell fusions include adam (a disintegrin and metalloproteinase domain) 12 (meltrin alpha), which has been associated with myoblast and osteoclast cell fusions (abe et al . 1999; in addition, adams 1 and 2 (fertilins) may be involved in sperm oocyte fusions, but do not seem indispensable for this function and, in man, the fertilin alpha gene is dysfunctional (chen and olson 2005; cho et al . Cd9 is also expressed by bewo trophoblast tumor (choriocarcinoma) cells and has been linked both to bewo invasiveness and to invasion during mouse embryo implantation (hirano et al . 1999; liu et al . Cd9 has, together with another tetraspanin family member, cd81, also been linked to myoblast fusion and myotube maintenance (tachibana and hemler 1999). Moreover, antibodies to either cd9 or cd81 have been shown to block fusions induced by mason pfizer monkey virus a d - type retrovirus (duelli et al . 2005). Tetraspanins are known to organize other proteins into membrane microdomains and may link to the actin cytoskeleton via ewi (glu - trp - ile) and erm (ezrin radixin moesin) proteins (hemler 2003; sala - valds et al . Possibly, their role as organizers of other proteins into microdomains may play a role in their involvement with cell fusions (zivyat et al . They do not express characteristics of fusogenic proteins like the syncytins, snares and class i viral envelope proteins, but are coexpressed with syncytin-1, at least in bewo cells . A macrophage fusion receptor (mfr, sirpalpha), resembling cd4the cell surface receptor for hiv has been identified in macrophages (saginario et al . Mfr, which belongs to the immunoglobulin superfamily, binds another member of this family, cd47 . Cd47 is also structurally related to proteins expressed by vaccinia and variola viruses (chen et al . Whereas expression of mfr is restricted to myeloid cells and neurons, cd47 is ubiquitously expressed . Mfr is transiently induced in macrophages at the onset of fusion while cd47 expression is constant . It has been hypothesized that cd47 initially binds to a long form of mfr to secure recognition and then switches to bind a shorter form to bring the plasma membranes closer (510 nm) for fusion (vignery 2005). Additionally, or alternatively, cd47 may promote calcium entry by forming a membrane pore (reviewed in chen et al . Also the hyaluronan receptor, cd44, is induced transiently when macrophages start to fuse . Mice lacking dc - stamp are osteopetrotic and lack multinucleated osteoclasts and giant cells (yagi et al . 2005). Dc - stamp is a seven - transmembrane receptor, somewhat similar to the hiv co - receptor cxcr4, but a ligand has yet to be identified . These and many more molecules, including integrins, vacuolar atpase and receptors and their ligands, as well as different signaling intermediates have been associated with cell fusions . With the exception of the syncytins, most of the mammalian molecules so far studied do not fulfill strict criteria for fusogens and several are dispensable for fusions (reviewed by chen and olson 2005; chen et al . However, this may reflect molecular redundancy and does not definitely exclude that these molecules may participate in fusions . It is noteworthy that, in both man and mouse, two different syncytins are expressed and are both fusogenic . It is possible that, in the mammalian system, fusion requires a flotilla of molecules organized into membrane microdomains by proteins like cd9, encompassing receptors / ligands, signaling entities, proteases and fusogens of retroviral origin that are capable of forming alpha - helical bundles, which bring membranes closer . Syncytins and related retroviral envelope sequences may, in this connection, function both as fusogens and receptor ligands . However, it seems unlikely that as irreversible an event as a cell cell fusion should depend upon a single receptor ligand interaction . Thus, both facilitatory and inhibitory factors are expected to be part of the flotilla . Studies by mortensen et al . (2004) documented that human breast cancer cells fused with endothelial cells in culture . Stimulated by studies showing that syncytin-1 was involved in cytotrophoblast cell fusions (blond et al . 2000; mi et al . 2000), we examined whether a similar mechanism could account for cancer endothelial cell fusions . Expression of syncytin-1 was documented in the breast cancer cell lines examined (mcf-7 and mda - mb-231 cells; bjerregaard et al . 2006, sk - br-3 cells: talts, bjerregaard and larsson: unpublished data). Moreover, we found that both tumor cells and endothelial cells expressed the syncytin-1 receptor asct-2 . Use of phosphorthioate - protected syncytin-1 antisense oligonucleotides downmodulated syncytin-1 expression as measured by either quantitative rt - pcr or western blotting and inhibited breast cancer endothelial cell fusions, whereas a scrambled oligonucleotide control was without effect . Additionally, the syncytin-1 chr peptide, referred to above, also inhibited the fusions whereas a control peptide was without effect (bjerregaard et al . However, neither the antisense nor the chr peptide experiments effected a total inhibition of cancer - endothelial cell fusions . Thus, the antisense oligonucleotide did not totally downmodulate syncytin-1 levels and could therefore not be expected to decrease cell fusions to zero and the lack of total inhibition by the chr peptide could potentially be ascribed to proteolytic degradation . However, we also detected that the breast cancer cells produced syncytin-2 and cannot exclude that also this molecule contributed to the cancer further experiments using shrna - directed downmodulation of both syncytin-1 and -2 as well as of additional putative fusogenic retroviral sequence are now underway to test this . We next examined two series of human breast cancer patients for tumoral expression of syncytin-1 using a polyclonal antiserum raised to a synthetic nonapeptide derived from the syncytin-1 sequence . In addition, tumors were also screened for expression of asct-2 using a peptide antiserum . Preabsorption of the antisera with the corresponding peptides, but not with irrelevant peptides, abolished staining (larsson et al . The results showed that 38% of all breast cancer samples showed detectable staining for syncytin and that endothelial cells expressed asct-2 (fig . 6). Moreover, significant expression of asct-2 was detected also in many tumor cells (fig . . The degree of syncytin immunostaining was visually graded using coded specimens and statistical analysis showed that it correlated positively with disease - free survival of the patients (larsson et al . 2007b). Multivariate analysis included age dichotomized at 40 years, tumor size dichotomized at 20 mm, grade and adjuvant therapy and identified syncytin expression as an independent prognostic indicator of increased disease - free survival also when used as a continuous variable, syncytin expression emerged as a significant prognostic indicator for disease - free survival in the cox model (p = 0.02) (larsson et al . 6human breast cancers immunocytochemically stained for syncytin-1 (a) or with antigen - preabsorbed antiserum (b) and for asct-2 (c, e) or with antigen - preabsorbed antiserum (d). Note presence of syncytin immunoreactivity in tumor cells and of asct-2 immunoreactivity in endothelial cells (arrows) and in tumor cells human breast cancers immunocytochemically stained for syncytin-1 (a) or with antigen - preabsorbed antiserum (b) and for asct-2 (c, e) or with antigen - preabsorbed antiserum (d). Note presence of syncytin immunoreactivity in tumor cells and of asct-2 immunoreactivity in endothelial cells (arrows) and in tumor cells the involvement of syncytin-1 in tumor cell fusion events was subsequently confirmed by strick et al . (2007), working with endometrial carcinomas . In their study, downmodulation of synctytin-1 expression also inhibited fusions between endometrial tumor cells . In agreement with findings on placental cells (vide supra), both camp elevating agents and estrogens upregulated syncytin-1 expression . However, only camp elevating agents stimulated cell fusions (strick et al . This apparent discrepancy may possibly be ascribed to the fact that estrogen treatment upregulated tgfbeta, which interfered with syncytin - induced fusions . Conversely, additions of tgfbeta 1 or 3 reduced fusions induced by camp - elevating agents . This effect was observed both in endometrial carcinoma cells and in trophoblast - derived cells . These results show that both camp and estrogens positively may regulate syncytin-1 expression in tumor cells and that the tgfbeta family may negatively regulate the fusogenic effects of syncytin-1 in both trophoblasts and cancer cells (strick et al . 2007). The role of syncytin-1 and cell fusions in cancer needs further study . Thus, our data show that syncytin-1 is not the only fusogenic protein expressed by breast cancer cells and the results presented by strick et al . (2007) show that additional regulators, such as tgfbeta isoforms may be important modulators of cell fusions . So far, our breast cancer data indicate that syncytin-1 expression constitutes a positive prognostic factor . This is not the same as to say that cell fusions may be universally beneficial to cancers . First, syncytins may have additional effects within the tumor environment (larsson et al . Secondly, it seems likely that several factors within the cancer and its stroma (inactivated tumor suppressor genes, activated oncogenes, expression of fusogens and of cd9 and cd81 as well as tgfbeta) may act together to bring about a tumor profile that may be as diverse as the one demonstrated by the cell fusion experiments referred to above . Interestingly, however, expression of cd9 has been analyzed in a number of tumors and seems, like syncytin expression, to predict a good prognosis (funakoshi et al . We propose that syncytins may be fusogens of importance to both trophoblast and cancer cell fusions and that they possibly also may mediate additional cell fusion events, acting in concert with other molecules with both enhancing and inhibitory regulatory effects.
The stability of the shoulder joints is the result of the combination of static - dynamic balance and the interaction between various joints1, 2 . In addition, coordination around the shoulder joints plays an important role in stabilization of the shoulder joints3 . Thus, strengthening exercise programs for these muscles can help with recovery from instability in the shoulder joints or help prevent instability from occurring4 . Moreover, vibration is regarded as an effective exercise method to improve muscle strength and endurance . According to bosco et al ., applying dynamic vibration to arm flexor muscles increased the muscle power significantly5, 6 . In addition, it has been shown that vibration stimuli in the lower extremity improved balance ability7 . Furthermore, han et al . Reported that vibration stimuli decreased knee joint reposition error8 . In order to apply vibration more safely and effectively, it has been used to apply a low frequency vibration stimulus to a local area while performing an oscillating movement9 . In addition, the upper quarter y balance test is a method for examining shoulder stability by measuring the distance a fingertip can reach while in a closed - chain position, which is known to be highly reliable . This method is a preprogram test through which the exercise limit and asymmetry can be tested10 . Nonetheless, few studies have been done on the acute and chronic effects of a vibration stimulus applied to the upper extremities . Therefore, the aim of this study was to determine the initial effect of local vibration on the stability of the shoulder joints by applying local vibration to the shoulder joints . They were chosen because they had a suitable range of motion and muscle strength to perform the exercises required in this test and had no problems or diseases related to the shoulder joints in the past . The mean age, height, and weight of the subjects were 24.22.7 years, 165.47.9 cm, and 62.213.5 kg, respectively . Also, this study was approved by the daegu university faculty of rehabilitation sciences human ethics committee . To apply the local vibration to the subjects, a flexbar (hygenic corporation, akron, oh, usa) was used11 . A flexbar is an oscillating device that weighs 0.59 kg and is 0.3 m long . For the test, the subjects held a flexbar with one hand, at about 10 cm from one end, and performed the oscillation movement with the shoulder at 90 flexion and the elbow in the full - extension position in scaption; the vibration stimulus was set to 5 hz11, 12 . In total, three sets of the oscillation movement were completed, with each set consisting of three exercises lasting three minutes each . About 10 minutes of rest was provided to the subjects between the sets to minimize muscle fatigue . Once all exercise sets were complete, the subjects underwent the upper quarter y balance test to evaluate the stability of the shoulder joints . The mean value of the three test results was used for the upper quarter y balance test, and the distance a subject could reach with a fingertip was divided by the arm length of the subject to find the percentage value, as this removed the bias due to the physical structure differences between the subjects . For the data analysis, spss for windows (ver . 18.0) was used, and a paired t - test was employed to compare the results before and after the oscillation movement . The moving distances in the left, right, and upper directions after the oscillation movement were 95.59.3, 85.111.9, and 63.814.8 cm, respectively; all increased significantly compared with the results before the oscillation movement (82.99.2, 75.711.4, and 52.511.5 cm, respectively) (p<0.05). The total moving distance also increased significantly after the oscillation movement, rising from 211.126.8 cm to 244.330.1 cm (p<0.05). The objective of this study was to determine the initial effect of the vibration stimulus on the stability of the shoulder joint . Scaption is an ideal exercise plane that is used to strengthen various muscles around the shoulders, as these muscles are required to lift the arms in daily life13 . Furthermore, the rotator cuff and deltoid are two dynamic stabilizers that surround the shoulder, and they press the humeral head toward the glenoid fossa or offset the superior gliding of the humeral head during shoulder movement, thereby preventing impingement14 . In the present study, the vibration stimulus in scaption effectively strengthened these muscles, thereby increasing the stability of the shoulder joints . Vibration has normally been used in resistance exercises to improve muscle strength and power by strengthening the neuromuscular performance, but different effects have been identified depending on the vibration characteristics12 . According to bongiovanni, a suppression effect was displayed if the duration and strength of vibration continued to increase gradually . Therefore, if a vibration stimulus is applied using a suitable amplitude and frequency, highly effective resistance training will be achieved . In conclusion, a vibration stimulus is effective as an exercise method to increase the stability of the shoulder joints . Furthermore, application of a vibration stimulus for patients with impaired shoulder joints can help support effective rehabilitation programs.
Genome mapping has become an essential tool in order to identify and characterize the genetic basis of phenotypic traits of organisms as well as to detect single genes of interest (e.g. Disease related genes) or to improve selective breeding . Genome mapping also offers the possibility of comparative mapping, which brings valuable knowledge production potential about genome structure, function, and evolution . Several mapping methods exist that have shaped the work of geneticists in the past decades . Genetic linkage maps or radiation hybrid maps give a global view of genetic markers mapped to groups that in a best case scenario correspond to whole chromosomes [1, 2]. While these approaches characterize a species genome with relatively low marker density (about one marker per mbp genomic sequence), today mapping approaches that use large insert clone libraries such as bacs (bacterial artificial chromosomes) [3, 4] have a higher impact, not only offering very dense maps but also revealing a part of the genome sequence and gene content . In the age of genomics mapped clones are of special value for the analysis of distinct genomic regions by a variety of molecular techniques . Especially, the sequencing of large genomes takes profit from bac maps, as they enable the reconstruction of chromosomal sized sequences from draft whole genome shotgun assemblies or sequencing strategies that apply a hierarchical first map then sequence strategy . Among the methodologies to construct bac maps, fingerprinting by restriction analysis [57] or hybridization of labeled marker sequences to bac colony filters, as well as pcr - based methods [9, 10], have been widely used for a variety of species to build de novo maps of overlapping bac clones . Today, protocols for high - throughput sequencing of bac end sequences (bac - es) enable comparative mapping strategies that rely on sequence alignment to closely related species, which have been sequenced to completeness [11, 12]. This strategy is based on automated pipelines for sequencing and bioinformatics that have been established worldwide during the last decade enabling the genome analysis of a wide range of species . Genomic information of species spanning a wider range of evolutionary distance will contribute to the complex questions of duplication and divergence . Furthermore, comparison between genomic - rich species and genomic - poor species has been a powerful tool for unravelling genes as well as specific genome regions of interest . In addition, comparative genomics is broadly used to identify and characterize functional regions in vertebrate genomes by the detection of evolutionary constrained sequences (purifying selection on functional sequences), or for revealing directional selection operating on different parts of the genome . The present work together with work on the european seabass, dicentrarchus labrax, adds genomic information to much shorter distances . In addition, due to their interest for aquaculture these species have already piled up an important body of biological knowledge: they are dwelling in about the same geographic range with a relatively stable gradient of physicochemical parameters, but have distinct ecology, behaviour, sex determination systems, contrasting reproductive biology, differential stress tolerance to a set of stressors, differential tolerance to pathogens, physiology, and so forth . These are applicable to closely related species for identifying adaptive evolution and functional variation by fine scale comparisons of genes and genomic regions and by simultaneous investigation of sequence polymorphism and divergence . Whereas the assignment of homologues groups between closely related species can be achieved already by low density maps [1517] rearrangements take place by processes such as translocation, inversions, duplications, and deletions . Therefore, in addition to synteny mapping (on the same chromosome), reconstructing homologous colinearity by comparative bac maps will enhance significantly the detection of new regions in the genome that are important for function as well as it will contribute to a better differentiation of lineage - specific constraint . To date the comparative bac mapping method is particularly suitable to map fish genomes, because several good quality draft reference fish genomes are already available in public databases for single or multispecies comparison . Five teleost fish genomes have been nearly completely sequenced (http://www.ensembl.org): the zebrafish (danio rerio (hamilton)), the medaka (oryzias latipes (temminck and schlegel)) and the three - spined stickleback (gasterosteus aculeatus l.) belonging to the order of cypriniformes, beloniformes, and gasterosteiformes, respectively, as well as the two pufferfishes (takifugu rubripes (temminck and schlegel) and tetraodon nigroviridis (marion de proc)) belonging to the order of tetraodontiformes . Sequencing of fish genomes is of particular interest as among vertebrates the ray - finned fishes (actinopterygians) are the most diverse groups comprising approximately 23,700 extant species . Genome projects in teleosts were first focused on model species either due to their compact genome size (pufferfishes), their importance in developmental research (zebrafish), or as ecological model (three - spined stickleback). The genome analysis of the three main model fish species, the zebrafish (danio rerio), pufferfish (tetraodon nigroviridis) and medaka (oryzias latipes), have shown that two rounds of genome duplications took place early in vertebrate evolution as well as a third round of genome duplication occurred in the ray - finned fish before the teleost radiation [2325]. The interest to study nonmodel fish species first was based either on their importance in evolution or on their high commercial impact . The latter enhanced the research in the two main species of commercial interest in the mediterranean, the gilthead sea bream sparus aurata l. and the european seabass dicentrarchus labrax l. the gilthead sea bream belongs to the order perciformes and sparidae family which comprises about 100 species of which 24 are described from the north / eastern atlantic and mediterranean coasts but also in the red sea and australia - new zealand . The family of sparidae also includes several species such as dentex dentex, pagrus pagrus, diplodus puntazzo which are important to mediterranean aquaculture or pagrus major which is routinely produced in japan . Sparus aurata is one of the main target species for aquaculture and fisheries in europe with 133,000 tons aquaculture production in 2008 (http://www.fao.org/fishery/culturedspecies/sparus_aurata/en). Sea bream is a mass spawner having a 2 - 3 months sequential spawning of batches of eggs and a particular sex determination system that is characteristic also for other candidates in aquaculture . Sea bream belongs to the protandrous hermaphrodite turning into females after the 2nd year [2628]. Another characteristic behaviour of sea bream is cannibalism between juveniles of different size . In general sea bream we have recently published a comparative bac map of the european seabass, which is closely related to the gilthead sea bream (both order of perciformes). The three - spined stickleback gasterosteus aculeatus genome was successfully used as reference genome in this regard . Stickleback does not belong to the order perciformes, but is the most closely related genome to seabass among the currently available five sequenced teleost genomes . The comparative bac mapping has already shown that some stickleback chromosomes have a nearly complete synteny with those of seabass . Here we present the results of comparative mapping of bac end sequences of the gilthead sea bream to the three - spine stickleback as well as to contigs of the european seabass genome project (kuhl et al . The establishment of a dataset containing information about similarities, synteny relationships as well as colinearity will enhance significantly future approaches to identify and characterize new and novel functional genome regions . It also represents an important first step for gilthead sea bream whole - genome sequencing by second generation techniques as well as high - throughput snp detection analysis, precise mapping of ests of specific interest, the identification of members of gene families and the differentiation of orthologous from paralogous genes . The sparus aurata bac - library was constructed by amplicon express and was provided by the institute of marine biology and genetics of the hellenic centre for marine research (imbg - hcmr). The total genome coverage of the library is 7 - 8-fold with an average insert size of 120 kbp per bac - clone . For template preparation, bac - clones were inoculated in 2 384 deep well plates containing 190 l of 2yt media and 12.5 mg / l chloramphenicol and cultivated for 18 hours at 37c with rigorous shaking at 1100 rpm in titramax 1000 incubators (heidolph instruments). E. coli cells were centrifuged at 2750 g for 5 minutes (rt) and the supernatant was removed . The bacterial pellets were resolved in 20 l buffer 1 (50 mm tris hcl, 10 mm edta, ph 8 supplemented with 25 mg / l rnase). Cell lysis was performed with 20 l of buffer 2 (0.2 m naoh, 1% sds) for 5 minutes and for neutralization 20 l of buffer 3 (0.933 m kacetat, ph 4.8) was added . Cell debris was separated from dna containing supernatant by centrifugation at 2750 g for 30 minutes (rt). Supernatants from both plates (40 l each) were combined in a new 384 deep well plate for size selective dna precipitation with 38 l of a polyethylene - glycol 6000/2-propanol mixture (75% 2-propanol(v / v)/100 g / l peg-6000) and centrifuged at 2750 g for 30 minutes at 4c . Supernatants were depleted by centrifugation of the inverted plates at 172 g for 1 minute (rt). The remaining dna pellets were washed with 40 l of 70% ethanol (v / v) to remove residual salts and peg . Most of the bac - dna template preparations were done by an automated process that was developed at the mpi for molecular genetics . Bac - dna templates were end sequenced using abi bigdyev3.1 terminator chemistry and t7 or sp6 primers . After postsequencing cleanup by ethanol / natrium acetate (naoac) precipitation, sequence analysis was performed on abi3730xl capillary sequencers with 36 cm capillary arrays . Processing of raw sequencing data was done by the phred basecaller, quality clipping, and vector - clipping by lucy . Initially, bac end sequences (bac - es) were aligned to the stickleback genome using blastn with low stringency parameters (-w 7 -q -1 -e 0.01). The output tables were further screened for the best hits of bacs aligned with both ends that matched the same chromosome in the reference . A further screening step involved orientation () and distance between paired bac ends (insert size smaller than 300 kb). To improve the number of mapped bacs we did a multiple genome alignment approach . We aligned all assembled contigs from the advanced seabass genome project (unpublished results, for a brief description see) to the stickleback genome and rebuild the stickleback genome with the seabass contigs . We then performed the same alignment steps as above for the sea bream bacs on the virtual seabass chromosomes and transformed the hit coordinates back to the stickleback sequence . Again read pairs were checked for consistency and alignments of both approaches were combined to result in an increased number of mapped bacs . For those bacs that mapped in both approaches we calculated the distance between the coordinates found by direct mapping and by mapping via seabass sequence, to check the placement error distribution induced by the coordinate transformation . If there were discrepancies between the same bacs in the two alignment approaches we considered those results that had better matches according to our consistency criteria . The start and end coordinates of the bac clones were converted to gff format and included in the ensembl genome browser for the stickleback genome (http://www.ensembl.org/gasterosteus_aculeatus/info/index). Bacs that form a minimal tiling path were chosen as described in and may be browsed independently of the other placed bacs . The number of bacs sequenced from both ends was 41,509 (physical coverage ~5.5x-6.5x). Bacs sequenced from one direction accounted for 5,485 (only t7 dir./~0.7x-0.8x) and for 3,965 (only sp6 dir./~0.5x-0.6x). The average, the sequences were submitted to embl nucleotide database [embl: fr502695-embl: fr595162]. Mapping all sequences directly to the stickleback genome resulted in 70,425 hits (76.2%/just best hit was counted). Further screening for paired bac ends matching to a single reference chromosome resulted in 19,776 hits (23.8% of paired end sequenced bacs). This indicates that many hits in the raw blast are false positives as expected due to the low stringency parameters used . After checking for read orientation and distance 17,184 hits (8,592 bacs/20.7% of paired bac es) were consistently mapped . These bacs (1.16x1.3x physical genome coverage) were already useful for building contigs of overlapping bac - clones, but these covered only 58.1% of the stickleback chromosomes . To further improve the mapping results we tried a multiple genome alignment approach . Unpublished results) were concatenated according to the order of their ortholog sequences in the stickleback genome . As seabass belongs to the order of perciformes, additionally, synteny of genes is more conserved than nucleotide sequence in distantly related teleosts, which legitimizes the use of the nucleotide sequence of a closer relative of sea bream ordered by a species that is not as close in the phylogeny . After aligning sea bream bac es to this sequence the hit coordinates this way results from directly mapping to stickleback and indirectly mapping to stickleback (via orthologous seabass sequence) can be combined . A total of 87,614 bac ends (94.8%) had hits with low stringency blast in the multiple genome alignment approach . Paired bac ends that mapped to the same reference chromosome accounted for 34,174 reads (41.2% of paired bac es). Consistency checks of read pairs resulted in 29,392 (14,696 bacs/35.4% of paired bac es). The combination of both mappings resulted in 28,530 bac es (14,265 bacs) with consistent mapping to the 21 stickleback chromosomes (1,488 bac es from direct and 27,042 from indirect mapping). The insert size distribution of mapped clones and its implications on genome size are shown in figure 1 . (see excel sheet with mapped bac es in supplementary material available online at doi: 10.11552011329025].) As the transformation of the seabass sequence coordinates to coordinates on stickleback sequence introduces some placement error, we compared the coordinates of bac clones (7,646) that were consistently mapped in both mapping approaches . The median deviation of start and end between the two mapping strategies (see figure 2) was 66 bp, the average deviation was 293 bp, and 96.7% of the mapped bacs had a deviation of less than 1,000 bp . As the placed bacs and their overlaps are much larger than these values, the fuzziness introduced by the seabass to stickleback coordinate transformation seems to be negligible . The minimal tiling path calculated from consistently placed bacs covered 73.5% of stickleback chromosomes with about 13,415 gene loci predicted (70.2% of 19,121 genes predicted in stickleback chromosome sequences). The largest bactig covers 2.15 mbp and n50 bactigs size is 337,253 bp . These sizes refer to the stickleback genome, as there is a large size difference between stickleback and sea bream the bactig sizes should be multiplied by a factor of 1.72 to estimate their sizes in sea bream . The mapped clones may be browsed alongside the stickleback genome and the ordered seabass bac clones at www.ensembl.org (login: heiner7@hotmail.com/passw: breambacmap2010). For a comparison of sea bream and seabass minimal tiling path clones mapped to stickleback chromosome vi and xxi, see figure 3 (for all stickleback chromosomes see supplementary material . Clones with inconsistent matches of both ends in terms of distance or orientation were screened manually for potential intra - chromosomal rearrangements . If an inconsistency was indicated by two or more independent bac clones it was considered a potential rearrangement . Studying the genomes of important commercial species is of direct value to the efficiency and exploitation of the species . Despite the fact that today access to other fish genomes, including the sister species of the gilthead sea bream the european seabass dicentrarchus labrax, is available, additional genome information is of importance concerning identification of species specific characteristics like disease resistance, salinity, and temperature tolerance by comparative genomics additionally, it will offer the possibility to use population genomics approaches combining interspecific divergence with intraspecific diversity for identifying adaptive variation, or comparative genomics for identification of small noncoding regions of functional importance . The power of such approaches is in general decreasing with increasing evolutionary distance, either because of intervening confounding effects and increased complexity or because of alignment problems . The identification of ultraconserved elements (uces), similar genomic elements as well as conserved noncoding elements (cnes) have pin pointed to genomic regulatory blocks, developmental regulatory target genes (s) and phylogenetically relevant and functionally genes . Comparative mapping of bac - es is a fast and efficient method to deduce genome maps of nonmodel organisms from sequenced genomes of model organisms due to conserved synteny . Thus the tremendous efforts that have been undertaken for model organisms in the past decade pay out now for the genomics of farmed animals . We have recently shown for the european seabass that the stickleback genome is a good choice for comparative studies in fish belonging to the order of perciformes . In this study nucleotide alignments in the present study indicate that sea bream and stickleback may be more diverged than seabass and stickleback . The resulting mapping data represents a valuable tool for genomic research in this important aquaculture species . Bac clones covering regions of interest may be easily chosen for advanced analysis using the ensembl genome browser tool . Besides these benefits some conclusions can be drawn on genome size of sea bream, when comparing the distribution of distances between two corresponding bac ends mapped to the stickleback chromosomes and their real distance observed by pulse field gelelectrophoresis during bac library construction . To estimate an organism's genome size flow cytometry of cells with stained nuclei has been widely applied . According to www.genomesize.com the resulting c - value found by this method for g. aculeatus is 0.58 pg of haploid dna content . In d. labrax the c - value accounts for 0.78 pg and in s. aurata it is even higher with 0.95 pg . If one relates the c - values to the lowest value observed in stickleback, the seabass genome should be 1.34-fold larger and the sea bream genome should be 1.64-fold larger than the stickleback genome . These values are in good concordance with values found by bac insert size distributions of the comparative mappings: s. aurata / g . Aculeatus = 1.3 . Nevertheless predicting the genome size by sequencing - based methods results in lower values for each genome compared to the size estimations of flow cytometry - based methods (g. aculeatus 462 mbp versus 567 mbp; d. labrax 601 mbp versus 763 mbp; s. aurata 791 mbp versus 929 mbp). On the one hand, sequencing - based methods tend to underpredict genome sizes as some regions are hard to assemble and are later on missing in the published genome assemblies . On the other hand, flow cytometry might overestimate genome sizes due to including ongoing replication of dna in the measurements or some staining background of rna: rna or rna: dna hybrids . Thus, we believe that the true genome size of the three teleosts lies in between the borders defined by the two approaches . The differences in genome - size among species may arise from insertion or deletion (indel) of large dna fragments and/or insertion or deletion of small dna sequences . Given their frequency particularly the small deletions largely outnumber small insertions [37, 38], small indels have been recognised as major mechanisms responsible for genome - size evolution in eukaryotic organisms . Several studies have also shown that the wide range of variation in genome size among eukaryotic organisms is correlated with the amount of repetitive elements in the genome [3941]. In fishes for example, the contribution of repetitive elements to genome - size difference between medaka and takifugu is estimated about 54% . Although the above interpretations are coherent and in agreement with results previously reported in fishes on the forces driving the evolution of genome size, they are still speculative since they are not based on comparisons at nucleotide sequence level . Further analysis of indels and repetitive elements are required to clarify the respective contribution to the differences in genome size between these species . This could be done when the genome of seabass and sea bream will be completely sequenced or in near future via a targeted analysis of large conserved sequence regions between the three species . The mapping of the bac inserts was done in a similar way as described here for the sea bream . When comparing both maps, the seabass map outperforms the sea bream map in most parameters like reference coverage (87.0% versus 73.5%), covered stickleback gene predictions (85.4% versus 70.2%), n50 bactig size (1.2 mbp versus 0.34 mbp), or total number of bactigs (588 versus 1,485). Differences in map quality may be based on the lower physical genome coverage and average insert size of the gilthead sea bream bac library than the european seabass bac library . The lower coverage of mapped bacs for sea bream complicates the detection of chromosomal rearrangements . Nevertheless, we could identify a number of 202 potential intrachromosomal rearrangements between stickleback and sea bream that were spanned by two or more clones . This number is similar to what was found, when comparing stickleback to seabass (214 potential rearrangements,). Seabass shares 114 of the 202 rearrangements with sea bream, thus the phylogenetic relationship between the three species seems to be reflected by the number of rearrangements . Although the alignment coordinates of additional 1,402 sea bream bacs imply other large, scale intra - chromosomal rearrangements, their evidence is weak as they are covered by a single bac clone only . . Nevertheless, inconsistently mapped bacs (see supplementary material) may be subject to further investigations (e.g., checking overlaps by pcr) to proof further potential rearrangements . The comparative sea bream bac map described here is a valuable tool for researchers in the field . It allows the fast selection of bac clones that cover genes or genomic loci of interest by simple blast searches in the stickleback genome and picking one of the bacs covering the hit location . According to the coverage of the stickleback genome we estimate that about 75% of the sea bream genome is covered by the mapped clones . Additional genes, which are not represented by mapped bacs, may be found by blast searches against the bac end sequences that comprise about 60.6 mbp of the sea bream genome, the largest set of genomic sequences published for sea bream so far . These data will be also of great value in future projects like a sea bream genome project and will allow scaffolding of wgs contigs or a sequencing strategy based on mapped clones, which will be especially useful, if short read sequencing technologies will be applied . Thus, taking also into account the comparative seabass bac map, ordered large insert clones for the two main aquaculture species in the mediterranean are now available and may contribute to answer basic biological questions like what makes up the difference between hermaphrodite (sea bream) and temperature - driven sex differentiation (seabass) as well as aquaculture - related questions that aim at improved breeding stocks in both species.
Several studies comparing emergency physicians (ep) skills at intubation with other practitioners, primarily anaesthesiologists, found similar proficiency [1, 2]. Complications of emergency department (ed) intubations have been studied, but most only examine the peri - intubation period . While there are several studies that provide data as to rates of success of ed intubation and need for cricothyrotomy, there is little information as to the long - term prognosis of these patients . Patients who are intubated in the ed are some of the most critically ill patients in the ed; the mortality after ed intubation has not been well studied . Previous studies have shown that the lay public have a lack of understanding of the dismal outcomes associated with cardiac arrests . Understanding the outcomes of ed intubation can be useful in discussion with families of patients as well as provide information for future research . The purpose of this study is to identify the rate of ed intubation and the mortality associated with ed intubation . We conducted a retrospective chart review for all ed intubations performed over a 1-year period between 1 january 2004 and 31 december 2004 at an urban level one trauma centre with approximately 50,000 ed visits annually . Ed intubations were identified retrospectively by reviewing the hospital financial databases for intubations for all ed patients . Patients that had critical care billing for an intubation procedure or for ventilator use in the ed were obtained . Ed patient logs were also examined to identify all patients who were admitted to the intensive care unit (icu), the operating rooms or died . Patients who were intubated prior to arrival in our ed either by another hospital or by prehospital personnel were excluded from our cohort . Two investigators reviewed all charts and abstracted the following data: age, sex, final disposition from hospital, reason for intubation using a standardized list of terms and trauma versus non - trauma . If all data were not available from the discharge summary, the original chart was reviewed to gather the necessary data . De - identified data were entered into a microsoft excel 2003 (redmond, wa, usa) database and analysed using stata v8.2 (college station, tx, usa). Confidence intervals were calculated and comparisons were made using unpaired t - tests and fisher s exact tests . One hundred and sixty - three patients were intubated in our ed during the 1-year study period . The total 163 patients, 44 (27.0%) died prior to discharge from the hospital, and 72 (44.2%) patients were discharged to home or left against medical advice (ama) (1 patient). Forty - two (25.8.%) patients were discharged to a skilled nursing facility (e.g. Nursing home, rehabilitation and extended care facility) for further care . Dividing this patient population into trauma and non - trauma subgroups, 6/37 (16.2%) of the trauma patients died and 38/136 (30.2%) of the non - trauma patients died . The mean age for all patients was 61.3 years, with trauma patients being younger (49.0 versus 65.0) than the non - trauma subgroup . The mean age of the survivors was lower than the non - survivors: 57.3 versus 72.2 (p <0.0001). No children were identified as having been intubated in our study, as our institution does not routinely care for paediatric patients . Table 1age and mortality of patients intubated in the ed overalltraumanon - trauman16337126age61.3 (58.064.7)49.0 (40.557.5)64.5 (61.768.3)mortality27.0% (20.033.9)16.2% (4.128.3)30.2% (22.238.3)table 2comparison of age of intubated patients survived vs died alltraumanon - traumasurvivor (mean age)57.3 (53.361.3)44.0 (34.953.0)62.0 (58.166.0)death (mean age)72.2 (67.476.9)74.8 (65.684.0)71.7 (66.477.1) age and mortality of patients intubated in the ed comparison of age of intubated patients survived vs died of the deaths, 38.6% occurred within 24 h of intubation . An additional 22.7, 29.5 and 9.1% of deaths occurred at 72 h, 7 days and 30 days, respectively, after intubation . No deaths were found to occur beyond 30 days of hospitalization . In our search of the literature no large studies reporting mortality after intubation in the ed were found . The near database, the largest ed airway registry at present, provides insight into the ed course of these patients, but does not collect post - intubation data or outcomes on their patients . Most of the emergency medicine literature concentrates on the mechanics of the intubation and the time surrounding the event . Considerable emergency medicine research focuses on the tools of airway assessment and techniques to improve intubation success such as the lemon score and the burp maneuver [5, 6]. Alternatively, there is a lack of information on how these patients do once they leave the ed, and how the various techniques impact on the mortality associated with ed intubation . While the patients who require ed intubation certainly represent a sicker population, our study demonstrates a relatively high mortality rate . Now that we know the mortality rate of 27%, we can begin to examine those interventions that not only improve the success of ed intubation, but also ultimately improve mortality . As an initial step, we chose to do a retrospective chart review of all ed intubations for a 1-year period . While recognizing the limitations of such an approach, we thought this initial study would provide some useful information and indications for future research . The considerable mortality rate following intubation is not surprising as the need for intubation in the ed probably represents a marker for severity of disease . Like mortality from sepsis or after a myocardial infarction, establishing a baseline mortality rate after ed intubation will provide us with an outcome measure we can use to test the benefit of specific interventions . Due to the limited number of patients in our cohort, many questions remain unanswered . Stratification of the patients by disease entity could not be achieved in a meaningful way . For example, should head injuries be classified as neurological or as traumatic? The one major subdivision of patients we could perform analysis on was trauma versus non - trauma patients . This finding could be explained by the lower age of trauma patients and their ability to respond to medical interventions . In both groups, this is most evident in the trauma group where the mean age was 44.0 years, but the mean age of those who died was 74.8 years as compared to the non - trauma group . Older patients with myocardial infarction and stroke have been demonstrated to have worse outcomes in the literature [7, 8]. Older patients tend to have less physiological reserve so the same insult would be expected to have a greater deleterious effect upon prognosis . Yanagawa et al ., in a study comparing intubation without medications versus with medications for non - traumatic ich, reported the mortality of the two groups as 46.8 and 72.9%, respectively . The study had 70 patients for their control and study group combined and the main outcome measure was the glasgow outcome score, not mortality . If we select non - traumatic ich from our patients, 9/16 (56.2%) died . The high mortality rate in the ich subgroup, and the differences in the trauma and non - trauma groups, suggest that with a large enough number of patients subgroup analysis may reveal differences in mortality by specific disease entity . Survival to hospital discharge was chosen over 30-day mortality since it eliminated the problem of having patients that could not be followed up while still providing a meaningful endpoint . This study was conducted as a pilot study to provide background data as to what directions would be of interest in future studies . As a single - centre retrospective study we are limited in our ability to make general statements based on our study population . As we saw no paediatric patients, we cannot make any inferences as to their mortality rate after intubation . The small sample size limits our ability to provide subgroup analysis for outcomes based on the reason for intubation other than the fairly broad trauma / non - trauma division . Since there is no central way to identify patients who were intubated in our ed, we used multiple methods to identify cases; however, there is a small chance we still could have overlooked a particular group that could alter the outcome . Future studies should attempt to identify predictors of mortality among ed intubations as well as look at this question in a multicentre setting.
Osteoporotic vertebral compression fracture (ovcf) is a common cause of pain and disability in the elderly population, affecting nearly one - quarter of individuals over the age of 50 during their lifetime . Worldwide, there are approximately 1.4 million new ovcfs reported each year, with approximately 750 000 annually in the united states alone [13]. Ovcfs often result in significant pain that often leads to decreased mobility, loss of independence, and subsequent loss of bone density, and have negative effects on the respiratory and digestive systems . The direct medical costs associated with ovcfs have been estimated at $13.8 billion annually in the us alone, and indirect costs in lost productivity, pain, and suffering are even greater . Conservative management, including bed rest, painkillers, and bracing, may help to reduce pain over weeks or months; however, in frail elderly patients, long periods of inactivity are associated with higher rates of pneumonia, decubitus ulcers, venous thromboembolism, and even death . Invasive surgery is not the optimal treatment due to their usually old age and associated comorbidities . Today, vertebral augmentation procedures, including percutaneous vertebroplasty (pvp) and percutaneous kyphoplasty (pkp), are commonly used in the treatment of ovcfs [810]. Pkp utilizes inflatable bone tamps to reduce fracture, restore vertebral anatomy, and control cement injection . Since the first report of pvp in 1987, the use of pvp and pkp for the treatment of ovcfs has been dramatically increased during the last 20 years . Potency and effectiveness of these procedures recently, a us large claims database analysis showed that pkp was associated with a significant reduction in mortality compared to conservative treatment and pvp . Pkp is now considered to be as minimally invasive and effective as conventional pvp for rapid pain relief in patients with painful ovcfs resistant to conservative treatment . In established osteoporosis, the definition of a vertebral fracture is usually based on the presence of a deformation of the vertebral body on lateral radiographs . Early diagnosis of ovcfs is crucial and the detection of vertebral fractures is based primarily on the identification of vertebral collapse . However, this can be misleading in the presence of a fracture without radiologic collapse, and the definition of occult osteoporotic vertebral fractures (oovfs) has been established . Patients highly suspected to have a fresh occult vertebral fracture, who have persistent pain and normal x - ray and ct images, should undergo additional radiologic tests, especially mri . Mri is the most confirmative screening examination used to determine the presence of painful oovfs; t2 and stir sequences of mri images show hyperintensity signal because of bone edema in the affected vertebra . Oovfs are disabling causes of severe low back pain, requiring bed rest and increasing the risk of comorbidities . Moreover, these patients usually require high doses of painkillers, especially for multiple oovfs, which can cause serious adverse effects . Thus, it is essential and necessary to enhance the vertebral body with occult fracture to prevent further vertebral collapse . Because the number oovfs patients was small and usually mixed with other ovcfs cases, clinical treatment of oovfs has been rarely discussed . Few reports exist documenting the outcomes of vertebral augmentation procedure as an alternative to traditional conservative therapy for painful oovfs, most of which are limited to case reports and small series of patients [1719]. In this report, we present a series of 89 patients with painful oovfs treated with pkp, and we assess the radiological and clinical outcomes . The aim of the research was to assess the effectiveness and safety of pkp in the management of painful oovfs . The research was approved by ethics committee of the first affiliated hospital of soochow university (suzhou, china). From january 2011 to december 2013, 923 kyphoplasties were performed in 688 patients at our institution . Among them, 89 patients (55 females and 34 males), aged 5679 years, had 1 or 2 oovfs, and underwent 142 pkp procedures . The mean time since onset of pain to the intervention was 1.25 months in all patients . Bone mineral density was preoperatively measured as an indicator of osteoporosis with dual - energy x - ray absorptiometry in all cases . We only included the osteoporotic patients (according to the world health organization; these patients have a t - score 2.5 or lower, meaning a bone density that is 2.5 standard deviations below the mean of a 30-year - old man / woman). Occult osteoporotic vertebral fractures (oovfs) were defined as vertebral fractures without radiographic measurable compression . The indications for pkp were oovfs with acute pain and not responding to nonoperative treatment, which included all types of medications, nerve blocks, exercises, and osteoporotic medications to strengthen the bone . Patients included in our research underwent a plain x - ray, magnetic resonance imaging (mri), and multislice computed tomography (msct) to evaluate the main cause of pain and to plan the treatment . Mri was the most confirmative screening examination used to determine the presence of painful oovfs; stir sequences of mri images show hyperintensity signal because of bone edema in the affected vertebra . Patients with local infections, non - correctable coagulation disorders, and other systemic diseases were excluded from the treatment . Patients received general anesthesia and were positioned prone on a radiolucent operation table . Using image guidance x - rays, a small incision was made and a probe was placed into the vertebral space at the fracture site . The bone was drilled and a balloon (kyphon inc ., sunnyvale, california) was inserted on each side . The balloon was then inflated slowly for injecting bone cement . During the cement injection, radiographic assessment and clinical examinations were performed preoperatively, and at 1 month, 6 months, and 2 years after treatment . The anterior and middle vertebral heights were obtained from standing lateral radiographs for compromised and adjacent uncompromised control vertebrae . For each vertebra, the normal height of the compromised vertebrae was estimated from the mean of the measurements from the closest uncompromised vertebral cephalad and caudalad to the treated level . Vertebral body height variations were calculated by the following method: (fractured vertebral body height / normal vertebral body height) 100% . Back pain was measured using a visual analog scale (vas scores) with values from 0 to 10, where 0 indicates no pain and 10 indicates the worst pain imaginable . Oswestry disability index (odi) scores, which are used to assess functional capacity, were also documented, in which a lower percentage indicates a better health status . The odi has been shown to have high test - retest reliability and is the most commonly recommended condition - specific outcome measure in patients with acute and chronic low back pain . The sf-36 questionnaire is an in - depth and broad - ranging health - related quality of life evaluation consisting of 36 questions, where a higher score indicates a better health status . The results are categorized into 8 domains; bodily pain (bp), vitality (vt), physical function (pf), and social function (sf) have been shown to have high reliability and responsiveness in spinal injury patients and were selected for evaluation in our study [2224]. Complications such as bleeding, infection, stroke, pulmonary embolism, and cardiac arrest were recorded . The rates of cement leakage outside the vertebral body were measured postoperatively using radiographs and ct scans . Mean changes, including mean values and standard deviations, in anterior vertebral body height variation and middle vertebral body height variation, sf-36 score, vas and odi score, were evaluated and analyzed with spss software (spss 19.0, inc ., comparisons between different time points were performed using a paired student s t test and tested by the repeated - measures analysis of variance (anova). The research was approved by ethics committee of the first affiliated hospital of soochow university (suzhou, china). From january 2011 to december 2013, 923 kyphoplasties were performed in 688 patients at our institution . Among them, 89 patients (55 females and 34 males), aged 5679 years, had 1 or 2 oovfs, and underwent 142 pkp procedures . The mean time since onset of pain to the intervention was 1.25 months in all patients . Bone mineral density was preoperatively measured as an indicator of osteoporosis with dual - energy x - ray absorptiometry in all cases . We only included the osteoporotic patients (according to the world health organization; these patients have a t - score 2.5 or lower, meaning a bone density that is 2.5 standard deviations below the mean of a 30-year - old man / woman). Occult osteoporotic vertebral fractures (oovfs) were defined as vertebral fractures without radiographic measurable compression . The indications for pkp were oovfs with acute pain and not responding to nonoperative treatment, which included all types of medications, nerve blocks, exercises, and osteoporotic medications to strengthen the bone . Patients included in our research underwent a plain x - ray, magnetic resonance imaging (mri), and multislice computed tomography (msct) to evaluate the main cause of pain and to plan the treatment . Mri was the most confirmative screening examination used to determine the presence of painful oovfs; stir sequences of mri images show hyperintensity signal because of bone edema in the affected vertebra . Patients with local infections, non - correctable coagulation disorders, and other systemic diseases were excluded from the treatment . Patients received general anesthesia and were positioned prone on a radiolucent operation table . Using image guidance x - rays, a small incision was made and a probe was placed into the vertebral space at the fracture site . The bone was drilled and a balloon (kyphon inc ., sunnyvale, california) was inserted on each side . The balloon was then inflated slowly for injecting bone cement . During the cement injection, radiographic assessment and clinical examinations were performed preoperatively, and at 1 month, 6 months, and 2 years after treatment . The anterior and middle vertebral heights were obtained from standing lateral radiographs for compromised and adjacent uncompromised control vertebrae . For each vertebra, the normal height of the compromised vertebrae was estimated from the mean of the measurements from the closest uncompromised vertebral cephalad and caudalad to the treated level . Vertebral body height variations were calculated by the following method: (fractured vertebral body height / normal vertebral body height) 100% . Back pain was measured using a visual analog scale (vas scores) with values from 0 to 10, where 0 indicates no pain and 10 indicates the worst pain imaginable . Oswestry disability index (odi) scores, which are used to assess functional capacity, were also documented, in which a lower percentage indicates a better health status . The odi has been shown to have high test - retest reliability and is the most commonly recommended condition - specific outcome measure in patients with acute and chronic low back pain . The sf-36 questionnaire is an in - depth and broad - ranging health - related quality of life evaluation consisting of 36 questions, where a higher score indicates a better health status . The results are categorized into 8 domains; bodily pain (bp), vitality (vt), physical function (pf), and social function (sf) have been shown to have high reliability and responsiveness in spinal injury patients and were selected for evaluation in our study [2224]. Complications such as bleeding, infection, stroke, pulmonary embolism, and cardiac arrest were recorded . The rates of cement leakage outside the vertebral body were measured postoperatively using radiographs and ct scans . Mean changes, including mean values and standard deviations, in anterior vertebral body height variation and middle vertebral body height variation, sf-36 score, vas and odi score, were evaluated and analyzed with spss software (spss 19.0, inc ., comparisons between different time points were performed using a paired student s t test and tested by the repeated - measures analysis of variance (anova). Significant improvements in sf-36 score, vas, and odi score were observed postoperatively and at 1 month, 6 months, and 2 years after treatment . Five new adjacent vertebral fractures in 89 patients were detected during the 2-year follow - up period . The levels were t5 (n=4), t6 (n=5), t7 (n=6), t8 (n=9), t9 (n=8), t10 (n=10), t11 (n=18), t12 (n=17), l1 (n=18), l2 (n=15), l3 (n=13), l4 (n=12), and l5 (n=7) (figure 1). The injected cement volume was 5.21.3 ml and the time of fluoroscopy was 10.51.4 minutes for each level . There were 50 cases (81 vertebral bodies) done with bilateral access and 39 cases (61 vertebral bodies) with unilateral access . A representative case of a 77-year - old female patient with l2 and l4 oovfs who received pkp treatment using unilateral access is shown in figure 2 . There were no treatment - related complications such as bleeding, infection, stroke, pulmonary embolism, and cardiac arrest . General properties of the patients are summarized in table 1 . On the basis of comparison of pre - operative and every postoperative data, anterior vertebral body height and middle vertebral body height were maintained in all cases . The mean anterior vertebral body height variation changed from 96.53.4% preoperatively to 97.22.5% postoperatively (p>0.05) and maintained at 95.73.4% at 2 years postoperatively . The mean middle vertebral body height variation changed from 96.32.8% preoperatively to 97.93.1% postoperatively and maintained at 96.74.4% at 2 years postoperatively . Statistically, no significant difference was detected at the immediate postoperative time and every follow - up assessment compared with the preoperative value . The mean vas score of these patients decreased from 8.31.2 preoperatively to 2.90.7 postoperatively and maintained at 3.01.2 at 2 years postoperatively, and the odi score decreased from 76.412.5 preoperatively to 26.75.6 postoperatively and maintained at 27.88.3 at 2-year postoperatively (table 2). The sf-36 scores for vitality (vt), bodily pain (bf), social functioning (sf), and physical function (pf) all showed notable improvement (p<0.05) (table 3). Statistically significant differences were observed at the immediate postoperative time and each follow - up assessment compared with the pre - operative value . The postoperative ct images of the pkp - treated vertebrae indicated a cement extravasation in 12 vertebras, representing 8.45% of all treated levels . Cement extravasation was located in the paravertebral soft tissues (6 leaks), vertebral venous plexus (2 leaks), and adjacent disk area (4 leaks). The levels were t5 (n=4), t6 (n=5), t7 (n=6), t8 (n=9), t9 (n=8), t10 (n=10), t11 (n=18), t12 (n=17), l1 (n=18), l2 (n=15), l3 (n=13), l4 (n=12), and l5 (n=7) (figure 1). The injected cement volume was 5.21.3 ml and the time of fluoroscopy was 10.51.4 minutes for each level . There were 50 cases (81 vertebral bodies) done with bilateral access and 39 cases (61 vertebral bodies) with unilateral access . A representative case of a 77-year - old female patient with l2 and l4 oovfs who received pkp treatment using unilateral access is shown in figure 2 . There were no treatment - related complications such as bleeding, infection, stroke, pulmonary embolism, and cardiac arrest . On the basis of comparison of pre - operative and every postoperative data, anterior vertebral body height and middle vertebral body height were maintained in all cases . The mean anterior vertebral body height variation changed from 96.53.4% preoperatively to 97.22.5% postoperatively (p>0.05) and maintained at 95.73.4% at 2 years postoperatively . The mean middle vertebral body height variation changed from 96.32.8% preoperatively to 97.93.1% postoperatively and maintained at 96.74.4% at 2 years postoperatively . Statistically, no significant difference was detected at the immediate postoperative time and every follow - up assessment compared with the preoperative value . The mean vas score of these patients decreased from 8.31.2 preoperatively to 2.90.7 postoperatively and maintained at 3.01.2 at 2 years postoperatively, and the odi score decreased from 76.412.5 preoperatively to 26.75.6 postoperatively and maintained at 27.88.3 at 2-year postoperatively (table 2). The sf-36 scores for vitality (vt), bodily pain (bf), social functioning (sf), and physical function (pf) all showed notable improvement (p<0.05) (table 3). Statistically significant differences were observed at the immediate postoperative time and each follow - up assessment compared with the pre - operative value . The postoperative ct images of the pkp - treated vertebrae indicated a cement extravasation in 12 vertebras, representing 8.45% of all treated levels . Cement extravasation was located in the paravertebral soft tissues (6 leaks), vertebral venous plexus (2 leaks), and adjacent disk area (4 leaks). Osteoporosis is a systemic bone disorder characterized by reduced bone mass and degradation of skeletal microarchitecture . Osteoporotic vertebral compression fractures (ovcfs) are one of the most common fractures associated with osteoporosis . In addition to acute or chronic pain, ovcfs are also associated with progressive spinal deformity, impaired physical function, decreased quality of life, and increased mortality in this population . The direct medical costs associated with ovcfs have been estimated at $13.8 billion annually in the us alone, and indirect costs in lost productivity, pain, and suffering are even greater [2527]. Conservative management includes analgesics, bed rest, external bracing, and a combination of these treatments . However, there are still limitations of these methods . Long - term bed rest often leads to further demineralization and may predispose the patients to future ovcfs . As for medication of anti - inflammatory drugs and certain types of analgesics, it may be difficult for patients to tolerate the adverse effects . In frail elderly patients, long periods of inactivity are associated with higher rates of pneumonia, decubitus ulcers, venous thromboembolism, and even death . In case of failure of these non - surgical methods, until the beginning of the 1980s the only options left were surgical stabilization and spine straightening via dorsal instrumentation . However, there were limits to the application of this type of surgery, especially with patients in relatively poor general condition . Pvp and pkp are 2 minimally invasive surgical procedures in which bone cement is injected into the fractured vertebral body . Clinical results indicate both are effective in relieving back pain caused by the ovcfs, especially those that are refractory to conservative treatment . The main difference between the 2 procedures is that pkp involves the use of an inflatable bone tamp, in an attempt to create a cavity in the vertebral body prior to cement injection, while pvp does not [3133]. Recently, a us large claims database analysis showed that pkp was associated with a significant reduction in mortality compared to conservative treatment and pvp . Pkp is now considered to be as minimally invasive and effective as conventional pvp for rapid pain relief in patients with painful ovcfs resistant to conservative treatment . Early diagnosis of ovcfs is important, and the detection of vertebral fractures is based primarily on the presence of a deformation of the vertebral body on lateral radiographs . Occult osteoporotic vertebral fractures (oovfs), which imply symptomatic vertebral fracture without measurable radiographic compression, can be misleading . The typical mri finding in acute compression fracture is hypointensity in t1-weighted image, heterogeneous intensity or hyperintensity on t2-weighted image, and hyperintensity on fat - suppressed t2-weighted image or on short - inversion time - inversion recovery image . In our study, numerous fractures would have been missed on standard radiographs, only being confirmed by mri . Therefore, patients highly suspect of acute osteoporotic vertebral fracture, with acute symptomatic pain and normal x - ray and ct images, should undergo an additional imaging test, especially mri . Histological study demonstrated trivial microtrabeculae fracture and end - plate fracture in the involved vertebral bodies . . Showed that back pain was equally likely to occur with each type of vertebral fracture . Lee et al . Reported that there was little correlation between the degree of collapse of the vertebral body and the level of pain . Oovfs are important causes of severe back pain, leading to chronic disability similar to that in patients with severe vertebral collapses . Therefore, it seems essential and necessary to enhance vertebral bodies with oovfs to prevent possible vertebral collapse . Clinicians may miss oovfs, which leads to delayed diagnosis or misdiagnosis . Current treatment recommendations vary from analgesia to surgery . Despite the benefits of vertebral augmentation procedures, reports discussing the vertebral augmentation procedure as an alternative to traditional medical therapy for the treatment of oovfs are scarce . There are several reports on the results of pvp and pkp in treating oovfs; favorable results were reported, but, unfortunately, the literature data is mostly limited to case reports and small series of patients, and high - quality assessment methods are lacking . Pham et al . Reported that 21 cases of oovfs in 16 patients presented with a typical history of acute back pain with no substantial vertebral deformation on the initial plain radiographs . Reported that 10 out of 95 ovcfs showing signal intensity changes on mri were difficult to identify on plain radiographs due to almost no collapse of the vertebral body . Mao et al . Reported results in 45 patients with oovfs who underwent pvp and pkp . They found notable pain alleviation and improved functional activity after pkp intervention and did not detect delayed collapse of the treated vertebral bodies . Pereira et al . Evaluated the clinical outcome and technical feasibility of percutaneous sacroplasty in treating insufficiency fractures of the sacrum . They performed 67 sacroplasty in 58 consecutive patients with intractable pain from osteoporotic fractures or from sacral tumors . The purpose of our study was to describe our experience and to assess the safety and effectiveness of pkp in patients with painful oovfs . In a consecutive series of 89 patients with painful oovfs, who had 142 levels of treatment in a 2-years follow - up period, we found pkp leading to a remarkable remission of pain and improvement of function . Significant improvement in vas score, odi score, and sf-36 score was observed postoperatively and at each follow - up time . It is likely that pain relief is obtained through stabilization of the fracture by the cement . Another explanation proposed is that the injected bone cement causes thermal necrosis and chemotoxicity of the intraosseous pain receptors . There have been numerous studies evaluating the anatomical distribution of ovcfs, which were consistently showing 2 prevalent peaks of vertebral fractures: the first in the mid - thoracic spine region (t7/t8) and another in the thoraco - lumbar junction [3537]. Vertebral fractures in the lower lumbar area, including l3, l4, and l5 level, are quite uncommon [3941]. Our study revealed the peak of oovfs is the thoracolumbar junction area, representing 55% of overall spine fractures, which was consistent with ovcfs . It also shows that the lower lumbar vertebrae are frequently affected, representing 22.5% of overall spine fractures . It is likely that the lower lumbar area suffers less biomechanical stress, which could explain the temporary preservation of vertebral bodies weakened by a fracture . In these patients, oovfs are disabling causes of severe low back pain, and conservative treatments often fail . Minimally invasive procedures, such as pkp and pvp, are effective treatment options that can result in rapid pain relief and rehabilitation, and prevent delayed vertebral collapse . The less frequent cement leakage with pkp is probably because this technique creates a cavity that allows for a more viscous cement to be injected under lower pressure . Moreover, the expansion of the balloon compacts cancellous bone in the intravertebral cavity, which may also reduce the rate of cement leakage . In our collective, asymptomatic cement leak was seen in 8.45% of treated cases, notably lower than the documented average for ovcfs . This may be attributed to the relatively intact vertebral cortex and end - plate of the occult fractured vertebral body . In our experience, some leakage via the deficiency in the vertebral wall can be avoided by good operative technique . Once cement leakage is detected, the operator may adjust the needle direction or stop the injection immediately . The injection process was stopped when the cement reached the margins of the vertebral body . Based on these individual skills, no symptomatic cement extravasation occurred in our cases; however, the significance of an asymptomatic leak remains unclear . It had been suggested that cement leaks into the adjacent intervertebral disc space affect the mechanical loading of either the disc or adjacent vertebrae, which might predispose the patient to an adjacent - level fracture [4446]. In our study, there were 5 new adjacent vertebral fractures during the follow - up period: 3 fractures were associated with cement leakage nearby and 2 fractures were related to sandwich vertebra . A sandwich vertebra is an intact vertebral body located between 2 previously cemented vertebras . Because of double load shifts, the sandwich vertebra was prone to an increased risk of adjacent vertebral fracture however, adjacent - level fracture can also reflect systematic weakening of bone in the osteoporotic spine . We therefore believe this phenomenon may also represent the natural progression of osteoporosis in this series . Hence, the long - term impacts of these asymptomatic cement leaks and sandwich vertebral fractures need further research . First, this study was performed retrospectively, which produces weaker evidence than a prospective study . Second, only patients who underwent pkp were included in our study; there was no control or alternative treatment, such as pvp . Further additional prospective studies are needed to assess the effectiveness and safety of pkp in treatment of painful oovfs . To achieve earlier detection and diagnosis of painful oovfs, suspected patients with osteoporosis who have symptomatic pain and normal x - ray and ct images should undergo further mri testing . Patients with incapacitating vertebral pain can be treated with, and benefit from, pkp.
Children experience rapid changes in their diets during early childhood at a time that they are also forming their eating habits . By the time children are 2 years old, picky eaters are typically characterized as consuming a narrow range of food, as well as rejecting several new and familiar food items . These behaviors can cause frustration, worry, or anxiety from the caregiver that the child is not consuming the appropriate nutrients needed for healthy growth . Eating behaviors are shaped in a variety of ways, including what food the child is introduced to, the environment in which the food is served, and the way in which the food is prepared or typically consumed . Strategies that caregivers use during mealtimes to encourage the child to eat can also affect their eating habits and behaviors . According to the us census bureau, 33% of children under age 5 are cared for in nonparental childcare arrangements for an average of 35 hours per week . The most popular form of nonparental childcare is center - based childcare (cbcc), encompassing 67% of children in nonparental childcare arrangements . Home - based childcare (hbcc) settings represent 30% of children in nonparental childcare arrangements . Center - based childcare centers are a structured, school - like environment; typically, they contain multiple classrooms comprising children of similar ages separated in each classroom with a set teacher to student ratio . Home - based childcares are environments where children are cared for in the care provider s house . There are usually fewer children in hbcc than in cbcc settings, and typically, there is only 1 caregiver . Home - based childcares have more freedom in terms of policies that need to be followed depending on their licensing status or involvement in federal reimbursement programs . Although children are being cared for in hbcc and cbcc, they typically consume at least 1 meal . The academy of nutrition and dietetics (formerly the american dietetic association) recommends that children in part - time programs receive foods and beverages that provide at least one - third of the daily nutrient requirements, whereas those in full - term programs receive foods and beverages that meet at least one - half to two - thirds of daily nutrient needs . Consequently, children are exposed to different eating environments and potentially different feeding strategies . However, most of the literature in this field focuses on parental feeding strategies, leaving a gap that focuses on how mealtime strategies used at different childcare locations may be varied from those at home . In addition, there is a gap in the literature that addresses how perceptions of child eating behavior, specifically picky eating, differ between caregivers . It is also unknown whether parents and childcare providers of the same child agree in their perceptions of child pickiness and whether pickiness perception has any impact on the mealtime strategies that are utilized . Finally, little is known regarding the differences between hbcc and cbcc parents and providers, even though these are 2 of the most popular forms of nonparental childcare in the united states . Therefore, the objectives of this study were to (1) compare perceptions of child pickiness between parents and childcare providers, (2) compare percent agreement in pickiness perception between the dyads of cbcc providers and parents and hbcc providers and parents, and (3) identify mealtime strategy utilization of each caregiver group (ie, hbcc parents and hbcc providers) based on pickiness perceptions . Due to the differences in mealtime environments between the family home, hbcc, and cbcc, it was hypothesized that perceptions of child pickiness would differ among caregivers in these 3 locations . Due to the considerable differences between hbcc and cbcc centers, especially in terms of structure and policies, it was further hypothesized that hbcc and cbcc caregivers would have different perceptions of child pickiness . Finally, it was hypothesized that differing perceptions of child pickiness would affect the mealtime strategies that were utilized . This study was approved by the institutional review board at the university of illinois . Parents and their families in champaign - urbana, il, a total of 27 families and 7 cbcc providers were recruited from the child development laboratory (a cbcc on the university of illinois campus), and 25 families and 12 hbcc providers were recruited from hbcc centers . Childcare providers were recruited via recruitment phone calls, mailed flyers, word of mouth, and advertising during local childcare provider workshops . Participation requirements included having at least 1 child aged 3 - 5 years with no food allergies . The 3- to 5-year - old age range for the study was determined based on the literature findings that this age range is when picky eating behaviors peak . If families had 2 children in the age range of 3 - 5 years, they could enroll both children if desired . Only 1 family from cbcc and 1 family from hbcc enrolled 2 children in the study; all others enrolled only 1 child . This resulted in 26 parents from cbcc enrolled and 24 parents from hbcc enrolled in the study . Two hbcc providers who were also mothers of children participating in the study were removed from the analysis . Parents and teachers completed 2 surveys either online or in paper: the mealtime assessment survey (mas) and the parent / teacher mealtime strategies survey (pms / tms). For all surveys, parents and childcare providers responded to questions using a 5-point likert scale with never, rarely, sometimes, often, or always response options . It was developed through a series of focus groups and conjoint analyses examining actions displayed by pe, nonpicky eater (npe), and parents during feedings and adapted from questionnaires found in the literature regarding toddler mealtime behaviors . Parent and teacher perceptions of the child s pickiness were determined via the question how often is your child / student a picky eater? On the mas . Responses were dichotomized to classify a child as pe (always, often, and sometimes) or an npe (rarely and never). This method of dichotomization is well accepted and has been reported previously in the literature . Questions on the tms were similar to those on the pms, although some questions were tailored for applicability to a childcare setting or were removed . To test the first objective, the mcnemar test for paired, binary data was used to determine differences in pickiness perceptions (pe / npe) between childcare providers and parents within each childcare setting . The chi - square test is not an appropriate test for these data because 2 perceptions per child were compared, thus violating the assumption of independence . For more detailed analysis that accounted for the clustering of responses within childcare setting, a multinomial cumulative logit model was used to explore the association of childcare setting (cbcc vs hbcc), caregiver type (parent vs provider), and their interaction on pickiness perception as measured using the full 5-point categorical scale . The proportional odds assumption was confirmed using the rao score test (chi - square score statistic) for testing equality of slope parameters . The resulting beta coefficient is interpreted as the increase in the log odds of higher perceived pickiness rating associated with a 1-unit change in a covariate after holding all other covariates as constant . To obtain the odds ratio (or) and the 95% confidence interval (95% ci), the exponent of the beta coefficient was determined . To test the second objective, percent agreement in pickiness perception between parents and childcare providers was determined based on whether or not the caregivers (parent and childcare provider) perceived the same child as a pe . If the 2 caregivers perceived the same child as a pe, their responses were recorded in the agreed category . Across - childcare differences in percent agreement between parent - childcare provider pairs were compared using chi - square test . To test the third objective, the frequency of the use of mealtime strategies by the parents and the childcare providers was identified using chi - square test via child pickiness perception (pe / npe) that was dichotomized as previously described . All statistical analyses were performed using microsoft excel (version 15.0.4727.1000; microsoft, redmond, wa, usa) or statistical analysis software (sas) version 9.3 (sas institute, cary, nc, usa). A 2-tailed, significance level of p <.05 was considered statistically significant . This study was approved by the institutional review board at the university of illinois . Parents and their families in champaign - urbana, il, a total of 27 families and 7 cbcc providers were recruited from the child development laboratory (a cbcc on the university of illinois campus), and 25 families and 12 hbcc providers were recruited from hbcc centers . Childcare providers were recruited via recruitment phone calls, mailed flyers, word of mouth, and advertising during local childcare provider workshops . Participation requirements included having at least 1 child aged 3 - 5 years with no food allergies . The 3- to 5-year - old age range for the study was determined based on the literature findings that this age range is when picky eating behaviors peak . If families had 2 children in the age range of 3 - 5 years, they could enroll both children if desired . Only 1 family from cbcc and 1 family from hbcc enrolled 2 children in the study; all others enrolled only 1 child . This resulted in 26 parents from cbcc enrolled and 24 parents from hbcc enrolled in the study . Two hbcc providers who were also mothers of children participating in the study were removed from the analysis . Parents and teachers completed 2 surveys either online or in paper: the mealtime assessment survey (mas) and the parent / teacher mealtime strategies survey (pms / tms). For all surveys, parents and childcare providers responded to questions using a 5-point likert scale with never, rarely, sometimes, often, or always response options . It was developed through a series of focus groups and conjoint analyses examining actions displayed by pe, nonpicky eater (npe), and parents during feedings and adapted from questionnaires found in the literature regarding toddler mealtime behaviors . Parent and teacher perceptions of the child s pickiness were determined via the question how often is your child / student a picky eater? On the mas . Responses were dichotomized to classify a child as pe (always, often, and sometimes) or an npe (rarely and never). This method of dichotomization is well accepted and has been reported previously in the literature . Questions on the tms were similar to those on the pms, although some questions were tailored for applicability to a childcare setting or were removed . To test the first objective, the mcnemar test for paired, binary data was used to determine differences in pickiness perceptions (pe / npe) between childcare providers and parents within each childcare setting . The chi - square test is not an appropriate test for these data because 2 perceptions per child were compared, thus violating the assumption of independence . For more detailed analysis that accounted for the clustering of responses within childcare setting, a multinomial cumulative logit model was used to explore the association of childcare setting (cbcc vs hbcc), caregiver type (parent vs provider), and their interaction on pickiness perception as measured using the full 5-point categorical scale . The proportional odds assumption was confirmed using the rao score test (chi - square score statistic) for testing equality of slope parameters . The resulting beta coefficient is interpreted as the increase in the log odds of higher perceived pickiness rating associated with a 1-unit change in a covariate after holding all other covariates as constant . To obtain the odds ratio (or) and the 95% confidence interval (95% ci), the exponent of the beta coefficient was determined . To test the second objective, percent agreement in pickiness perception between parents and childcare providers was determined based on whether or not the caregivers (parent and childcare provider) perceived the same child as a pe . If the 2 caregivers perceived the same child as a pe, their responses were recorded in the agreed category . Across - childcare differences in percent agreement between parent - childcare provider pairs the frequency of the use of mealtime strategies by the parents and the childcare providers was identified using chi - square test via child pickiness perception (pe / npe) that was dichotomized as previously described . All statistical analyses were performed using microsoft excel (version 15.0.4727.1000; microsoft, redmond, wa, usa) or statistical analysis software (sas) version 9.3 (sas institute, cary, nc, usa). A 2-tailed, significance level of p <.05 was considered statistically significant . A total of 50 child, parent, and childcare provider triads participated in the study . There were approximately equal proportions of boys and girls (48% and 52%, respectively). 35% of the children were 3 years old, 40% were 4 years old, and 25% were 5 years old . Most (72%) of the parents in the study were women, 46% were between the age of 26 and 35 years, and 61% were white . Most (47%) childcare providers were between the age of 46 and 55 years and white . Abbreviations: cbcc, center - based childcare; hbcc, home - based childcare . The results from objective 1 showed that 56% of cbcc parents and 44% of cbcc providers perceived their child or student as a pe, whereas 57% percent of hbcc parents and 48% of hbcc providers perceived their child or student as being a pe (figure 1). Although these proportions were not statistically significant within each childcare setting, using a multinomial cumulative logit model, the results showed that parents are about 1.4 times more likely than providers to rate the child as being more picky (or, 1.4; 95% ci, 1.3 - 1.5). In addition, hbcc parents and providers are also 1.4 times more likely to rate a child as being picky than cbcc parents and providers (or, 1.4; 95% ci, 1.3 - 1.4) (table 2). Differences in percent of caregivers who perceive the child as being a picky eater between cbcc providers (n = 7) and parents (n = 26) and hbcc providers (n = 11) and parents (n = 22). Proportions not significantly different within each childcare setting . Multinomial cumulative logit regression model for the association of childcare type and caregiver with the outcome of increasing child pickiness perception . Abbreviations: cbcc, center - based childcare; ci, confidence interval; hbcc, home - based childcare; or, odds ratio . Or of 1.4 for site indicates that hbcc parents and providers are 1.4 times more likely to perceive a child as being a pe . Or of 1.4 for caregiver indicates that parents are 1.4 times more likely than childcare providers to perceive a child as being a pe . Results from objective 2, percent agreement in pickiness perception between hbcc and cbcc parents and childcare providers, showed that parents and providers do not agree in their perception of the same child s pickiness, supporting our hypothesis . Center - based childcare parent / provider pairs significantly disagreed more than hbcc parent / provider pairs; 41% of cbcc parent / provider pairs did not have the same perception of child pickiness compared with 26% of hbcc parent / provider pairs abbreviations: cbcc, center - based childcare; hbcc, home - based childcare . Significant at p <.05 using chi - square . In total, 41% of cbcc parents and teachers did not agree in their perception of the same child s pickiness as opposed to 26% of hbcc providers and parents not agreeing . Results of objective 3 identified 2 strategies to be significantly different between cbcc parents who perceived their child as being picky compared with those parents who do not (figure 2). In contrast, no mealtime strategies were found to be significantly different when a cbcc provider perceived a child to be a pe vs when they did not . In other words, the mealtime strategies used by cbcc providers were consistent, regardless of their perception of a child s pickiness . Association of mealtime strategies with child s pickiness perception (pe [n = 15] and npe [n = 12]) among center - based childcare parents . Significance according to chi - square test (* p <.05). Parents who perceived the child as a pe used both mealtime strategies more often than those who perceived the child as an npe . Npe indicates nonpicky eater; pe, picky eater . Also, in objective 3, 3 strategies were shown to be significantly different between hbcc parents who perceived their child to be a pe vs those who did not (figure 3). Among hbcc providers, 2 strategies were found to be differently utilized based on differing perceptions of child pickiness (figure 4). Association of mealtime strategies with child s pickiness perception (pe [n = 13] and npe [n = 10]) among home - based childcare parents . Significance according to chi - square test (* p <.05, * * p <.01). Parents who perceived the child as a pe used all mealtime strategies more often than those who perceived the child as an npe . Npe indicates nonpicky eater; pe, picky eater . Association of mealtime strategies with child s pickiness perception (pe [n = 11] and npe [n = 12]) among home - based childcare providers . Significance according to chi - square test (* p <.05, * * p <.01). Parents who perceived the child as a pe used all mealtime strategies more often than those who perceived the child as an npe . The findings of this research showed that parents are more likely to identify their children as being pes than those children s care providers . It could be that parents are more sensitive to the child s eating behavior and are therefore more likely to perceive a child as being picky . Because most parents worry about their child s growth, any indication of hesitance to eat from the child may lead the parent to perceive their child as a pe . On the contrary, childcare providers may not be as sensitive to their student s eating habits because they are caring for multiple children or focus on other objectives during mealtime . Another theory as to why parents are more likely than childcare providers to rate their children as pes is that children may, in fact, display more picky eating behaviors at home than at childcare . Children are likely aware that at home other food is available if they do not prefer what is served to them and are more inclined to reject or avoid that food until they are given an item they do enjoy . In addition, children are also aware of their parent s sensitivity to their eating habits and may know that if they avoid or refuse an item, they will be offered a different food . At childcare, especially cbcc, it is speculated that these options do not exist, therefore resulting in less pe behavior from the child . It was also found that between cbcc and hbcc caregivers (both parents and childcare providers), hbcc caregivers are about 1.4 times more likely to rate a student as a pe . Due to the nature of hbcc, in that it is typically a neighbor or friend of the parents, these caregivers may have similar ways of thinking or may talk more about the child s eating habits and, therefore, have a more similar perception of the child s pickiness than cbcc caregivers . In addition, the hbcc environment is more similar to the family home and there may not as many children as a typical cbcc, which lead children to act in a more similar manner during mealtimes in their hbcc location and their home vs cbcc children . Similarly, hbcc parents and childcare providers were in greater agreement with each other in their perception of child pickiness than cbcc parents and providers . This may also reflect the fact that the hbcc environment is usually more similar to the home environment and cbccs . This similarity in environment could result in comparable behavior from the child and therefore similar perception of child pickiness . In addition, hbcc providers and parents oftentimes know one another on a personal level, which could result in sharing or coordination of mealtime strategies and therefore in more agreement in perceptions of child pickiness . Regarding behavioral strategies around mealtimes, cbcc parents with perceived pes used a greater variety of mealtime strategies than parents with perceived npes, which confirmed our hypothesis . Previous research found that using food as a contingency factor negatively affected a child s preference for that food . In contrast, parental modeling of positive eating behaviors has been shown to be effective in establishing healthy eating habits in children . Parents with perceived pes may use more mealtime strategies than parents of perceived npes in efforts to improve food consumption in their child . Center - based childcare providers did not change their mealtime strategies, regardless of their perception of child pickiness . Strategies utilized by this group of caregivers were consistent, not only from student to student for the same childcare provider but also across childcare providers . This may be due to the policies and procedures surrounding mealtime that have been put into place by the cbcc that providers must follow . As with cbcc parents, hbcc parents with perceived pes were more likely to use different strategies, including rewards and making the meal into a game, to encourage eating . The consensus on whether the strategies of using foods / nonfoods as a reward for eating are beneficial are inconclusive . Some studies have shown that these are negative strategies in that the child can develop dislike for the food that is being used as a means to receive the reward, whereas others show that using rewards can be motivating to children . The strategy of making a meal into a game to encourage eating may also be negative because it distracts the child from focusing on the meal and their satiety cues . Similar to cbcc parents, these results indicate that parent perception of child pickiness does have an effect on mealtime strategy utilization . Based on these results, hbcc parents with perceived pes are more likely to use ineffective strategies at mealtime in attempts to improve child eating . Home - based childcare providers with perceived pes were found to use the strategy of spoon - feeding more than hbcc providers who did not perceive to have pes . Children between the age of 3 and 5 years should have the ability to feed themselves, rendering this strategy inappropriate for children in this age range, but hbcc providers with perceived pes may not have this knowledge, and therefore, use this strategy . In addition, hbcc providers with perceived pes were found to offer a child a reward for eating more than providers who did not perceive to have pes . Although a consensus on the benefits or consequences of using rewards for eating has not been reached, it may be that hbcc providers with perceived pes use this strategy because in a setting such as hbcc, this strategy is effective . Although only 2 strategies were found to be different, these results indicate that pickiness perception does affect mealtime strategy utilization in hbcc providers . The findings from this study provide insight to not only elucidate differences regarding pickiness perception between parents and childcare providers and how that affects utilized mealtime strategies but also further our understanding regarding differences in mealtime between home and childcare overall . The caregivers with perceived pes, regardless of location, are more likely to use a variety of mealtime strategies, even potentially ineffective ones . These results can be shared with parents trying to decide which childcare to choose, be used as a basis for mealtime strategy interventions with parents and childcare providers, and aid educators when trying to create educational materials for parents or childcare providers on mealtimes . First, due to the difficulty of recruitment of this study population, our sample size is small . The study population is novel in that we enrolled pairs of parents and providers for the same child, meaning recruitment was 2-fold . Not only did the childcare provider have to want to participate in the study, but so did the parents whose children attended the childcare . In addition, our study population included hbcc, a group that is not routinely investigated . Contacting, locating, and building enough rapport with future research should focus on achieving a greater sample size in multiple states to validate findings . Second, although these findings show that there are differences in pickiness perception among caregivers who care for the same child, we do not know the reason why . Without understanding why differences in perceptions exist therefore, future research should focus on determining why parents and childcare providers of the same child have differing perceptions of child pickiness to develop appropriate feeding strategy interventions for caregivers based on their location.
Toxoplasmosis is one of the most common human parasitic infections worldwide with an estimated prevalence in 1 - 2 billion people . Toxoplasmosis is a zoonotic disease caused by a sporozoan parasite, toxoplasma gondii, distributed throughout the world . The definitive hosts are domestic cats and various species of wild felids and the intermediate hosts are mammals and birds . Transmission occurs following ingestion of sporulated oocysts or bradyzoites within cysts present in the tissues of numerous food animals . Seroprevalence in the human population reportedly ranges from 0 to 90% and infection is more common in warm climates and in low - lying areas than in cold climates and mountainous regions, where conditions for sporulation and survival of oocysts are less favourable . The prevalence of t. gondii infection also varies between ethnic groups, and it is thought that this is largely due to sanitary and cooking habits rather than genetic differences . A seroprevalence of 80% has been reported from paris where undercooked meat is often consumed . Lower seroprevalences (1040%) have been reported in countries from southeast asia where meat is cooked thoroughly . There is limited evidence supporting the hypothesis that cattle are not a favoured host for t. gondii but in general infection is more likely to occur from consumption of pork, lamb's and goat's meat than from beef [68] and consumption of cured pork has indeed been recognized as an important source of t. gondii infection . Limited information is available on the prevalence of toxoplasmosis in man and animals in sudan . Camel toxoplasmosis was first reported in sudan by el din et al . Who recorded an infection rate of 54% . Afterwards bornstein and musa and elamin et al . Reported 22.5% and 11.8% in sudanese camels, respectively . A more recent study recorded prevalence rates of toxoplasma antibodies of 20%, 32%, and 57.5% in camels, cattle, and sheep, respectively . Prevalence rates of up to 73% were recorded in childbearing age women and up to 100% in camel herders in sudan [1417]. The present study was carried out to assay seroprevalence of t. gondii in dairy cattle herds with reproductive problems and to determine risk factors associated with the infection in two states, namely, khartoum and gazira of sudan . A total of 181 (168 females and 13 males) serum samples were taken from dairy cattle herds with reproductive problems such as abortion, infertility, and stillbirth . Dairy herds were raised under semi - intensive husbandry systems in farms around khartoum state (khartoum, khartoum north, and omdurman) and gazira states (al kamleen and wad madani). Sera were harvested following centrifugation of clotted blood, labeled, and stored at 20c until tested . Antibodies to t. gondii were tested by indirect enzyme linked imunosorbent assay (ielisa). A herd was considered positive for t. gondii when at least one animal with positive antibody reaction was detected . Positive serum samples will present yellow colour; the colour visualized in each well is proportional to the titer of antibody specific to t. gondii present in the diluted sample (1/400). The serological results and other information gathered during this investigation such as season, location, breed, sex, and age of the sampled animals were edited and analyzed statistically using statistical package (spss version 13). To identify the association of the risk factors with the specific seroprevalence, the chi - square (test) and out of 29 herds tested at both states, 13 (44.8%) proved to be positive for t. gondii antibodies by elisa . The result showed that 10 (50%) out of the 20 herds in khartoum state and 3 (33.3%) out of nine herds in gazira state were positive with the highest herd prevalence (57.1%) being detected in khartoum north locality (khartoum state) (table 1). One hundred and eighty one serum samples from khartoum and gazira states were assayed for the presence of anti - toxoplasma gondii antibodies using elisa . The prevalence rate was 12.7% (17/134) in khartoum state and the prevalence according to the locality was as follows: 12.9% (8/62), 14% (6/43), and 10.3% (3/29) were recorded for khartoum, khartoum north and omdurman localities, respectively . In gazira state the prevalence rate was 14.9% (7/47) and positive samples were detected in alkamleen 25% (2/8) and wad madani 12.8% (5/39) localities (table 2). The prevalence of t. gondii infection was significantly (p <0.05) higher (36.4%) in animals less than one year old than those above two years (12.8%). Moreover, significantly (p <0.05) higher seroprevalence of t. gondii was observed in males (30.8%) than in females (11.9%), but there was no significant relationship between antibodies prevalence and breed, location, season, or signs of reproductive disease (table 3). Until now insufficient data are available on cattle toxoplasmosis in the world, and there have been limited number of reports on cattle toxoplasmosis from sudan . The overall seroprevalence of toxoplasmosis reported in the present study in sudan using elisa (13.3%) is higher than that reported for cattle in china (2.3%) and vietnam (10.5%) but is comparable to that reported in iran (15.91%) and lower than that (27.9%) recorded in the netherlands . The differences in prevalence rates between countries may be due to the samples size of different studies besides the different techniques used . However, data on the seroprevalence in cattle show great variation, ranging from 0 to 99% [21, 22]; hence, such data should be analyzed with caution as geographical variations occur not only among different countries but also within countries . In the present study out of the 20 herds tested in khartoum state, nine (45%) herds proved to be positive for t. gondii antibodies and three (33.3%) of nine herds in gazira state were positive . The highest infection rate (57.1%) among herds was observed in khartoum north in khartoum state . Although no statistically significant association was found regarding signs of reproductive disease, this high prevalence rate (four herds out of seven were infected) is in concordance with high abortion rates reported from farms in this particular area . The seroprevalence of t. gondii infection reported in the current study in khartoum state was 13.3% using elisa . Gondii antibodies in cattle in khartoum state using latex agglutination test (lat) was 32% . The difference in the prevalences between the two studies could be attributed to the different techniques used in estimating these prevalences and to the lower number (50) of cattle investigated by khalil and intisar . Positive cases were more frequent among cattle less than one year old (36.4%, p <0.05) than cattle above two years old . This is in concordance with nematollahi and moghddam who reported similar results in iran in 2008 . This may reflect dominance of maternally acquired antibodies in this age group or that the cattle unless reinfected, deplete antibodies as their age increases . Cattle would harbour few parasite tissue cysts, which may not persist for the lifetime of the host [2325]. The prevalence was also observed to be significantly (p 0.05) higher in males (30.8%) than females . However, the numbers of males assayed for t. gondii antibodies in the present study were too low (13) to allow for any concrete conclusions regarding association of gender with the seroprevalence of this parasite . It could be concluded that toxoplasmosis is prevalent in dairy herds in khartoum and gazira states . Age and gender were observed to be the main risk factors involved, while no association with signs of reproductive disease such as abortion, stillbirth, and infertility was discerned . The results also suggest that toxoplasma oocysts and reservoir host are widely distributed in the environment . Hence, further epizootiological and parasitological investigations on toxoplasmosis in cattle and other farm animals and human at the country level are required to determine the magnitude of the infection and to estimate its economic impact; the relative importance of different species in the epidemiology of toxoplasmosis; and its public health hazard in sudan.
Epidemiological studies suggest a considerably high rate of overlap between functional dyspepsia (fd) and irritable bowel syndrome (ibs). According to the recent systematic review and metanalysis,1 the prevalence of ibs in subjects with dyspepsia is 37% (95% ci, 30%-45%) as compared to 7% (95% ci, 5%-10%) in those without . The pooled odds ratio for ibs in subjects with dyspepsia was 8 (95% ci, 5.74 - 11.16) as compared to that in those without dyspepsia . However, the original studies used an older classification such as manning's, rome i or ii, than rome iii and also did not exclude organic diseases, possibly resulting in the contamination with peptic ulcer diseases or reflux esophagitis . There are a few evaluations of overlap between fd and ibs based on the rome iii classification . Kaji et al2 conducted a cross - sectional study to examine the prevalences of gastroesophageal reflux, fd and ibs, their overlap rates, and the health - related quality of life (hrqol) for each disease and each overlap syndrome, as compared with the corresponding values in healthy controls in a japanese health check - up population . Of the 2,680 eligible subjects, 269 (10.0%) were diagnosed as having fd, and 381 (14.2%) as having ibs . Overlap significantly worsened the hrqol in most domains, except in the " role emotional " domain . The hrqol was particularly poor in the mental component summary for overlapping ibs.2 on the other hand, nakajima et al3 conducted a survey in a general gastroenterology outpatient clinic of 1,378 consecutive patients . Among them, 29 (2.1%) were diagnosed as having fd, and 33 (2.4%) as ibs . Overlaps between fd and ibs were found in 12 (0.9%). Wang et al4 investigated 3,014 patients who responded to their questionnaires (male: female = 47.2:52.8, response rate 89%) at a general gastroenterology outpatient clinic . Fd - ibs overlap was observed in 151 (5.0%) patients, while 457 (15.2%) and 329 (10.9%) were classified as having fd alone and ibs alone, respectively.4 as compared with that in the non - ibs cohorts, the or of having fd among ibs was 2.09 (95% ci, 1.68 - 2.59). Patients with fd - ibs overlap had higher severity scores for the symptom of postprandial fullness (2.4 1.5 vs 1.7 1.6, p <0.001) and overall fd symptoms (6.7 2.9 vs 5.8 2.8, p <0.05) than those with fd alone . The only independent risk factor for fd - ibs overlap versus fd alone was the presence of postprandial fullness (or, 2.67; 95% ci, 1.34 - 5.31).4 lee et al5 investigated the differences in depressive mood and quality of life (qol) among korean patients with fd, ibs and fd - ibs overlap diagnosed by the rome iii definition . According to their report,5 out of 279 subjects, 70 (25.1%) and 124 (44.4%) patients with fd - ibs overlap and those with fd alone showed higher beck depressive inventory scores than normal subjects (p <0.001 and p = 0.02, respectively), whereas those with ibs alone showed no differences in scores from normal subjects (p = 0.17). All of the sf-36 (the 36-item short form general health survey) subscores of the fd - ibs overlap cohorts were significantly lower than those in normal subjects (p <0.05). Depressive mood was significantly related to fd and fd - ibs overlap, but not to ibs alone . Patients with fd - ibs overlap had a worse qol than patients with fd alone or ibs alone.4 furthermore, according to the recent report by park,6 the sensitivity and specificity of the rome iii classification in discriminating functional gastrointestinal disorders (fgids) from organic diseases of the upper gastrointestinal (gi) tract were 60% and 53%, respectively, while the values of the lower gi tract were 80% and 50%, respectively, partially supporting the use of the rome iii criteria in korea.5 data on the impact of fd on the hrqol in the general population are scarce . Aro et al7 explored the impact of fd based on the rome iii classification on the hrqol in the general population . Among 1,001 cohorts, 202 (20%) individuals reported uninvestigated dyspepsia (uid), and 157 (16%) reported fd . Fd - ibs overlap had a significant impact on bodily pain (p <0.01) and general health (p <0.05).7 although hori et al8 used the rome ii criteria, they examined concurrent gi symptoms in fd and ibs in a total of 186 college students who filled out a questionnaire administered to determine whether they had uid or ibs . The diagnosis of uid, ibs and uid + ibs overlap was made in 12 (6.7%), 40 (22.1%) and 8 (4.4%) patients, respectively and a significant prevalence of uid + ibs overlap was observed (66.7% ibs in uid; 20.0% uid in ibs).8 although corsetti et al9 also used rome ii criteria for the diagnosis of fd in their questionnaire survey of 309 consecutive fd patients to assess the dyspepsia and ibs symptom patterns, 54% of the patients had fd alone, whereas 46% had fd + ibs . Fd - ibs overlap patients were more likely to be female (75% vs 60%, p <0.01) and to have greater weight loss (5.4 0.6 vs 3.5 0.4 kg, p <0.05). Coexisting ibs did not increase the risk of dyspepsia, however, the overall symptom severity was significantly higher in the patients with fd - ibs overlap (12.4 0.4 vs 9.8 0.3, p <0.01). Fd - ibs overlap patients had a lower threshold for first perception (2.9 0.3 vs 3.8 0.3 mmhg, p <0.05) and for discomfort (7.9 0.4 vs 9.5 0.5 mmhg, p <0.05) and a greater prevalence of hypersensitivity to gastric distention (44% vs 28%, p <0.05).9 recently, we performed a web - based survey comprised of rome iii criteria for fd, the gastrointestinal symptom rating scale, and questions to determine demographic information among subjects registered for japanese clinical trial programs.10 cluster analysis revealed 3 distinct clusters: cluster associated with diarrhea, cluster associated with constipation and cluster associated with neither diarrhea nor constipation . Cluster associated with constipation and cluster associated with diarrhea were significantly linked to the presence of fd, suggesting that fd was more prevalent among participants with bowel symptoms than in those without . Furthermore, fd patients with bowel symptoms had more severe dyspepsia symptoms than those without . Although gi symptoms are quite common in the general population, different methods for the survey show different epidemiologies, and the effects of psychosocial and behavioral factors on the symptoms have been studied, mainly by subgroup analysis . According to the japanese questionnaire survey focusing on gi symptoms and the psycho - behavioral background in members of a registered panel via e - mail and postal mail, despite the difference in the prevalence of gi symptoms, that is, 47% in the electronic survey and 25% in the postal one, similar proportions of symptom subtypes and patterns of overlaps were obtained with such 2 methods.11 while 56% were diagnosed as having fd, and 58% as having ibs in the electronic survey, 57% were diagnosed as having fd, and 55% as having ibs in the postal one . Fd - ibs overlap was found in 24% in the electronic survey and in 23% in the postal one . Subjects who have higher scores for psycho - behavioral problems had a higher prevalence of fd and ibs symptoms . The data suggest that psycho - behavioral conditions may affect the development of functional gi symptoms, regardless of the subtype of gi disorders, and can explain the high proportion of overlap among the subtypes.11 taken together, the above - mentioned prevalence of fd - ibs overlap would be altered depending on the selected study population . The rate of fd - ibs overlap could be in the same range, such as 11.4%,6 16.5%,2 23.8%,4 24.0%3 and 27.6%5 (table). A general definition of fd, to be used mainly for clinical purposes, and although further research on more specific definitions is ongoing, is provided under category b1: functional dyspepsia (fd) in rome iii classification . However, particularly for pathophysiological and therapeutic approach, it is recommended in rome iii that new entities of meal - induced dyspeptic symptoms as postprandial distress syndrome (pds): b1a, and epigastric pain and burning as epigastric pain syndrome (eps): b1b, should be used.12 these 2 subcategories such as pds and eps seem very useful in clinical practice as well as in the investigative settings . According to the report by wang et al,4 more patients with pds alone had ibs than those with eps alone . Patients with fd - ibs overlap were more likely to be classified as the pds + eps subtype, had more frequent presence of the postprandial fullness symptom, one of major symptoms of pds (p <0.001), and overall fd symptom (p <0.01) than those with fd alone . Furthermore, patients with fd - ibs overlap were less likely to be classified as the eps alone (p <0.01) compared to those classified as fd alone . By the multivariate logistic regression analysis, only the presence of postprandial fullness (or, 2.67; 95% ci, 1.34 - 5.31; p <0.01) had a statistically significant and independent effect on the probability of fd - ibs overlap . On the other hand, subtypes of ibs did not differ between patients with constipation type - ibs (ibs - c) and diarrhea - type ibs (ibs - d). In addition, neither ibs - d (or, 1.44; 95%ci, 0.93 - 2.22; p = 0.10) nor mixed - type ibs (ibs - m) (or, 1.62; 95% ci, 0.77 - 3.40; p = 0.21) was identified as risk factor for fd - ibs overlap versus ibs alone in the multivariable analysis . Aro et al7 reported the data on the impact of pds and eps on qol . The impact of pds is statistically significant compared with controls except for role emotional and the results are consistently the same when analyzing for the possible confounders such as anxiety, depression and use of proton pump inhibitors . Patients with eps had a statistically relevant impairment of hrqol only in 2 domains such as bodily pain and vitality compared with nondyspeptic cohorts . Namely, pds seems to damage hrqol in all sf-36 domains more than eps . In addition, non - erosive relux disease is more frequently overlapped with fd, especially with eps and presents with significantly increased frequency of ibs.13 fd and ibs are common functional disorders without defined pathophysiology and are known as multifactorial syndromes . The pathophysiological factors for the generation of fd or ibs would be listed as visceral hypersensitivity, central abnormal deregulation for sensory perception, gi dysmotility, and abnormal alteration of the intestinal flora, gi inflammation, and psychosocial factors . Although such a mechanistic investigation is further preceded in the research field in ibs, the detail of pathophysiology in fd - ibs overlap has not been fully explored . However, as these 2 syndromes (fd and ibs) would have common causative factors, the high prevalence of fd - ibs overlap is considered to be easily acceptable . Abnormalities of psychosocial or central nervous factors, which would affect the whole gi tract, could be a possible causative factor in the pathogenesis of fd - ibs overlap . Savas et al14 examined the 1-year prevalence of ibs and dyspepsia symptoms and their associations with depression, anxiety and post - traumatic stress disorder (ptsd) among women veterans receiving primary care at a women's clinic, veteran affairs (va) medical center . They reported that women with ibs - dyspepsia overlap showed higher scores of anxiety (ibs: 24 vs 12, p <0.001 and dyspepsia: 26 vs 12, p <0.001), depression (ibs: 22 vs 11, p <0.001 and dyspepsia: 23 vs 11, p <0.001) and ptsd (ibs: 87 vs 69, p <0.001 and dyspepsia: 86 vs 69, p <0.0005), and age- and ethnicity - adjusted logistic regression analyses revealed a 3- to 46-fold increase in the or of ibs and dyspepsia among women with depression, anxiety or ptsd.14 although gastric sensorimotor dysfunction, psychosocial factors and somatization are all implicated in the development of fd symptoms according to the report by van oudenhove et al,15 symptom severity and weight loss in fd are determined by psychosocial factors (depression or history of abuse) and somatization, and to a lesser extent by gastric sensorimotor function, but not by fd - ibs overlap . On the other hand, kindt et al16 in belgium investigated the 5-year evolution of symptoms in a clinical fd population to identify factors associated with the outcome and indicated that the dyspepsia symptom score (dss) at the initial visit and trait anxiety were longitudinally associated with the dss at follow - up, with a trend found for weight loss; depression, chronic fatigue and ibs at follow - up were cross - sectionally associated with dss . On the other hand, gi inflammation and altered immune responses could be another candidate as the cause of fd - ibs overlap . Mast cells and eosinophils interact with t lymphocytes and may alter the enteric nerve and smooth muscle functions . Examination of 48 ibs patients with either diarrhea or constipation,17 12 patients with microscopic colitis, 20 patients with ulcerative colitis, and 24 healthy controls, indicated that as compared to male ibs patients, female ibs had greater numbers of mast cells (p = 0.066), but lesser numbers of cd3 + and cd8 + t cells (p <0.01 and p <0.001, respectively). Such mucosal mast cell infiltration in ibs patients was significantly associated with the frequency of abdominal bloating (p <0.05) and with dysmotility - like dyspepsia (p = 0.001), but not with ulcer - like dyspepsia.17 according to the report by walker et al,18 intraepithelial lymphocytes in ibs - c were significantly increased (p = 0.005) and mast cells were significantly increased in the second part of the duodenum in ibs (p <0.001), while eosinophils were significantly increased in the duodenal bulb and second part of the duodenum in fd (p <0.001), suggesting that duodenal mast cell hyperplasia is linked to ibs, and eosinophilia to fd . Recently, it has been revealed that previous infectious gastroenteritis is often followed by postinfectious fd (pi - fd)19 or postinfectious ibs (pi - ibs).20 porter et al21 recently conducted a matched, case - control study describing the epidemiology and risk determinants of ibs, functional constipation, functional diarrhea and dyspepsia using electronic medical encounter data in active - duty us military personnel, and demonstrated a significant association between infectious gastroenteritis and all fgids (or, 2.64; p <0.001), with the highest frequency of functional diarrhea (or, 6.28; p <0.001) and ibs (or, 3.72; p <0.001), and a moderate frequencies of functional constipation (or, 2.15; p <0.001) and fd (or, 2.39; p <0.001). According to kindt et al22 and suzuki23, pi - fd is associated with persistent focal t cell aggregates, decreased cd4 + cells and increased macrophage counts surrounding the crypts, indicating an impaired ability of the immune system to terminate the inflammatory response after an acute insult . Since the etiology of fd is still uncertain, it is not surprising that helicobacter pylori, a major pathogen in the stomach,24 - 26 has been implicated in the pathophysiology of so called dyspepsia . Many trials reporting the efficacy of h. pylori eradication therapy for fd including non - ulcer dyspepsia have given conflicting results but there is a clear indication that h. pylori eradication treatment is effective in at least a subset of patients with fd or non - ulcer dyspepsia.27,28 the recent meta - analysis also suggests that h. pylori eradication at 12 months has a small but statistically significant benefit in the treatment of fd (relative risk [rr] of remaining dyspepsia with h. pylori eradication therapy = 0.91; 95% ci, 0.87 - 0.99). While statistically significant, the clinical significance of this finding is less clear because the effect is small, that is, 15 h. pylori - positive dyspeptic patients will need to be treated to achieve just one cure.29 on the other hand in asia, gwee et al30 conducted a double blind, randomized, placebo - controlled trial of h. pylori eradication for fd was conducted in singapore population . Forty - one patients received active treatment consisting of a 1-week course of omeprazole 20 mg once daily, clarithromycin 250 mg twice daily and tinidazole 500 mg twice daily whereas another 41 patients received matching placebo tablets . They reported on itt analyses, that while symptom resolution was observed in 24% of patients on active treatment, only 7% was found on the placebo (p = 0.02; 95% ci, 1.1 - 17.7). In addition, their report shows that among patients with h. pylori eradicated on active treatment the symptom resolution rate was 39%, whereas it was 3% among patients in the placebo who had persistent h. pylori infection,30 suggesting that patients with fd in asian population might benefit more from treatment for h. pylori infection . On the aspects of pathophysiology of dyspepsia in h. pylori infection, we recently reported in mice that after long - term h. pylori infection, gastric emptying was significantly accelerated with a significant thickening of the muscular layers of the stomachs that was manifest in the hyperplasia of myocytes.31 we also examined the expression profile of micrornas (mirs), which is recently reported to be involved in the pathogenesis of gi disorders including cancers.32,33 in h. pylori - infected mice, the muscle - specific mirs such as mir-1, mir-133a and mir-133b were significantly down - regulated in the stomachs with an enhancement of the expression of histone deacetylase 4 and serum response factor, which are reported target genes of mir-1 and mir-133 and to enhance the muscular hyperproliferation . Accelerated gastric emptying may be possibly through the disturbed gastric accommodation due to the alterations in muscular layer modification, providing a novel insight into the molecular pathogenesis of dyspepsia associated with h. pylori infection.31 there might be a reason to consider h. pylori - associated dyspepsia as an organic disease and to deal with it as a different disease entity from fd.34,35 a new classification based on the pathophysiology and specific symptoms needs to be considered to further the diagnostic and therapeutic advances in this field . There is a paucity of data on racial differences in the epidemiology of ibs alone and uid alone as compared to that of the " overlap syndrome . " According to a random survey of 990 subjects who completed a questionnaire which included the rome ii criteria for ibs and fd,35 among african americans, the prevalences of ibs alone, uid alone and their overlap were 0.6%, 17% and 7.3%, respectively, while among caucasian americans, they were 0%, 13% and 13%, respectively . Overlap syndrome was seen in 30% of african americans, as compared to 50% of caucasian americans . African americans below the poverty line were more likely to have uid than overlap (22% vs 10%). As compared to african americans, caucasians with overlap syndrome were likely to be married and to be living in an urban area . There was a higher prevalence of overlap syndrome (uid - ibs) among caucasians with lower levels of education . Uid - ibs overlap was 2.5 times more likely to occur among caucasians than among african americans.36 however, among uid patients, there might be considerable numbers of patients with peptic ulcer disease or erosive esophagitis . The concept of fd - ibs overlap syndrome is important for determining the independent therapeutic approach for such disease conditions of fd alone or of ibs alone, by treating them as a disease of the entire gut as a single organ . Under such concept of disease overlap, fd and ibs can be recognized as being frequently coexisting and not mutually exclusive syndromes.
Lymphatic filariasis is common in tropical countries and is endemic in india with most infections caused by wuchereria bancrofti . The microfilaria are not just confined to the lymphatic system, but also associated with other organs, subcutaneous tissues, and serous cavities like pleura and pericardium . Demonstration of microfilariae (mf) in the peripheral blood smear or in the tissues is the mainstay of diagnosis . Filariasis acutely presents with fever, adenolymphangitis, funiculitis, epididymitis, orchitis, or pericarditis . Lymphedema, hydrocele, elephantiasis and chyluria, and pleural or pericardial effusion are features of chronic infection . Our case was a rare as recurrent hemorrhagic pericardial effusion and tamponade, which was first treated with diethyl carbamazine and finally albendazole . She complained of cough and breathlessness on exertion and low grade fever since 3 weeks and generalized weakness since 1 week . On examination, she was severely dyspneic and had pulsus paradoxus . Echocardiogram showed massive pericardial effusion with cardiac tamponade [figure 1]. Apical four - chamber view showing large pericardial effusion with right atrial collapse (left) and parasternal short axis view showing large pericardial effusion with right ventricular outflow tract (rvot) diastolic collapse (right) pericardiocentesis was done and 1,300 ml of hemorrhagic fluid was collected . Results of complete blood picture were: total count - 6,800/mm, polymorphs - 67%, lymphocytes - 23%, eosinophils - 06%, monocytes - 04%, platelet count - 160,000/mm, hemoglobin - 11 g%, erythrocyte sedimentation rate (esr) - 40 1 h., human immunodeficiency virus (hiv)-nonreactive, and hepatitis b surface antigen (hbsag)-negative . Complete urine examination, blood sugar, urea, creatinine, and electrolytes; all were in normal range . Cytological analysis of fluid showed - 7,000 cells / mm, (neutrophils - 35%, lymphocytes - 65%, plenty of red blood cells (rbc)), negative for malignant cells, sugar - 61 mg%, proteins - 6 g%, albumin - 2.5 g%, adenosine deaminase (ada) - 23 u / l, acid - fact bacilli (afb) - negative; microscopy and routine cultures were negative for bacteria . While screening the smears, a few large, long, smooth - walled, refractile unstained worm - like structures were observed . The organisms were identified as mf of w. bancrofti in the smears stained by leishman's stain [figure 2]. Cytology showed plenty of rbc, occasional pus cells, and microfilaria of w. bancrofti [figure 3]. The patient improved rapidly by next day and effusion was grossly reduced by the 2 day . Pericardial fluid cytology smear showing microfilaria of wuchereria bancrofti with occasional pus cells (leishman stain, 1000) pericardial fluid cytology smear showing microfilaria of w. bancrofti with occasional pus cells (leishman stain, 40) it is transmitted by the culex mosquito and is caused by two closely related nematodes, w. bancrofti and brugia malayi, which are responsible for 90 and 10% of the cases, respectively . Microfilaria have been confined not only to the lymphatic system but also associated with other organs, subcutaneous tissues, and serous cavities like pleura and pericardium . Pericardial effusion is a rare manifestation of filariasis, and recurrent effusion with tamponade is even more rare . Our case was of massive pericardial effusion established as filarial in origin on demonstration of mf in pericardial aspirate . It might have lodged itself through blood stream subsequent to development of pericarditis . As mf are capable of penetrating the tissues of the vector, they can freely move in and out of small, delicate blood, and lymphatic capillaries and tissues of the vertebrate host . Peripheral blood smears may be negative for mf in cases of secondary manifestations of filariasis . In this case patient developed effusion despite being treated with dec suggests that it may not be 100% effective in all cases as dec does not act on microfilaria residing in lymphatics, and they might have migrated to pericardial space causing recurrent effusion . She was finally cured with alternate combination of ivermectin and albendazole, which makes it even more interesting . Although mf in unusual sites are considered incidental findings, on the basis of existing evidences we suggest that some of the cases of recurrent pericardial effusion, especially those from endemic areas, might be of filarial origin . It is therefore emphasized that a thorough examination of the aspirated fluid for mf is warranted in all patients with unexplained pericardial effusion.
Myanmar is one of the countries hardest hit by the human immunodeficiency virus (hiv) epidemic in asia . The first case of hiv was detected in myanmar in 19881 and of acquired immunodeficiency syndrome (aids) in 1991.2 the adult hiv prevalence reached a peak at 0.9% in 2000,3 followed by a downward trend reaching 0.6% in 2009 and 0.5% in 2011.1 it is estimated that around 216,000 people were living with hiv in myanmar in 2011, of which, 36% were female . In the same year, an estimated 18,000 people died of aids - related illnesses . The incidence of new infections was estimated at well above 8,000 in 2011.4 the hiv / aids epidemic in myanmar has been mostly concentrated in men . However, the number of infected women has been increasing gradually.1 the hiv epidemic in myanmar is largely concentrated among subgroups of populations with high - risk behaviors . As of 2011, over 60% of new hiv infections occurred among female sex workers, their clients, people who use drugs, and men who have sex with men . It is estimated that by the year 2015, these groups will constitute over 70% of new infections.4 according to the 2011 hiv sentinel sero - surveillance, hiv prevalence rates among female sex workers in myanmar has dropped, from 18.4% in 2008 to 9.4% in 2012.5 it is estimated that there are 40,000 to 80,000 sex workers in myanmar.3 although illegal and culturally unacceptable, the sex trade exists in most of the major cities, like yangon and mandalay, and in mining and border areas.6 this widespread existence of sex trade could be due to low levels of education, unemployment, poverty, drug abuse, and migration, especially among women from remote regions and border areas.710 sex industry - related jobs may include direct, brothel - based work as well as various other indirect types of sex work . Most direct sex workers are highly mobile within the country but are usually concentrated in brothels in crowded major cities, mining areas, or along the country borders.6 some brothels provide condoms to the sex workers to protect them from pregnancy and sexually transmitted diseases (stds). The usual rate for a sexual experience with teenage sex workers may range from 20,000 to 40,000 kyats a night . According to the findings of the 2011 hiv sentinel sero - surveillance, hiv prevalence among female sex workers was the highest among singles, aged 30 to 39 years and plying their trade in urban areas like yangon (18%), followed by kyaingtong (11%), myitkyina (9%), taunggyi (7.5%), mandalay (5%), and lashio (4.7%). The prevalence was found to be significantly higher among direct sex workers (12.2%) than indirect sex workers (6%).5 because prostitution is illegal in myanmar, sex workers generally do not have access to hiv prevention and treatment programs, for fear of prosecution . The objective of this study was to determine the prevalence of hiv among the female sex workers in the major cities in myanmar and the risk behaviors associated with it . This cross - sectional study was conducted in 2009 in the major cities of myanmar, namely: yangon, mandalay, taunggyi, muse, tachileik, and lasho . These locations were chosen because of the high number of sex workers and sex - related businesses in these cities . Due to the sensitive nature of the study, it was not possible to use a sample frame to conduct probabilistic sampling, hence sex workers in this study were defined as female adults and young people who receive money or goods in exchange for sexual services, either regularly or occasionally, and who may or may not consciously define those activities as income - generating.11female sex workers who openly defined themselves as sex workers and who earned a living buying and/or selling sex were classified as sex workers, and women for whom sex work was not the first source of income and who did not consider themselves as sex workers were classified as indirect sex workers.12 both direct and indirect female sex workers aged 15 years or older who had traded sex for money in the month prior to the study were recruited from brothels (yangon, mandalay, taunggyi, muse, tachileik, and lasho), karaoke (yangon, mandalay, and taunggyi) and massage parlors (yangon, mandalay, and lasho). A quantitative questionnaire was developed and field tested prior to the actual study . Besides the sociodemographic questions, the knowledge and awareness of hiv and sexually transmitted infections (sti), and the risk - taking behaviors of the participants were questioned . The interviews were conducted by trained research assistants who were able to communicate in the local languages . The interviewers were trained for 3 days and had an additional 2 days of field practice . The hiv status of the respondents was asked and confirmed with their blood test reports from the laboratories of the myanmar national aids programme sti / aids teams and nongovernmental organizations (ngos). An informed verbal consent was taken before commencing the interview and reviewing the blood test report . This cross - sectional study was conducted in 2009 in the major cities of myanmar, namely: yangon, mandalay, taunggyi, muse, tachileik, and lasho . These locations were chosen because of the high number of sex workers and sex - related businesses in these cities . Due to the sensitive nature of the study, it was not possible to use a sample frame to conduct probabilistic sampling, hence, a convenience sampling process was used . Sex workers in this study were defined as female adults and young people who receive money or goods in exchange for sexual services, either regularly or occasionally, and who may or may not consciously define those activities as income - generating.11female sex workers who openly defined themselves as sex workers and who earned a living buying and/or selling sex were classified as direct sex workers, and women for whom sex work was not the first source of income and who did not consider themselves as sex workers were classified as indirect sex workers.12 both direct and indirect female sex workers aged 15 years or older who had traded sex for money in the month prior to the study were recruited from brothels (yangon, mandalay, taunggyi, muse, tachileik, and lasho), karaoke (yangon, mandalay, and taunggyi) and massage parlors (yangon, mandalay, and lasho). Refusal to participate was the main exclusion criterion . A quantitative questionnaire was developed and field tested prior to the actual study . Besides the sociodemographic questions, the knowledge and awareness of hiv and sexually transmitted infections (sti), and the risk - taking behaviors of the participants were questioned . The interviews were conducted by trained research assistants who were able to communicate in the local languages . The interviewers were trained for 3 days and had an additional 2 days of field practice . The hiv status of the respondents was asked and confirmed with their blood test reports from the laboratories of the myanmar national aids programme sti / aids teams and nongovernmental organizations (ngos). An informed verbal consent was taken before commencing the interview and reviewing the blood test report . A total of 226 sex workers were identified as eligible participants, however 17 were excluded for refusal to give consent, and the data of nine respondents were excluded because of numerous missing and unreadable data . The age of the participants ranged from 15 to 33 years, with a mean age of 25 years . Most were myanmars (60.5%), buddhist (83.0%), between the ages 22 and 29 (59.0%), not married and either living alone (40.0%) or living with spouses (40.0%), and with the highest level of education up to middle school (40.5%). Most of the participants were previously unemployed (73.0%), and at the time of the study, most were working in brothels as direct sex workers (52.5%). Almost all of them were mobile, moving from one place to another working as sex workers, and 40% of the respondents were ethnic minorities . Most were on the move alone (41.0%), for more than 5 years (49.5%), and had never returned home (53.5%). There were varied methods of contraceptives used, but only 23 (11.5%) of the participants used condoms (table 1). As seen in table 2, almost everyone was aware of sti (99.5%) and hiv (100.0%) and had a fair knowledge of hiv transmission; however, only 70 (35.0%) had hiv counseling . Out of the 136 (68.0%) who tested for hiv, 25 (18.4%) were hiv - positive . Only 30 (15.0%) respondents had a history of taking drugs, mostly yaba, (67.5%) and at the time of the study, no one was injecting drugs . The age range during which the respondents first started having sex was from 13 to 29 years, with the mean age of 19 years; most were within the age group of 1821 years (54.5%). The number of times the respondents had paid sex per month was between 10 and 90, with a mean of 35 . Most of the respondents had always (65.0%) used condoms during the past year and during their last encounter with paid clients (67.0%). The most common reason for not using a condom was because the client refused (54.5%). Most of the respondents had one regular partner who was not their client during the period of the study (87.0%) and had regular sex with him (57.5%). Most had not used condoms in the last sexual encounter (69.6%) nor used condoms regularly with the regular partners (27.0%) because they did not consider it necessary (86.3%). As shown in table 3, respondents of other ethnic groups than myanmars and other religions than buddhist were about six times (or 5.9; 95% confidence interval [ci] 2.215.9) and five times (or 4.6; 95% ci 1.712.3), respectively, at higher odds of being hiv - positive . Those who were earning an income of less than 200,000 kyats were almost three times (or 2.9; 95% ci 1.27.0) at higher odds of being hiv - positive . The differences in the age groups was found to be statistically significant (p = 0.001). As shown in table 4, respondents who did not have hiv counseling (or 7.3; 95% ci 2.818.9), who did not use a condom (or 1.3; 95% ci 1.11.4), and with regular partners who refused the use of condoms (or 6.0 95% ci 3.2;11.3) were at higher odds of being hiv - positive . The prevalence of hiv among the female sex workers in this study (18.4%) was higher than that reported by the hiv sentinel surveillance reports of the national aids program, myanmar, which showed a declining trend from 2008 (18.4%), 2009 (11.2%), 2010 (11.4%), and 2011 (9.4%).5 the prevalence in this study, was also higher than that reported in thailand, which reported a prevalence of 2.8% among direct sex workers and 1.7% among indirect sex workers.13 similarly, the prevalence of hiv reported among brothel - based sex workers in cambodia (14.7%)14 and among indonesian direct sex workers (10.4%) and indirect sex workers (4.6%)15 was lower compared with that in this study . In this study, the prevalence of hiv was higher in the younger age group and other minority races than myanmar and in those with a lower income . Ethnic minorities are known to be more vulnerable to hiv infections.16 large numbers of shan (eastern region of myanmar) women and girls work in the thai sex industry, and they have been shown to be more likely to be infected with hiv.17 studies among asian masseuses in san francisco,18 among minorities in laos,19 african americans and hispanics in the usa,20,21 and among minorities in north america, asia, and europe22 have shown that ethnic minorities have higher prevalence of hiv . The reason for this higher prevalence could be due to poverty, substance abuse, homelessness, unequal access to health care, and unequal treatment once in the health care system.2325 the finding of the present study that sex workers with a lower income were at higher odds of being hiv - positive is consistent with studies conducted elsewhere.26,27 low income is associated with poverty - related factors, like no permanent place of residence, poor access to food and other basic necessities, and limited social support networks, all of which can potentially drive the lower - income sex workers into risk - taking sexual behaviors, which expose them to hiv . Economic need is a compelling reason to accept a client who refused to use a condom.28 according to a study conducted in 2007 by the international organization for migration among myanmar migrant female sex workers, there were incidences reported when clients had paid extra not to use a condom.29 female sex workers with a good income may be more insistent on the use of condoms by their clients, whereas sex workers who do not make enough money for their daily needs may be more inclined to give in to the demands of the clients who do not wish to use condoms.29 there is evidence that counseling, with or without voluntary confidential testing, is an effective tool for risk - reduction behavior change in the prevention of hiv / sti transmission.3034 these studies show that respondents who received counseling show substantial positive risk - behavioral change, with increasing usage of condoms, resulting in lower transmission of sti and hiv compared with control groups . In addition to the prevention of hiv transmission, counseling also helps to reduce stigma and fear and creates an opportunity for receiving treatment . Studies have shown that sex workers with higher knowledge concerning hiv and its mode of transmission have a lower prevalence of hiv.3537 a study in china reported that lack of sexual knowledge and condom use was associated with a higher prevalence of various stds.35 this could facilitate the sexual transmission of hiv . A study conducted by ding et al35 found that female sex workers with lower education level, less knowledge about hiv / aids, and lower rate of condom use were more vulnerable to hiv infection and stds . Another study conducted among female sex workers in yangon, myanmar found that new entrants to the industry, especially teenage girls, rarely used condoms, and they had lesser knowledge about hiv and sti.28 it has been reported that among the myanmar migrant sex workers, condoms were rarely used with regular noncommercial partners, such as boyfriends and husbands.29 and according to the behaviour surveillance survey,38 about half of female sex workers have sex with regular partners in addition to their clients, and condom use at the last sex encounter with regular partner was reported to range from 55% to 62% . This risk behavior is a cause of concern, especially when a significant proportion of those with hiv or at high risk of being infected are spouses or regular partners.5 it is because of this consistent finding that the hiv sentinel sero - surveillance 2011 called for vigilant prevention services targeting the regular sexual partners or intimate partners (spouses) of these high - risk population groups.5 the possible reason for the lack of condom use could be because the sex workers perceive themselves as lovers of their regular partners and not using condoms is an expression of their love or intimacy . Possibly, they want to show themselves as clean and safe . For the part - time sex workers, maintaining the confidentially of their profession from their spouses could be another reason . The prevalence of hiv among the female sex workers in this study (18.4%) was higher than that reported by the hiv sentinel surveillance reports of the national aids program, myanmar, which showed a declining trend from 2008 (18.4%), 2009 (11.2%), 2010 (11.4%), and 2011 (9.4%).5 the prevalence in this study, was also higher than that reported in thailand, which reported a prevalence of 2.8% among direct sex workers and 1.7% among indirect sex workers.13 similarly, the prevalence of hiv reported among brothel - based sex workers in cambodia (14.7%)14 and among indonesian direct sex workers (10.4%) and indirect sex workers (4.6%)15 was lower compared with that in this study . In this study, the prevalence of hiv was higher in the younger age group and other minority races than myanmar and in those with a lower income . Ethnic minorities are known to be more vulnerable to hiv infections.16 large numbers of shan (eastern region of myanmar) women and girls work in the thai sex industry, and they have been shown to be more likely to be infected with hiv.17 studies among asian masseuses in san francisco,18 among minorities in laos,19 african americans and hispanics in the usa,20,21 and among minorities in north america, asia, and europe22 have shown that ethnic minorities have higher prevalence of hiv . The reason for this higher prevalence could be due to poverty, substance abuse, homelessness, unequal access to health care, and unequal treatment once in the health care system.2325 the finding of the present study that sex workers with a lower income were at higher odds of being hiv - positive is consistent with studies conducted elsewhere.26,27 low income is associated with poverty - related factors, like no permanent place of residence, poor access to food and other basic necessities, and limited social support networks, all of which can potentially drive the lower - income sex workers into risk - taking sexual behaviors, which expose them to hiv . Economic need is a compelling reason to accept a client who refused to use a condom.28 according to a study conducted in 2007 by the international organization for migration among myanmar migrant female sex workers, there were incidences reported when clients had paid extra not to use a condom.29 female sex workers with a good income may be more insistent on the use of condoms by their clients, whereas sex workers who do not make enough money for their daily needs may be more inclined to give in to the demands of the clients who do not wish to use condoms.29 there is evidence that counseling, with or without voluntary confidential testing, is an effective tool for risk - reduction behavior change in the prevention of hiv / sti transmission.3034 these studies show that respondents who received counseling show substantial positive risk - behavioral change, with increasing usage of condoms, resulting in lower transmission of sti and hiv compared with control groups . In addition to the prevention of hiv transmission, counseling also helps to reduce stigma and fear and creates an opportunity for receiving treatment . Studies have shown that sex workers with higher knowledge concerning hiv and its mode of transmission have a lower prevalence of hiv.3537 a study in china reported that lack of sexual knowledge and condom use was associated with a higher prevalence of various stds.35 this could facilitate the sexual transmission of hiv . A study conducted by ding et al35 found that female sex workers with lower education level, less knowledge about hiv / aids, and lower rate of condom use were more vulnerable to hiv infection and stds . Another study conducted among female sex workers in yangon, myanmar found that new entrants to the industry, especially teenage girls, rarely used condoms, and they had lesser knowledge about hiv and sti.28 it has been reported that among the myanmar migrant sex workers, condoms were rarely used with regular noncommercial partners, such as boyfriends and husbands.29 and according to the behaviour surveillance survey,38 about half of female sex workers have sex with regular partners in addition to their clients, and condom use at the last sex encounter with regular partner was reported to range from 55% to 62% . This risk behavior is a cause of concern, especially when a significant proportion of those with hiv or at high risk of being infected are spouses or regular partners.5 it is because of this consistent finding that the hiv sentinel sero - surveillance 2011 called for vigilant prevention services targeting the regular sexual partners or intimate partners (spouses) of these high - risk population groups.5 the possible reason for the lack of condom use could be because the sex workers perceive themselves as lovers of their regular partners and not using condoms is an expression of their love or intimacy . Possibly, they want to show themselves as clean and safe . For the part - time sex workers, maintaining the confidentially of their profession from their spouses could be another reason . This study has shown there is a gap in the hiv prevention programs in myanmar that needs to be addressed, especially with respect to poverty, which is believed to be the main reason for involvement in the sex industry by the ethnic minorities . The lack of use of condoms by sex workers when intimate with their regular partners also is a very serious cause of concern . Due to monetary and time constraints as well as the sensitive nature of the study, recruiting sex workers to participate in this study was difficult, resulting in the researcher employing a nonprobabilistic sampling process . The sample size of the sex workers was far from optimal . Due to monetary and time constraints as well as the sensitive nature of the study, recruiting sex workers to participate in this study was difficult, resulting in the researcher employing a nonprobabilistic sampling process.
The largest and most conspicuous exocrine system in the acariform mite suborders oribatida and astigmata is the paired opisthonotal glands (syn . Oil glands). These glands, according to a current and widely accepted hypothesis, evolved only once within ancient oribatida (after the near - basal oribatid groups, palaeosomata and enarthronota, had split - off), and are considered characteristic of the four more - derived oribatid groups, parhyposomata, mixonomata, desmonomata, brachypylina, and also of astigmatid mites . The presence of opisthonotal glands not only constitutes a major argument for a proposed monophyletic unit of so - called glandulate oribatida, including astigmata (norton 1998), but also provides evidence for the evolutionary origin of astigmatid mites in an ancient opisthonotal gland - bearing oribatid group . In addition to the presence or absence of opisthonotal glands, the secretions of these glands provide phylogenetic information as they include a rich variety of hydrocarbons, terpenes, aromatics, and alkaloids in species - specific or group - specific patterns . These secretions, derived from a homologous gland system, allow the tracing of evolutionary lineages from near - basal to more - derived glandulate oribatida . Apart from the well - studied opisthonotal gland secretions of astigmata (kuwahara 2004), the secretions of a selection of species of several major groups of glandulate oribatids have been investigated, including representatives of parhyposomata (sakata and norton 2001), a few species of mixonomata and desmonomata (sakata et al . 2001, 2005a, b; shimano et al . 2002; sakata and norton 2003), and also examples of brachypylina (takada et al . 2005; saporito et al . 2007). While parhyposomata produce phenols and naphthols, the so - called astigmatid compounds (sensu sakata and norton 2001) evolved within ancient mixonomatans, and hence, are possibly found in all the above groups . These astigmatid compounds comprise a set of terpenes and aromatics, namely neral, geranial, neryl formate, 2-hydroxy-6-methyl - benzaldehyde, and -acaridial . The distribution of astigmatid compounds in oribatids is of special interest since these compounds not only provide chemical support for the evolutionary origin of astigmatid mites within ancient oribatida (norton 1998), but also define a presumably monophyletic subunit within glandulate oribatida, the astigmatid compounds - bearing oribatids (raspotnig 2006). Several chemotaxonomic studies that examined these presumptive astigmatid compound - bearing groups have been undertaken and have yielded a fragmented picture of the distribution of astigmatid compounds . These compounds are difficult to detect in the more derived groups of desmonomata (apart from trhypochthoniidae) and generally in brachypylina, suggesting that, in these groups, astigmatid compounds have been prone to evolutionary reduction and replacement . In some cases, desmonomatan and brachypyline species are chemically dimorphic, with astigmatid compounds present only in juveniles and completely replaced in adults by novel compounds such as toxic alkaloids (takada et al . In contrast, the full (or nearly full) set of astigmatid compounds seems to characterize highly derived mixonomatans, low to moderately derived representatives of desmonomata, and astigmata . Specifically, these compounds have been detected in opisthonotal gland secretions of collohmannia gigantea (raspotnig et al . 2001), in all trhypochthoniidae so far investigated (e.g., sakata et al . 1995, 2003), and in astigmatid mites (e.g., kuwahara 2004). However, there are large gaps in our knowledge of the distribution of astigmatid compounds: the most conspicuous gap being in the euphthiracaroidea, a speciose group of box mites whose opisthonotal gland chemistry has yet to be investigated . Euphthiracaroids are considered a group of highly derived mixonomatans that, together with phthiracaroids, constitute a possible sister group of collohmannia . Thus, euphthiracaroids are likely candidates for producing astigmatid compounds . The study of the chemistry of opisthonotal glands in euphthiracaroids is important not only to further our understanding of the evolution of astigmatid compounds, but also because this group is a crucial link for tracing a possible evolutionary lineage from oribatida to astigmata . With respect to the latter, some (not all!) Recent molecular studies (e.g., murell et al . 2005; domes et al . Therefore, studying the opisthonotal gland chemistry may resolve this problem . In the present paper, we provide the first chemical analysis of the opisthonotal gland secretion of oribotritia berlesei, a large - sized oribatid mite from the near - basal euphthiracaroid family oribotritiidae . Mites and mite extracts specimens of o. berlesei (michael 1898) (mixonomatan oribatida: euphthiracaroidea, oribotritiidae) were collected from the litter and fermentation layer of a mixed forest near ferlach (carinthia, austria). Individuals were collected by hand or extracted from soil samples by using berlese - tullgren funnels . In all, 35 adults (all in the course of one collection in september 2006) were used for chemical analyses, five adults were used for scanning electron microscopy, and 30 adults were transferred to petri dishes (equipped with plaster of paris and dead wood) and kept in the dark for a period of about 12 months either at room temperature (one petri dish) or at 10c (second petri dish), respectively . Individuals of both petri dishes laid eggs, and the offspring were reared to deutonymphal and tritonymphal instars . Since immatures are burrowers in dead wood, pieces of dead wood (provided in the rearing dishes) were checked for immatures at weekly intervals . Ten deutonymphs and eight tritonymphs were used in the chemical studies.extracts (containing opisthonotal gland secretions) of adults were prepared by immersing freshly collected, living, individuals in hexane (one individual per 100 l) for 30 min (raspotnig et al . 2001, 2005a, b). For juveniles, individual and pooled extracts (containing two to six individuals) were prepared similarly . Mass spectrometry a trace gas chromatograph (gc) coupled to a voyager mass spectrometer (ms; both from thermo, vienna, austria) and equipped with a zb-5ms fused silica capillary column (30 m 0.25 mm i.d ., 0.25 m film thickness, phenomenex, germany) was used for the analyses . Injection was splitless with helium (at a constant flow rate of 1.5 ml min) as a carrier gas . The column temperature was programmed from 50c (held for 1 min) to 200c at 10c min, and then to 300c at 15c min . The ion source of the mass spectrometer and the transfer line were kept at 150c and 310c, respectively . Derivatization, syntheses, reference compounds, and other chemicals tri-, penta-, and heptadecane, as well as citral (60% geranial, 40% neral) and selenium dioxide for the preparation of oxocitral (see bellesia et al . For determination of double bonds in the heptadecadiene, dimethyl - disulfide (dmds) adducts were generated according to the method described by vincenti et al . 2005b) was used as a natural source for 6,9-heptadecadiene and 8-heptadecene for chromatographic comparisons . In order to clarify further the structure of chrysomelidial, the o - methyl - oxime derivatives were prepared with methoxylamine hydrochloride (mox.hcl; 2% in pyridine): 200 l of mox was added to 50 l of extract and left at 70c for 1 h. products were cleaned in h2o, extracted with hexane, and analyzed . . Secretions from eversible defense glands of larvae of the chrysomelid beetles, gastrophya viridula and plagiodera versicolora (collected in the surroundings of graz, austria), known to contain natural (3s,8s)-chrysomelidial (e.g., pasteels et al . 1982), were collected on small filter paper pieces, extracted in hexane (one individual in 100 l), and used as sources for authentic reference compounds . Hexane extracts of fresh leaves of actinidia polygama (obtained from the botanical garden in tbingen, germany) were taken as a natural source for (3r,8s)-dehydroiridodial (yoshihara et al . Scanning electron microscopy air - dried mites were mounted on aluminum stubs, sputtercoated (agar sputtercoater, grpl, tulln, austria), and examined with a philips xl30 esem (philips / fei, vienna, austria) at high vacuum mode and 20 kv accelerating voltage . The main component in extracts, component c (rt = 13.36 min; ca . 45% of the total extract, based on peak areas) had an ei fragmentation pattern resembling a methyl - cyclopentene monoterpene (fig . 2a), with a molecular ion at m / z 166 (7) and characteristic fragments at m / z 151 (6), 148 (46), 138 (25), 133 (9), 123 (15), 120 (12), 119 (10), 109 (49), 108 (40), 105 (30), 95 (19), 91 (27), 81 (100), 79 (78), and 77 (38). The spectrum was consistent with that of chrysomelidial from the literature (e.g., blum et al . 1978; oldham et al . 1996) and with a compound from the nist library (2-formyl-,3-dimethyl-2-cyclopentene-1-acetaldehyde; cas no . 75332 - 42 - 2: 2-methyl-5(1-methyl-2-oxoethyl)-1-cyclopentene-1-carbaldehyde). In order to prove the dialdehyde structure, we generated o - methyl - oxime derivatives, resulting in the elimination of peak c and the formation of two new peaks, c and c, with longer retention times (rt = 14.92 and 15.11 min). The two compounds had identical mass spectra, with molecular ions at m / z 224 (4), and fragment ions at m / z 193 (13), 178 (22), 146 (25), 138 (100), 107 (38), 106 (95), 91 (18), 87 (21), 79 (47), and 77 (29). This pattern indicated o - methyl - oximation of compound c, forming two isomeric dimethyloximes, which confirmed the proposed dialdehydic structure (addition of two o - methyl - oxime groups to compound c = 166 plus 2 29 = new molecular ion at m / z 224). Peaks a (tridecene), b (tridecane), c1 (epi - chrysomelidial = 3s,8s - chrysomelidial), d (pentadecene), e (pentadecane), f (6,9-heptadecadiene), g (8-heptadecene), h (heptadecane), i (tentatively, -springene), j (tentatively, 9,17-octadecadienal)fig . B compound c1, tentatively identified as epi - chrysomelidial = (3s,8r)-chrysomelidial . Note the differences in relative intensities of ions at m / z 148 (m h2o) and m / z 138 (m co). Further diagnostic fragments are assigned to: m h2o co (m / z 120); c7h9o (m / z 109), c7h7 (m / z 91), and c6h9 (m / z 81: base peak = the methyl - cyclopentene moiety) typical mass chromatograms of extracts of oribotritia berlesei: a adult individual . B deutonymph . Peaks a (tridecene), b (tridecane), c1 (epi - chrysomelidial = 3s,8s - chrysomelidial), d (pentadecene), e (pentadecane), f (6,9-heptadecadiene), g (8-heptadecene), h (heptadecane), i (tentatively, -springene), j (tentatively, 9,17-octadecadienal) electron impact mass spectra of chrysomelidials in extracts of oribotria berlesei . B compound c1, tentatively identified as epi - chrysomelidial = (3s,8r)-chrysomelidial . Note the differences in relative intensities of ions at m / z 148 (m h2o) and m / z 138 (m co). Further diagnostic fragments are assigned to: m h2o co (m / z 120); c7h9o (m / z 109), c7h7 (m / z 91), and c6h9 (m / z 81: base peak = the methyl - cyclopentene moiety) for a comparison of compound c to authentic chrysomelidial and its characterization, we used (1) a synthetic mixture of chrysomelidial - stereoisomers, and (2) naturally occurring (3s,8s)-chrysomelidial . The synthetic mixture was assumed to contain four stereoisomers of chrysomelidial and dehydroiridodial (according to bellesia et al . 1986), namely two diastereomeric pairs of enantiomers, i.e., (3s,8s)-chrysomelidial, (3r,8r)-dehydroiridodial, and (3s,8r)-chrysomelidial (= epi - chrysomelidial), (3r,8s)-dehydroiridodial . However, the mixture exhibited only two, poorly separable peaks at rt = 13.32 and 13.36 min, each peak corresponding to one pair of enantiomers, with single enantiomers not separable under the chromatographic conditions . The later - eluting peak of the mixture corresponded to compound c from the oribotritia extract, with respect to both retention time and ei fragmentation pattern . Larval secretions of the leaf beetle gastrophysa viridula (see methods and materials), contain mainly (3s,8s)-chrysomelidial (original chrysomelidial). Analysis of this secretion showed only one abundant component that eluted at rt = 13.36 min, corresponding to the later - eluting peak of the synthetic mixture and to compound c of the mite extracts . Thus, the later - eluting peak of the synthetic mixture was assigned as either (3s,8s)-chrysomelidial or its enantiomer, (3r,8r)-dehydroiridodial . Since dehydroiridodials are considered of plant origin only (see discussion), compound c was tentatively identified as (3s,8s)-chrysomelidial . The mass spectrum of peak f (rt = 16.36 min), the second most abundant compound in extracts (about 25% of total), suggested a doubly unsaturated c17-hydrocarbon, based on a molecular ion at m / z 236 and a series of unsaturated hydrocarbon fragments . The dmds adduct of the heptadecadiene showed a molecular ion at m / z 362 and diagnostic ions at m / z 231, 203, 183, 159, 155 (base peak), and 131, consistent with 6,9-heptadecadiene (see raspotnig et al . Compound i (rt = 18.70 min; ca . 20% of the total) showed a terpene fragmentation pattern with a molecular ion at m / z 272 (4) and further m / z of 257 (3), 229 (4), 203 (5), 187 (10), 161 (17), 133 (31), 120 (15), 119 (17), 107 (20), 93 (50), 81 (37), 69 (100), 55 (16), 53 (17), and 41 (59). The compound was tentatively identified as -springene on the basis of mass spectral comparison to -springene spectra from literature and from the nist library . The remaining peaks a, b, d, e, g, and h, all of them minor components, appeared to be a series of hydrocarbons, with compounds b (rt = 11.69), e (rt = 14.32 min), and h (rt = 16.65 min) exhibiting the spectra of saturated n - alkanes with molecular ions at m / z 184, 212, and 240, respectively . The compounds were identified as tri-, penta-, and heptadecane by comparison of retention times to authentic standards . Compounds a (rt = 11.54), d (rt = 14.10 min), and g (rt = 16.44 min) had mass spectra of mono - unsaturated analogs, with molecular ions at m / z 182, 210, and 238, corresponding to tri-, penta-, and heptadecene . The position of the double bonds was not determined because, due to their low amounts, the compounds were not recovered after dmds derivatization . The retention time of compound g was the same as that of 8-heptadecene (from extracts of p. peltifer, see methods and materials). Compound j (rt = 19.27 min), also a minor component, had the following mass spectrum: m / z 264 (11), 235 (3), 221 (4), 207 (4), 165 (4), 151 (7), 137 (10), 123 (10), 109 (22), 95 (61), 81 (16), 79 (52), 67 (100), 55 (38), 54 (32), 44 (32), and 41 (69). The compound was tentatively identified as 9,17-octadecadienal on the basis of these mass spectral data . The pattern of the ten compounds was consistently detected in 30 of the 35 individuals of adult o. berlesei (fig . 1a), with relatively low inter - individual variation with respect to the relative abundance of components (data not shown). Extracts of juvenile o. berlesei extracts of juveniles (exclusively deuto- and tritonymphs) contained only two compounds in a 1:2 ratio (peak c1 and c in fig . 1b): the major component c corresponded to tentatively identified (3s,8s)-chrysomelidial from the adult extracts, based on identical retention time and mass spectrum . The minor component c1 eluted earlier (rt = 13.32 min) but was not fully separable from (3s,8s)-chrysomelidial . The mass spectrum of compound c1 appeared to be similar to that of (3s,8s)-chrysomelidial (fig . 2); one difference, however, was the greater intensity of the ion at m / z 138 in relation to the ion at m / z 148 [c.f ., in (3s,8s)-chryomelidial the ion at m / z 148 is more intense]. These data suggested the compound to be one of the other possible stereoisomers of chrysomelidial, most likely an epimeric diastereomer of (3s,8s)-chrysomelidial . Moreover, the retention time and mass spectrum of compound c1 fully corresponded with those of the earlier - eluting peak of the synthetic chrysomelidial mixture, limiting the possibilities to epi - chrysomelidial (3s,8r - chrysomelidial) and its enantiomer, (3r,8s)-dehydroiridodial . In addition, its retention time and mass spectrum matched those of plant - derived (3r,8s)-dehydroiridodial . Thus, again assuming that dehydroiridodials are exclusively plant - derived compounds, compound c1 was tentatively identified as epi - chrysomelidial (3s,8r - chrysomelidial).from a total of eight extracts of juvenile o. berlesei investigated (derived from 18 individuals), seven extracts consistently showed these two stereoisomers only; the other extract (an extract of a single individual) apparently did not contain any compounds at all . Potential secretory glands the most conspicuous exocrine gland system of o. berlesei is the paired opisthonotal glands that, in light microscopic observations, are visible through the cuticle of the notogaster as kidney - shaped sacs of considerable size (maximal diameter about 0.5 mm: i.e., ca . The glands open to the notogastral surface via one single flap - provided pore orifice on either side of the body (fig . The flap, presumably an external closing mechanism, was found either in an opened (fig . Opisthonotal glands are present in adults and in all juvenile stages . Fig . 3topography and external morphology of opisthonotal glands in oribotritia berlesei . A scanning electron micrograph (sem) of an adult individual, lateral view . D sem of the pore of another individual, orifice closed topography and external morphology of opisthonotal glands in oribotritia berlesei . A scanning electron micrograph (sem) of an adult individual, lateral view . Occurrence of chrysomelidial even though iridoid monoterpenes are widely distributed in exocrine secretions of a diverse range of arthropods, chrysomelidial, so far, has only been reported from leaf beetle larvae (chrysomelidae) and from certain rove beetles (e.g., pasteels et al . Originally, chrysomelidial was found as a major constituent of the larval eversible glands of the chrysomelids, gastrophysa cyanea (blum et al . 1978) and plagiodera versicolora (meinwald et al . 1977), and its stereochemistry later elucidated by meinwald and jones (1978). According to these studies, and to current knowledge, chrysomelidial in leaf beetles is a mixture of two diastereomers, (3s,8s - chrysomelidial (= the original chrysomelidial) and (3s,8r)-chrysomelidial (= epi - chysomelidial), with the former being the predominant isomer in the majority of chrysomelidial - producing species (e.g., sugawara et al . In contrast, the remaining two possible stereoisomers (there are four stereoisomers due to the two asymmetric carbon atoms at c3 and c8) are termed dehydroiridodials and are considered of plant origin only (yoshihara et al . 1978; sugawara et al . 1979). Under the conditions used in this study, we were able to separate the diastereomers but not the respective enantiomers of chrysomelidial and dehydroiridodial . For instance, bellesia et al . (1986) described the chrysomelidials as a pair of enantiomers, whereas they are in fact diastereomers . In the present paper, (1979) for these compounds, but also use the more common nomenclature of bellesia et al . Thus, assuming dehydroiridodials are found exclusively in plants, the compounds in o. berlesei extracts are (3s,8s)-chrysomelidial and (3s,8r)-chrysomelidial (epi - chrysomelidial).our analysis of o. berlesei is not only the first example of the chemistry of exocrine glands of euphthiracaroidea but also the only report of chrysomelidials found outside the coleoptera . In o. berlesei, chrysomelidials most likely originate from the opisthonotal glands: these glands are quite large in o. berlesei, and thus probably capable of producing compounds in the amount detected . Furthermore, some of the components, especially 6,9-heptadecadiene and some other hydrocarbons, found in the extract are characteristic constituents of opisthonotal glands of many oribatid (and astigmatid) mite species (kuwahara 2004; raspotnig 2006). Functionally, at least in chrysomelidae, iridoid monoterpenes such as chrysomelidials are considered defensive or for microbial protection; these functions are also generally attributed to opisthonotal glands of oribatida (raspotnig et al . Chemosystematic impact with respect to chemosystematic studies that use opisthonotal gland profiles of oribatida, the finding of chrysomelidials in o. berlesei is significant . In preliminary investigations, chrysomelidials have been found also in other species of euphthiracaroidea (data unpublished), suggesting that this chemical class may be characteristic for species of oribotritiidae, possibly representing an important chemical synapomorphy of this family . Also, two further compounds in extracts of o. berlesei, if our tentative identifications are correct, would be novel for oribatid and astigmatid mite opisthonotal gland secretions . The diterpene, -springene, which we tentatively identified as one of the three main components of the secretion, was originally found in secretions of springboks (burger et al . 1978), and has also been detected in exocrine secretions of further vertebrates, especially mammals and reptiles (e.g., waterhouse et al . 2003), and invertebrates such as certain hymenopterans (e.g., howard et al . 2003; cruz - lopez et al . The other novel compound (for oribatid and astigmatid mite opisthonotal gland secretions) is the tentatively identified 9,17-octadecadienal, a minor constituent of the o. berlesei extract . Both tentatively identified compounds, -springene and 9,17-octadecadienal, may be of chemosystematic value in oribatida . Their unequivocal identification and possible distribution across other species of euphthiracaroidea remain to be studied . Astigmatid compounds perhaps the most surprising result of our study is the lack of astigmatid compounds in the opisthonotal gland extract of o. berlesei . These compounds are common secretion constituents in mixonomata desmonomata and astigmata, and are regarded as characteristic of all groups above middle - derived mixonomatans . In collohmanniidae (proposed close relatives of euphthiracaroids), astigmatid compounds, at least in some higher - derived desmonomatans and in brachypylines, may be reduced and replaced by other (novel) compounds in adult secretions of certain groups as in, for example, hermannia convexa (raspotnig et al . 2005a) and scheloribates ssp . Thus, there is a chemical dimorphism between juveniles and adults, with juvenile secretions being distinguished by the plesiomorphic, astigmatid compounds - rich condition . This chemical dimorphism was tested in o. berlesei; however, neither adults nor juveniles contained any astigmatid compounds . Even though the lack of astigmatid compounds in o. berlesei may be explained by complete evolutionary reduction, this situation may suggest a possible polyphyletic origin of astigmatid compounds in opisthonotal glands in general . The lack of astigmatid compounds is even more surprising in light of the classification system proposed by haumann (1991), in which oribotritiids are regarded as basal euphthiracaroids . Astigmatid compounds - free opisthonotal gland secretions in adults and juveniles also occur in several groups of brachypylines (raspotnig, unpublished data), but these groups are considered highly derived . The lack of astigmatid compounds in oribotritiidae could be reconciled by a systematic replacement of euphthiracaroids below the collohmanniidae, corresponding to a possible split from the oribatid stem - line before astigmatid compounds evolved . . The lack of astigmatid compounds may not necessarily be true for all oribotritiidae, and any extrapolation from data for o. berlesei remains speculative . Our major aim for future studies is the search for astigmatid compounds in other representatives of euphthiracaroid families.
Trypanosoma brucei, trypanosoma cruzi and leishmania major are unicellular protozoa of considerable medical importance since they are the etiologic agents of sleeping sickness (african trypanosomiasis), chagas disease (american trypanosomiasis) and leishmaniasis, respectively . The geographic range of these parasites is determined by their insect vectors: in the case of sleeping sickness, a blood sucking fly of the genus glossina, also known as the tsetse fly, for chagas disease, a reduviid bug known as the kissing bug and for leishmaniasis, a phlebotomine sandfly . While sleeping sickness occurs in sub - saharan africa, chagas disease is prevalent in latin america . Leishmaniasis is considered to be endemic in 88 countries, 72 of which are developing countries in asia, south america and africa . Together, these three parasitic diseases represent a huge burden since approximately 0.5 million people are infected with t. brucei, 10 million with t. cruzi and an estimated 12 million with different species of leishmania . In addition to the two human - infective subspecies, t. brucei gambiense and t. b. rhodesiense, other species and subspecies of african trypanosomes cause the disease known as nagana in domestic animals, imposing a further economic burden on several african countries . Different forms of leishmaniasis are caused by at least 20 leishmanial species: cutaneous leishmaniasis, with an estimated 1.5 million cases, and visceral leishmaniasis, with about 500,000 new cases annually, are the most common . Although control of the arthropod vectors of these diseases is an achievable goal and has been successful against the t. cruzi vector in parts of latin america, the alarming resurgence of sleeping sickness in africa and of leishmaniasis in parts of asia and latin america is a constant reminder of the need for better forms of chemotherapy and prevention of these diseases . More detailed information on african and american trypanosomiasis and the different forms of leishmaniasis, including vector distribution, disease control and treatment protocols can be found at http://apps.who.int / tdr/. Trypanosoma brucei, t. cruzi and leishmania spp . Are hemoflagellates of the family trypanosomatidae (order kinetoplastida) that is characterized by the presence of a single flagellum and one mitochondrion containing a unique organelle known as the kinetoplast which contains the mitochondrial dna (simpson et al ., 2006). Each parasite has a complex life cycle that involves humans as one of their various hosts (figure 1). As some of the earliest divergent members of the eukaryotae (haag et al ., 1998), these parasites have peculiar aspects of gene expression, including polycistronic transcription of most of their genomes (martnez - calvillo et al ., 2010), rna polymerase i - mediated transcription of protein - coding genes (gunzl et al ., 2003), rna trans - splicing to generate mature, capped mrnas (lebowitz et al ., 1993) and extensive rna editing to generate functional mrnas transcribed from mito - chondrial genes (hajduk et al ., 1993). Apart from their medical relevance, these peculiar characteristics make these parasites very interesting models for studying genome evolution and other aspects of genome function . On the other hand, the early evolutionary divergence of these organisms has resulted in biochemical characteristics that are not common in higher eukaryotes, such as enzymes related to antioxidant metabolism (olin - sandoval et al ., 2010) as well as sterol and glycosylphosphatidylinositol (gpi) biosynthesis (lepesheva et al ., 2011; koeller and heise, 2011) that have been exploited as promising drug targets . Genome sequencing of tri - tryp parasites began in the early 90s with the analyses of 518 expressed sequence tags (ests) generated from mrna isolated from bloodstream forms of t. b. rhodesiense (el - sayed et al ., 1995). Shortly thereafter, a comparison between est and genomic sequences showed that sequencing random dna fragments was as efficient as est analyses for discovering new genes in the african trypanosome (el - sayed and donelson, 1997). In 1996, an est analysis of cdna libraries constructed with mrna from l. major promastigotes was published (levick et al ., 1996), and the first est analysis of t. cruzi epimastigote forms was published in 1997 (brando et al ., 1997). During this period, pulsed - field gel electrophoretic analysis of chromosomes and the sequencing of large dna fragments from cosmid, bacterial artificial chromosome and yeast artificial chromosome libraries were also undertaken to generate physical maps of tri - tryp genomes (blackwell and melville, 1999). In 1999, the sequence of a 257-kilobase region spanning almost the entire chromosome 1 of l. major revealed the unusual distribution of protein - coding genes that was later found to be characteristic of all tri - tryp genomes . The complete sequence of l. major chromosome 1 revealed 79 protein - coding genes, with the first 29 genes all encoded on one dna strand and the remaining 50 genes encoded on the opposite strand (myler et al ., 1999). The tri - tryp gene organization is reminiscent of bacterial operons, with protein coding genes densely packed within directional clusters in one strand separated by strand switch regions (i.e., changes in the coding strand) (figure 2). Experimental evidence suggests that transcription initiates bi - directionally between two divergent gene clusters (martnez - calvillo et al ., 2003, 2004) to produce polycistronic pre - mrnas that are subsequently processed . Remarkably, with the exception of the spliced leader (sl) promoter, no promoter is recognized by rna polymerase ii and only a few transcription factors have been identified (cribb and serra, 2009; cribb et al ., 2010). Even more surprisingly, although orthologs of all conserved components of the rna polymerase ii complex were identified in the tri - tryp genome (ivens et al ., 2005), the transcription of some trypanosomatid genes such as vsg (variant surface glycoprotein) and the procyclin genes of t. brucei, as well as several exogenous genes transfected into t. cruzi, are mediated by rna polymerase i once the polycistronic pre - mrna is produced, two coupled reactions (trans - splicing and poly - adenylation) result in mature monocistronic transcripts . Trans - splicing means that every mature mrna has an identical capped sequence of 39 nucleotides, known at the spliced leader (sl), at the 5 end (liang et al ., 2003). Whilst no sequence consensus for polyadenylation or sl addition has been found, several studies have demonstrated that polypyrimidine - rich tracts located within intergenic regions guide sl addition and poly - adenylation, resulting in mature mrnas (lebowitz et al ., 1993) intergenic sequences involved in the processing of t. cruzi, t. brucei and leishmania mrna have been thoroughly investigated by comparing mrna with genomic sequences, initially using est databases (benz et al ., 2005; campos et al ., 2008; 2008) and, more recently, using high - throughput rna - sequencing (rnaseq) (siegel et al . In addition to providing valuable information on the mechanisms of gene expression in these organisms, these analyses also yielded data that allowed the optimization of transfection vectors used to express foreign genes and genetic manipulation in trypanosomatids . Comparative genomic analyses using the tri - tryp sequences have already provided interesting insights into the genetic and evolutionary bases of the distinct and shared lifestyles of these parasites . Probably the most striking finding is that the three genomes display high levels of synteny and share a conserved set of ~6,200 genes, 94% of which are arranged in syntenic directional gene clusters (el - sayed et al ., 2005a). Alignment of the deduced protein sequences of the majority of the clusters of orthologous genes across the three organisms reveals an average 57% identity between t. cruzi and t. brucei and 44% identity between t. cruzi and l. major that reflected the expected phylogenetic relationships (lukes et al ., 1997; the majority of species - specific genes occurs on non - syntenic chromosomes and consists of members of large surface antigen families . Structural rnas, retroelements and gene family expansion are also often associated with breaks in the conservation of gene synteny (el - sayed et al ., 2005a). Multi - gene family expansions are generally species - specific and most pronounced in the t. cruzi genome . As discussed below, a number of t. cruzi multi - gene families encode surface proteins, such as trans - sialidases, mucin - associated surface proteins (masp) and mucins tcmuc and gp63 that likely play important roles in host - parasite interactions (di noia et al ., 1995; vargas et al ., 2004; baida et al ., 2006; bartholomeu et al ., 2009). Based on their location in regions of synteny breaks these arrays may be subject to extensive rearrangements during the parasite s evolution and are thus directly associated with the specificities of each of the three parasitic diseases . Chagas disease, caused by t. cruzi, is endemic in more than 20 latin american countries, where an estimated 10 million people are infected and the domiciliation of the triatomines exposes at least 90 million individuals to the risk of infection . With no vaccine or effective drug treatment available, the main strategy for control must rely on the prevention of transmission by the insect vectors and blood transfusions . The parasite proliferates in the midgut of several species of a triatomid hematophagous vector . After reaching the insect s hindgut, epimastigote forms differentiate into non - dividing, infective metacyclic trypomastigotes that are excreted in the insect s feces . Trypomastigotes can infect a mammalian host by passing through mucous membranes or skin lesions during feeding by the insect . Once inside the mammalian host, trypomastigotes invade different types of cells where they transform into proliferative intracellular amastigotes . After a number of cell divisions in the host cell cytoplasm, amastigotes differentiate into trypomastigotes that are released into the bloodstream after host cell rupture and, after being taken up by an insect during a blood meal, they start a new cycle (brener, 1973) (figure 1). The highly heterogenous t. cruzi population consists of a large number of strains with distinct characteristics related to morphology, growth rate, parasitemia curves, virulence, pathogenicity, drug sensitivity, antigenic profile, metacyclogenesis and tissue tropism (buscaglia and di noia, 2003). Despite the broad genetic diversity observed among different strains and isolates, early studies based on different genotyping strategies identified two major lineages in the parasite population, named t. cruzi i and t. cruzi ii (souto et al ., 1996; momen 1999). These divergent lineages occupy distinct ecological environments, namely, the sylvatic cycle (t. cruzi i) and the domestic cycle (t. cruzi ii) of chagas disease (zingales et al ., 1998), as well as further analyses led some authors to propose the sub - division of t. cruzi ii into five sub - groups: t. cruzi iia, iib, iic, iid and iie (brisse et al ., 2000). Phylogenetic analyses of the t. cruzi strains became more confusing when additional data indicated the existence of not just two, but three major groups in the t. cruzi population, in addition to hybrid strains (miles et al ., 1978; augusto - pinto et al ., 2003; de freitas et al ., 2006). After intense debate, in 2009 an international consensus recognized the existence of six major strains, also known as discrete typing units (dtus) i vi (zingales et al ., 2009) (table 1). Since chagas disease spawns a variety of clinical forms, these studies are highly relevant: understanding the genetic variation among strains can potentially explain differences in disease pathogenesis, host preferences and, most importantly, provides essential information for the identification of new drug targets and good antigenic candidates for better diagnosis and vaccine development . For instance, t. cruzi ii strains and the hybrid strains belonging to t. cruzi v and vi are the predominant causes of human disease in south america (zingales et al ., 2009), whereas t. cruzi i strains are more abundant among wild hosts and vectors . Although detailed analysis of the biological and molecular factors underlying t. cruzi population structure and the epidemiology of chagas disease are beyond the scope of this review, one must keep in mind that the genetic variability found in the t. cruzi population is an essential aspect to be considered when analyzing this parasites genome . Cl brener, a clone derived from a hybrid t. cruzi strain belonging to t. cruzi vi, was chosen as a reference strain for the initial t. cruzi genome project . The hybrid nature of the cl brener clone became clear only after the genome sequencing had begun, when analyses of nuclear and mitochondrial sequences showed that this strain resulted from a fusion event that had occurred between ancient genotypes corresponding to strains belonging to t. cruzi ii and iii groups (el - sayed et al . Prior to this knowledge, the choice of the clone cl brener, initially classified as a member of sub - group iie, was based on five characteristics: (1) it was isolated from the domiciliary vector triatoma infestans, (2) its pattern of infectivity in mice was very well known, (3) it had preferential tropism for heart and muscle cells, (4) it showed a clear acute phase in accidentally infected humans, and (5) it was susceptible to drugs used to treat chagas disease (zingales et al ., 1997). In addition, several genomic studies had previously used this strain for karyotype analyses (branche et al ., 2006) and the generation of physical maps and ests from all three stages of the parasite life cycle (cano et al ., 1995; henriksson et al ., 1995; brando et al ., 1997; verdun et al ., 1998; the t. cruzi cl brener haploid genome, estimated to be 55 mb, was sequenced using the wgs (whole genome shotgun) strategy . Because of its hybrid nature and the high level of allelic polymorphism, a 14x coverage, much higher than the usual 810x coverage, was required to distinguish the ambiguities derived from allelic variations from those produced by sequencing errors . In contrast to the other two tri - tryp genomes, the t. cruzi draft sequence (el - sayed et al ., 2005b) was published as an assembly of 5,489 scaffolds built by 8,740 contigs . Four years later, based on synteny maps for the t. brucei chromosomes, weatherly et al . (2009) assembled the t. cruzi contigs and scaffolds initially in 11 pairs of homologous t. brucei - like chromosomes and, ultimately, in 41 t. cruzi chromosomes . Since trypanosomatid chromosomes do not condensate during mitosis and are therefore not visualized in metaphasic cells the predicted number of t. cruzi chromosomes was based on studies of pulsed - field gel electrophoresis (pfge) analyses (branche et al ., 2006), which turned out to be similar to the number of assembled chromosomes . As mentioned above, the genome organization in t. cruzi is largely syntenic with the other tri - tryp (t. brucei and l. major) genomes, with most species - specific genes, such as surface protein gene families, occurring in internal and subtelomeric regions of non - syntenic chromosome (el - sayed et al ., 2005a). Because of its hybrid nature, the cl brener genome is represented by a redundant dataset since homologous regions displaying a high level of polymorphism were assembled separately, generating two set of contigs, each corresponding to one haplotype . To identify the two haplotypes, reads from the genome of the cloned esmeraldo strain, a member of t. cruzi ii, and representing one of the cl brener parental strain (de freitas et al . Thus, in the annotation data of the cl brener genome, the two haplotypes are referred to as esmeraldo - like or non - esmeraldo - like sequences (aslett et al ., 2010). The haploid cl brener genome has an estimated 12,000 genes . As with the other tri - tryps, the t. cruzi genes are organized in long polycistronic clusters that are transcribed by rna polymerase ii and processed into monocistronic mrnas that accumulate differentially during the various stages of the parasite life cycle . As indicated before, one of the main characteristics revealed by the complete sequence of the t. cruzi genome was the dramatic expansion of families encoding surface proteins (el - sayed et al ., 2005a). Compared to t. brucei and l. major, t. cruzi has the largest set of multi - gene families, perhaps because of its unique capacity to invade and multiply within different types of host cells . Long terminal repeat (ltr) and non - ltr retroelements and other sub - telomeric also contribute to the large proportion of repetitive sequences (50% of the genome) in this genome . The largest protein gene family encodes a group of surface proteins known as trans - sialidases (ts), with 1,430 members . Tss are surface molecules identified as virulent factors of t. cruzi that are responsible for transferring sialic acid from host sialogly - coconjugates to the terminal - galactose on t. cruzi mucins . Mucin - associated surface proteins (masp) are the second largest t. cruzi gene family, with a total of 1,377 members . Although masp sequences correspond to ~6% of the parasite diploid genome, they were only identified during annotation of the t. cruzi genome . Masps are glycosylphosphatidylinositol (gpi)-anchored surface proteins that are preferentially expressed in trypomastigotes; these proteins are characterized by highly conserved n- and c - terminal domains and a strikingly variable and repetitive central region (bartholomeu et al ., 2009). Together with the mucin and gp63 gene families, these four gene families account for ~17% of all protein - coding genes and are organized as dispersed clusters of tandem and interspersed repeats . Other large families consist of the previously described rhs and dgf-1 genes whose functions are unknown and which, like the ts genes, occur mostly at subtelomeric locations . Examples of other gene families with more than 10 members also present in the t. cruzi genome include glycosyltransferases, protein kinases and phosphatases, kinesins, amino acid transporters and helicases, in addition to several gene families encoding hypothetical proteins (el - sayed et al ., 2005a). The collapse of nearly identical repeats in some gene families, such as the gene cluster encoding - and -tubulins, meant that not all copies of the family were included in the original genome assembly . 2007) described an analysis of the total genomic repetitive content of protein coding sequences and concluded that 18% of all protein coding sequences existed in 14 or more copies . In addition to the need to evade the host immune system, the existence of highly repetitive gene families in the t. cruzi genome in which a large number of gene copies can lead to the enhanced expression of various proteins may help to overcome a major problem in this genome, namely, the lack of strong promoters capable of generating high levels of mrna from single copy genes . It is also likely that many of the striking polymorphisms among t. cruzi isolates that are reflected in several epidemiological and pathological aspects of chagas disease are partly attributable to variability within regions containing gene families . Whole genome comparisons of distinct t. cruzi lineages are beginning to improve our understanding of this question . Soon after the cl brener genome was completed several groups began sequencing the genome of representative strains of other major t. cruzi lineages . As indicated above, the hybrid nature of the cl brener genome provided data for two genomes, with esmeraldo - like and non - esmeraldo contigs making it possible to distinguish information from t. cruzi ii and iii groups, respectively (see table 1). 2011) published a draft genome sequence of sylvio x10, a strain belonging to t. cruzi i group, which is the predominant agent of chagas disease in central america and in the amazon . Although rarely isolated from humans in endemic areas in southern countries of latin america where most cases of chagas disease with mega - syndromes occur, t. cruzi i strains are highly abundant among wild hosts and vectors (zingales et al . Thus, the distinct ecological niches occupied by t. cruzi i and ii strains, together with the fact these strains are highly divergent in terms of phylogenetic analysis, prompted franzn et al . (2011) to sequence the genome of a representative of t. cruzi i group and to undertake a comparative analysis with the cl brener genome . In agreement with previous analyses, the sylvio x10 genome was estimated to be ~44 mb in size, i.e., smaller than the cl brener genome . Indeed, smaller genomes seems to be a general feature of t. cruzi i strains (branche et al . As expected, the architectures of the two genomes were very similar, with highly conserved syntenic regions corresponding to the gene - dense core of the coding regions organized for long polycistronic transcription . As with the cl brener genome, the presence of repetitive sequences meant that the sylvio x10 genome was represented as fragmented contigs . The technical difficulties associated with the assembly of repetitive sequences meant that only about 49% of the generated sylvio x10 sequence data was incorporated into contigs, leaving 710,109 reads that were not included in the assembly . Consequently, the draft genome of sylvio x10 was assembled into 7,092 contigs, which is slightly less than the number of contigs reported for the draft genome of cl brener . The alignment of these contigs to both cl brener haplotypes showed that the mean nucleotide identity was greater between sylvio x10 and non - esmeraldo (98.2%) than between sylvio x10 and esmeraldo (97.5%). This finding agrees with previous phylogenetic analyses indicating that sequences from t. cruzi i strains are more closely related to t. cruzi iii (represented by the non - esmeraldo cl brener haplotype) than to t. cruzi ii (represented by the esmeraldo - like haplotype) (cerqueira et al ., 2008; ruvalcaba - trejo and sturm, 2011). In contrast to the hybrid cl brener genome, for which the amount of heterozygosity in the core genome was estimated to be 5.5% (el - sayed et al ., 2005a), the diploid sylvio x10 genome was homozygous (<0.08% heterozygosity). Most importantly, analysis of the core gene content of cl brener and sylvio x10 revealed six open reading frames that were missing in the sylvio x10 genome . Besides these six genes, estimations based on total sequence reads indicated that several multicopy gene families, including dgf, mucin, masp and gp63 contained substantially fewer genes in sylvio x10 than in cl brener . A 5.9 mb size difference between the sylvio x10/1 and cl brener genomes largely reflected the expansion of these gene families . However, the extent to which these genomic variations are related to strain differences in host preference and the ability to cause chagas disease remains to be determined . The advent of next - generation sequencing technologies has ushered in a new era in comparative sequencing by allowing the exploration of a wide range of evolutionary and pathological questions within the t. cruzi lineage . A consortium of laboratories funded by the national institutes of health / national institutes of allergy and infectious diseases (niaid) and the national human genome research institute (nhgri) is sequencing t. cruzi strains representative of each one of the six main groups, such as esmeraldo (t. cruzi ii), 3869 (t. cruzi iii), can iii (t. cruzi iv), nrcl3 (t. cruzi v) and tula cl2 (t. cruzi vi) (n. el - sayed, personal communication). Our laboratory has been involved in the sequencing of another t. cruzi i strain (dm28c) and cl-14, a non - virulent strain that belongs to the t. cruzi vi group (s. teixeira, unpublished). In contrast to cl brener, balb / c mice injected with cl-14 trypomastigotes showed no parasitemia but developed high resistance against a lethal challenge with virulent trypomastigotes from the cl brener or y strains (lima et al ., 1995). Our goal in this work is to use comparative analyses of the cl brener and cl-14 genomes to identify potential sequences that can restore the virulence of cl-14 and then test these in transfection protocols . In addition to investigations of the nuclear genome, several studies have examined the mitochondrial genome of kinetoplastids which contains a mass of concatenated dna known as kinetoplast dna (kdna) that is easily identified near the insertion of the flagellum (brener, 1973). In t. cruzi, kdna consists of a highly structured disk - shaped network of thousands of concatenated mini - circles 0.510 kb in size and dozens of concatenated maxicircles 2040 kb in size . Whereas minicircle sequences are present exclusively in kinetoplastids, maxicircles are the homologues of mtdna molecules found in other eukaryotes (lukes et al ., 1997). (2006) described the complete sequences of maxicircle dnas corresponding to groups t. cruzi ii (from sequences of the esmeraldo strain) and iii (from cl brener sequences). As with other trypanosomatid mitochondrial genes, sequence analyses showed that t. cruzi maxicircle dna contained frameshift errors in most of its genes that were corrected at the rna level by a complex u - insertion / deletion process known as rna editing (hajduk et al ., 1993). Key elements of this repair process include grnas (guide rnas) which are encoded mainly by minicircles, although a few grna sequences are also present in maxicircles . The grnas hybridize to the 3 end of a target message and undertake direct u insertion and deletion by the so - called editosome machinery (stuart and panigrahi, 2002). The complete sequences of the 25 kb t. cruzi maxi - circles revealed 18 tightly clustered mitochondrial protein - coding genes and two rrna genes that were syntenic with previously sequenced maxicircles of t. brucei and leishmania tarentolae . Fifteen of the 18 protein - coding genes were edited . Outside the coding region, strain - specific repetitive regions and a variable region that was unique for each strain were identified (westenberger et al ., 2006). More recently, comparative analyses of the mitochondrial genomes of t. cruzi i, ii and iii were reported after ruvalcaba - trejo and sturm (2011) generated the sequence of the coding region of the maxicircle from sylvio x10 . In agreement with the nuclear genomic analysis, phylogenetic analysis of the maxicircle coding regions supported a close evolutionary relationship between t. cruzi i and iii . Based on their mitochondrial dna analyses, these authors proposed a model in which an ancestral strain belonging to t. cruzi i provided the maxicircle for the progeny of a tci - tcii hybridization event that resulted in the generation of t. cruzi iii and t. cruzi iv strains . A subsequent back - cross hybridization between t. cruzi ii and t. cruzi iii strains resulted in the t. cruzi v and vi strains, such as cl brener, that carry the maxicircle from their t. cruzi iii ancestor . Leishmania spp . Are parasitic protozoa transmitted by the bites of phlebotomine sand flies that are endemic in tropical and subtropical regions worldwide . More than 20 species are responsible for a wide spectrum of diseases, known as leishmaniasis (murray et al ., 2005). Parasites in this genus are classified into two subgenera according to the part of the sandfly gut where colonization and development occur: the subgenus leishmania (leishmania) consists of parasites with mid and foregut development, whereas the subgenus leishmania (viannia) consists of parasites that undergo hindgut development (lainson et al ., 1977; bates, 2007). Depending on the species of leishmania, infection of humans may result in diverse clinical forms of leishmaniasis with symptoms ranging from self - healing cutaneous lesions (l. major / l . Infection by leishmania can also result in mucosal leishmaniasis (mainly caused by l. braziliensis) and diffuse cutaneous leishmaniasis (mainly caused by l. in addition to the species of leishmania, other factors such as the genetic variability of the human host may determine the disease tropism and clinical manifestations in leishmaniasis (blackwell et al . The world health organization (who) estimates that there are over two million new cases of leishmaniasis each year, with more than 360 million people at risk of contracting this disease in 88 countries on five continents (asia, africa, europe, north america and south america) (www.who.int/tdrdiseases/leish). As part of their life cycle, leishmania spp . Alternate between the alimentary tract of the sandfly vector, where they grow as extracellular flagellated promastigotes and differentiate into infective non - dividing metacyclic forms, and the phagolysosome of the vertebrate host macrophages, where they differentiate into aflagellated, replicative amastigotes (figure 1). There is no effective vaccine against leishmania and the available therapeutic arsenal is extremely limited (mauel, 2002). Thus, completion of the genome sequences of several leishmania species (ivens et al ., 2005; peacock et al ., 2007) represents a long awaited aspiration for groups involved in the discovery and development of new drugs and vaccine targets . Leishmania major friedlin was chosen as the leishmania reference strain for the tri - tryp genome project . The l. major haploid genome (~32.8 mb) is distributed among 36 relatively small chromosomes ranging from 0.28 to 2.8 mb in size (wincker, 1996) and was sequenced after shotgun cloning of large dna fragments derived from chromosomal bands separated in agarose gels . Prior to publication of the tri - tryp genome sequence, the complete sequences of chromosomes 1 and 3 from l. major were published (myler et al ., 1999; worthey et al ., 2003) and an optical map of the entire genome was generated (zhou et al ., 2004). In 2007, the complete genomes of two other leishmania species, l. infantum and l. braziliensis, were also described (peacock et al ., 2007). Leishmania infantum, also known as l. chagasi in latin america, was chosen as the second leishmania species to have its genome sequenced on the basis of its virulence in animals, transmissibility in sandflies and adaptability to laboratory experimentation (denise et al ., 2006). This species is the causative agent of visceral leishmaniasis, the most serious form of the disease and frequently fatal if left untreated . The new world species l. braziliensis, within the subgenus l. (viannia), is the third and most divergent species sequenced . The l. infantum and l. braziliensis genome sequences were obtained by the whole - genome shotgun approach with five- and six - fold coverage, respectively (peacock et al ., 2007). Importantly, the three complete genomes are from strains that cause distinct types of leishmanial diseases, are adapted for maintenance and manipulation in the laboratory and are also frequently used in studies in vitro and in animal models of infection (laurentino et al ., 2004; ivens et al ., 2005; denise et al ., 2006). The complete sequences of all three leishmania genomes can be accessed in the tri - tryp database; sequencing of the genome from a fourth species (l. mexicana) is in progress . Surprisingly, comparative genomic studies of the three evolutionarily and geographically distinct species, l. major and l. infantum (old world species) and l. braziliensis (new world species), showed very little divergence among the species in terms of genomic sequence and organization (peacock et al ., 2007; lynn and mcmaster, 2008), despite a divergence of 20100 million years within the leishmania genus (lukes et al ., 1997). The haploid genome of the three species has an estimated 8,300 genes, with more than 99% of them maintaining synteny in the three leishmania genomes (peacock et al as with the other tri - tryps, the majority of leishmania genes are annotated as genes of unknown function . Of the 8,300 genes, only 200 were identified as being differentially distributed between the three genomes . The l. braziliensis genome possesses 47 genes that are absent from the other two species, while 27 and 5 genes are specific for the l. infantum and l. major genomes, respectively . Such high conservation in overall genome sequences and genome synteny indicates that the leishmania genome is highly stable and has not undergone large genomic rearrangements during speciation . This finding also suggests that only a few species - specific parasite genes may contribute to differential pathogenesis and tissue tropism (peacock et al . Thus far, the only gene for which there is experimental evidence indicating direct involvement in the differential tropism among leishmania diseases is the a2 locus . Initially identified as an amastigote - specific gene family in l. donovani, a2 has been shown to play a major role in parasite virulence and visceralization (zhang et al ., multiple copies of the a2 gene alternating with a distinct gene termed the a2rel gene are found in the genome of several species of the l. donovani group that is responsible for visceral diseases . Although its precise function is still unknown, the product of the a2 gene, an endoplasmic reticulum protein with a large repetitive domain, may be related to the parasite stress response (mccall and matlashewski, 2010). In l. major, which does not cause visceral leishmaniasis, the a2 gene is a pseudogene and introduction of the l. donovani a2 gene into l. major enhanced the ability of l. major to survive in visceral organs of susceptible balb / c mice (zhang et al ., 2003). More recently, the expression of a2 in l. tarentolae, a lizard parasite that is not pathogenic in mammals, significantly increased the infectivity of this species and enhanced its ability to survive in the liver of balb / c mice (mizbani et al ., 2011). In addition to experiments suggesting a possible role for the a2 gene in the differential tropism of cutaneous and visceral leishmania parasites, the a2 gene has been used as a promising vaccine candidate and diagnostic antigen (fernandes et al ., 2008). Another locus possibly involved in macrophage invasion and that varies considerably among the three leishmania genomes is the gp63 locus . Also known as the major surface protease (msp), gp63 constitutes a family of surface metalloproteases expressed in all trypanosomatids examined so far (yao et al ., 2003). Gp63 sequences identified in the three leishmania genomes and in the t. cruzi and t. brucei genomes vary in their gene copy number, and this may have implications for differences in the disease phenotype (voth et al ., 1998). A similar conclusion may apply to the amastin multi - gene family that encodes a family of amastigote - specific, highly glycosylated hydrophobic surface proteins also present in t. cruzi but which has been greatly expanded in the genus leishmania (teixeira et al ., 1995; interestingly, in the t. brucei genome, which does not have an intracellular stage, only two copies of a highly divergent amastin sequence are present (jackson, 2010). The identification of these species - specific genes represents an initial step towards the characterization of parasite factors that may determine the specificities of each type of parasitic infection . On the other hand, antigens common to all leishmania species could be used as potential vaccine candidates (peacock et al ., 2007). However, apart from differences in gene sequences, it is possible that the distinct clinical manifestations observed in the different parasitic diseases may be a consequence of differential gene expression that occurs throughout the various stages of life cycle in each parasite species . As discussed above, leishmania genes are arranged in the genome as directional gene clusters that resemble prokaryotic polycistronic transcription units (martnez - calvillo et al ., 2004). This type of gene organization and polycistronic transcription have profound implications on the regulation of gene expression, which must rely on post - transcriptional mechanisms (boucher et al ., 2002; myung et al ., 2002; holzer et al ., 2006; leifso et al ., 2007). Since the dependency on promoter - based transcription initiation mechanisms for the control of mrna levels is greatly reduced, greater emphasis is placed on post - transcriptional regulatory mechanisms controlling mrna stability and translation, as well as protein turnover (clayton, 2002). Despite significant differences in life stage morphology, biochemical properties and disease phenotypes, comparative gene expression studies have revealed surprisingly few differences in gene expression when mrna levels in different life - cycle stages or in the same stage but in different leishmania species were compared (peacock et al . Global interspecies analyses in l. major and l. infantum have shown that only 10%12% of differentially expressed genes are unique to each species (rochette et al ., 2008; depledge et al ., 2009). A more careful examination of the protein expression patterns and the elucidation of regulatory mechanisms will provide unique insights into this important aspect of the parasite s biology . This information will also improve our understanding of the host - parasite interaction and lead to the development of new strategies for contolling leishmaniasis . Eukaryotic genomes contain an abundance of repeated dna and some of these repeated sequences are mobile elements . Transposable elements (tes) are defined as dna sequences that are able to move from one location to another in the genome and have been identified in all organisms (prokaryotic and eukaryotic) examined so far . Retroposons, also known as non - long - terminal - repeat (ltr) retrotransposons, are ubiquitous elements that transpose through an rna intermediate and are found in the genomes of most eukaryotes (ivens et al ., 2005; peacock et al ., 2007). Trypanosoma brucei and t. cruzi contain long autonomous retroposons of the ingi clade (tbingi and l1tc, respectively) and short nonautonomous truncated versions (tbrime and nartc, respectively), as well as degenerate ingi - related retroposons devoid of coding capacity (dires) that represent the most abundant transposable elements in these genomes (<3% of the nuclear genome). In contrast, l. major contains only remnants of extinct retroposons (lmdires) and short nonautonomous heterogenous elements (lmsiders). Signature known as lmsiders (for short interspersed degenerate retroposons) have also been identified in the genomes of l. major (ivens et al ., 2005), l. infantum and l. braziliensis (peacock et al ., 2007). Unexpectedly, in l. braziliensis, the site - specific non - ltr retroposon slacs / czar, which is associated with tandemly repeated spliced leader sequences in an arrangement similar to that of the slacs or czar element in t. brucei or t. cruzi, respectively, is fully active (aksoy et al ., 1987; villanueva et al ., 1991 however, in contrast to the african trypanosome genomes and similar to l. major (ivens et al ., 2005) and l. infantum, no potentially active ingi - related retroposons were detected in the l. braziliensis genome (bringaud et al ., 2009). Mobile elements are involved in creating mutations and genomic rearrangements and, in many eukaryotes, these effects can be regulated through an rna silencing mechanism such as rna interference (rnai) (shi et al ., 2004b; girard and hannon, 2008). Since first reported in 1998 (fire et al ., 1998), rnai has swept through all fields of eukaryotic biology and has proven to be a very useful tool for analyzing gene function in a variety of organisms in which the introduction or expression of short double - stranded rnas leads to the rapid destruction of cognate mrnas (figure 4a) (ngo et al ., 1998). This approach has a number of advantages: it is fast, requires very little sequence information, and reduces the expression of multiple gene copies, an action that is especially advantageous in asexual diploid organisms (lacount et al ., soon after it was described in c. elegans, rnai was rapidly identified in t. brucei (ngo et al ., 1998). As discussed below, rnai has proven to be a powerful new tool for functional genomic studies in t. brucei . Unexpectedly, experimental evidence as well as searches of genome databases quickly showed that rnai is absent in l. major and t. cruzi (robinson and beverley, 2003; darocha et al ., 2004). (2007) revealed that the genome of l. braziliensis retained key genes involved with rnai . These authors showed that l. braziliensis genome contains orthologs carrying domains characteristic of the dicer protein as well as genes with the typical argonaute domains paz and piwi, the latter containing conserved amino acid residues that are essential for functional tbago1 (shi et al ., 2004a). In addition, an n - terminal rgg domain, present in tbago1 and shown to be essential for its association with polyribosomes, is also present in the l. braziliensis ago1 gene (shi et al ., 2004a). (2010) demonstrated that the rnai pathway is functional in l. braziliensis and in other species within the leishmania subgenus viannia (l. guyanensis and l. panamensis) (figure 4b). Thus, this divergent species of leishmania appear to have retained not only the mechanisms for (rnai)-mediated regulation but also potentially active retroposons (peacock et al ., 2007), which might have assisted to create the greater divergence within the l. braziliensis genome compared with the other leishmania species . For molecular parasitologists, these findings came as very good news since, as shown in t. brucei, the efficacy of rnai knockdown as a tool for systematic analysis of gene function is now also applicable in some species of leishmania . Infection by t. brucei occurs after metacyclic trypomastigotes are injected into the bloodstream by the bite of a tsetse fly . In the mammalian host, the parasite differentiates into long slender trypomastigotes (bloodstream form -bsf) that divide, colonize the body fluids and can transform into a nonproliferative bloodstream form, also known as the short stump form . After a blood meal, trypomastigotes acquired by the insect vector differentiate into procyclic trypomastigotes in the gut, replicate and then migrate to salivary glands where they transform into metacyclic trypomastigotes (matthews, 2005) (figure 1). The ability of the bsf to invade the central nervous system leads to the neurological manifestations associated with sleeping sickness . In contrast to t. cruzi and leishmania, t. brucei develops extracellularly throughout its entire life cycle . Its direct exposure to a strong antibody response in the bloodstream requires a sophisticated immune evasion protocol, known as variant surface protein (vsg) switching (pays et al ., 2007). Vsgs are bloodstream - specific surface proteins with a hypervariable n - terminal domain that is exposed extracellularly and a more conserved c - terminal domain buried in the parasite s surface coat (van der ploeg et al ., 1982; since they are tightly packed at the surface, vsgs are able to shield other invariant surface proteins from attack by the immune system . The tactic for evasion is based on antigenic variation in which the expression of a specific vsg is replaced by another gene from a supply of almost thousand copies at a rate of approximately one event per 100 cell divisions (van der ploeg et al ., 1982; turner and barry, 1989). Vsg genes are thus a family of t. brucei - specific genes whose monoallelic expression distinguishes african trypanosomes from the other two groups of pathogenic trypanosomatids; the lack of antigenic variation in t. cruzi and leishmania spp . Means that these species must evade the host s immune system by entering and multiplying inside host cells . After completion of the t. b. brucei genome, it was found that of the 9,068 predicted genes, including 904 pseudogenes, there were 1,700 t. brucei - specific genes, 806 of them (or 9% of the whole genome) corresponding to vsgs (el - sayed et al ., 2005b). Several vsg genes occur within hundreds of mini - chromosomes (50150 kb in size) that are also part of the t. brucei genome . Surprisingly, analyses of all vsg sequences showed that only 57 are fully functional genes and have all the recognizable features of a typical vsg (berriman et al ., 2005). It has been long known that vsg variability can be generated in t. brucei by creating mosaic genes that include pseudogenes (roth et al ., 1989). Besides producing variability in vsg epitopes, these rearrangements may have the capacity to generate protein with new functions, as in the case of the sra gene discussed below (vanhamme et al ., 2003). As part of their life in the bloodstream, african trypanosomes that are pathogenic to humans have developed a mechanism to withstand other types of attack from the host immune system . In contrast to t. brucei brucei, which do not infect humans, t. b. gambiense and t. b. rhodesiense are resistant to trypanolytic factors (tlf) present in normal human serum (nhs). Tlf activity has been attributed to apolipoprotein l1 (apol1) and haptoglobin (hp)-related protein found in high density lipoprotein (for a review, see wheeler, 2010). Nhs resistance in t. b. rhodesiense is conferred by a truncated vsg known as serum resistance associated (sra) protein which is located in endosomes and binds and neutralizes tlf (de greef and hamers, 1994; de greef et al ., 1992;, 2003; prez - morga et al ., 2005; wheeler, 2010). The killing of t. b. brucei requires the binding of tlf-1 and trafficking to the parasite acidic lysosome . It has been proposed that, in t. b. gambiense, changes in the receptor that binds tlf, as well as decreased expression of this gene, are responsible for the resistance of this t. brucei sub - species to attack by the human innate immune system (kieft et al ., 2010). Since t. b. brucei is harmless to humans and t. b. gambiense is the most clinically relevant subspecies, jackson et al . (2010) sequenced the genome of t. b. gambiense using the whole - genome shotgun approach combined with bacterial artificial chromosome large insert sequencing . Comparison of the sequences from a draft genome assembly of 281 contigs (~22.1 mb) with the t. b. brucei genome data (berriman et al ., 2005) revealed a very similar composition in terms of gene content, gene synteny and sequence identity (86.4% of t. b. gambiense coding sequences vary by <1% from their orthologs in t. b. brucei). Even though these subspecies behave differently in humans, there were very few differences in their genomes . One of these differences involved a gene encoding a putative iron - ascorbate oxidoreductase that is specific to a few t. b. brucei strains and is also absent in other trypanosomatids (l. major and t. cruzi). Thus, it seems that not only differences in gene content per se, but also individual single nucleotide polymorphisms (indels) and variations in gene expression, or a combination of these factors, may contribute to this phenotypic variation (jackson et al ., 2010). Coordinated changes in gene expression are vital for trypanosomes since they must deal with rapid changes in their environment (nutrient availability, temperature, host defenses, the presence of drug, etc .) And be able to disseminate by alternating through different hosts . In addition, unique mechanisms for regulating gene expression are required since there is an almost complete absence of transcriptional control at the level of initiation . The lack of transcriptional regulatory elements, including a typical rna polymerase ii promoter, led to the conclusion that most factors involved in controlling gene expression act at the post - transcriptional level . By using approaches such as differential display, rna fingerprinting, differential screening of cdna libraries, random sequencing of cdna clones and dna microarrays, several groups have shown that significant changes in mrna levels occur during the life cycle of all tri - tryps (teixeira et al ., 1995; el - sayed et al ., 1995; mathieu - daud et al . Experimental evidence also indicates that, in addition to mrna stability, changes in polysomal mobilization constitute an important mechanism for regulating gene expression (alves et al ., 2010). With the advent of next generation sequencing technologies such as rnaseq, a global description of gene expression patterns in t. brucei has finally been achieved (kolev et al ., 2010; nilsson et al ., 2010; siegel et al ., 2010; veitch et al . This approach has provided much more complete information about mrna structure and expression levels, including the patterns of spliced leader (sl) and poly - a additions, as well as alternative pre - mrna processing . Moreover, these global gene expression analyses have confirmed that only two t. brucei genes (poly - a polymerase and dna / rna helicase) contain introns . 2010) showed that 2,500 alternative splicing events occur during processing of t. brucei genes, with a large number of these being regulated during the life cycle (a total of 600 genes have transcripts with more than one trans - spliced variant). The alternative splicing reactions can alter the message dramatically, as shown by nilsson et al . (2010) and represented in figure 3, with changes in the sl addition site leading to alterations in regulatory elements in the 5utr (resulting in the modification of gene expression) or the initiator aug (generating a different n - terminus or even causing a complete change in the translated orf). Aminoacyl trna synthetase transcripts are interesting examples of trans - spliced variants since differences in the enzyme n - terminus result in changes in the protein targeting signal: distinct mrnas produce proteins that are directed to mitochondria or remain in the cytoplasm (nilsson et al ., 2010). Likewise, alternative splicing is a potential mechanism for dual localization of the t. cruzi lyt1 gene (benabdellah et al ., 2007). In addition to providing a mechanism for generating changes in the proteome, such flexibility in the selection of splicing sites is compatible with evidence that polycistronic transcription may also initiate at internal sites in gene clusters since correct processing of the 5 end of mrna can occur at various points in the primary transcript (kolev et al ., 2010). Recently, chromatin immunoprecipitation in combination with conventional sanger sequencing (respuela et al ., 2008) or next - generation sequencing technology (siegel et al ., 2009; thomas et al ., 2009; wright et al ., 2010) has provided a much awaited analysis of the distribution patterns of modified histones throughout the t. brucei genome . As summarized in figure 3, the divergent strand - switch regions (ssr) have been found to contain an enrichment of histone variants (h2az and h2bv), acetylated histone 4 at lysine 10 (h4k10ac), histone 3 acetylated at residues k9 and k14 and trimethylated at k4, the bromodomain factor bdf3, and the transcription factors trf4 and snap50 (respuela et al ., 2008; siegel et al ., 2009; thomas et al ., 2009; wright et al ., 2010). The histone variants h3v and h4v are present at transcription termination sites (siegel et al ., 2009). Similar patterns of chromatin modifications from transcription start sites (tss) located at the beginning of polycistronic units were also found internally in the same polycistronic unit, suggesting the presence of internal tss, in agreement with rnaseq data (siegel et al ., 2009; kolev et al ., 2010; the increasing number of published genomes and transcriptomic data, partly as a consequence of the introduction of new sequencing platforms, has created enormous challenges for the field of functional genomics . One of the most useful techniques that has been used to identify gene function in t. brucei is a combination of the inducible system mediated by t7 rna polymerase and the tetracy - cline repressor (wirtz et al ., 1999) in combination with rnai gene knockdown . As indicated before, in contrast to t. cruzi and l. major, rnai is functional in t. brucei (ngo et al ., 1998; robinson and beverley 2003; darocha et al ., 2004; lye et al ., 2010). The first large scale functional genomics study using rnai was reported by morris et al . (2002), who transfected t. brucei procyclic forms with a random rnai library . This library was generated by using a construct to inducibly express dsrna via two head - to - head tetracycline - regulated t7 rna polymerase promoters . By selecting parasites that expressed the dsrna but were unable to bind lectin, these authors identified clones with a reduced glycosylation of surface proteins and showed that these clones had lower expression of hexokinase (morris et al ., 2002). A few years later, the functions of 197 orfs from t. b. brucei chromosome i were tested by rnai . Rnai - induced parasites were tested for growth, nuclear and kinetoplast abnormalities by dapi staining, and a pleio - tropic phenotype, morphology and motility . At least one of these phenotypes was found in 68 individual knockdowns (subramaniam et al ., 2006). In a similar approach, portman et al . (2009) identified novel components of the para - flagellar rod (pfr) structure by comparing proteomic profiles before and after ablating the expression of individual pfr proteins . The silencing of pfr1 and pfr15 was evaluated by two - dimensional difference gel electrophoresis, and the spots with a two - fold change in volume were subjected to tandem ms protein identification . Proteomic analysis combined with rnai knockdown led to the identification of 30 proteins as potential pfr components, 20 of which were novel proteins . High - throughput cloning systems also enhance functional analyses . By using the gateway technology to create yeast two - hybrid vectors, eight non - redundant protein - protein interactions were detected among proteins with a pfr structure (lacomble et al ., 2009). These authors were able to construct a map showing the complex interaction of pfr proteins . The availability of efficient methods for genetic manipulation and for testing gene function through rnai knockdown has led to major advances in genomic, transcriptomic and proteomic analyses of t. brucei, and has made this species a model organism for studying basic aspects of trypanosomatid biology . More recently, with the discovery of functional rnai machinery in l. braziliensis and other leishmania species, gene function studies can now be conducted in this group and will soon provide valuable new information about leishmania - specific genes . However, as is becoming increasingly apparent from the data generated by comparative genomic analyses, each of the trypanosomatid species has its peculiarities . Researchers thus face the challenge of developing new protocols specific for studying each parasite and its corresponding diseases . With our current knowledge of each tri - tryp disease and the new research methods that are being developed we can expect many new studies to emerge from hypotheses based on the tri - tryp genomic data . As a consequence of these new findings,
Cardiovascular disease (cvd) is the most prevalent cause of death in developed nations and it is increasing in prevalence in developing countries . While many factors contribute to the development of this disease in adults, such as, smoking, male gender, blood pressure, elevated cholesterol, diabetes, and renal failure, the mechanisms underlying cvd are still not fully understood [14]. One of the main problems in clinical practice is that the symptoms become evident late in the course of the disease . In fact, asymptomatic processes, that are associated with plaque formation, develop causing silent yet progressive tissue damage . If atheroma plaques finally rupture, highly thrombogenic material is released and an atherothrombotic event occurs . In this context, there is an urgent need to find out novel biomarkers of practical value for clinical intervention which, alone or combined with existing ones, allow cardiovascular risk prediction at individual level . Currently, controversy exists regarding contribution of biomarkers to the information derived from conventional risk factors . When novel markers utility for predicting cvd was investigated in a wide cohort of more than 5000 individuals without cvd, the gains over conventional factors resulted to be minimal . However, positive outcomes are expected when high - risk populations are investigated; thus, the risk level of selected patients, the chosen biomarkers to be investigated and other factors, such as, statistics, highly influence expected results . Combination of multiple biomarkers in assessment of individual responses adds only moderately to standard risk factors . Therefore, there is a substantial interest in the discovery and use of newer biomarkers, to complement the best existing ones and to identify persons who are at risk for the development of cardiovascular disease and who could be targeted for preventive measures . In particular, finding biomarkers that predict the risk of rupture will provide the opportunity to institute a preventive life style and permit timely pharmacological treatment . Currently, the improvements in outpatient and inpatient care, diagnosis and biomarker discovery have reshaped the landscape of cvd . It is important to note that the new diagnostic methods currently available are based on noninvasive techniques that, although they present a number of benefits, may be limited in terms of specificity, sensitivity, availability and cost . -omics technologies has provided sensitive, fast and robust tools to analyze biomarkers in cvd . Metabolites are small molecules that participate in general metabolic reactions and that are required for the maintenance, growth and normal function of a cell . The term metabolome, derived from the word genome, refers to the complete set of metabolites in an organism and its organelles [7, 8] or the total complement of metabolites in a cell . In this way, metabolomics and metabonomics refer to the use of analytical methods to identify and quantify all metabolites in a biological system, as well as the monitoring of changes in the metabolome of a biofluid, cell culture or tissue sample following perturbation [8, 10]. In parallel with genomics, transcriptomics and proteomics, application of metabolomic technologies to the study of cvd will increase our understanding of the pathophysiological processes involved and this should help us to identify potential biomarkers to develop new therapeutic strategies [11, 12]. Indeed, the identification and quantification of these low molecular weight molecules (e.g., lipids, amino acids, and sugars) will define the phenotype of these diseases . From a clinical perspective, the study of metabolic changes that occur in response to different physiological processes will help establish the mechanisms underlying the disease . In terms of personalized medicine, pharmacometabonomic approaches can serve to predict the action of specific drugs in a particular individual based on the predose urinary metabolite profile . Furthermore, the gut bacterial fauna influences drug efficacy, which could be deliberately modified to optimize the benefits and minimize adverse effects of a given treatment . In addition, this approach will help understanding how drugs act during patients' recovery or how they influence outcome . There are several analytical strategies that can be used to analyse the metabolome, such as nuclear magnetic resonance (nmr), fourier transformation infrared spectroscopy (ft - ir) [17, 18], and mass spectrometry (ms) coupled to separation techniques such as high performance liquid chromatography (hplc), gas chromatography (gc), or capillary electrophoresis (ce). The combination of these different analytical techniques offers important advantages when analyzing the complete metabolome . High field h nmr is one of the preferred platforms for urine and plasma analysis [19, 20], as it is a nondestructive technique that does not require prior separation of the analytes and it provides detailed information on molecular structure . For example, the capacity to predict the occurrence of exercise - induced ischemia in patients with suspected cad was investigated by nmr blood analysis, demonstrating lactate, glucose, lipids, and long - chain fatty acids to be the main metabolites involved . Xanthine and ascorbate were proposed as possible markers of plaque formation in an atherosclerotic mouse model and lipoprotein subclasses can now be analyzed by a commercial nmr - based protocol called nmr lipoprofile [23, 24]. However, one of the main limitations of nmr is the poor sensitivity, although this can be improved enormously when it is combined with mass spectrometry . Coupled to a separation technique, ms has recently been introduced into the metabolomics field and its use in such studies will constitute the main focus of this paper . Indeed, gas chromatography / mass spectrometry (gc - ms), liquid chromatography / mass spectrometry (lc - ms) and capillary electrophoresis / mass spectrometry (ce - ms) are the most powerful techniques for metabolite separation and analysis . Gc - ms provides an extraordinary resolution, permitting the separation of structurally similar compounds that would otherwise be very difficult to separate by hplc . However, this technique requires the analyte to be volatile and thermally stable . In some cases, a chemical derivatization step is required prior to the chromatographic separation in order to render polar metabolites volatile . Some of the metabolites best suited for gc - ms include fatty acids, organic acids, steroids, di - glycerides, sugars and sugar alcohols . For those metabolites that are not volatile and which cannot be derivatized, thus, lc - ms can analyze a much wider range of chemical species (polar and nonpolar metabolites) with ample selectivity and sensitivity . Apart from reversed phase chromatography (rp - lc), which is widely used in metabolomics applications, hydrophilic interaction chromatography (hilic) is a complementary approach suitable for very polar metabolites (nonvolatile). Indeed, the metabolites suited to analysis by gc or hplc can be represented according to their polarity (see scheme in figure 1). Similarly, capillary electrophoresis (ce) can be coupled to a mass spectrometer (ms), with the particular advantage of improving the resolution of separation as narrower peaks than with lc are obtained . Accordingly, different approaches have been described in combination with ion trap (it), triple quadrupole (qqq), time of flight (tof), and q - tof instruments . The main advantage of qqq and q - tof instruments is that they provide the possibility of identifying the compounds by tandem ms / ms analyses . In order to obtain a full overview of the detectable molecules, electrospray ionization (esi) irrespective of the analytical approach used in metabolomics, particular care has to be taken in preparing the sample . Bearing in mind that the typical half - lives of metabolic reactions in an organism are less than 1 s, it is important not to monitor metabolic changes extrinsic to the pathology or drug effect under study, producing misleading interpretations of the situation . Evidence - based epidemiological studies have led to the discovery of well - established biomarkers . These studies now tend to be complemented by control - case investigation using a different methodology, based on two main stages: the discovery phase, resulting in a set of novel biomarkers candidates and the validation phase, where discovered potential biomarkers are further validated in a different cohort of samples . In this context, biological variation would be expected to be higher than the analytical variability and thus, it is essential to pay particular attention to: (a) the precise definition of a clinical phenotype (in this sense, network - based analysis on associations among genes, proteins, metabolites, and environmental factors would be encouraged to increase sensitivity and selectivity of the diagnosis) and (b) group matching in terms of sex, age, lifestyle, diet, or pharmacological treatment, otherwise attempts may fail in terms of disease prediction . For instance, gender and statins treatment strongly influence the findings in studies of cvd and when individuals with normal coronary arteries were compared with cvd patients, a> 99% confidence limit was only obtained for 6% of the predictions in the treated groups . Technical reproducibility and sampling time are also critical to minimize external factors that will influence the results and their clinical relevance . Ideally, snapshots of different conditions should be taken so that they can be quantitatively compared . If all these considerations are kept in mind, metabolomic research can set out to identify characteristic patterns that can be used for diagnostic purposes and risk prediction, substituting traditional, more expensive clinical approaches (e.g., angiography). In principle, metabolites can be measured in several body fluids or tissues, although plasma and urine are the most commonly used biological matrices in cardiovascular research due to their availability and clinical relevance as a source of potential biomarkers . Almost all cells in the body communicate with the plasma, either directly or through different tissues and biological fluids, releasing at least part of their intracellular content . By contrast, urine is produced by renal filtration of the plasma and it is widely considered as one of the most important samples for diagnosis as it contains not only many plasma components but also the catabolic products of different metabolic pathways . Sample pretreatment varies depending on the analytical platform chosen (see the common strategies employed in figure 2). Metabolites from frozen tissue samples can be extracted and simultaneously fractionated by treating the ground tissue with mixtures of organic solvents, such that molecules are extracted in different fractions according to their polarity . If a biological fluid is the starting material (urine, serum, plasma), metabolite fractions are usually obtained after proteins are removed by precipitation . The crude or diluted sample can then be injected directly, although matrix effects causing ion suppression should be expected . If analyzed by lc - ms(/ms), it may be desirable to preconcentrate (e.g., by lyophilisation) or fractionate the sample prior to chromatographic separation . In case of gc - ms(/ms), preconcentration can be performed by solid phase microextraction (spme) with or without head space (hs) procedures, which are particularly useful when analysing volatile organic compounds (vocs). For ce analysis three complementary approaches are used for metabolic research (see figure 3): metabolic fingerprinting, metabolic profiling, and metabolic footprinting . In the first case, and like proteomics strategies, an unbiased analysis is performed that is oriented towards defining clinically relevant differences rather than identifying all the molecules present in a sample . Alternatively, metabolic profiling involves a preselection of a set of metabolites, or a specific class of compounds, that might participate in a targeted pathway . Metabolic footprinting does not rely on the measurement of intracellular metabolites but rather, on monitoring those that are secreted or fail to be taken up by a cell or tissue [31, 32]. Below, we will discuss relevant findings from these approaches in cvd (a compilation of the main studies is shown in table 1). Metabolic fingerprinting does not aim to identify the entire set of metabolites but rather to compare patterns or fingerprints of metabolites that change in response to a disease state, pharmacological therapies or environmental alterations, for example . A wide variety of biological matrices can be used for metabolic fingerprinting, such as urine, plasma / serum, tissues / cells and saliva . This approach can be used as a diagnostic tool to evaluate the disease state by comparing healthy controls and disease subjects, or to assay the success of a particular treatment (prognosis / recovery). However, if we want to understand the mechanisms underlying a disease, qualitative and quantitative analyses are required . Once a differential pattern is discovered, which provides information that can be considered as the pathological phenotype, further steps to identify the participating compounds (qualitative) and to determine the absolute amounts of metabolites that participate in the processes studied (quantitative) must be followed . This is not a trivial issue and prior to embarking on the task of discovering metabolic biomarkers, sufficiently sensitive and selective instruments and extensive compound libraries for metabolite identification should be available, while certain expertise in data analysis and interpretation will be necessary . One of the few metabolomic studies in the field of cvd involved a comparison of the metabolomic fingerprint obtained by gc - ms of plasma samples from non - st - segment elevation acute coronary syndrome (nsteacs) patients, stable atherosclerosis patients and healthy patients . Citric acid, 4-hydroxyproline (4oh - pro), aspartic acid and fructose were found to decrease in nsteacs patients, whereas lactate, urea, glucose, and valine increased . Both lactate and glucose are also involved in prediction of exercise - induced ischemia in patients with suspected cad . The decreased in 4oh - pro was especially interesting because circulating 4oh - pro is thought to prevent the binding of ldl to lipoprotein previously deposited in the vascular wall, as well as releasing already deposited ldl from the atherosclerotic lesions . It is also a component of collagen, which confers stability to the atherosclerotic plaque . The high resolution of ce - ms makes it a powerful technique to separate and analyse charged metabolites, although only a few metabolomic applications have been published to date . The isolation of polypeptide fraction from urine or plasma was analyzed by ce - ms and used to discriminate between coronary artery disease (cad) and non - cad patients with clinical symptoms and who had been subjected to coronary angiography . The stability of urine samples and their resistance to oxidation or precipitation reflect the advantages of this biological fluid for proteomic analysis . Polypeptide profiling in urine is more reproducible than in plasma, with no significant loss of polypeptides over time when performing consecutive analyses over a 24-hour period, which also demonstrates the reproducibility of the ce - ms . In total, 200 of the most abundant polypeptides were detected and a set of 17 urinary polypeptides permitted cad and non - cad patients to be distinguished . Among them, collagen -1 (i and iii) was augmented in cad samples, which was corroborated by their increased expression found in atherosclerotic plaques . Metabolite profiling focuses on the analysis of a group of metabolites related to a specific metabolic pathway [38, 39]. In this approach, technological advances have increased the number of metabolites that can be quantified simultaneously . Moreover, the results of metabolic profiling are quantitatively independent of the technology used for data acquisition . Metabolite profiling has been applied to cvd in order to identify and quantify metabolites that might serve as new biomarkers . A metabolite profile of peripheral blood from individuals undergoing planned myocardial infarction (pmi) has been established . Serial blood from 36 patients were obtained before and at various intervals after pmi, and the changes in circulating levels of metabolites were identified by mass spectrometry - based metabolite profiling . Most alterations produced by pmi were observed in the tricarboxylic acid cycle, in purine and pyrimidine catabolism, and in the pentose phosphate pathway . Indeed, 7 metabolites were significantly affected immediately 10 minutes after the onset of myocardial injury (p <.005): alanine, aminoisobutyric acid, hypoxanthine, isoleucine / leucine, malonic acid, threonine, and trimethylamine n - oxide (tmno). All these alterations were especially interesting as they were observed before any significant rise in the clinically available biomarkers in plasma (ckmb and troponin t). After 60 minutes, six new metabolites had also changed significantly (p <.005): 1-methylhistamine, choline, inosine, serine, proline, and xanthine, with the later being a candidate of a marker for plaque formation in an atherosclerotic mouse model . The anatomic origin of the early metabolic changes observed was further explored in a subgroup of 13 patients by simultaneously comparing the metabolite levels obtained in samples from peripheral blood and from a catheter placed in the coronary sinus . A further 8 metabolites were transmyocardially enriched at least 1.3-fold 10 minutes after pmi (taurine, ribose-5-phosphate, dcmp, lactic acid, amp, malic acid, glutamine and glutamic acid) and once 60 minutes had passed, six additional metabolites augmented (glycerol-3-phosphate, orotic acid, succinic acid, glycerate-2-phosphate, taurine and malic acid). Plasma and urine samples from atherosclerotic and control rats have been compared by ultra fast liquid chromatography coupled to ion trap - time of flight (it - tof) mass spectrometry (uflc / ms - it - tof). Accordingly, 12 metabolites were identified as potential biomarkers in rat plasma and 8 metabolites in rat urine . The concentration of leucine, phenylalanine, tryptophan, acetylcarnitine, butyrylcarnitine, propionylcarnitine and spermine decreased in plasma, and 3-o - methyl - dopa, ethyl n2-acetyl - l - argininate, leucylproline, glucuronate, n(6)-(n - threonylcarbonyl)-adenosine and methyl - hippuric acid were diminished in the urine of atherosclerosis rats . Conversely, ursodeoxycholic acid, chenodeoxycholic acid, lpc (c16:0), lpc (c18:0) and lpc (c18:1) increased in plasma and hippuric acid augmented in the urine from atherosclerosis rats . The alterations to these metabolites reflected the abnormal metabolism of phenylalanine, tryptophan, bile acids and amino acids . Lysophosphatidylcholine (lpc) plays an important role in inflammation and cell proliferation, highlighting the relationship between lpc with the progress of atherosclerosis and other inflammatory diseases . The lipidomic profile of mice liver homogenates from cholesterol - free, low cholesterol and high cholesterol diets demonstrated the influence of dietary cholesterol intake and atherosclerosis . To obtain individual metabolite fingerprints, nearly 300 metabolites were measured in plasma samples by lc - ms / ms, including di- and tri - glycerides, phosphatidylcholines, lysophosphatidylcholines and cholesterol esters . When dietary cholesterol intake increased, the liver compensated for the elevation in plasma cholesterol by adjusting metabolic and transport processes related to lipid metabolism, which leads to an inflammatory, pro - atherosclerotic state . A cholesterol - free diet did not induce early atherosclerosis, while the low cholesterol diet only mildly induced early atherosclerosis . By contrast, intense early atherosclerosis was induced by the high cholesterol diet, in association with proinflammatory gene expression . Indeed, a relationship appears to exist between cholesterol intake (measured as cholesterol plasma levels) and atherosclerotic lesion size . The lipidome of cell membranes and tissues has been studied by measuring the plasmalogens contained in rabbit and rat myocardial nuclei by esi - ms . Plasmalogen is an ether lipid where the first position of glycerol binds a vinyl residue with the double bond next to the ether bond . The second carbon has a typical ester - linked fatty acid and the third carbon usually has a phospholipid head group, which can protect cells against the damaging effect of singlet oxygen . This seems to be the reason for the strong enrichment of plasmalogens found in the membrane of myocardial cells . Metabolic changes associated to atherosclerosis have also been investigated through nmr and gc - ms metabolite profiling . There are clear biochemical explanations to these findings, and the alterations to these metabolites, which cause the final atherosclerotic lesion, can be related to different disorders . For instance, insulin resistance in diabetic patients increases the activity of transaminases, which are critical enzymes in amino acid metabolic pathways . Hence, if the insulin response is deficient, these amino acid pathways will be altered, and many others metabolites will be affected such as glutamate, ketoglutarate, succynyl - coa, 4-oh - l - proline (4ohpro), 2-hydroxybutyrate, creatinine, pyruvate, oxaloacetate, malate, glycolate and 2,3,4-trihidroxybutirate . These effects could indicate damage to tissue at the intima artery walls . The myocardial metabolic response has been investigated in cad and left ventricular dysfunction (lvd) patients, both at baseline and following ischemia - reperfusion (i / r). Accordingly, glucose, lactate, free fatty acids, total ketones, 3-hydroxybutyrate, pyruvate, leucine / isoleucine and glutamate are present at lower concentrations in a preischemia state in the coronary sinus (cs) than in arterial samples (reflecting myocardial uptake). By contrast, the alanine concentration is higher (reflecting release). A principal components analysis (pca) shows several potentially important postoperative metabolic changes during the clinical course of the disease . Ventricle dysfunction are associated with the global suppression of metabolic fuel uptake, and limited myocardial metabolic reserves and flexibility following global i / r stress is associated with cardiac surgery . The citric acid cycle plays an important role in oxidative phosphorylation and atp production in cardiomyocytes, and citric acid cycle intermediates are supplied by glycolysis and -oxidation of fatty acids . Metabolomic profiling based on quantitative mass spectrometry was also used to study the heritability of premature coronary disease in 117 individuals unaffected by cad but with a family member affected . There was a string heritability of amino acid levels such as arginine, ornithine, alanine, proline, leucine / isoleucine, valine, glutamate / glutamine, phenylalanine and glycine, free fatty acids such as arachidonic, palmitic, linoleic and acylcarnitines . Hence, it was concluded that metabolic changes associated with cad can be inherited and they are strongly related to age . This would indicate that metabolic processes could be controlled genetically, implying a correlation between genotype and phenotype in families with cad . More recently, a subset of 69 metabolites was shown to have diagnostic value, such that some derived factors showed discriminative capability for cad after pca . Moreover, a signature composed of dicarboxyacylcarnitines was predictive of further cardiovascular events in those patients and most significant differences persisted after adjustment for cad risk factors . Metabolic changes in human atrial fibrillation (af) have been investigated by nmr, performing a quantitative analysis of 24 previously selected metabolites . Significant differences were found for beta - hydroxybutyrate, ketogenic amino acids and glycine, all of which augmented in af patients when compared to control subjects, suggesting a pathological role for ketone bodies . Metabolic profiles enable more than 80% of patients at risk of af at the time of coronary artery bypass grafting to be classified, as a discordant regulation of energy metabolites was found to precede post - operative af . The effect of drug treatment on apoe mice was investigated by nmr analysis of metabolites in urine, showing allantoin to act as a marker for drug treatment, and xanthine and ascorbate as possible markers of plaque formation (both were elevated in untreated mice). The application of metabolic analysis to cardiovascular diseases is an emerging field, and at this incipient stage it is not possible to clearly define a metabolic picture which is responsible for cvd prediction and progression . Further metabolomic investigation promises to improve researchers and clinicians knowledge of these diseases in three critical ways . Firstly, a complete description of the metabolites altered in a disease will better define the pathophysiology of the disease . Secondly, metabolic profiling will enhance the feasibility of high - throughput patient screening to diagnose the disease state or risk evaluation . Indeed, the identification of clinically relevant changes in circulating metabolites that may be considered as potential new biomarkers will also help with the evaluation of prognosis and will contribute to the development of new therapeutic strategies . Thirdly, metabolite profiling will enable the effects of pharmacological treatments to be monitored, in particular, assessing the individual's response to a particular drug . In contrast to genomics, metabolomics defines dynamic states that reflect the actual status of an organism, which requires the control of many variables (from an individual's status to metabolite degradation following sample collection). Failure to do so may lead to the production of erroneous results and misleading conclusions . Minimal protocol - specific differences can produce inconsistent findings, which must be clearly overcome prior to proposing the use of a biomarker to the scientific community . Similarly, the results must be confirmed in a validation cohort composed by a different set of samples than that used in the discovery phase . Adequate follow - up studies must corroborate earlier predictions, and adjustment for conventional risk factors to assess significant contribution of a discovered metabolite to current knowledge should be included . To date, there have been considerable efforts in improving instrumentation (e.g., mass spectrometry) and the analytical methods suitable to complement these approaches (e.g., based on nmr), resulting in an expansion of the metabolites with potential roles in the development of atherosclerosis that can be quantified . However, further research is still needed prior to proposing an ideal platform for metabolite analysis that can replace conventional cvd diagnosis in clinical practice . With the growth of public metabolite databases, further improvements in the sensitivity and selectivity of analytical techniques and the development and routine use of novel platforms of demonstrated potential,
A 13-year - old, castrated male, domestic cat that lived indoors in a single - cat household was brought to the iowa state university lloyd veterinary medical center because of depression, inappetance, and respiratory signs of 4 days duration . The family members noted that the cat was reluctant to lie in lateral recumbency and instead rested in sternal recumbency with neck extended, which was indicative of dyspnea . The cat s vaccination status was up to date . Before the onset of clinical signs in the cat, 2 of the 3 family members had experienced an undiagnosed influenza - like illness an upper respiratory tract infection characterized by fever, coughing, and myalgia that lasted 3 days . Onset of the cat s clinical signs was noted 6 and 4 days after onset of illness for the first and second family members, respectively . At the time of examination, the cat had bilateral adventitial lung sounds (wheezes), was afebrile, and was clinically dehydrated . Cytologic and microbiologic examination of bronchoalveolar lavage (bal) fluid showed foamy macrophages (65%), nondegenerate neutrophils (25%), and small lymphocytes (10%). Standard microbial culture of bal aliquots yielded no substantial growth of aerobic or anaerobic bacteria . Radiographic and cytologic findings were inconsistent with bacterial or parasitic pneumonia and not supportive of allergic airway disease . A viral cause was considered most likely; however, the cat was given amoxicillin with clavulanate (125 mg orally 2/day) to reduce the possibility of secondary bacterial pneumonia . Notable findings from laboratory testing (complete blood count, serum biochemistry, urinalysis, and total thyroxine measurement) were moderate leukopenia characterized by a moderate lymphopenia, modest hemoconcentration, and a slightly elevated thyroxine level . Radiographs of the thorax of a cat with confirmed influenza a pandemic (h1n1) 2009 virus infection . An alveolar pattern, composed of air bronchograms with border - effaced (indistinct) adjacent pulmonary vessels, is most pronounced in the left caudal lobe . A small gas lucency in the pleural space appears in the right caudal and dorsal thoracic cavity . An endotracheal tube is visible at the thoracic inlet on the lateral view in this moderately obese cat . Pcr testing (feline urd panel; idexx laboratories, westbrook, me, usa) of a bal sample showed negative results for chlamydophila felis, feline calicivirus, feline herpesvirus-1, bordetella bronchiseptica, and mycoplasma felis . Results of feline immunodeficiency virus (antibody) and feline leukopenia virus (antigen) testing (idexx snap fiv / felv combo test; idexx laboratories) were also negative, ruling out the potential that viral - induced immunosuppression was a concurrent factor . For the following reasons we included pandemic (h1n1) 2009 on our list of differential diagnoses: recent history of respiratory disease in household family members, known widespread community prevalence of pandemic (h1n1) 2009 influenza in humans, paucity of common viral infections causing infectious caudodorsal alveolar pneumonia in adult cats, and documented susceptibility of felids to avian influenza (h5n1) (12,13). We therefore submitted a bal sample to the iowa state university veterinary diagnostic laboratory for molecular screening and typing for influenza a and the pandemic (h1n1) 2009 virus . Rna was obtained from the bal fluid by using the magmax viral rna isolation kit (applied biosystems, austin, tx, usa) and a semiautomated magnetic particle processor (kingfisher 96; thermo electron corp ., pcr (rrt - pcr) influenza a screening assay specific for the nucleoprotein gene . Preliminary differentiation of pandemic (h1n1) 2009 virus from other h1 or h3 types of influenza a was performed by using an in - house rrt - pcr assay that distinguishes between pandemic (h1n1) 2009 [eurasian matrix (10)] and endemic (to north america) swine h1n1 influenza viruses (north american matrix). Pcrs were conducted by using the agpath - id multiplex one - step rt - pcr kit (ambion / applied biosystems) according to manufacturer s recommendations; 10 units of multiscribe reverse transcriptase (applied biosystems) were added per reaction . Thermocycling was performed by using the applied biosystems 7500 fast real - time pcr system according to manufacturer s recommendations . * primers and mgb probes were obtained from integrated dna technologies (coralville, ia, usa) and applied biosystems inc . Pcr testing showed the bal sample to be positive for influenza a virus (nucleoprotein gene), and the virus was determined to contain the matrix (m) gene of the pandemic (h1n1) 2009 virus strain . A bal sample was submitted to the us department of agriculture national veterinary services laboratories (ames, ia, usa) for confirmatory testing . Rrt - pcr confirmed that the bal sample was positive for the m gene of influenza a virus and the neuraminidase (n) gene of pandemic (h1n1) 2009 virus . A cytolytic virus was isolated by using mdck cells (8) and was designated as a / feline / ia / nvsl026991/2009 . Pcr testing of the isolate for influenza a virus (m gene) and n1 gene of pandemic (h1n1) 2009 showed positive results . Sequence analyses for hemagglutinin (ha), n, and m genes confirmed that the virus was pandemic (h1n1) 2009 virus (genbank accession nos . Gu332630 (for ha), gu332632 (for na), and gu332631 (for m). Nucleotide homologies with the first us human pandemic (h1n1) 2009 isolate (a / ca/04/2009) were 99.4%, 99.4%, and 99.8% for the ha, na, and m genes, respectively . * primers and probes were obtained from integrated dna technologies (coralville, ia, usa) and biosearch technologies, inc . M, matrix; ai, avian influenza; n, neuraminidase . The cat was discharged from the medical center after diagnostic testing and correction of dehydration . A veterinarian (b.a.s .) Visited the home to monitor the cat s clinical status and administer subcutaneous fluids (120160 ml) until the cat s appetite improved; adventitial lung sounds resolved within 3 days . Reassessment 1 week later showed marked improvement of clinical signs but only modest improvement of the lymphopenia and radiographic findings . Because the cat was from a single - animal household and remained indoors, he was presumably infected through contact with the family members . Attempts to retrospectively confirm pandemic (h1n1) 2009 infection in the family members have been unsuccessful, but additional testing of archived biologic samples is being conducted . Although more surveillance and studies are needed to determine susceptibility of companion animals to the pandemic (h1n1) 2009 virus, possible reverse zoonotic transmission (humans to animals) remains a concern . Indeed, cases in a domestic dog and other felids have been confirmed (11) (www.cdc.gov/h1n1flu/qa.htm,www.avma.org/public_health/influenza/new_virus/default.asp, www.usda.gov/wps/portal/?navid=usda_h1n1). Implications of pandemic (h1n1) 2009 virus infection in companion animals are 1) apparent human - to - animal transmission; 2) broader host range for the virus; 3) potential endemic establishment of influenza in companion animals; 4) possible transmission of influenza from companion animals to other species, including humans; and 5) the need to reevaluate companion animals as potential reservoirs or intermediate hosts for reassortment of influenza virus . This case emphasizes the need for close monitoring for interspecies transmission of influenza virus and reinforces the need for collaboration among many disciplines, a cornerstone of the one health initiative (www.onehealthinitiative.com).
Methods for the study to assess hepatitis c risk have been summarized (7). In brief, during march 2009june 2010, persons 1840 years of age who were residents of san diego county, california, and who had injected drugs in the previous 6 months were recruited to participate in this study . Eligibility screening and acquisition of informed consent for potential participants were followed by a behavioral risk assessment and serologic testing . Data collected included participant demographics, substance use, injection practices, diagnosis with sexually transmitted infections, exchange of sex for money, homelessness, travel to mexico, and hiv status . Serologic testing included detection of antibodies against hepatitis a virus (hav), hepatitis b virus core antigen, and hepatitis c virus (hcv) by using the vitros immunodiagnostic system (ortho clinical diagnostics, rochester, ny, usa), and igg against hev by using a commercial assay (dsi, saronno, italy). We performed a comparative analysis of all persons on the basis of their status for igg against hev by using demographics, seropositivity for other viral hepatitides, travel to mexico, history of incarceration, homelessness, hiv status, and high - risk sexual behavior . We used bivariate logistic regression to calculate odds ratios; 95% cis; and p values, which were set at 0.05 to determine significance for factors associated with hev prevalence . All data were analyzed by using sas version 9.2 (sas institute, cary, nc, usa). Of 508 pwid, 72% were men, their mean age was 29 years (range 1840 years); and 62% were white . Fourteen (2.7%) persons had igg against hev; none of these persons were positive for hev rna by pcr (all were negative for igm against hev). Of the 14 persons with igg against hev, 11 (79%) were men; their mean age was 33.4 years (range 3036 years); and 57% were white (table). Relative to participants <30 years of age, persons 30 years of age were more likely to be positive for igg against hev (odds ratio 3.61, 95% ci 1.319.94). Travel history and presence of antibodies against hav, hepatitis b virus, or hcv were not associated with presence of antibody against hev . Bivariate logistic regression showed that there was no association between presence of igg against hev and a history of incarceration, sharing of injection drug equipment, homelessness, high - risk sexual behavior, and hiv status . Nd, not determined; hav, hepatitis a virus; hbc, hepatitis b core antigen; hcv, hepatitis c virus . We found an overall hev seroprevalence of 2.7% in young pwid in the united states . This seroprevalence was higher among participants 30 years of age than in participants <30 years of age . Variables typically associated with hcv / hiv transmission (i.e., high - risk sexual behavior, incarceration, or sharing of injection drug use equipment) were not associated with presence of antibodies against hev . These findings were consistent with results of a study that found no association between antibodies against hev and co - infection with other hepatitis viruses or sharing of drug paraphernalia (2). Because of the common mode of fecal oral transmission of hav and hev, other studies have also investigated an association between hav and hev infections, but results have been inconclusive (1,5,6). As in previous studies, we found an association of presence of antibodies against hev and age (1,6). Higher prevalence among older pwid suggests that there may be age - related exposures independent of injection drug use that increases the likelihood of hev infection . This birth cohort effect has been seen in other low - prevalence countries, such as denmark (8), and decreased possible exposure may help explain the lower prevalence rates in our study . Our small sample size reduced the potential to detect significant differences between hev - negative and hev - positive persons . In addition, we did not have information about other exposures that have been associated with hev infection, including particular dietary or zoonotic exposures or history of travel to a country to which hev is endemic . Information about hev genotype was not available for seropositive persons, which might have provided clues as to the mechanism of exposure . Lower prevalence estimates may also reflect the fact that our population only included persons 1840 years of age . Previous data have suggested that increasing age is associated with higher hev positivity (6), particularly in countries in which prevalence is low and infection is caused mainly by hev genotype 3 (9). Although our data cannot be generalized to the us population, seroprevalence in this study appears to be low, which is similar to time trends in the general population of other low - prevalence areas (8). Variability in assay types used may account for discrepancies seen with previous seroprevalence studies of hev . In a study evaluating the performance and concordance between various assays for detection of igg against hev available at the time, overall concordance ranged from 49% to 94% (median 69%), and concordance among reactive serum samples ranged from 0% to 89% (median 32%) (10). Evaluation of the performance characteristics and concordance of currently available assays for detection of antibodies against hev, including the assay used in this study, remains to be determined . Overall, our data showed an increase in antibodies against hev for pwid 30 years of age and no other association with other reported risk factors . Future research is needed to explore other marginalized populations in hev - endemic areas to determine whether there are other risk factors that have not been identified in low - prevalence areas.
Heavy metal contamination in the environment is a major concern worldwide because of the toxicity of these metals and their potential threat to human health . Currently, quantification of heavy metals relies upon collection of liquid discrete samples for subsequent laboratory analysis using techniques such as icp - ms, aas, gc, hplc, ft - ir, and gs / ms . Electrochemical detection has several advantages over these methods in their simplicity, fast response, and suitability for the preparation of inexpensive and portable instrumentations . Adsorptive stripping voltammetry (asv) is not only an extremely sensitive electrochemical technique for measuring trace metals but also can carry out simultaneous detection of several metals in various matrices [3, 4]. Chemically modified electrodes (cmes), with surfaces designed for reacting and binding of target analyte, hold great promise for chemical sensing [57]. There are different directions by which cmes can benefit for analytical application of environmental samples . These include acceleration of electron transfer reactions, preferential accumulation, or selective membrane permeation . Such steps can impart higher selectivity, sensitivity, and stability on electrochemical devices . As a member of the flavonoid family, quercetin (qu) can form stable complexes with various metal cations . Many researches have focused on the application of qu modified carbon paste electrode (cpe) [9, 10]. However, there are no reports about simultaneous determination of copper, lead, and cadmium by using qu modified electrode . Ionic liquids (il) are liquid electrolytes composed entirely of ions . In recent years, il have emerged as a research frontier due to their unique chemical and physical properties [1113]. Il, as the modifier, can offer improvements in the stability and reproducibility of electrode, and has attracted much attention in electrochemical field [1416]. To our knowledge, there is no report about il modified electrode to determine heavy metal ions . Ordered mesoporous materials are highly unusual in their textural characteristics: uniform pore sizes, high surface areas, and long - range order of the packing of pores [1720]. Hexagonal mesoporous silica (hms), possessing hexagonal array of pores, is one of the most important mesoporous sio2 materials as adsorbents, gas storage materials, separation membranes, catalyst supports, and so on . Yantasee [2227] and coworkers have reported on simultaneous determination of copper, lead, and cadmium using a carbon paste electrode modified with mesoporous silica . In published documents, polymeric monolayers assembling on the surfaces of ordered mesoporous materials were the focus due to the superior stability and durability of electrode . However, because of the micropore size of hms limits the entry of large chelating agent leads to reduce the chelation of chelating agent toward metal ions . Otherwise, time - consuming synthesis procedures and preparation of the electrode are also difficult . In this study, we developed a more simple and sensitive electrochemical method for simultaneous determination of copper, lead and cadmium utilizing the excellent properties of hms and qu . In this work, a new quercetin modified carbon paste electrode (qu / cpe), quercetin ionic liquid modified carbon paste electrode (qu - il / cpe), and an hms immobilized quercetin carbon paste electrode (hms - qu / cpe) were fabricated, respectively . The electrochemical responses of copper, lead and cadmium at cmes were investigated in detail . The results show that hms - qu / cpe as a working electrode exhibits higher sensitivity and selectivity toward the detection of copper, lead and cadmium than qu / cpe, and qu - il / cpe . A novel electrochemical method is developed for simultaneous determination of copper, lead, and cadmium, and applied to the analysis of soil samples with satisfying results . Qu (purity> 98.9%) was obtained from shanghai reagent co. (shanghai, china), and graphite powder (spectral pure) was obtained from sinopharm group chemical reagent co. (shanghai, china). Tetrafluoroborate 1-butyl-3-methylimidazolium ([bmim]bf4) was obtained from shanghai ling feng reagent co. (shanghai, china) and used without further purification . Hms was synthesized as described previously . A stock standard solution of cu(ii) (0.01 m) was prepared by dissolving 0.1589 g of copper powder (shanghai reagent company, china) in 4.0 m nitric acid 20 ml, then the resulting solution was moved into the 250 ml volumetric flask, added twice - distilled water to the mark and stirred . A stock standard solution of pb(ii) (0.01 m) was prepared by dissolving 0.5179 g lead powder (tianjin reagent company, china) in 4.0 m nitric acid 20 ml, then the resulting solution was moved into the 250 ml volumetric flask, added twice - distilled water to the mark and stirred . A stock standard solution of cd(ii) (0.01 m) was prepared by dissolving 0.2810 g of cadmium powder (shrc, china) in 3.0 m hydrochloric acid 20 ml, then the resulting solution was moved into the 250 ml volumetric flask, added twice - distilled water to the mark and stirred . An oxygen free nitrogen (ofn) gas (99.9995%, jinan gas company, china) cyclic voltammetries (cv) and differential pulse voltammetries (dpv) were performed on electrochemical analyzer (chenhua instrumental co., china) chi 842b controlled by a personal computer . Electrochemical impedance spectroscopy (eis) was carried out by electrochemical analyzer (zahner co., germany) im6ex and the frequency range was from 60 khz to 0.1 hz with an amplitude of 5 mv . A three - electrode system was employed, including a modified carbon paste electrode as the working electrode, a platinum wire as the auxiliary electrode, and an ag / agcl (3 m kcl) electrode as the reference electrode . The conventional cpe was prepared by mixing graphite powder and paraffin oil in a ratio of 5: 2 (w / w) in a mortar . The resulting paste was packed firmly into the cavity (5 mm diameter) of a glass tube . A copper wire fixed to a graphite rod and inserted into the glass tube served to establish electrical contact with the analyzer . Qu - il / cpe was prepared by dissolving 0.0240 g qu in 50 ml ethanol and uniformly mixing with graphite powder . 0.05 g paraffin oil and 0.6 ml [bmim] bf4 were added, and mixed until obtaining a uniformly wetted paste . A portion of the resulting paste was packed firmly into the cavity (5 mm diameter) of a glass tube . Qu / cpe was prepared in the same fashion, but no [bmim] bf4 was added . Hms - qu / cpe was fabricated by dissolving 0.0240 g qu in 50 ml ethanol and mixed the solution with 0.1 g hms . The paraffin oil was mixed with graphite powder until a uniformly wetted paste was obtained . A portion of the resulting paste was packed firmly into the cavity (5 mm diameter) of a glass tube and the electrical contact was established by a copper wire . The surface of the electrodes was smoothed on a weighing paper and rinsed carefully with twice - distilled water prior to the each measurement . The samples were dried, ground, and sifted by the 60 mesh nylon sieve . 0.5 g of powder was accurately weighed into the decomposing cell . 5.0 ml nitric acid, 5.0 ml hydrochloric acid, and 5.0 ml peroxide were added . The sample solution into decomposing cell is decomposed by microwave . The resulting solution was moved into the 50 ml volumetric flask, and water was added to the mark and stirred . Placed 1 ml of sample solution was placed into 10 ml volumetric flask, and 0.1 m hcoona - hcl buffer solution was added to the mark and stirred . Unless otherwise stated, 0.1 m hcoona - hcl solution (ph4.7) was used as a supporting electrolyte for the determination . The measurements were carried out after a preconcentration step, in which the solution was stirred during preconcentration time of 120 s at preconcentration potential of 0.6 v. after a rest period of 30 s, the response curve was recorded by scanning the potential in the negative direction with differential pulse voltammetry (dpv) technique . The electrochemical behaviors of qu / cpe and cpe were investigated in ph4.7 hcoona - hcl buffer solution . Figure 1 shows the cyclic voltammograms of qu / cpe (figure 1, curve a) and cpe (figure 1, curve b). A pair of well - defined redox peaks of qu was observed at qu / cpe, the anodic peak potential epa at 0.394 v, cathode peak potential epc at 0.284 v, and peak - to - peak separation (ep) of 0.11 v. the result indicates that qu can undergo redox reaction at modified electrode . The redox peaks of qu were also observed at qu - il / cpe and hms - qu / cpe . Figure 2 shows the electrochemical responses of 1.0 m (each) multicomponent cu(ii)/pb(ii)/cd(ii) solution at qu / cpe, qu - il / cpe, and hms - qu / cpe . At the qu / cpe (figure 2, curve a) and qu - il / cpe (figure 2, curve b), two reduction peaks are observed for copper and lead at 0.11 v and 0.48 v, respectively, and no obvious reduction peak of cadmium is observed . At the qu - il / cpe, the reduction peak currents of copper and lead are largely increased, indicating that the chemical and physical properties of il improve the sensitivity of electrode . At the hms - qu / cpe (figure 2, curve c), the reduction peak of cadmium appears at potential of 0.69 v and peak currents of 60.9 a . The peak currents of copper and lead are also obviously increased from 31.8 a to 33.6 a and from 82.8 a to 102.3 a compared with that of qu - il / cpe . This tremendous increase in the current density for copper, lead, and cadmium with the hms - qu / cpe is apparently due to the cooperative effect of hms and qu . Meanwhile the reduction currents of qu at different modified electrodes are remarkably lower in multicomponent cu(ii)/pb(ii)/cd(ii) solution than those in the blank solution . The reduction current change (ip) of qu at different modified electrodes was in a sequence of: (1)qu / cpe(ip=97.4 a) <qu - il / cpe(ip=117.4 a) <hms - qu / cpe(ip=153.8 a). According to above comparison, the concentration of free qu at hms - qu / cpe is the lowest, suggesting that hms - qu / cpe can produce more complexes than other modified electrodes . The chelation of qu with metal ions in acidic solution has been studied in the previous literatures [9, 10]. When qu was immobilized on the surface of hms, effective reaction sites of qu with metal ions on hms are remarkably increased due to the high surface area and strong adsorption ability of hms . Therefore, hms - qu / cpe exhibits better capability of adsorptive preconcentration, higher sensitivity and selectivity than qu / cpe and qu - il / cpe . The result indicates that hms - qu / cpe is very satisfactory for the development of the simultaneous determination of copper, lead, and cadmium . Eis experiments were carried out using different modified electrodes at the frequency range from 60 khz to 0.1 hz at the perturbation amplitude of 5 mv . Figure 3 shows that the semicircle in high frequency region was not obvious and the straight lines in low - frequency region were obvious in different modified electrodes . The result indicates that the electron transfer rate is fast and the process of electrode reaction is controlled mainly by adsorption and diffusion . The variety of linear slopes at different modified electrodes may be attributed to the different captance characteristic of electric double - layer at modified electrodes, which indicated that the surfaces of different electrodes had different specific adsorptions . The specific adsorption of hms - qu / cpe was the intensest, qu / cpe and qu - il / cpe showed no obvious difference . These results suggested that hms - qu / cpe can effectively improve selectivity and sensitivity and achieved simultaneous determination of copper, lead, and cadmium by the strong chelation and adsorption of hms - qu / cpe with metal ions . Voltammetric behavior of the metal ions at hms - qu / cpe was evaluated in terms of the influence of stripping parameters such as effect of ph, preconcentration time, and preconcentration potential . The effect of ph was studied by using different types of supporting electrolytes for their suitability in the simultaneous detection of these metals . The hcoona - hcl buffer solution was found to be more suitable for its better sensitivity . The effect of ph on peak currents was studied in hcoona - hcl buffer solution . As shown in figure 4, the peak currents of copper, lead, and cadmium is maximum at ph4.7 . The effect of preconcentration time on the magnitude of the stripping peak current was examined in the range of 20150 s, and shown in figure 5 . Taking into account the sensitivity and resolutions of adjacent peaks, preconcentration time is optimized to 120 s in the experiments . Preconcentration potential was also varied starting from 0.3 v to 0.8 v. the result showed that lower preconcentration potential might increase the possibility of interference between ions in this study, therefore, the preconcentration potential was applied at 0.6 v. at optimized experimental condition, three peaks are observed for copper, lead, and cadmium at 0.11 v, 0.48 v and 0.69 v, respectively . In ph4.7 hcoona - hcl buffer solution, the relationship between the peak current and concentration was studied by adsv . Linear range and detection limits of copper, lead, and cadmium were shown in table 1 . The interference experiment of some cations and anions was also investigated . Under the optimum conditions, relative error of determination was chosen in the order of 5% or smaller in 1.0 m (each) multicomponent cu(ii)/pb(ii)/cd(ii) solution . The results showed that 1000-fold al(iii), k (i), sulfate, phosphate and nitrate, 200-fold mg(ii), sc(iii), co(ii) and ni(ii), 50-fold mo(vi), v(v) and in(iii), 25-fold ag(i) and cr(vi), did not interfere with the determination . Under the optimum conditions, the stability and reproducibility of hms - qu / cpe were studied in 1.0 m (each) multicomponent cu(ii)/pb(ii)/cd(ii) solution . After continuous determination 20 times, average stripping peak currents of copper, lead, and cadmium were 31.8 a, 104.8 a and 59.5 a, respectively . The relative standard deviations (rsd) were 3.9%, 3.2% and 7.8%, respectively . In addition, after 30 days of storage in dry conditions, no significant change in current response was observed, suggesting that the modified electrode has an excellent reproducibility and stability . In order to ascertain the potential application, this newly developed method was employed to determine copper, lead, and cadmium in soil sample . The concentration of copper, lead, and cadmium was determined by the standard addition method, and the results were listed in table 2 . In order to testify the accuracy of this method, the concentration of copper, lead, and cadmium was also detected by faas . The results obtained by two methods are in good agreement, indicating that this method is reliable and accurate . The electrochemical responses of copper, lead, and cadmium at hms - qu / cpe were improved in comparison with qu / cpe and qu - il / cpe . The hms - qu / cpe exhibited dramatically high sensitivity and selectivity due to high surface area, numerous active sites, and strong adsorption ability of hms . After optimizing the parameters such as ph value, preconcentration time, and preconcentration potential, a novel electrochemical method is simple, fast, sensitive, and selective, and can simultaneously determine three metals with high accuracy and stability . Moreover, the hms - qu / cpe was successfully used as a sensor for simultaneous determination of copper, lead, and cadmium in soil sample.
Spirituality and medical ethics has recently emerged as an important topic in the health care provision and there exists a heated debate surrounding the issue . Medical ethics is in the curricula of virtually all medical schools all around the world . On the other hand, spirituality and religion therefore, one could suggest that patients religious ideology and beliefs should be taken into account in any clinical setting (1, 2). Spirituality is usually defined as the aspect of humanity that refers to the way an individual seeks to follow in life . It is supposed to add meaning and purpose to life, and facilitate the connection with the moment, self expression, and attitude towards other human beings, nature and the sacred (3). Spirituality and religious beliefs have been shown to have an enormous impact on how people can cope with serious illness and life threatening stresses (4, 5). Spirituality if often claimed to give people a certain feeling of wellbeing, improve the quality of life, and provide social support through spiritual social activities (6, 7). New advances in medical sciences have made it more than obvious that including moral issues in this profession is indispensable . As medical profession deals with individuals bodies and lives, it needs to consider morality and dignity of human beings (8). Therefore, medical practice is interlinked with a great deal of responsibility as it deals with human beings physical and mental health; consequently, it should take all considerations into account including spirituality and religion (9). In today s world, medical ethics has been the subject of considerable attention due to the mentioned issues and it is the subject of heated debates . Medical ethics include a set of values and codes which aim to build trust and confidence in physicians, patients, and the society as a whole . A physician s competence depends not only on his medical knowledge, clinical decision making, and practical skills, but also, on his ideology and practice of medical ethics (10, 11). As one of the four basic principles of modern ethics is respect to patients choice in clinical practice, physicians should respect patients religious and spiritual choices (12). Patients are only one side of the interaction in a clinical setting; physicians too, may have religious ideas and beliefs which may influence their practice . The religious ideology and beliefs of physicians can even more complicate the issue when patients religious choices come into play (12). As argued, the concept of morality and medical ethics has become the focus of attention in medical practice, and, its implementation is furiously advocated (13). Therefore, hospitals need to consider all aspects of patient s rights including the right to be treated based on their religious ideology . In fact, respecting all aspects of patients rights such as their participation in decision making, respect for privacy and dignity, and interpersonal relationships are considered intertwined with several different aspects of health care delivery (14). The u.s . Legal system has been pioneer in implementing a robust approach to a health care provision system respecting all aspects of patients rights such as their special religious demands, while pursuing excellence in all aspects of health care delivery (15). In addition to considering moral issues and respecting religious beliefs of patients, public and governmental hospitals need to promote accountability . Respect for patients rights and enhanced communication with patients can pave the way for development of moral competence (16). In fact, it is argued that undermining medical and moral accountability in some countries has resulted in patients loosing their confidence in the system regarding moral, legal, monetary, and political affairs (17). Moral accountability in governmental hospitals is defined as the extent to which they implement and monitor adhering to moral principles by the authorities and staff (18). The need for defining a role for religion in human beings everyday tasks is not limited to a specific religion, a certain geographical area, or a particular time period in the history . In fact, it can be argued that man has always felt this need all through the human history including the present time . Islam, as both an ideology and a practical way of life, is argued by its followers, to provide solid foundation for different aspects of life (19, 20). The clinical setting and medical affairs seem to be in priority when implementing the religious law is concerned . Islam is all about faith, dignity, honesty, and mutual trust, medical ethics is of paramount importance for muslims as it needs to protect them from anything that might endanger the islamic principles (21, 22). In this study, we aimed to conduct a poll to find out what female iranian patients think about the implementation of the same sex health care delivery system (sshcd) in accordance with the religious law in a number of teaching general hospitals of tehran, the capital of iran . This plan was first proposed by ministry of health and medical education of the islamic republic of iran due to increasing demand of patients and policy makers, and, consequently, the supreme council of sshcd was established in 1997 . The parliament of the islamic republic of iran passed the bill of this plan in 1998, and, in the same year, it was communicated to all healthcare organizations in iran . This study is the result of all endeavors to clarify whether the project can be implemented in iranian hospitals or not, and whether the patients would prefer and embrace it . This project is supposed to encompass all aspects of religious law regarding medical practice including the need for female patients to be visited and cared for, exclusively, by health care providers from the same sex (24, 25). The plan of sshcd project also includes a different part which is designed to define medical practice codes based on the religious law . This project is supposed to be followed by all medical practitioners practicing in the islamic republic of iran (26). The first step of the project is intended to separate women and men in hospitals (21). Moreover, physicians should be required to visit patients from the opposite sex only when it is inevitable . This way, any unnecessary contact of the health care provider with the patient from the opposite sex is minimized (24). Finally, physicians bedside manner and their conduct toward their patients need to be according to the religious law (22). This research was a cross - sectional, descriptive analytical study which was conducted at teaching general hospitals in tehran, iran . In this study, we chose four hospitals from different neighborhoods of tehran which were similar in terms of size, number of beds, number of patients admitted during a particular period of time, number of staff, and the health care services delivered . The questionnaires were to be answered anonymously, and, in order to protect their confidentiality, the names of the participating hospitals were not disclosed . The cochran formula was used to calculate the number of female patients as the research cohort . In each hospital, 30 female patients were selected from different wards using cluster sampling method . The assessment tool was a questionnaire comprised of 18 questions and the questions had a focus on demographic information, the preferred gender of the visiting physician, the importance of being visited by a physician from the same sex, and the most important moral issue they faced during their hospitalization . Before the commencement of the study, a pilot study was performed to check the reliability of the questionnaire . In order to ensure that, some of the patients were asked to complete the questionnaire randomly two weeks prior to the study . The results obtained from test- retest method were studied and compared with those of the main study . The questionnaires were distributed among 120 women who were admitted to different wards of the hospitals . Also, we asked the patients to mention their husbands opinion about their wives being visited exclusively by female physicians and they were asked to express their opinion about the questions . In order to specify the degree of the agreement the three level likert scale was used as agree, disagree and neutral . The statistical analysis, chi - square and pearson tests were used to analyze the data . As it was difficult to analyze patients opinions by direct observation, the attitude was assessed using measurable bits of evidence such as the expression of beliefs and emotions (27). Most of the patients were 20 40 years old, 89% were married and 79.3% were housewives . 30% of them were illiterate and the remaining had high school diploma, bachelor s degree, master s degree or doctorate degree . 60% of the patients lived in the capitals of the provinces and 40% lived in towns and rural areas . The results indicated that most of the hospitalized women s husbands preferred female physicians to visit their wives . In contrast, 14.8% believed it was not necessary, and 36.5% did not answer this question . Almost, half of the patients believed that female physicians were available, 37.2% of them expressed that female physicians were not available, and the rest expressed that they had not noticed it . Most of the patients believed that they mostly felt embarrassed when male medical students were present by their bedsides . As it is tabulated in table 1, more than half of the hospitalized women believed in the competency of the female physicians and staff and preferred to be visited by them even when male physicians were being present . Most of the hospitalized women in this study, expressed their concern and dissatisfaction about a number of issues in the questionnaire . Moreover, they expressed their feeling embarrassed by being visited by male physicians of their own family members or relatives (to whom marriage is prohibited by the religious law). They also opposed to a number of services delivered by male staff, including nursing care, housekeeping services, changing position, being helped with personal activities, urethral catheterization and intramuscular injections . They also were against presence of male medical students during medical examinations, failure of patient s covering during radiology and laboratory services provided by the technicians or technologists, and the presence of male nurses and staff members of close relationship (to whom marriage is prohibited) during physical examinations . In addition, the majority of the hospitalized women were in favor of the separation of men and women s rooms at the hospital wards and the presence of a chaperone during medical examinations . According to table 2, 3/4 of the hospitalized women were in favor of implementation of the sshcd project in tehran, iran . We analyzed the dependency or independency of the variables based on pearson test, and, it was demonstrated that there was significant relationship between the level of education of the studied women and their opinion about the gender of their physician (p=0.041) and being delivered nursing care by male nurses (p=0.045). Therefore, it can be concluded that more educated patients preferred to be visited by male physicians and received health care by male nurses . Also, there was a significant correlation between the age of the participants and their opinion of the presence of male physicians during physical examinations (p=0.002), being examined by male medical students (p=0.002), and receiving personal services by male hospital staff (p=0.019). Older patients were in favor of being visited by male physician, being examined by male medical students, and receiving personal services by male hospital staff . Moreover, there was a significant correlation between the incidence of hospitalization and the patient s opinion about receiving nursing care provided by male nurses (p=0.04), and sharing their personal private issues with male physicians and other medical staff (p=0.01). It was observed that increased incidence of hospitalization was positively correlated with preferring to receive nursing care by male nurses and the other male medical staffs . There was also considerable correlation between the occupation of patients and their attitude towards the presence of male staff during their examinations by male physicians (p=0.01) and the implementation of adjustment plan . There was also correlation between marital status of the hospitalized women and their attitude toward being examined by male medical students (p=0.025). Thus, the hospitalized women with higher job positions preferred to be examined by male physicians; in other words, woman with higher job positions were not in favor of the implementation of the sshcd project . Most interestingly, there was a direct relationship between the opinion of the husbands and the patients, and the patients themselves regarding the preferred gender of physicians as well as being examined by male medical students . Firstly, it had a focus on sshcd according to the religious law from a different point of view which is based on medical ethics and patients rights . Secondly, this study is one of the few studies conducted on the need for implementation of the sshcd according to the free will and respect to the choice of iranian female patients and their family . Thirdly, in this article, the researchers analyzed the demographic characteristics of the hospitalized women (such as age, education, occupation, the incidence of admission to hospitals, marital status and the opinion of their husbands) with regard to the implementation of the sshcd . Approximately, half of the hospitalized women believed it necessary to be visited by female health care professionals and they considered it as their indisputable right . Zare dehabadi has suggested that patient s rights to choose their physician is in accordance with the who declaration and that of the world medical association . This is also considered in patient s rights charters in malaysia, new zealand, and slovakia (28). Waseem et al . Have analyzed several points regarding patients preferences for the gender of their health care deliverer . Among the study subjects, 80 percent of the women preferred a female doctor, and when it came to the choice between more experienced and female physicians, none of them chose the doctor who had more experience . They researchers hypothesized some potential underlying basis for the results and suggested the possible implications . However, several studies conducted on hospitalized women s opinion about their preferences for the gender of health care deliverers found that characteristics such as interpersonal communication and clinical skills had priority over physician s gender . The aforementioned results are in congruence with those of the current study (29, 30). The subject of the gender related issues in medical practice and research has been the subject of considerable amount of debate recently . Sex and gender related laws and regulations do not only affect the patient, but also include the relationship amongst practitioners themselves . Considering the important role it plays in daily activities of medical practitioners and its legal, political, philosophical, and moral consequences, the gender related interpersonal communication issues should be taken into account more seriously (31). The most frequently expressed concern of the patients in this study was the issue of a male medical student s being present during physical examinations and ward rounds . The participants also expressed their dissatisfaction with the health care services rendered by male nurses and allied health professionals . Moreover, they were strongly in favor of the separation of female patients rooms in hospitals . It is universally acknowledged that patients and their families should have the right to choose their health care providers (32). Therefore, hospitals bear the responsibility of providing patients with their preferred health care services . These services need to respect patients choice of the clothing, head cover or hijab, and carrying religious relics or other symbolic items, as long as they do not interfere with diagnostic procedures or treatment options . Many even argue that such issues need to have priority over medical procedure with more favorable outcomes . Privacy, confidentiality of clinical information, the presence of a chaperone during clinical examinations, and having the right to be visited and cared for by health care providers of one s own sex are usually considered as patient indisputable rights (33,34). Yarsis surakarta hospital in indonesia that is a private healthcare facility with 219 beds and zhejiang woman hospital in china that was founded in 1951 and fit, the local healthcare demand for women and babies, with nearly 1000 staffs, 750 bed, for women and 250 beds for babies, are examples of the world s attention to human rights (35, 36). Although the results of our study clearly demonstrated that most of the hospitalized women were in favor of the implementation of the sshcd project according to the religious law, one should consider several different factors when its implementation is concerned . Azari argues that one of the most important strategies in this regard is to employ proportionate workforce, especially nursing staff and allied health care professionals (37). Shojaie and ghofranipour indicated that a conservative and tolerant approach should be considered so that to avoid fierce opposition by those who disagree with this plan, and, also, to prevent extremism . It should be also taken into account that the implementation of the sshcd requires deep and sincere faith of both policy makers and health care providers, and, the strategy of its implementation must be similar to that of the islamic ethics . Taking these into consideration, sshcd seems easy to be implemented and requires sincere faith and dedication (38, 39). One of the most important limitations of the present study was that it was conducted over a specific time period . It could be argued that an increased duration of the study might have demonstrated different results . Secondly, as all patients were recruited by means of a questionnaire, it is possible that during the information collection process some data are missed . Finally, one could suggest that some other factors influencing health care provision for the hospitalized patients that have not been taken into account in our study . Considering mentioned limitations further studies with a larger cohort and extending over a longer period of time seems to be of great importance before implementation of the sshcd or any such comprehensive plan at a national scale . It is noteworthy that written informed consent was obtained from all patients and every single aspect of academic honesty has been taken into account to avoid plagiarism, misconduct, data fabrication and / or falsification, and double publication . Apparently, although a decade is passed since the proposal of the implementation of the sshcd in iranian hospitals, iranian patients and their families are still in favor of its implementation . In fact, the advocacy of its implementation has increased during the recent decade . Moreover, the results of previous studies and those of the present one conclude that not only patients are in favor of the sshcd project, but also a great majority of health care providers advocate it . As a result, we hope that a new common issue about patients rights and medical ethics has opened . Therefore, the results of this study, in accordance with those of the previous ones, indicate that iranian health care policy makers need to consider accelerating the implication of the sshcd plan.
Ever since humans began to walk upright, some of their muscles have always been activated to maintain the balance of the body on narrow support surfaces . If these posture - maintaining muscles lose their function, low back pain will be induced due to the maintenance of unstable postures1 . Compared to healthy persons, patients with low back pain have reduced proprioceptive sense in the deep lumbar muscles, which makes the spine unstable, and this may lead to the recurrence of low back pain2 . When healthy persons without any low back pain move their upper or lower limbs quickly, their transversus abdominis (tra) muscles contract first to contribute to lumbar stabilization . However, in the case of patients with low back pain, the tra muscles do not contract first3 . Delayed contraction of the tra makes the trunk unstable, leading to reduced motor and postural control abilities and inefficient actions of the spinal muscles4 . Richardson et al.5 reported that the low back pain recurrence rate was reduced from 75 to 35% for three years after abdominal drawing - in exercises were performed to strengthen the tra and multifidus muscles . Page6 advised that rather than exercises using stable bearing surfaces, lumbar stabilization exercises using an unstable support surface are more effective for the maintenance of postural balance and the recovery of proprioceptive senses . Kang et al.7 reported that according to the measurements taken using ultrasonography and the visual analogue scale after patients who had received herniated disc surgery had performed lumbar stabilization exercises using slings, which are unstable support surface, the activity of the tra muscle increased and pain was relieved . Therefore, these previous studies indicate that closed chain exercises on an unstable support surface have the effect of activating lumbar stabilization muscles . The local vibration used for the intervention in this study took the form of isometric exercises that have been attracting great attention after results indicating that they positively affect motor function improvement and increase energy metabolism and blood flow rates8 . In addition, rittweger et al.9 reported that when vibration and exercises were performed by a group of low back pain patients, there was evidence of pain relief and improvement in their neuromuscular control ability . This study compared and analyzed the changes in the thickness and length of the tra muscle when vibration was applied to low back pain patients while they performed closed chain exercises on an unstable support surface . The study subjects were 64 healthy university students residing in daegu, korea, who were randomly divided into a bridge exercise with sling and vibration group (besvg) of 30 students, and a bridge exercise with sling group (besvg) of 34 students . The besvg s mean age was 21.81.6 years, their mean height was 166.17.7 cm, and their mean weight was 61.211.2 kg . The besg s mean age was 22.51.8 years, their mean height was 169.67.2 cm, and their mean weight was 60.79.4 cm . Those who had neurologic or orthopedic problems or could not perform supine / prone bridge exercises due to pain or other problems were excluded from the study . All of the participants understood the purpose of this study and provided their written informed consent prior to their participation in this study, in accordance with the ethical principles of the declaration of helsinki . When the subjects in the besvg performed the bridge exercise in supine positions, each adopted a supine position on the table . For the starting posture, the subjects placed their two hands on the floor to support the body, maintained 90 flexion postures of the hip and knee joints, and placed both ankles in a narrow sling . During the exercise, the subjects loaded their weight on both lower limbs, avoiding pelvic tilt and lifted their hip so that the trunk, pelvis, and lower limbs made a straight line . When the subjects in the besvg performed the bridge exercise in the prone position, each adopted a prone position on the table . For the starting posture, the subjects placed their two ankles in a narrow sling while maintaining the shoulder and elbow joints at 90. during the exercise, the subject loaded their weight on both lower limbs and elbows, avoiding pelvic tilt and lifted their hips so that the trunk, pelvis, and lower limbs made a straight line . The performance of each exercise four times constituted one set, and nine sets of the bridge exercise were performed in both the supine and prone positions . The besg performed the same bridge exercises without local vibration in the supine and prone positions . Local vibration was applied by suspending the two ropes of the sling that supported both lower limbs from vibration equipment (redcord stimula, norway), so that vibration was applied only while the subjects were performing bridge exercises and not during rest times . Vibration was applied at 15 hz in the first 3 sets, at 20 hz in the next 3 sets, and at 30 hz in the last 3 sets . An hd11xe ultrasound system (philips, netherlands) was used for ultrasonic imaging, and measurements were conducted by one person who was skilled in ultrasonic measurement . Before the ultrasonic measurements were taken, subjects lay comfortably in the supine position . A pressure biofeedback unit (pbu) was placed between the umbilicus and the floor, and a triangular pillow was placed under the subject s knees for support, thereby relaxing the subject s lower limbs . An assistant adjusted the pressure of the pbu to 40 mmhg, and the tester then placed the probe transversely on the top of the iliac crest and moved it to the center of the abdomen to align the end point of the tra muscle with the end point of the screen . Once this had been done, the subject drew in the abdomen within the range in which the gradation of the pbu did not change, and the distance moved by the end point of the tra muscle was measured . In this way, the distance was measured three times, both before and after the intervention and the average values were recorded . Since the total length of the tra muscle could not be measured due to the nature of ultrasonography, the sliding distance from the abdomen to the outside was measured instead . For the statistical analyses, intra - group changes in tra thicknesses and lengths were analyzed using the paired t - test, and intergroup comparisons were performed using the independent sample t - test . Statistical processing was conducted using spss 12.0ko (spss, chicago, il, usa) with a significance level of =0.05 . According to the results of this study, in the intragroup comparison, the besvg showed a significant increase in tra thickness and a significant decrease in tra length (p<0.05). The besg showed a significant increase in tra thickness (p<0.05). In the intergroup comparison, the besvg showed a significant increase in tra thickness and a significant decrease in tra length compared to the besg (p<0.05) (table 1table 1.intergroup comparison of tra thickness and lengthtragroupprepostthickness (cm)besvg0.30.10.50.1besg0.30.10.40.1length (cm)besvg0.90.20.80.2besg1.00.20.90.3besvg: bridge exercise with sling and vibration group, besg: bridge exercise with sling group, : paired t - test,: independent sample t - test,: p<0.05, * : p<0.01). Besvg: bridge exercise with sling and vibration group, besg: bridge exercise with sling group, : paired t - test,: independent sample t - test,: p<0.05, * *: p<0.01 bridge exercises for lumbar stabilization use the performer s weight, increase joint alignment and stability, and improve proprioceptive stimuli and dynamic stabilization10 . In a study involving 51 patients with chronic low back pain, saliba et al.11 demonstrated that bridge exercises on an unstable support surface provided by slings were more effective at increasing tra muscle activity than bridge exercises on stable bearing surfaces as measured by ultrasonography . Eom et al.12 reported that bridging exercises performed by healthy persons using slings as an unstable support surface showed larger increases in tra thicknesses than bridging exercises performed by the same persons on stable bearing surfaces . In a study conducted by brumagne et al.13 with chronic low back pain patients, in which proprioceptive sense exercises and local vibrations were additionally applied, proprioceptive stimuli and deep muscle control abilities of the subjects improved . The studies cited above indicate that closed chain exercises on an unstable support surface are effective lumbar stabilization exercises for activating the tra muscle . Hides et al.14 reported that according to mri and ultrasonography conducted after athletes had performed drawing - in exercises, the tra muscle became thicker and shorter . Hides et al.15 reported that weight - bearing stabilization exercises performed by low back pain patients greatly increased the thickness of the internal oblique muscles, and shortened the tra muscle . According to the results of this study, in the intragroup comparisons the besvg showed a significant increase in tra thickness and a significant decrease in tra lengths; and the besg showed a significant increase in tra thicknesses . In the intergroup comparison, the besvg showed a significant increase in tra thickness and a significant decrease in tra lengths compared to the besg . Given these results, we consider the local vibration applied to the besvg during closed chain exercises on an unstable support surface improved tra muscle strength and power and lumber / pelvic tilt repositioning senses, thereby affecting tra thickness and length more than in the besg . Fontana et al.16 reported that when healthy persons performed weight - bearing exercise while vibration was applied, the subjects lumber / pelvic tilt repositioning senses were improved by as much as 39% . Therefore, applying local vibration during bridge exercises using slings in clinics should be an effective method for lumbar stabilization, because it improves tra muscle activation . In addition, it can be presented to patients with chronic low back pain as an exercise intervention method in clinics . Although many previous studies have measured tra muscle activity through the thickness of the muscle using ultrasonic imaging, those that used local vibrations are few in number . Therefore, it is important that related studies in this field are undertaken in the future.
A great variety of animal viruses encode for proteases that accomplish crucial functions during the biological cycle of the virus . Usually, the main function of these proteases is to proteolyze viral polypeptide precursors to render mature viral proteins that form part of viral capsids or participate in virus vegetative processes . Although both dna and rna viruses can encode proteases, the proteolytic tailoring of polypeptide precursors is most common among viruses with positive single - stranded rna genomes, such as picornaviruses, flaviviruses, caliciviruses, and retroviruses [37]. This mechanism of gene expression by proteolytic processing serves to compress the genetic information of viruses in the limited space provided by the genome . In this manner, viruses reduce the genetic space occupied by 5 and 3 untranslated regions (utrs), the signals devoted for mrna transcription and to initiate translation are minimal, such that, for instance, in the case of picornaviruses or flaviviruses, only one 5 and 3 utr is necessary for viral replication, transcription, translation, and morphogenesis, despite the fact that several viral proteins are synthesized by the infected cells . In addition, a number of polypeptide precursors may exhibit functions that differ from those present in their mature products . In the case of poliovirus (pv), eleven mature proteins are produced from a single translation initiation event, and at least two precursors, 2bc and 3cd, accomplish functions which are not present in their mature proteins . Taking together all these considerations, the proteolytic strategy provides the small rna viruses with an advantageous and efficient mechanism for distribution of the genome to accomplish all the viral biological functions with the smaller genetic space . Apart from generation of active viral proteins that participate in capsid morphogenesis and genome replication, viral proteases may also target a number of cellular proteins . Proteolysis of these cellular substrates can very much affect a variety of cellular processes and play an important role in virus - induced cytopathogenesis [8, 9]. In this regard, productive poliovirus infection induces rapid morphological alterations in host - cell . Among them, the most prevalent is the accumulation of numerous membranous vesicles in the cytoplasm, derived from endoplasmic reticulum where the viral proteins 2c and 2bc play a central role . In addition, cellular shape is modified upon viral replication giving rise to cell rounding, which is most probably induced by disorders in the cytoskeletal network . Finally, chromatin condensates at late times postinfection, associated with the nuclear envelope except for sites where nuclear pores are placed . Interestingly, individual expression of the viral proteases 2a and 3c leads to the induction of most of these cytopathic effects, supporting the idea that these proteases actively contribute to the viral - induced morphological changes . Indeed, long - term expression of either 2a or 3c triggers the activation of caspases and, thus, cell death by apoptosis [11, 12], reflecting the strong cytotoxicity of both proteases . In addition to the cytopathic effects induced by 2a and 3c, hydrolysis of host proteins may impact on other cellular functions such as the antiviral responses to virus infection . Activation of innate immunity pathways, as well as the establishment of an antiviral response, is absolutely dependent on signals traversing the nuclear membrane through the nuclear pore complex . Therefore, many viruses block cellular gene expression at different levels, that is, translation, transcription or protein and rna trafficking between nucleus and cytoplasm . The blockade of active trafficking can inhibit the nuclear import of antiviral signals or prevent the export of cellular mrnas detrimental to virus processes . The precise number of cellular proteins degraded by a viral protease, which is known as the degradome, still remains unknown for a given viral protease . Perhaps, one of the best - studied proteases in this respect is pv 2a . The discovery that pv 2a bisects the initiation factor of translation eif4 g leading to the regulation of translation in the infected cells has attracted much attention from many laboratories during the past three decades [13, 14]. More recently, 2a has been involved in the alteration of rna and protein trafficking between the nucleus and the cytoplasm upon proteolysis of several nucleoporins [1517]. The present paper focuses on the multifaceted activities of 2a and its regulation of different viral and cellular processes . Pv is a prototype member of the picornaviridae family that infects cells of human or simian origin cytolytically or persistently and is responsible for poliomyelitis in humans . The rna genome is housed in a naked capsid formed by 60 copies of each of the four structural proteins: vp1, vp2, vp3, and vp4 . The infectious cycle commences by the attachment of a viral particle to cellular receptors present at the cell surface [19, 20]. This interaction leads to virion internalization and destabilization of the capsid, which adopts a less compact structure . Once the rna is released in the cytoplasm, it interacts with the translational machinery, directing the synthesis of viral proteins during the early phase of infection . The pv genome is composed of a single - stranded rna copy of positive polarity of about 7.4 kb [21, 22]. This rna molecule is uncapped and contains a poly(a) tail at its 3 end and a single open reading frame, which encodes for a polyprotein of about two thousands amino acid residues . This polyprotein is proteolytically processed giving rise to the mature viral proteins (figure 1(a)). Three different cleavages can be distinguished on the viral polyprotein: (i) polysomal cleavages that are produced on the nascent polypeptide chain . The first of these cleavages is catalyzed by 2a at its amino terminus separating the p1 precursor that encodes for the structural proteins from the rest of nonstructural polypeptides (figure 1(b)). The second cleavage still on polysomes is performed by 3c, releasing the p2 precursor (2abc) from p3 (3abcd); (ii) cytoplasmic cleavages that are mostly exerted by 3c and (iii) hydrolysis of vp0 (vp4vp2), which is concomitant with the morphogenesis of virus particles [2, 23]. All these hydrolytic events lead to the formation of eleven mature proteins and several precursors such as p1, p2, p3, vp0, vp3, vp1, 2bc, 3ab, and 3cd . This last precursor, 3cd, can be used as substrate by 2a or 3c . The alternative cleavage carried out by 2a renders the mature products 3c and 3d, whereas 3c generates the canonical proteins 3c and 3d . However, the biological significance of this alternative cleavage is obscure because pv mutated at 2a - cleavage site on 3cd does not exhibit defects in virus replication . The nonstructural proteins that are generated participate in the replication of viral genomes [25, 26]. To this end, the positive rna genome is recognized at its 3 end by proteins of the replication complex to synthesize the complementary rna strand of negative polarity . In this process, 3b protein, also known as vpg, acts as a primer to initiate viral rna transcription . This leads to the formation of a double - stranded rna molecule, also known as the replicative form . The negative rna synthesized serves in turn as a template to direct the synthesis of several copies of positive rna, so this process leads to the production of several nascent rna molecules with a vpg molecule bound to their 5 ends on the negative rna molecule forming a replicative intermediate . The positive rna molecules synthesized may participate in three processes (i) to serve as templates for synthesizing more negative rna molecules; (ii) as mrnas that will be engaged in translation, and (iii) as genomes that will be encapsidated in new viral particles . In picornaviruses, the only type of mrna molecule known is exactly the same as the genome . Once the synthesis of several thousands of positive rna molecules is performed, the late phase of translation takes place, giving rise also during this period to a great amount of viral proteins, some of which will participate in virus morphogenesis . This late phase of infection is preceded by the abrogation of cellular mrna translation, such that only viral proteins are being synthesized late in the pv life cycle . In the case of picornaviruses thus, inhibition of viral mrna translation provokes the sudden blockade of viral rna synthesis . Moreover, translation is coupled to transcription, such that viral rnas transfected into picornavirus - infected cells are not able to direct protein synthesis . Therefore, these two processes of viral macromolecular biosynthesis are tightly coupled, making it difficult to determine exactly the function affected in some pv mutants . Notably, continuous lipid and cellular membrane synthesis is also necessary for pv rna synthesis . The morphogenesis of progeny virions in pv - infected cells is observed concomitantly with viral rna translation and replication . The release of new viral particles takes place by cell lysis, due to membrane permeabilization that occurs at the late phase of infection . Viroporin 2b and its precursor 2bc are responsible for this permeabilization upon the formation of pore channels in cellular membranes [32, 33]. Pv has represented a useful model to gain insight into diverse aspects of molecular biology and gene expression . A number of discoveries concerning animal viruses with rna genomes were initially made in pv . For example, the presence of uncapped mrna, the sequencing and development of an infectious cdna clone, the three - dimensional structure of a virus particle, the discovery of the ires elements, the synthesis of an infectious virus in a cell - free system, the chemical synthesis of a complete viral genomes, among others, were initially reported in pv [3438]. In addition, the first time that eif4 g was found proteolytically cleaved was in pv - infected cells . Picornaviruses encode different proteases depending on the virus species although it is common to all of them to encode 3c and its precursor 3cd (figure 2). In pv, both these exhibit protease activity, and they execute most of the hydrolytic events on the viral polyprotein [2, 23, 40]. Apart from these two proteases, 3c and 3cd, picornaviruses also contain a 2a gene, whose product in some species exhibits proteolytic activity, as is the case for pv (figure 2). 2a has a limited proteolytic effect on the polyprotein and its function is most probably one of altering cellular functions by the cleavage of a number of cellular proteins . In this regard, the best studied of these cleavages is the bisection of eif4 g (figure 3). The general organization of the picornavirus genomes is to encode for p1-p2-p3 precursors giving rise to 4 - 3 - 4 mature products . Some picornavirus species, in addition, encode a leader protein (l) placed before p1 (figure 2). In the case of aphthoviruses, such as foot - and - mouth disease virus (fmdv), the l protein has proteolytic activity and it is known as l [42, 43]. During polyprotein synthesis l is the first protein synthesized and its autoproteolytic activity releases itself from the rest of the polypeptide chain . Thus, the only known hydrolysis executed by l on the polyprotein is to hydrolyze between its carboxy terminus and the amino terminus of vp4 (figure 2). Because l does not play a direct role in viral replication and its protease activity has a limited impact in the viral polyprotein, this protease may be involved in the interaction with the host - cell [4547]. Indeed, l also exhibits proteolytic activity on eif4 g acting at a position close to that of pv 2a (figure 3) [4850]. In the case of fmdv, the 2a protein is reduced to a small peptide of 18 residues that does not hydrolyze eif4 g; instead it induces the release of 2a from its carboxy terminus by a ribosomal skip mechanism [51, 52]. This model proposes that fmdv 2a modifies the activity of the ribosome to promote hydrolysis of the peptidyl(2a)-trna(gly) ester linkage at the c - terminus of 2a, thereby releasing the polypeptide from the translational complex . However, not all l or 2a proteins from picornaviruses exhibit protease activity, since in the case of emcv, which encodes both proteins, neither has been demonstrated to possess proteolytic activity . All known proteases have been classified in four classes and many subgroups according to three parameters: (i) their catalytic center, (ii) their substrate specificity, and (iii) their three - dimensional structure . The classification of the different picornavirus proteases initially relied upon the effect of protease inhibitors . Compounds that blocked sulphydryl groups abrogated the proteolytic activity of 2a and 3c, suggesting that the nucleophilic aminoacid in the active site was cysteine [54, 55]. However, another cysteine inhibitor such as e64 had no effect on these proteases, while l activity was inhibited not only by sulphydryl - active compounds but also by e64 [56, 57]. These findings together with structural observations imply that l belongs to the class of papain - like cysteine proteinases [43, 5860]. Comparison of the structure of picornavirus proteases with prototypes of cellular ones revealed that both 2a and 3c are similar in structure to the chymotrypsin like group [6163]. Picornavirus 3c reflects similarities to the staphylococcus aureus proteinase, whereas 2a is more akin to streptomyces griseus proteinase a. pv 2a is a protein composed of 149 amino acids that belongs to the cysteine protease group . Pv 2a is autocatalytically processed at its amino terminus between the capsid protein vp1 and 2a (see figure 1(b)). The determinants of substrate specificity of picornaviral pv 2a have been investigated in detail by identification of cleavage sites by n - terminal edman degradation, mutational analysis and using synthetic peptides as substrates [34, 6567]. Pv 2a can recognize a wide variety of amino acid residues at the p1 position . The determinants of substrate specificity for pv 2a lie at positions p4, p2, p1, and p2, which are preferentially occupied with ile / leu, thr / ser, gly, and pro, respectively . Moreover, the determinants of substrate specificity of hrv and coxsackievirus 2a are very similar to those found for pv 2a [6567]. The yeast two - hybrid system has been used to identify the substrate sequence interacting with pv 2a . All the sequences identified contain the leu - x - thr - z motif (x for any amino acid; z for a hydrophobic residue) in positions from p4 to p1 suggesting the presence of a common interacting site on pv 2a substrates . Several 2a variants have been generated in the entire pv genome or in the isolated 2a gene . Generation of pv 2a mutants was initially used to identify the cys106, his18, and asp35 as the residues that form part of the catalytic triad of 2a [69, 70]. The role of the conserved cys and his residues in the structure - function relationship has also been studied by mutagenesis . The residues cys55, cys57, cys115, and his117 play a critical role in the cis and trans proteolytic activity by maintaining 2a structure . The structure of hrv2 2a shows that the zn ion is coordinated tetrahedrally by the side chains of these conserved cys and his residues . This zn ion is tightly bound near to the c - terminal domain and may be important for the stability of the 2a [71, 73, 74]. The yeast saccharomyces cerevisiae has been used as a system to obtain pv 2a variants . The fact that this protease is very toxic for yeast has been exploited to generate 2a variants devoid of this cytotoxicity . Using this approach, the characterization of these mutants revealed a region in 2a involved in the interaction with substrates but none of the mutations were found in the catalytic triad . A parallelism has been observed between the ability of these pv 2a variants to block protein synthesis and to cleave eif4 g . Pv mutants in 2a gene that lack trans but not cis proteolytic activity have been also identified . Normal processing of the viral polyprotein is observed upon infection with these pv variants, whereas eif4 g remains intact in these cells . Interestingly, rna replication of those mutant viruses is hampered, suggesting that there is a correlation between pv rna replication and the trans activity of 2a . Although for many years it was thought that pv 2a plays a direct role in pv replication, more recent studies have shown that a full - length dicistronic pv construct lacking 2a is capable to give rise to progeny viruses . Moreover, virus yields of pv variants lacking the p1 coding region is partially restored when p1 is expressed in trans, suggesting that cleavage of the viral polyprotein by pv 2a is not essential for viral replication . . However, it is known that 2a is important for inducing the cytophatic effect and for avoiding the inhibition of pv replication in interferon (ifn) treated cells . In agreement with the idea that 2a participates in viral rna replication, a fraction of this protease localizes in pv replicative foci although the majority of 2a is associated with the matrix structure in the cytoplasm of infected cells . However, the presence of 2a in the proximity of replication complexes does not demonstrate that it participates directly in the replication process . The process of translation can be divided in different steps: initiation, elongation, termination and ribosome recycling . The synthesis of cellular proteins is highly regulated, and in this sense, the most precisely controlled step is the initiation of translation (for a recent review, see [81, 82]). For most eukaryotic mrnas, the initiation of translation commences with the recognition of the cap structure (mgpppn) and the poly(a) tail by the heterotrimeric complex eif4f and the poly(a)-binding protein (pabp), respectively, followed by the recruitment of the 43s preinitiation complex containing the 40s ribosomal subunit, the ternary complex met - trnai - eif2-gtp, and the eukaryotic initiation factors (eifs), 1, 1a, 3, and 5 . Then, the preinitiation complex scans along the 5 untranslated region until an aug initiation codon is encountered in a favourable context . The perfect complementarity between the aug start codon and the anticodon of met - trnai leads to the arrest of scanning and the hydrolysis of gtp in the ternary complex . The release of eif2-gdp and other factors triggers the interaction of the preinitiation complex with the 60s ribosomal subunit to form the 80s initiation complex, proceeding to translation of the mrna coding region . A number of eukaryotic initiation factors participate in both mrna binding and scanning of the 5 utr of the mrna by the small ribosomal subunit . The cap - binding protein eif4e, together with the dead - box helicase eif4a and the translation initiation factor eif4 g, forms the protein complex eif4f . The heterotrimeric complex eif4f is required for recruiting the 43s preinitiation complex onto the cap structure located at the 5 end of the mrna . In this sense, eif4 g, the larger polypeptide of eif4f, functions as an adaptor molecule that bridges the mrnas to ribosomes via interactions with factors eif4e (which binds the 5cap structure), pabp (which binds the poly(a) tail), and eif3, which interacts with the 40s ribosome subunit (figure 3) [81, 8486]. In addition, eif4 g also contains binding sites for other polypeptides involved in translation, such as the rna helicase eif4a (figure 3), which is required to unwind the secondary structure within the mrna 5 leader sequence that would otherwise inhibit ribosome scanning . The simultaneous interaction of eif4 g with eif4e and pabp promotes circularization of the mrna in a closed loop that facilitates the initiation of new rounds of translation by the proximity of the 5 and 3 ends [88, 89]. Furthermore, eif4 g also interacts with the mitogen - activated protein kinase 1 (mnk1) (figure 3), which phosphorylates eif4e, although the role of this phosphorylation in the initiation of translation in still unclear [9094]. Two forms of eif4 g, known as eif4gi and eif4gii, have been identified in mammalian cells . Both forms show only 46% amino acid sequence identity but they are thought to be functionally interchangeable due to the high homology in key domains that interact with other factors . Evidence obtained by specific depletion of each eif4 g form or differential cleavage of each of them by specific proteases (see below) points to the idea that both factors should be lacking for complete abolition of protein synthesis [95, 96]. Eif4gi is the dominant form in hela cells, in which the ratio between eifgi and eif4gii is 9: 1 . However, the specific role of each form of eif4 g in the initiation of translation remains unknown . It was proposed that both eif4 g forms are differentially regulated by different kinases, supporting the hypothesis that eif4gi and eif4gii could drive differentially translation initiation . Eif4gi is phosphorylated in response to serum and in a rapamycin - dependent manner at ser 1148, 1188, and 1232, although the role of these posttranslational modifications is still under investigation . In addition, eif4gi is phosphorylated by p21-activated protein kinase (pak-2) that is induced under stress conditions . This phosphorylation takes place in the eif4e - binding site of eif4 g and avoids the interaction between these two factors, inhibiting cap - dependent initiation of translation . On the other hand, therefore, activity of eif4gi and eif4gii might be tightly and reversibly regulated by phosphorylation under different physiological conditions . Nevertheless, this factor is also subjected to irreversible modifications such as caspase - mediated proteolysis, which is triggered during apoptosis and leads to shutoff of protein synthesis . Cleaves directly eif4gi in positions 532 and 1175 removing pabp, mnk1, and one eif4a - binding domain from the eif4gi core (figure 3) [103, 104]. In contrast, eif4gii is degraded during apoptosis with a delayed kinetics in relation to eif4gi proteolysis, correlating with the shutoff of the protein synthesis . Furthermore, many eif4gi isoforms have been detected in hela cells and these are synthesized from several distinct mrnas via alternative promoter usage and alternative splicing . The largest is the eif4gi - a isoform, which contains 1,600 residues, while the eif4gi - b, -c, -d, and e are shorter variants . It has been described that the longer isoforms are more active in translation initiation, most probably because they contain the pabp - binding site . Infection of cells with pv results in a rapid shutoff of host - cell protein synthesis, whereas viral mrna translation takes place efficiently . It was initially observed that the inhibition of host - cell translation in pv - infected cells correlated with the proteolysis of a component of the eif4f complex with a molecular mass of about 220 kda (later identified as eif4 g) [39, 109]. This cleavage is exerted by 2a and can be prevented by both insertion of mutations that abolish the protease activity and addition of 2a inhibitors [76, 110, 111]. Interestingly, this proteolysis is more effective when eif4e is interacting with eif4 g, suggesting that pv 2a preferentially acts on the eif4 g pool involved in translation . Cleavage of eif4gi also occurs in cells infected with other picornaviruses such as hrv, coxsackievirus, and fmdv [48, 113, 114]. Interestingly, 2a from pv, hrv, and coxsackieviruses cleave eif4gi at positions 681/682 (figure 3), suggesting the conservation of the specificity of enterovirus 2a proteases for the substrate determinants present in eif4gi [114, 115]. Cleavage at position 681/682 separates eif4e- and eif3-binding sites of eif4gi, contained in n - terminal and c - terminal fragments respectively, thus decoupling mrna and ribosome recruiting activities . The pv 2a cleaves eif4gi directly and does not require any additional proteins for this process to occur [116, 117]. However, the fact that pv 2a is not copurified with eif4gi fragments from pv - infected cell extracts suggest that 2a induced the activation of a host protease, which in turn cleaves eif4 g during pv infection . In addition, it has been proposed that eif3 and an unknown host - cell protein could act as cofactors for eif4gi cleavage by pv 2a . In this sense, zamora and colleagues suggested that pv infection activates at least two host - cell proteases, which together with pv 2a, cleave eif4gi . Nevertheless, no additional evidence has been put forward to support this hypothesis and the identity of these host proteases has not yet been determined . Many reports have demonstrated that the kinetics of protein synthesis shutoff and eif4gi cleavage are not correlated in pv - infected cells [120122]. These data clearly indicate that additional translation factors may be cleaved to achieve an efficient inhibition of cellular mrna translation . In this regard, additional reports showed that eif4gii is also proteolyzed by 2a during pv and hrv infections and that this cleavage is exerted between amino acids 699/700 leading to a proteolytic pattern similar to eif4gi . Interestingly, eif4gii is significantly more resistant to 2a - mediated cleavage than eif4gi and the kinetics of protein synthesis shutoff close correlates with eif4gii cleavage in pv- and rhv - infected cells [122, 123]. In those studies, gradi and colleagues proposed that hydrolysis of both eif4gi and eif4gii is required for achieving pv- and hrv - mediated inhibition of host - cell mrna translation and that the cleavage of eif4gii is the rate - limiting step in the shutoff of host - cell translation after infection with those viruses [122, 123]. A variety of approaches have been devised to express pv 2a in order to cleave eif4 g in culture cells or in cell - free systems . Of these approaches, the most straightforward system has been the addition of the purified pv 2a, usually as a hybrid protein such as mbp-2a, to cell - free systems such as rabbit reticulocyte lysates (rrls), hela and krebs-2 extracts [76, 118, 124127]. In this sense, the addition of about 1 to 5 g mbp-2a suffices to hydrolyze eif4 g in those cell - free systems [76, 125127]. An alternative method to cleave eif4 g in an in vitro system is the translation of an mrna encoding pv 2a in translation competent extracts . This assay has the advantage of providing genuine and freshly made pv 2a, leading to total cleavage of eif4 g in the test tube after several minutes of translation . Many different approaches have been explored in culture cells, the most popular being transfection of plasmids encoding pv 2a in different eukaryotic cell types [75, 76, 128134]. Several plasmids have been utilized in this respect, and perhaps the most successful one is ptm1 - 2a, which is transfected in mammalian cells that transiently express t7 rna polymerase by infection with a recombinant vaccinia t7 virus [76, 130, 131, 133, 134]. The amount of protease synthesized in this system is similar to that found in pv - infected cells at late times of infection, but these amounts are reached 1 - 2 hours after transfection . Similar results have been obtained in cells constitutively expressing t7 polymerase, which comprises a less pleiotropic system, because vaccinia virus proteins are not expressed (unpublished data). Since pv 2a targets a number of different cellular proteins, which affect several cellular functions depending on the amount of protease synthesized (see below), in some instances, it is useful to express 2a at low levels . We have explored many alternative methods trying to get a system that allows us to control the levels of pv 2a into the cells . Novoa and colleagues developed a protocol based on the addition of hybrid proteins bearing pv 2a . These recombinant proteins enter into the cytoplasm on cell membrane permeabilization by different methods such as addition of mbp-2a mixed with replicationally inactive chicken adenovirus particles . Cleavage of eif4 g following these protocols takes place after incubation for 810 hours, suggesting that the amount of protease internalized is probably low but sufficient to hydrolyze eif4gi in virtually all culture cells . Probably one of the most attractive systems is a stable cell line that inducibly express pv 2a, obtained in two different laboratories including ours [137, 138]. In these cell lines, pv 2a is synthesized when tretracycline is removed from the culture medium, leading to low expression of pv 2a that induces efficient cleavage of eif4 g after 13 h post induction correlating with a potent inhibition of cellular translation . Finally, long term expression of pv 2a in tet off cell lines triggers apoptosis [12, 137, 138]. The main drawback of this cell line is the low pv 2a escape under repression conditions that gives rise to a basal cytotoxicity . Probably, the most efficient method is electroporation of an mrna encoding 2a under the control of emcv leader sequence (ires-2a). The biggest advantage of this method is the capacity to regulate levels of 2a expression by controlling the amounts of ires-2a transfected . For example, electroporation of 9 g of ires-2a into ~1.5 10 hela cells leads to total cleavage of both eif4gi and eif4gii in only 2 h, resulting in an almost complete shutoff of cellular protein synthesis . In contrast, electroporation of low amounts of ires-2a (1 g) into hela cells induces efficient cleavage of eif4gi, whereas eif4gii remains largely intact . Therefore, 9-fold more ires-2a mrna is required to cleave eif4gii compared to eif4gi . Based on the ires-2a mrna electroporation method we were able to induce the differential proteolysis of eif4gi and eif4gii in a time- and dose - dependent manner kinetics of protein synthesis shutoff and eif4gii cleavage is closely correlated in hela cells, resembling what was found in pv - infected cells . In agreement with what was observed with the addition of exogenous recombinant proteins, translation of de novo synthesized mrnas showed higher susceptibility to low doses of pv 2a than mrnas already engaged in translational machinery . These results suggested a possible specific role of eif4gi in the pioneer round of translation in agreement with a previous report . However, specific ablation of eif4gi using sirnas induced a moderate inhibition of luciferase synthesis from de novo synthesized and preexisting mrna (about 40% in both cases). These findings reported by welnowska and colleagues indicated that the higher susceptibility of de novo synthesized mrna translation to low doses of ires-2a might be produced by an additional effect of pv 2a on another gene expression step . In this regard, further studies demonstrate that the stronger impact of 2a on de novo synthesized mrnas is due to the concomitant inhibition of rna nuclear export by nucleoporin 98 cleavage, which is also achieved under these conditions (see below). Interestingly, cellular mrnas are able to initiate translation after a polysome runoff with high salt treatment when eif4gi is totally cleaved by pv 2a, whereas it is completely abolished when both forms of eif4 g are proteolyzed . Taken together these set of data from cell expressing 2a [95, 136] as well as from pv - infected cells [120122] we can conclude that complete shutoff of the protein synthesis induced by pv 2a is achieved when both eif4gi and eif4gii are completely cleaved . Therefore, when the levels of one of the two populations of eif4 g remain unaffected either because it is not cleaved by pv 2a or it is not depleted by sirnas, extensive host protein synthesis takes place . The infection of pv and coxsackievirus also leads to hydrolysis of pabp [140, 141]. This cleavage is carried out by pv 3c and coxsackievirus 2a and 3c and it might actively contribute to the host translational shutoff induced by these viruses [142, 143]. In conclusion, the proteolysis of different components of the translation initiation machinery by picornavirus proteases can account for the shutoff of host translation induced after infection although the specific contribution of hydrolysis of eif4gi, eif4gii, and pabp remains still unclear . Infection of animal cells with fmdv also leads to proteolysis of eif4 g and to rapid inhibition of cellular translation . The proteolysis of eif4 g is carried out by the two virally encoded proteases l and 3c [144, 145]. L cleaves both eif4gi and eif4gii extremely rapidly at positions 674 (figure 3) and 700, respectively, located seven and one amino acids upstream of the 2a cleavage sites on eif4gi and eif4gii [50, 146]. The cleavage of eif4 g by fmdv l results in the rapid shutoff of host - cell protein synthesis . Although the initial cleavage of eif4gi can be carried out by fmdv l in the absence of virus replication, a sequential cleavage of the c - terminal fragment of eif4gi by fmdv 3c also occurs in bhk cells at early stages of infection concomitant with the shutdown of viral translation . The 3c cleavage site on eif4gi has been located at position 712, 38 amino acids downstream of the l cleavage site although the role of this sequential cleavage is still unclear . The amino acid segment of eif4 g located between the l and 3c cleavage sites binds rna and was suggested to be critical for mrna scanning by the preinitiation complex . Interestingly, this secondary cleavage does not occur in human cell lines due to an amino acid substitution at the cleavage site on eif4gi . Infection of cells with other picornaviruses such as cardioviruses (emcv and mengovirus) leads to a shutoff of host - cell protein synthesis . These findings indicate that apart from eif4 g cleavage, there are other mechanisms that may block host translation by picornaviruses . In addition to picornavirus l and 2a, proteases from other viruses can also cleave eif4 g . The protease of human immunodeficiency virus type-1 (hiv-1) hydrolyzes eif4gi during infection of human cd4 + cells . The cleavage of eif4gi takes place at positions 718, 721, and 1125, separating it in three domains (figure 3) [149, 150]. Interestingly, hiv-1 protease efficiently cleaves eif4gi, but not eif4gii, both in cell - free systems and in mammalian cells . The differential sensitivity of eif4gi and eif4gii to hiv-1 protease is more selective than that observed with picornaviral 2a proteases [122, 123]. Hiv-1 protease also cleaves pabp at positions 237 and 477 separating the two first rna - recognition motifs from the c - terminal domain of pabp . Cleavage of eif4gi and pabp by hiv-1 protease is sufficient to inhibit the translation of capped and polyadenylated mrnas in cell - free systems, as well as in transfected cells [127, 151]. In contrast, ires - driven translation is unaffected or even enhanced by hiv-1 pr after cleavage of both eif4gi and pabp [127, 151]. Moreover, the translation of capped and polyadenylated hiv-1 genomic mrna remains unaffected in hela extracts under these conditions suggesting that viral protein synthesis might persist at late phases of hiv-1 infection where those factors are cleaved . In contrast, a previous report claimed that the hydrolysis of eif4gi impaired the translation of both capped and ires - driven mrnas in reticulocyte lysate assays . However, the different effect observed by these authors on ires - driven translation can be due to differences already reported between hela extract and rrl [143, 153]. In addition, eif4 g cleavage is executed by proteases from other retrovirus species, such as hiv-2, simian immunodeficiency virus (siv), human t - cell leukemia virus (htlv-1), moloney murine leukemia virus (momlv), and mouse mammary tumor virus (mmtv). These proteases hydrolyze eif4gi and eif4gii with different cleavage patterns and kinetics . And indeed, several retroviruses, including hiv, siv, and momlv, promote the translation of their gag gene products by internal ribosome entry, indicating that eif4 g cleavage could be compatible with viral protein synthesis in infected cells [154156]. Furthermore, cleavage of eif4gi and eif4gii also occurs in feline calicivirus - infected cells although the cleavages occur at different sites to those observed for picornavirus proteases . In addition, the 3c - like protease of two caliciviruses, like pv 3c, cleaves pabp perhaps as a complementary strategy to inhibit cellular translation . The fact that proteases from many picornaviruses, retroviruses and caliciviruses, target eif4 g, and, in some cases, pabp, strongly suggest that those viruses may share a common mechanism to regulate cellular and viral translation . However, further investigation is required to determine the specific contribution of eif4gi, eif4gii and pabp to the shutoff of host - cell translation and virus protein synthesis . The biological cycle of picornaviruses is confined to the cytoplasm of infected cells . However, some of the pv proteins are able to target nuclear proteins such as transcription and splicing factors and proteins involved in nuclear - cytoplasmic trafficking . One of the best studied cases in this regard is the cleavage of nucleoporins (nups), components of the nuclear pore complex (npc), by pv and hrv 2a, which directly impacts on nuclear - cytoplasmic trafficking of proteins and rnas [15, 16, 159]. Complementarily, emcv l protein affects on the phosphorylation status of nup62 and induces similar effects to those described for pv 2a in the transport of macromolecules through npc . Nevertheless, nups are not only targets for picornavirus proteins but also for matrix (m) protein from vesicular stomatitis virus (vsv) and nonstructural protein 1 (ns1) from influenza virus, which also impair components of the nuclear export - import host machinery following analogous mechanisms . Taking into account that proteins from different positive and negative strand rna viruses target nups, we highlight in this paper npc as a key target for viral proteins, although the possible role of npc in virus biological cycle is still under intensive research . Nucleus and cytoplasm are physically separated by a semipermeable barrier known as the nuclear envelope . Due to this compartmentalization, a large number of macromolecules traverse the nuclear envelope to reach their biological destination . For example, proteins are synthesized by ribosomes in the cytoplasm, but some of them such as polymerases, transcription factors, nucleosome components and splicing factors, have to traverse the npc to reach the nucleoplasm . Conversely, all rna species are transcribed and processed in the nucleus and later, most of mature rnas are exported through the npc to the cytoplasm, where their biological roles take place . Therefore, the regulation of rna and protein trafficking between nucleus and cytoplasm directly impacts on gene expression [163, 164]. Npc forms large structures (~125 mda) embedded in the nuclear envelope with a polarized eightfold symmetrical core . It is sandwiched by a cytoplasmic and nuclear ring, which projects eight filaments of about 50 nm into the cytoplasm and a basket - like structure of about 100 nm into the nucleoplasm [165, 166]. The npc is composed of multiple copies (8, 16 or 32) of ~30 different proteins, called nups, that are grouped in three major classes: (i) the phenylalanine - glycine (fg)-containing nucleoporins that actively work in the nuclear - cytoplasmic trafficking of macromolecules; (ii) the structural components, which lack fg - rich domains and (iii) the membrane integral proteins, which anchor the npc to the nuclear envelope [163, 167]. Whereas the two last groups of nucleoporins play a role in the architecture and localization of the npc, the fg - nucleoporins directly regulate the transport of rnas and proteins through the npc, and they are the main nucleoporin class targeted by viruses (figure 4). Movement of ions, metabolites and other small molecules between nucleus and cytoplasm takes place by passive diffusion; however, transport cargos larger than 40 kda require the participation of specific receptors and carriers [168, 169]. Fg nucleoporins are placed on both cytoplasmic and nucleoplasmic sides of the npc and play a central role in the active transport of macromolecules . The fg domains of nucleoporins are unfolded regions that participate in energy - independent transient interactions with the cargo receptors during the docking and translocation processes . Nevertheless, the delivery of the cargo and some of the directional steps require hydrolysis of gtp . Trafficking of proteins through npc is mediated by a family of conserved transport receptors named karyopherins, which recognize short peptides known as nuclear localization signal (nls) and nuclear export signal (nes) [169, 171]. In addition, karyopherins also recognize nucleotide sequences during the export of some classes of rnas . Due to their key role, karyopherins involved in cargo import are known as importins and those involved in export are known as exportins . The rangtp cycle also plays a central role in karyopherin activity and, therefore, in trafficking of macromolecules through the npc . Importins bind the cargo in the cytoplasm and release it on binding to rangtp in the nucleus . In contrast, exportins bind the cargo in the nucleus together with rangtp; and then the ran - associated gtp is hydrolyzed in the cytoplasm by rangap and cargo is liberated [163, 164]. Proteins containing constitutively active nls are predominantly nuclear; but in some cases, the accessibility of nls or the nls itself is modified to selectively regulate the localization of the protein . A similar regulatory mechanism is also exerted for control of nes activity . For example, the nls of the transcription factor nfb is masked by the interaction with the inhibitor ib. However, ib is degraded on proinflammatory stimuli, exposing the nls of nfb for importin recognition . Therefore, under proinflammatory conditions, nfb is imported to the nucleus, where it triggers a specific gene response . In addition to masking strategies, phosphorylation, ubiquitination or methylation of nls or nes also influences (negatively or positively) their recognition by importins or exportins . Therefore, trafficking of proteins between nucleus and cytoplasm could be finely regulated by posttranslational modifications in the nls and nes [164, 174]. Conceptually, export of most of nuclear rna follows a similar mechanism to that described above for protein trafficking . This process also involves cargo receptors, export factors, and nucleoporins to deliver mature rna to the cytoplasm, but in this case, structure and function of nuclear export signals are not well understood . Aminoacylated trnas are necessary in the cytoplasm for protein synthesis, but trnas are transcribed in the nucleus . Trna export to the cytoplasm is mediated by exportin - t, which belongs to the karyopherin family . Exportin - t forms a complex together with rangtp in the nucleus, and once the exportin - t - rangtp - trna complex reaches the cytoplasm, rangap induces the hydrolysis of the ran - associated gtp and the release of the trna [175, 176]. It has been proposed that exportin-5 could act as an auxiliary protein in this process . However, later reports in yeast have opened the possibility of alternative pathways for trna nuclear export . Snrnas are transcribed by the rna polymerase (pol) ii (with the exception of u6 snrna which is produced by poliii) and then they are capped but not polyadenylated . Cap - binding proteins (cbp) 80 and 20 interact with the cap of snrnas in the nucleus and recruit an export adaptor known as phax . This adaptor is phosphorylated and in this state, it is able to interact with crm1, another member of karyopherin family . Hydrolysis of rangtp and dephosphorylation of phax lead to the release of the snrna in the cytoplasm . Ribosomal proteins are assembled together with the different rrnas in the nucleolus following a complex process of maturation to give rise to the ribosomal subunits 40s and 60s . Although is known that preribosomal subunits are exported by separate routes that involve crm1 and rangtp, nowadays the exact mechanism followed by 40s preribosomal subunits to leave the nucleus remains unclear . However, 60s preribosomal subunit relies on nmd3 adaptor, which mediates the interaction with crm1 [179, 180]. The release of the 60s preribosomal subunit requires two gtpase steps: (i) the hydrolysis of the ran - associated gtp induces the liberation of crm1; and (ii) the hydrolysis of gtp mediated by the cytoplasmic gtpase lsg1, which induces the release of nmd3 . Finally, mrnas compose the most heterogeneous group of rnas, varying in length and structure . Mrnas are transcribed by polii, and, concomitantly with this process, a number of rna - binding proteins assemble with them . These rna - binding proteins exert different modifications in immature mrnas such as polyadenylation, splicing and capping . In addition, export factors and adaptor proteins are also recruited to nascent pre - mrnas, playing a further function in nuclear export . Trex (transcription - coupled export) complex is recruited to the 5 end of nascent pre - mrnas in a splicing - dependent manner by means of the interaction of one of its components, namely aly / ref, with the subunit of the nuclear cap - binding complex cbp80 . The trex complex also recruits the conserved rna - helicase uap56 that is important for mrnp biogenesis . Tap - p15, (also known as nxf1-nxt1) directly binds the mrna immediately after splicing and actively participates in mrna export . Aly / ref, tap - p15 and uap56 associate with exon junction complexes (ejc), which are deposited in a splicing - dependent manner at 2024 nt of every exon - exon junction [184, 185]. This interaction network makes spliced mrnas more susceptible to export and couple splicing and mrna - trafficking . In fact, unspliced mrnas are exported by an alternative and less efficient pathway that involves crm1 and rangtp . This alternative route is also followed by mrnas encoding some protoncogenes and cytokines [186, 187]. It is important to mention that although mrna can follow different nuclear export pathways, in all cases the interaction of export receptors with nucleoporins plays an essential role in the transport of the mrnps throughout the npc . Pv infection strongly impacts on host - cell protein localization, giving rise to an unusual cytoplasmic distribution of nuclear proteins . This particular effect has been characterized by different laboratories for a number of nuclear factors involved in several cellular processes and containing different types of nlss . Nevertheless, not all nuclear proteins are re - localized after pv infection, evidencing the presence of a viral - specific mechanism affecting protein subcellular distribution . Gustin and colleagues proposed the inhibition of nuclear protein import machinery as the cause of the cytoplasmic accumulation of nuclear proteins in pv - infected cells (figure 4). They demonstrated that pv impairs protein trafficking across the npc by expressing gfp proteins encoding classical or transporting nlss in mock and infected cultured cells [15, 16, 159]. These recombinant proteins accumulate in the cytoplasm after pv infection, being almost completely depleted from the nucleus . Nevertheless, pv does not affect gfp distribution when the nls is mutated or deleted, since the small size of gfp allows its inefficient efflux throughout the npc . Interestingly, cell - free nuclear import assays demonstrated that nls - containing gfp is unable to traverse the npc when cells are previously infected with pv . In agreement with these findings, shuttling endogenous proteins, such as heterogeneous nuclear ribonucleoprotein (hnrnp) a1 and hnrnp k, are detected in the cytoplasm of infected cells from 3 hpi but are undetectable in the nucleus at 4.5 hpi . However, cytoplasmic accumulation of nuclear resident proteins such as hnrnp c requires longer times of infection . These findings support the idea that the distribution of a protein that shuttles between nucleus and cytoplasm may be strongly altered by the disruption of protein trafficking pathways, as compared to nuclear resident proteins . However, not all the nuclear factors are redistributed to the cytoplasm of pv - infected cells . This is the case for sc35 (a serine / arginine - rich splicing factor), fibrillarin or tata - binding protein (tbp), which remain in the nucleus for the duration of infection . The different behaviour of these groups of proteins could arise as a consequence of different turnover; thus, the distribution of highly stable nuclear proteins might be less affected by the inhibition of protein nuclear import than those proteins with low stability . Alternatively, pv might not impair all protein import pathways, and some might remain operative, thereby allowing the nuclear import of several families of nuclear proteins . Interestingly, some nuclear factors such as la antigen, ptb, sam68, or nucleolin have been shown to interact with pv rna or viral proteins . These proteins accumulate in the cytoplasm of pv - infected cells and, consequently, their availability for viral replication is increased [188, 193195]. Cytoplasmic accumulation of nuclear factors might be produced mostly by the blockade of nuclear - cytoplasmic trafficking . However, loss of the nls after the cleavage of ptb and la proteins by pv 3c may also contribute to the subcellular relocalization of those proteins in pv - infected cells [193, 195]. Redistribution of nuclear proteins as well as impairment of cellular import machinery was also observed in cells infected with other picornaviruses such as hrv or emcv . These findings indicate that most picornaviruses might share similar strategies to impair nuclear - cytoplasmic trafficking machinery . An alternative hypothesis has been proposed as the cause of the redistribution of nuclear factors . Belov and colleagues reported that the nuclear envelope is permeabilized on pv infection, allowing nuclear proteins to diffuse across the nuclear membrane . However, this hypothesis did not clarify why other proteins resident in the nucleus do not diffuse to the cytoplasm after pv infection . Both protein nuclear import and permeabilization of nuclear envelope protein import blockade is detected early after infection, correlating with the first modifications in the npc (see below). However, prolonged expression of viral proteins might induce nuclear membrane leakiness, reflected by stronger alterations in npc architecture . Nevertheless, what might the biological relevance of these events be for a cytoplasmic virus? The most evident answer, which was extensively commented above, is that inhibition of nuclear protein import and further nuclear envelope leakiness might increase the presence of nuclear proteins in the cytoplasm of infected cells, which has been proposed in some cases to play a relevant role in pv replication . In addition, many transcription factors are arrested in the cytoplasm of uninfected cells (e.g., nf-b, irf7, and irf3), but they are immediately activated and imported to the nucleus after proinflammatory extracellular signals or on the activation of intracellular sensors as a consequence of the viral replication . Once in the nucleus, these factors trigger the transcription of a set of genes involved in the antiviral response . Inhibiting the import of these transcription factors, pv might prevent or, at least attenuate, the establishment of a hostile intracellular environment . Interestingly, nup98, nup153 and nup62, components of the npc belonging to fg nup family, were found to be degraded in pv- as well as hrv - infected cells (figure 4) [15, 16, 159]. These proteins are essential factors of the nuclear - cytoplasmic trafficking machinery since their n - terminal fg - rich domains serve as docking sites for soluble transport factors [163, 198]. Nup98, nup153 and nup62 are proteolyzed in pv - infected cells following different kinetics . Thus, nup98 is the cleaved early after infection (from 1 hpi), whereas nup153 and nup62 are targeted at late times after infection (from 4 hpi) [15, 16]. In agreement with these findings, cleavage of nup98 is induced even in presence of inhibitors of pv replication, suggesting that small amounts of viral proteins are sufficient for this proteolysis to occur . However, cleavage of nup153, and nup62 are efficiently prevented on arrest of viral replication, probably because they are only efficiently achieved when large amounts of viral proteins are produced . Therefore, nup98, nup153, and nup62 exhibit different susceptibilities to pv replication, thus pv might have a gradual impact on the nuclear - cytoplasmic trafficking machinery . Nup153 is also proteolyzed by caspase-3 and caspase-9 during apoptosis induction; however, the involvement of these cellular proteases in pv - induced nup cleavage has been ruled out by different laboratories . First, nup153 cleavage products generated upon caspase activation differ to those found in pv - infected cells . In addition, nup62 is cleaved in pv - infected cells, but it remains intact despite caspase activation . Most importantly, pv - induced npc structural damage takes place in cells lacking caspase 3 and 9, and nup153 and nup62 are efficiently cleaved in pv - infected cells even in presence of the caspase inhibitor z - vad . All together, these data support the idea that one or more viral proteins play a direct role in the cleavage of those nups . In agreement with this hypothesis, pv - induced npc damage is prevented by pv 2a inhibitors such as elastatinal, elastase, and mpcmk, suggesting an involvement of this viral protease in the alteration of npc . Indeed, individual expression of pv 2a in hela cells as well as addition of this protease to cell - free systems gives rise to nup98, nup62, and nup153 cleavage [16, 17, 200]. In agreement with the data obtained from pv - infected cells, on pv 2a expression in hela cells, nup98 is cleaved faster than nup62 and nup153, which suggests the presence of optimal cleavage sites in this protein . Proteolysis of nup98 in pv - infected cells as well as in cell - free systems generates two different cleavage products of around 5065 kda and 35 kda . There are two optimal cleavage sites in nup98 for pv 2a located between aminoacids 373 - 374 and 551 - 552, containing gly at p1, thr at p2, and leu at p4 . Hydrolysis at both sites results in n- and c - terminal products with predicted molecular masses of 37 and 53 or 55 and 35 kda, in good agreement with the size of the peptides detected experimentally in pv - infected cells and 2a - treated hela extracts [16, 17]. An explanation for the delayed kinetics of nup62 and nup153 with respect to nup98 is that optimal pv 2a cleavage sites were not found in these nups (unpublished data). Recently, park and colleagues have reported that pv 2a directly cleaves nup62 at six different positions rendering multiple proteolytic products . These cleavage sites are located between aminoacids 103 and 298, thus releasing the fg - rich region from the protein core . Functionally, loss of the fg - rich region might make nup62 inactive for interaction with cargo receptors . This hypothetical mechanism of nup62 functional decoupling could be extrapolated to nup98 and nup153 (figure 4). However, it remains unknown whether pv 2a is able to directly cleave nup98 and nup153 or where cleavage might occur . Nup98, nup153 and nup62 are also involved in rna export from the nucleus and therefore, cleavage by pv 2a might also impact on this process (figure 4). However, oligo d(t) hybridization studies showed that pv infection does not affect distribution of the polyadenylated mrna bulk after 3 hpi . A more detailed analysis revealed that expression of pv 2a in hela cells induces a number of disorders in rna location . Nuclear export of cellular mrnas is inhibited in 2a - expressing cells in a dose dependent manner concomitantly with nup98, nup62 and nup153 cleavage . Interestingly, mrna export of constitutively expressed mrnas such as -actin is less affected than that of newly synthesized mrnas . For example, tetracycline - induced luciferase mrna was almost totally retained in the nucleus when these nups are cleaved by pv 2a . This effect was also observed for endogenous mrnas such as il-6, c - myc or p53 mrnas which are induced on pv 2a expression . Therefore, pv 2a could counteract the induction of proapoptotic (c - myc and p53) and proinflammatory (il-6) responses by accumulating the c - myc, p53 and il-6 mrnas in the nucleus . This export blockage may prevent the establishment of a host - cell response against pv infection . These findings could explain why pv 2a is essential for replication of pv in cells pre - treated with ifn- . Furthermore, impairment of mrna export strongly alters the localization of mrnas with high turnover as compared to constitutively expressed and highly stable mrnas such as -actin . As observed in pv - infected cells, oligo d(t) hybridization revealed that pv 2a expression hardly affects the distribution of the polyadenylated mrna pool after short times of expression (8 h). However, nuclear accumulation of polyadenylated mrna bulk is detected when cells are exposed to pv 2a for longer times (16 and 24 h). In this regard, the progression of the alterations of polyadenylated mrna localization in 2a - expressing cells takes place as follows: (i) disruption of nuclear mrna - containing foci (ii) appearance of mrna - containing granules in the cytoplasm (most probably stress granules) and (iii) depletion of cytoplasmic mrnas . These events were more clearly observed when high amounts of pv 2a are synthesized, reflecting that nuclear accumulation of mrnas is a time- and dose - dependent process . Nevertheless, pv 2a is not only able to block mrna export, but also rrna and snrna transport . Both 18s rrna and u2 snrna accumulate in the nucleus of 2a - expressing cells in a dose - dependent manner . Most probably, cleavage of nup98, nup62 and nup153 is involved in these effects, since rrna, snrna, and mrna are exported using different cargo receptors and auxiliary proteins (see above) but all of them relay in nup activity to traverse npc . Importantly, trnas (val - trna) are exported normally despite pv 2a expression, indicating that some rna nuclear export pathways are not affected by this viral protease . In fact, nup98, nup62, and nup153 are not directly involved in trna export, reinforcing the idea that nucleoporin cleavage plays a central role in the impairment of protein and rna trafficking by pv (figure 4). Notably, ifn- induced a specific increase of nup98 levels in hela cells that counteracts the inhibition of mrna export by pv 2a [17, 201]. Collectively, these findings reflect the central role of nup98 in pv infection and in antiviral response, since its overexpression by itself prevents, at least in part, blockade of nuclear rna export . Therefore, secretion of ifn- by immune cells might allow the induction of antiviral response by neighbouring cells by increasing the levels of nup98 in order to protect nuclear - cytoplasmic protein and rna trafficking pathways . The physiological relevance of the crosstalk between nup98-pv 2a in pv (and other viruses) infection might be studied in the future with cellular and animal systems . As mentioned above, hrv also induces cleavage of nup62, nup153 and most probably nup98, leading to the impairment of nuclear protein import . Nevertheless, pv and hrv 2a exhibit high homology and both proteases are therefore expected to share common targets . In contrast, the 2a gene of cardioviruses encodes a short peptide with autoproteolytic activity but lacks trans - protease activity as occurs in pv - infected cells, these cardioviruses induce both protein nuclear import inhibition and late membrane leakiness but they do not induce the cleavage of nups [160, 202, 203]. Cardioviruses encode an additional protein known as l protein, which is highly cytopathic although it lacks protease activity (in contrast to aphthovirus l). L protein contains a zinc finger domain, and an acidic region, which is proposed to be phosphorylated in infected cells . Individual expression of this protein in cultured cells or in cell - free systems induces several cellular disorders including the inhibition of protein nuclear import that resembles that observed in emcv - infected cells [160, 203]. Several studies reported emcv l protein mutations that resulted in defective virus growth phenotypes in cell culture [202, 204]. In particular, mutations in the zinc finger domain (cys19ala and cys22ala) or in the acidic region (thr47ala) partially avoid blockade of protein trafficking between the nucleus and the cytoplasm . Taken together, these data support the involvement of cardiovirus l protein in npc damaging and in the inhibition of protein nuclear import, inducing nups phosphorylation rather than their cleavage . Indeed, nup62 is quickly and strongly phosphorylated after emcv infection (2 hpi), and it was clearly detected by conventional western blotting . Nevertheless, analysis with pro - q diamond phosphoprotein stain revealed that nup153 and nup214 are also phosphorylated to a certain extent upon emcv infection . Notably, nups phosphorylation was avoided when cys19ala mutation was inserted in the l protein sequence, suggesting that the zinc finger domain is essential for this posttranslational modification to occur . However, the exact role of nups phosphorylation in nuclear - cytoplasmic trafficking is still unknown . The idea that nups phosphorylation could regulate protein import and rna export as a switch that turns the different pathways on / off should be pursued in more detail . As mentioned above, ran is essential for the regulation of most of the nuclear export and import pathways, because it acts as a cofactor modulating the affinity of importins and exportins for the cargo . It has been described that emcv l directly interacts with ran, and this interaction is abrogated by the insertion of c19a mutation in l . However, the potential role of l / ran interaction in the modulation of rangtp cycle and its impact in nuclear import and export pathways have not yet been studied . Alteration of nuclear - cytoplasmic trafficking is not only restricted to picornaviruses but has also been observed with negative strand viruses such as vesicular stomatitis virus (vsv) and influenza virus . Her and collaborators reported that rna export and protein import are strongly inhibited by vsv matrix (m) protein, by microinjection of oocytes with radiolabeled rnas and proteins . Radiolabeled trnas, mrnas, u snrnas, and rrnas were injected directly into the nucleus of xenopus laevis oocytes, and the subcellular localization of those rnas was monitored by autoradiography . The conclusion of this work is that mrna, u snrna, and rrna but not trna trafficking is blocked by vsv m protein, in agreement with our findings on 2a - expressing hela cells . In addition, protein nuclear import was monitored by microinjection of radiolabeled proteins containing nls in the oocytes cytoplasm . This assay revealed that vsv m protein abrogates protein nuclear import to the same extent as treatment with specific inhibitors of this pathway such as wga . These interesting findings support the idea that pv 2a and vsv m protein could target similar host proteins to impair macromolecule trafficking between nucleus and cytoplasm . In agreement with this possibility, it was found that the vsv m protein interacts with nup98, which is one of the primary targets of pv 2a . The n - terminal domain of vsv m is sufficient to block rna nuclear export and aa 5254 may play an essential role in this blockade, because their mutations to ala completely abrogate this inhibitory effect . Indeed, the n - terminal domain of m protein is involved in the interaction with nup98 and, in particular, aa 5254, because their mutation to ala blocks the binding of vsv m to nup98 . In addition, binding of m to nup98 requires active mrna export pathways since, treatment with inhibitors such as wga hampers this interaction . Vsv m is also able to induce the accumulation of endogenous polyadenylated mrnas in the nucleus of hela cells, and this effect is prevented again by mutations in aa 5254 of m . Furthermore, vsv m interacts with rae1, which plays an essential role in mrna nuclear export by its interaction with nup98 and mrnps . Overexpression of either nup98 or rae-1 prevents the nuclear accumulation of polyadenylated mrnas, suggesting that both factors may play a role in the blockade of mrna nuclear export by vsv m protein . Interestingly, ifn- specifically increases the level of both nup98 and rae-1 and indicates a potential antiviral effect of these proteins . Indeed, overexpression of both proteins by ifn- treatment counteracts the inhibitory effects of vsv m protein on mrna nuclear export, highlighting the possibility of a crosstalk between m and ifn- that might control the fate of the viral replication in infected animals [201, 206]. Influenza virus replication also impacts on nuclear - cytoplasmic trafficking and leads to the nuclear accumulation of host mrnas [162, 207]. Influenza virus ns1 protein is a major virulence factor that is essential for pathogenesis, because it impairs innate and adaptive immunity by inhibiting host signal transduction and gene expression [208, 209]. Ns1 forms a complex with nxf1/tap, p15/nxt, rae1, and e1b - ap5, which are components of the mrna nuclear export machinery (see above). Individual expression of ns1 in 293 t cells induces the accumulation of polyadenylated mrnas in the nucleus, suggesting that the interaction of ns1 with these export factors yields an inactive complex for mrna export . Influenza virus also induces a strong reduction of nup98 steady - state levels although the viral mechanisms involved in this process are still unknown . Expression of reporter luciferase mrna synthesized from a nuclear plasmid, this inhibition is overcome by overexpression of nxf1, p15, rae-1 or nup98, evidencing the role of ns1 interaction with these factors in the impairment of mrna nuclear export . Furthermore, mouse cells expressing low levels of nup98 or / and rae-1 show greater susceptibility to influenza infection, resulting in a significant increase in cell death and virus production . In addition, mrnas encoding antiviral factors or immunomodulators such as irf-1, mhc i and icam1 accumulated more in the nucleus of those cells than in cells expressing normal levels of nup98 or rae-1 . All these data support the physiological role of ns1 interaction with rna export factors as well as the reduction of nup98 levels in influenza pathogenicity . Interestingly, vsv m, influenza ns1 and pv 2a expression gives rise to similar effects on mrna trafficking . All these viral proteins target nup98 and other components of the cellular machinery involved in nuclear - cytoplasmic trafficking . Survival of motor neurons (smn) complex is composed by smn and a class of proteins called gemins, which localize in both cytoplasm and nucleoplasm [210, 211]. Gemin7 and gemin8 constitute the core of the complex where the other gemins associate by means of numerous protein - protein interactions from the periphery . The smn complex is involved in the biogenesis of uridine - rich small nuclear ribonucleoprotein (u snrnp) in the cytoplasm and then the u snrnp carries out the splicing of pre - mrnas in the nucleus [212214]. The snrnps are composed of the major u snrnas u1, u2, u4, u5, and u6 as well as a group of seven proteins known as sm ribonucleoproteins that collectively make up the extremely stable sm core of the snrnp . Gemins (except gemin-2) associate with sm proteins to form a heptameric ring structure in the presence of u snrnas . After sm core assembly, the u snrnps are imported to the nucleus, localizing in foci known as cajal bodies, where further maturation processes take place . Gemin-3, one of the main components of smn complex, is cleaved in pv - infected hela cells leading to a 50 kda cleavage product . Scission of gemin-3 negatively impacts on the kinetics of sm core assembly, which is prevented in presence of inhibitors of pv replication . These results indicated that high levels of pv proteins are required for this process to occur, as is the case of nup153, nup62, and eif4gii, [16, 122]. Pv 2a is able to hydrolyze purified gemin-3 in vitro, rendering a cleavage product similar to that found in pv - infected hela cells . Only one potential 2a - cleavage site, between the amino acids tyr462 and gly463 (vhtyg), was found in this smn complex component . Proteolysis of gemin-3 at this position would render two cleavage products of about 5030 kda, in agreement with the polypeptide of about 50 kda found in pv - infected and 2a - expressing cells . In addition, g463e mutation avoids direct hydrolysis of gemin-3 exerted by pv 2ain vivo and in vitro . Taken together, these findings support the notion that vhtyg is the cleavage site for pv 2a in gemin-3 . Although hydrolysis of gemin-3 is exerted in cells transfected with plasmid encoding pv 2a, it does not take place when this protease is expressed from exogenous mrnas; contrary to that found with eif4gi, eif4gii, nup98, nup153 and nup62 (and unpublished data). A probable explanation for this difference is that pv 2a is expressed at lower levels from transfected mrnas than from plasmids (castello et al ., 2006). Thus, gemin-3 cleavage may be a very late event in pv - infected cells because it requires expression of high amounts of pv 2a . Gemin-3 hydrolysis may directly impact on pre - mrna splicing since this event reduces the availability of smn complexes, which is involved in u snrnps biogenesis . Nevertheless, alstead and colleagues could not detect any apparent effect of gemin-3 proteolysis in splicing of cellular pre - mrnas . In addition to eifs, nups and proteins from smn complex, pv 2a is able to cleave proteins involved in other cellular processes, such as transcription . Tbp is cleaved by pv 2a between amino acids tyr34 and gly35 in vitro, although this cleavage only removes the first 34 aa located at the n - terminus and does not inhibit transcription carried out by rna polymerase ii [217, 218]. These findings are in agreement with the fact that host mrna transcription takes place in 2a - expressing cells when both translation and rna nuclear export are inhibited, upon cleavage of eif4 g and nups . One attractive hypothesis is that pv 2a could cleave specific initiation factors affecting specific rather than general mrna transcription in order to modulate host - cell response to viral infection . Further studies in this direction can be carried out using microarray platforms to detect precise alterations in cellular transcriptome after pv - infection or 2a expression . These studies could be complemented by screening for new host factors cleaved by pv 2a using different in silico and experimental approaches . The study of viral proteases is crucial to understand the mechanism used by animal viruses to replicate their genomes and to translate viral mrnas at the molecular level . In addition, we wish to draw attention to the concept that viral proteases can be used as tools to reveal the exact functioning of their target cellular proteins . In this regard, pv 2a has been very useful for examining the requirements for eif4 g to translate different cellular or viral mrnas . In addition to this, in this paper, we have highlighted the role of pv 2a not only in the processing of the poliovirus polyprotein but also in the interaction with the host - cell . Interestingly, a single viral protein is able to modulate many steps of gene expression in order to generate an optimal intracellular environment for the viral biological cycle . In particular, pv 2a cleaves cell proteins involved in transcription, pre - mrna splicing, nucleus / cytoplasm transport and translation, in order to hijack those host functions and to concentrate the cellular resources on the production of the viral progeny . For example, pv 2a inactivates host translational machinery for capped cellular mrnas by cleaving eif4 g, whereas viral protein synthesis takes place under those conditions by ires - driven translation . Because of the decrease of cellular mrna translatability in pv - infected cells, host ribosomes are available for viral protein synthesis . Probably, the inhibition of host protein synthesis may prevent the production of antiviral proteins . Similarly, the blockade of nucleus / cytoplasm transport of macromolecules might isolate nuclear processes from cytoplasmic cellular ones, hampering the arrival of specific proinflammatory transcription factors to the nucleus and of mrnas encoding proinflammatory, antiviral or proapoptotic proteins to the cytoplasm of infected cells . Finally, transcription and pre - mrna splicing could be also modulated by pv 2a and might reduce the availability of mature mrnas . Nevertheless, very little is known about the potential role of pv 2a in transcription and pre - mrna splicing, and further studies of these steps of gene expression in pv - infected and 2a - expressing cells should be carried out . Making use of the omics technologies it would be possible to identify changes in the host transcriptome in those cells, allowing us to understand the readjustment of host gene expression to viral infection and to the cytotoxic effect of pv 2a . Gene ontology tools can be used to cluster the pathways (kegg), molecular activities and biological functions of the genes which are transcriptionally up- or downregulated or not affected after these unfavourable stimuli . In silico analysis could be carried out in order to identify whether these gene clusters belong to particular networks controlled by specific transcription factors . Complementarily, deep sequence (rnaseq), specific microarrays types and conventional rt - pcr could be used to screen for nonspliced or abnormal spliced mrna variants in pv - infected and 2a - expressing cells . Finally, microarrays can be employed to identify the cytoplasmic and nuclear transcriptome in those cells to determine whether mrna nuclear export inhibition induced by pv 2a impacts on the distribution of the entire host mrna bulk or in specific mrna pools . Taken together, all this information may provide us with a general and deep vision of the modification induced by pv 2a on the different steps of mrna metabolism . Additionally, the physiological role of nups and gemin-3 cleavage might be studied using different models . One possible and interesting approach is to engineer stable cell lines expressing noncleavable versions of nup98, nup62, nup153 and/or gemin-3 and then to analyze the fitness of pv in the different cell types, especially in presence of extracellular antiviral stimuli such as ifns or interleukins, which will activate different epigenetic programs . These studies will provide essential information to help us understand the specific role that those host factors play in pv infection . The total number of cellular proteins targeted by pv 2a (degradome) remains unknown, but it can be anticipated that with the expanding use of proteomic methodologies, this analysis will be known soon not only for pv 2a, but also for other viral proteases of interest . Furthermore, the analysis of the pv 2a - induced degradome in human cells will be of general interest for many researchers, including virologists and cellular biologists . This goal could be achieved combining in silico prediction of 2a cleavage sites and experimental tools such as proteomics . In the first case, blom and collaborators developed a bioinformatics tool using neural network algorithms to predict cellular targets for picornavirus proteases . This approach has been successfully used to predict the cleavage of dystrophin by coxsackievirus 2a although most of the predicted human targets for rhinovirus and enterovirus 2a have not been proved yet . In addition, the algorithm did not predict the cleavage of cellular targets that have been later demonstrated to be proteolyzed by picornaviral 2a such as nup98 and cytokeratin 8 [16, 17, 221]. Thus, it would be necessary to develop an improved algorithm able to find optimal cleavage sequences in the host proteome by implementing the proteolytic sites known for newly described 2a targets . Many parameters have to be taken into account, including the protein localization (cytoplasmic and nuclear protein will be considered, but not proteins resident in the lumen of other organelles such as re or peroxisomes), the exposure of the cleavage site to the solvent (the sequence must be accessible to the protease), and the secondary structure in which the proteolytic site is included (optimally, unstructured regions). Potential targets could be ordered by their degree of homology with optimal cleavage sequences, as well as with the degree in which they fulfill the above prerequisites . On the other hand, novel pv 2a targets can be identified by proteomic tools such as two - dimensional differential gel electrophoresis (dige) or quantitative proteomics such as stable isotope labeling by amino acids in cell culture (silac) coupled to monodimensional electrophoresis . Following these two methods, it will be feasible to identify proteins with reduced levels on 2a expression and, in addition, to detect the cleavage products that will appear as lower size peptides . By the uncovering of novel 2a targets we will be able to map the cellular networks impacted by pv 2a and to integrate them in the context of pv infection . In fact, the role of 2a hijacking host processes could be potentially expanded to other cellular pathways with direct impact on control of viral infection . Such knowledge will provide more insight into our understanding of the cytopathogenicity of viral proteases at the molecular level.
Developmental age is an indicator of the stage of development of a child as a proportion of chronological age . Various biological ages, such as skeletal age and morphological age, secondary sexual characteristics, as well as dental age have been proposed for determining developmental age . These criteria can be applied separately or collectively in order to assess the degree of physiological maturity of a growing child . Of all methods, dental age, several methods like radiographic or gingival emergence methods have been used to determine dental age . Gingival emergence represents only one stage in the continuous process of dental development or migration to reach the occlusal level . Emergence may be influenced by local factors: ankylosis, early or delayed extraction of the deciduous tooth, impaction and crowding of the permanent teeth . In contrast, the formation rate of the permanent teeth is not affected by premature loss of the deciduous teeth . Furthermore, clinical emergence of teeth can be used during some intervals because of the completion of deciduous dentition . Previous studies investigating the relationship between gingival emergence and tooth formation concluded that tooth formation is a more reliable indicator of dental maturity than gingival eruption . Most methods make use of panoramic radiographs obtained by full mouth periapical radiography because of the more radiation . Among radiological methods for dental age estimation in children, the demirjian method is widely used and accepted, mainly because it allows comparison of different ethnic groups . This was based on the ratings of radiographs of the seven left - side teeth of the mandible . In this method, each tooth is given a mark indicating a developmental stage . There are eight stages; a to h and each stage is assigned a given numeric value from tables prepared separately for boys and girls . The value obtained on the summation of the obtained values indicates the dental age of the patient, which is derived from standard tables or centile charts . Another dental age estimation method based on 4 teeth for patients with missing teeth in the mandible was developed . Dental age is of particular interest to orthodontists in planning the treatment of different types of malocclusion in relation to maxillofacial growth . It can also be of help in determining the age of cadavers or skeletal material where other parts of the body are missing . The difference between dental age and known chronological age is of interest, indicating an advancement or delay compared with the standard . Although comparative studies of chronological age and dental age have been conducted, no direct association between different malocclusions obtained using the demirjian method was found in the literature . Therefore, the purpose of this study was to investigate the relationship between chronological age and dental age in cases of different malocclusions by using the demirjian method . Panoramic and lateral cephalometric radiographs of 321 children (165 girls and 156 boys) referred to the department of orthodontics between 2007 and 2010 were obtained . Standard lateral cephalograms and panoramic films were taken at the same magnification (magnification error, <1.1) with the same equipment by using a cephalostat incorporated into a conventional x - ray device (proline 2002, planmeca oy, finland). Patients with any syndromes, missing teeth or cleft lip and palate were excluded from the study . The chronological ages of the patients were calculated on the basis of the time from the child's birth to the day the panoramic radiograph was obtained . The entire sample was divided into subgroups with the distribution based on the chronological age . Dental maturation on the mandibular left - side was evaluated according to the method described by demirjian et al . The range of the stages ascribed to the state of development of the teeth in the present study was between c and h. if the development of a tooth was found to be between two stages, a half - value was assigned . The development of the seven left permanent mandibular teeth was then assessed using panoramic radiographs by means of the eight - grade scale according to the demirjian system . The total dental maturity score can be converted into dental age by using a table of standards for boys and girls . The evaluation of the dental ages from 30 panoramic films was completed twice by the same author for intra - observer variability, evaluated by the pediatrician again for inter - observer variability, then calculated using dahlberg's formula . The intra - observer variability in dental age assessment was r = 0.93 and inter - observer variability was r = 0.81 . All patients were divided into three subgroups according to the anb angle: class i, class ii and class iii groups . Data were analyzed using the spss statistical program (version 15.5, spss inc ., descriptive statistics were calculated for each parameter . Whether the distributions of continuous variables were normal chronological and dental ages showed normal distribution while sna, snb, anb and gogn - sn did not show normal distribution . Therefore, inter - group comparisons regarding normally distributed data were analyzed by unpaired t - test, whereas mann - whitney u - test was used for abnormally distributed data . The comparison of dental age and chronological age according to gender was evaluated by the student's t - test . Predicted age was compared with actual age and the mean differences were calculated for each gender - specific age group . The comparisons between the dental and chronological age in each sagittal classification were assessed by paired sample t - test . The relationships between dental age and sna, snb, anb and gognsn parameters were evaluated using the spearman's rank correlation coefficient . Dental maturation on the mandibular left - side was evaluated according to the method described by demirjian et al . The range of the stages ascribed to the state of development of the teeth in the present study was between c and h. if the development of a tooth was found to be between two stages, a half - value was assigned . The development of the seven left permanent mandibular teeth was then assessed using panoramic radiographs by means of the eight - grade scale according to the demirjian system . The total dental maturity score can be converted into dental age by using a table of standards for boys and girls . The evaluation of the dental ages from 30 panoramic films was completed twice by the same author for intra - observer variability, evaluated by the pediatrician again for inter - observer variability, then calculated using dahlberg's formula . The intra - observer variability in dental age assessment was r = 0.93 and inter - observer variability was r = 0.81 . All patients were divided into three subgroups according to the anb angle: class i, class ii and class iii groups . Data were analyzed using the spss statistical program (version 15.5, spss inc ., chicago, illinois, usa). Descriptive statistics were calculated for each parameter . Whether the distributions of continuous variables were normal chronological and dental ages showed normal distribution while sna, snb, anb and gogn - sn did not show normal distribution . Therefore, inter - group comparisons regarding normally distributed data were analyzed by unpaired t - test, whereas mann - whitney u - test was used for abnormally distributed data . The comparison of dental age and chronological age according to gender was evaluated by the student's t - test . Predicted age was compared with actual age and the mean differences were calculated for each gender - specific age group . The comparisons between the dental and chronological age in each sagittal classification were assessed by paired sample t - test . The relationships between dental age and sna, snb, anb and gognsn parameters were evaluated using the spearman's rank correlation coefficient . Distribution of the patients according to gender and sagittal classifications are shown in table 1 . Gender distribution according to classes the chronological age range of the male patients was between 7.0 and 15.7 and the mean age was 11.84 1.57 years . Their dental ages ranged from 7.8 to 15.1 and the mean was 12.12 1.56 years . In male patients, the difference between chronological age and dental age was 0.33 years and this difference was statistically significant (t = 5.000, p <0.001). There was also a strong linear relationship between dental age and chronological age (p <0.001). The chronological ages of the female patients ranged from 7.0 to 15.9 years and the mean age was 11.38 1.70 years . Their dental ages ranged from 7.8 to 15.8 years and the mean age was 12.23 1.87 years . The dental age of female patients was therefore greater than that of the male patients by 0.94 years . This difference was also statistically significant (t = 11948, p <0.001). A stronger linear relationship between dental age and chronological age the difference between chronological age and dental age seen in the female patients was greater than the difference seen in the male patients . The mean chronological ages of patients with class i, class ii and class iii malocclusions were 11.71 1.65 years, 12.29 1.41 years and 10.98 1.44 years, respectively . The corresponding mean dental ages were 12.05 1.71, 12.49 1.31 and 11.35 1.60 years . Chronological age and dental age were compared in each group and were significantly different [table 2]. This was statistically significant for girls in all grades and male patients with class i and class ii malocclusions (p <0.01) while the statistical significance for male patients with class iii malocclusions was p <0.05 . Differences in chronological age and dental age according to gender and classes chronological ages by gender within each class were evaluated and the chronological ages of boys and girls with class i and class iii malocclusions were similar . The mean chronological age of the boys with class ii malocclusions, however, was significantly higher than that of the girls with class ii malocclusions (p <0.01). In terms of dental age, similar values were observed in boys and girls in each class . Dental age and chronological age differences between the groups were evaluated and the difference was found to be much greater in female patients than in male patients in both class i (p = 0.029) and class ii (p <0.001) groups, but not in the class iii group, in spite of the greater difference in female patients (p = 0.128). The difference was found to be similar between the classes in both females and males . Differences between dental and chronologic ages according to sub - age groups are shown in table 3 . There were statistically significant differences between the dental and chronological ages in all age groups ranging from 7 to 13.9 years in female patients, while there was no difference in 14 - 15.9 years age groups . In male patients, there were significant differences only in the age groups 10 - 10.9 and 11 - 11.9 years and the differences were not statistically significant in the other age groups . Differences between dental and chronologic ages in sex and age groups the distribution of classes in sna, snb, anb and gognsn measurements are shown in table 4 . The relationships between the dental age and these parameters were first evaluated in general and then evaluated separately for each class . Dental age did not show any significant correlation with the sna or gognsn angle, while a weak, statistically significant negative relationship was observed between dental age and the snb angle (= 0.205, p <0.001). There was a weak, linear and statistically significant correlation between dental age and the anb angle (= 0.313, p <0.001). Median values of sna, snb, anb and gognsn parameters when the dental age was evaluated according to gender and classes, only in boys did the anb angle shows a statistically significant correlation with dental age, although a weak linear correlation was found (= 0.346, p <0.05). The chronological age range of the male patients was between 7.0 and 15.7 and the mean age was 11.84 1.57 years . Their dental ages ranged from 7.8 to 15.1 and the mean was 12.12 1.56 years . In male patients, the difference between chronological age and dental age was 0.33 years and this difference was statistically significant (t = 5.000, p <0.001). There was also a strong linear relationship between dental age and chronological age (p <0.001). The chronological ages of the female patients ranged from 7.0 to 15.9 years and the mean age was 11.38 1.70 years . Their dental ages ranged from 7.8 to 15.8 years and the mean age was 12.23 1.87 years . The dental age of female patients was therefore greater than that of the male patients by 0.94 years . This difference was also statistically significant (t = 11948, p <0.001). A stronger linear relationship between dental age and chronological age (p <0.001) the difference between chronological age and dental age seen in the female patients was greater than the difference seen in the male patients . The mean chronological ages of patients with class i, class ii and class iii malocclusions were 11.71 1.65 years, 12.29 1.41 years and 10.98 1.44 years, respectively . The corresponding mean dental ages were 12.05 1.71, 12.49 1.31 and 11.35 1.60 years . Chronological age and dental age were compared in each group and were significantly different [table 2]. This was statistically significant for girls in all grades and male patients with class i and class ii malocclusions (p <0.01) while the statistical significance for male patients with class iii malocclusions was p <0.05 . Differences in chronological age and dental age according to gender and classes chronological ages by gender within each class were evaluated and the chronological ages of boys and girls with class i and class iii malocclusions were similar . The mean chronological age of the boys with class ii malocclusions, however, was significantly higher than that of the girls with class ii malocclusions (p <0.01). In terms of dental age, similar values dental age and chronological age differences between the groups were evaluated and the difference was found to be much greater in female patients than in male patients in both class i (p = 0.029) and class ii (p <0.001) groups, but not in the class iii group, in spite of the greater difference in female patients (p = 0.128). The difference was found to be similar between the classes in both females and males . Differences between dental and chronologic ages according to sub - age groups are shown in table 3 . There were statistically significant differences between the dental and chronological ages in all age groups ranging from 7 to 13.9 years in female patients, while there was no difference in 14 - 15.9 years age groups . In male patients, there were significant differences only in the age groups 10 - 10.9 and 11 - 11.9 years and the differences were not statistically significant in the other age groups . The distribution of classes in sna, snb, anb and gognsn measurements are shown in table 4 . The relationships between the dental age and these parameters were first evaluated in general and then evaluated separately for each class . Dental age did not show any significant correlation with the sna or gognsn angle, while a weak, statistically significant negative relationship was observed between dental age and the snb angle (= 0.205, p <0.001). There was a weak, linear and statistically significant correlation between dental age and the anb angle (= 0.313, p <0.001). Median values of sna, snb, anb and gognsn parameters when the dental age was evaluated according to gender and classes, only in boys did the anb angle shows a statistically significant correlation with dental age, although a weak linear correlation was found (= 0.346, p <0.05). Despite the development of dental maturation, prediction methods in the 1970's, studies conducted in many countries over the recent years show that there is still much to be investigated about this issue . The main reason this method is used is that the scoring is performed according to the shape of the tooth instead of the length of the tooth . Thus, the magnification between 3% and 10% in the panoramic film is eliminated as a possible source of error . In addition, depending on the length of the root, it may be difficult to provide an assessment of standardization . The reason for preferring the demirjian method is its high reproducibility . As with the many studies previously reported here, intra- and inter - observer variability assessment of dental maturation is lower . In this study, the upper age limit of the selected patients was 15.9 years, at which there is closure of the latest erupted permanent teeth apices (except the third molar), as in previous studies . The lower limit was determined to be 7 years, because only a very limited number of patients admitted to the orthodontics clinic were under 7 years of age . This age group is also the most common age group of patients in the practice of orthodontics . Clinical orthodontists are more likely to encounter patients with malocclusion or jaw malposition in this age group than in the normal population . Therefore, the relationship between malocclusion and dental age, as well as the relationship between chronological age and dental age needs to be investigated because dental age is one of the most important issues in orthodontic treatment planning . In cases with delayed maturation, orthodontic treatment may begin at a later stage and more stable results can be obtained . In this study, in girls, the dental age was greater than the chronological age by 0.94 years, the corresponding value in boys was 0.33 years this difference can be considered clinically insignificant in males, but it can be considered as clinically important in girls . Reaffirm that a difference of more than 6 months between chronological age and dental age is clinically important . This difference in girls may be attributed to the fact that girls attain puberty earlier than boys and the difference may be related to a significant development in facial structure . Similarly, in many previous studies, dental age was found to be more than chronological age in both girls and boys . However, compared with the dental maturation in french - canadian children, dental maturation of children in kuwait was found to be delayed by approximately 0.7 years . In addition, dental age in the demirjian method is exactly as predicted in certain populations . The age of the sample group, the statistical method, method reliability and each child's individual genetic and geographic variation can influence the differences reported in the results . Genetic research conducted on monozygotic and dizygotic twins showed that the effect of environmental factors is not as high as that of genetic factors, which influence more than 50% of dental maturation . In other words, the relationship between dental maturation and chronological age may remain stable during some age, after which it may accelerate . Reported that the position of tooth buds remained relatively stable inside the jaw until root formation started . Therefore, eruption is accelerated with the beginning of root formation . In a study of dental development related to the dental and skeletal maturation in children, uysal et al . Found that skeletal maturation was closely related to dental development . Contrasted this situation in their study of delayed dental maturation between the ages of 4 and 9 years . In another similar study conducted in the turkish population, a significant development in dental maturation previous studies have investigated the relationship between skeletal maturation and dental maturation; however, the relationship between dental maturation and malocclusion had not been investigated . This study shows that class i, class ii and class iii malocclusion groups have different chronological and dental ages [table 2]. However, dental and chronological age differences were not found to be significantly different between classes . In the present study, sna, snb and anb angles, which are the most common parameters used for classifying anomalies in the sagittal aspect, were used . The results of correlation studies indicate there is no relationship between dental age and the sna angle . However, there is a negative and weak statistically significant correlation between dental age and snb angle . A positive and weak correlation between anb angle and dental age is noteworthy . In other words, as a case presented a tendency to class ii, dental maturation increases . However, since the values indicate a weak correlation, the results should be interpreted carefully . Furthermore, teeth eruption and mastication play an important role in alveolar development . During normal growth and development of the maxillofacial area, there is a consistency between the vertical growth of the nasomaxillary complex, maxillary and mandibular vertical alveolar bone growth and the vertical growth of the mandibular condyle . Janson et al . Conducted the first study to investigate the influence of facial type on dental development in subjects of the same chronological age . They reported that compared to subjects with small faces, subjects with long faces showed a tendency to have an advanced dental maturation . In another study in dental maturation in subjects with small and long faces were evaluated, no clinically significant difference was found in terms of dental maturation . In our study, the mandibular plane angle (gognsn) showed no correlation with dental maturation . These findings suggest that dental maturation and the mandibular vertical growth pattern are probably controlled by different genes . Studies need to be conducted in bigger malocclusion groups as well as groupings that compare the vertical aspects of the jaws and face . Evaluation with the demirjian method revealed statistically significant differences in the chronological and dental ages for children in all classes . Dental age was significantly greater than chronological age in the 10 - 10.9 year and 11 - 11.9 year age groups in male patients and in all age groups between 7 and 14 years in female patients . Female patients were found to have significantly more advanced dental maturation than male patients . In individuals evaluated in terms of sagittal jaw relationship, dental maturation showed a weak and negative correlation with the snb angle and a weak linear correlation with the anb angle . In individuals evaluated according to the vertical plane, dental maturation did not reveal any relationship with the gognsn angle.
This is due to the fact that any alteration in the biological system as a result of mechanical loading results in strain within the biological system . This in turn, leads to release of various neurotransmitters resulting in remodeling and adaptation of the biological system to the newer environment . As a result of this principle, remodeling of the periodontal ligament (pdl) and research is constantly being done and updated regularly on the biology of orthodontic tooth movement and the tissue level response within the cellular level on application of orthodontic force . Orthodontic tooth movement may broadly be divided into three phases: the initial phase, the lag phase, and the post - lag phase . During the initial phase there is immediate and rapid tooth movement occurring within 24 to 48 hrs after force application to the tooth . The lag phase lasts approximately 20 to 30 days, showing relatively little to no tooth displacement . No subsequent tooth movement occurs until the cells complete the removal of all of the necrotic tissues . The post - lag phase follows the lag phase, during which the rate of movement again increases . Prostaglandins (pg- s) are local hormone - like chemical agents produced by mammalian cells, including osteoblasts and are derivatives of arachidonic acid . They are synthesized immediately in response to tissue injury . Pgs exist in two different iso - forms: the constitutive isoform or cyclooxygenase-1 (cox-1) and the inducible isoform or cyclooxygenase-2 (cox-2). Application of orthodontic force results in stress concentration in the extracellular matrix and the deformation of the osteocytes of alveolar bone . This deformation, in turn opens the hemi - channels in the strained osteocytes allowing the release of prostaglandins, which are responsible for orthodontic tooth movement . It has been proved that cox is also closely associated with periodontitis and that pgs are mediators of gingival inflammation and alveolar bone resorption . In addition, numerous studies have confirmed pge2 levels in periodontal tissues and gingival crevicular fluid are highly correlated with periodontal tissue destruction . Cox-2 has been proved to play an important role in gingival inflammation and alveolar bone loss during the progress of periodontitis . In vitro studies have proved that the expression and production of pge2 is promoted by mechanical stimulation of the periodontal ligament . Prostaglandins of the e series also play an important role in the pathogenesis of chronic periodontitis by regulating production of osteoclast activating factor in activated lymphocytes . They can also trigger a multilevel cascade of signal transduction pathways, including the prostaglandin e2 (pge2) pathway, which in turn initiates structural and functional changes in extracellular, cell membrane, and cyto - skeletal proteins . Subsequent changes in cyto - skeletal protein structure and function lead to the creation of new cells and bone matrix formation . Pge2 is one of the earliest biomarker for bone resorption, which can be used for monitoring orthodontic tooth movement (otm). The principal trigger factor responsible for orthodontic tooth movement (otm) is the strain experienced by the pdl cells and the extracellular matrix . This strain results in alteration in the gene expression within the cells and the extracellular matrix . Orthodontic force causes capillary vasodilatation within the blood vessels periodontal ligament, resulting in migration of inflammatory cells and cytokine production . These cytokines are actually proteins, acting as signals between the cells of the immune system, produced during the activation of immune cells . Cytokines like il-1, il-6, il-8 and tnf- have been proved to be associated with bone remodeling . On application of orthodontic force, the compression region within the pdl shows increased osteoclastic activity, whereas in the tension region, there is proliferation of osteoblasts and mineralization of the extracellular matrix . The osteoclastic cells involved in bone resorption are multinucleated giant cells originating from hematopoietic stem cells . Interleukin-1 beta (il-1), interleukin-6 (il-6), and other inflammatory cytokines facilitate osteoclastic bone resorption processes and have the potential to serve as one of the earliest biomarkers for monitoring and validating orthodontic tooth movement . These proteins regulate osteoclastic activity through the activation of the nuclear factor kappa b (rank) and of the nuclear factor kappa b ligand (rankl). Cc chemokines ligand 2 (ccl2) has been found to be involved in osteoclast activity and its expression is increased within the pdl on orthodontic force application . There is a reduction of osteoclast and osteoblast activities in the absence of ccl2 . Similarly ccr5 has been suggested to be a down regulator of alveolar bone resorption during orthodontic tooth movement . Matrix metalloproteinases (mmps) help in bone remodeling by breaking down the extracellular matrix . It has been found that, compression of pdl induces an increase in mmp-1 levels 1hr after mechanical loading . Tension within the pdl too resulted in significantly increased levels of mmp-1 protein after 1hr of force application which also subsequently disappeared . Mmp-2 protein was induced by pdl compression, which increased significantly in a time - dependent fashion, reaching a peak after 8 hrs after mechanical loading . Mmp-2 was significantly increased on the tension side 1 hr after force application, but gradually returned to basal levels within 8 hrs . This indicates that mmp-2 could be used as a biomarker for monitoring active tooth movement during the early stages of orthodontic treatment . The cleavages of procollagen produces procollagen type i c - terminal pro - peptide (picp) and procollagen type i n - terminal pro - peptide (pinp) and were proposed to be measured as bone formation markers . However, both picp and pinp are markers that can only indicate the formation of type i collagen and not totally bone specific . Therefore, they cannot be used to monitor otm . Bone morphogenetic proteins (bmps), transforming growth factor - beta (tgf-) and growth - factor- (gfs) associated internal signaling molecules are other bone - forming genes that encode proteins for gfs . Bmps bind to the surface receptors on progenitor and mature osteoblasts and subsequently trigger a signaling pathway which promotes the differentiation of osteoprogenitor cells and the up - regulation of osteoblast activity . Osteoblastic activity can also be promoted by the interaction of growth factors with specific surface receptors on osteoblasts, thereby stimulating insulin - like gf-1 . They have growth - promoting effects on bone, in addition to regulating cell growth and development . Studies have found that msx 1 and msx 2 are potential regulators of osteoblastic activity . The msx 1, protein is known as a critical modulator of bone development and remodeling, and msx 2 is an alternative regulator protein of cbfa1 expression in bone formation during otm . Msx 1 and msx 2 can be used as potential biomarkers during the development of stem cells into osteoblasts during otm . Tnf- is a pro - inflammatory cytokine that is expressed during periodontal disease and is responsible for alveolar bone resorption during periodontal breakdown . Rankl and its receptor rank, which are present on osteoblasts and precursor osteoclasts, respectively, have been recognized as the key factors that stimulate osteoclast formation . Recent studies analyzed the cytokine expression pattern in compression and tension sides of the pdl during orthodontic tooth movement in humans by means of real - time polymerase chain reaction (pcr). It was found that both the pressure and tension sides showed higher expression of all the cytokines when compared to the pdl of normal teeth . The compression side exhibited higher expression of tnf-, matrix metalloproteinase i (mmp-1) and rankl, whereas the tension side presented higher expression of type i collagen, il-10, tissue inhibitor of matrix metalloproteinase i (timp-1), osteoprotegerin (opg) and osteocalcin (ocn). The expression of tgf- was found to be similar in both pressure and tension sides . It is now clear that rankl, together with macrophage - colony stimulating factor, is required for osteoclast formation from precursor monocytes and macrophages . The natural inhibitor of rank - rankl interactions is the soluble tnf receptor - like molecule osteoprotegerin (opg), which binds to rankl and prevents its ligation, thereby preventing osteoclast differentiation and activation . Rankl protein was found to be predominant in inflammatory cells adjacent to areas of pathological bone loss in periodontal disease and is associated with the progress of periodontal disease . The local rankl gene transfer to the periodontal tissue using a hemagglutinating virus of japan (hvj) envelope vector was reported to accelerate otm in six - week - old male wistar rats . The activation of transferred rankl gene in the periodontal tissue indicates that rankl is involved during active otm especially in periodontal area . However, the activation of the opg gene through gene transfer to periodontal tissues neutralized rankl activity and hence inhibited osteo - clastogenesis . This decrease was found to be significant during the first month of active tooth movement and started to stabilize later . Batra et al ., found that the alkaline phosphatase activity in the gcf showed an increase during canine retraction which peaked during the 14 day . It is one of the important enzymes responsible for initiating orthodontic tooth movement and then sustaining it for a two week period . Nitric oxide (no) is produced through the activity of constitutive endothelial nitric oxide synthase (enos) or inducible nitric oxide synthase (inos) and plays an important role in regulating bone response to mechanical stress . High levels of no reduce the osteoclastic activity, while the inhibition of no production increases osteoclastogenesis and osteoclastic activity . The potential of tartrate - resistant acid phosphatase (trap) as a biomarker of bone resorption has been long recognized . Refinement of the activity assay to primarily measure trap 5b at a ph level of 6.1 was suggested for investigation of osteoclastic activity and bone resorption rates . However, controlled experimental studies are required to validate the use of trap as a biomarker to monitor otm . It is expressed by highly differentiated osteoblasts and is incorporated into the bony matrix during alveolar bone remodeling . Smaller osteocalcin fragments are found in areas of bone remodeling and are actually degradation products of the bone matrix . Lactate dehydrogenase (ldh) and aspartate aminotransferase (ast) are inflammatory biomarkers found outside cells during inflammation . Increased levels of lactate dehydrogenase and aspartate aminotransferase[4143] were detected in human gcf samples obtained during otm . Myeloperoxidase (mpo) is an enzyme found in polymorphonuclear neutrophil (pmn) granules and can be used to estimate the number of pmn granules in tissues . Mean mpo activity was increased in both the gcf and saliva of orthodontic patients at 2 hrs after appliance activation . Cathepsin b is an intracellular lysosomal cysteine proteinase, capable of degrading extracellular components including collagen . It is also known to play an important role in the initiation and perpetuation of inflammatory processes . The accumulation of cathepsin b in the gingival crevicular fluid has been shown to increase on mechanical loading . Cathepsin b is present in higher concentrations around osteoclasts and plays a major role in alveolar bone remodeling . Leptin and its receptor share structural and functional similarities with members of the long - chain helical cytokines including il-6, il-11, il-12 and oncostatin m. leptin stimulates the immune system by enhancing cytokine production and phagocytosis by macrophages . Several studies have observed that the levels of gcf leptin activity may play an important role in the development of periodontal disease . Karthikeyan reported that leptin levels in the gingival crevicular fluid decreased progressively as periodontal disease progressed . Prostaglandins (pg- s) are local hormone - like chemical agents produced by mammalian cells, including osteoblasts and are derivatives of arachidonic acid . They are synthesized immediately in response to tissue injury . Pgs exist in two different iso - forms: the constitutive isoform or cyclooxygenase-1 (cox-1) and the inducible isoform or cyclooxygenase-2 (cox-2). Application of orthodontic force results in stress concentration in the extracellular matrix and the deformation of the osteocytes of alveolar bone . This deformation, in turn opens the hemi - channels in the strained osteocytes allowing the release of prostaglandins, which are responsible for orthodontic tooth movement . It has been proved that cox is also closely associated with periodontitis and that pgs are mediators of gingival inflammation and alveolar bone resorption . In addition, numerous studies have confirmed pge2 levels in periodontal tissues and gingival crevicular fluid are highly correlated with periodontal tissue destruction . Cox-2 has been proved to play an important role in gingival inflammation and alveolar bone loss during the progress of periodontitis . In vitro studies have proved that the expression and production of pge2 is promoted by mechanical stimulation of the periodontal ligament . Prostaglandins of the e series also play an important role in the pathogenesis of chronic periodontitis by regulating production of osteoclast activating factor in activated lymphocytes . They can also trigger a multilevel cascade of signal transduction pathways, including the prostaglandin e2 (pge2) pathway, which in turn initiates structural and functional changes in extracellular, cell membrane, and cyto - skeletal proteins . Subsequent changes in cyto - skeletal protein structure and function lead to the creation of new cells and bone matrix formation . Pge2 is one of the earliest biomarker for bone resorption, which can be used for monitoring orthodontic tooth movement (otm). The principal trigger factor responsible for orthodontic tooth movement (otm) is the strain experienced by the pdl cells and the extracellular matrix . This strain results in alteration in the gene expression within the cells and the extracellular matrix . Orthodontic force causes capillary vasodilatation within the blood vessels periodontal ligament, resulting in migration of inflammatory cells and cytokine production . These cytokines are actually proteins, acting as signals between the cells of the immune system, produced during the activation of immune cells . Cytokines like il-1, il-6, il-8 and tnf- have been proved to be associated with bone remodeling . On application of orthodontic force, the compression region within the pdl shows increased osteoclastic activity, whereas in the tension region, there is proliferation of osteoblasts and mineralization of the extracellular matrix . The osteoclastic cells involved in bone resorption are multinucleated giant cells originating from hematopoietic stem cells . Interleukin-1 beta (il-1), interleukin-6 (il-6), and other inflammatory cytokines facilitate osteoclastic bone resorption processes and have the potential to serve as one of the earliest biomarkers for monitoring and validating orthodontic tooth movement . These proteins regulate osteoclastic activity through the activation of the nuclear factor kappa b (rank) and of the nuclear factor kappa b ligand (rankl). Cc chemokines ligand 2 (ccl2) has been found to be involved in osteoclast activity and its expression is increased within the pdl on orthodontic force application . Similarly ccr5 has been suggested to be a down regulator of alveolar bone resorption during orthodontic tooth movement . Matrix metalloproteinases (mmps) help in bone remodeling by breaking down the extracellular matrix . It has been found that, compression of pdl induces an increase in mmp-1 levels 1hr after mechanical loading . Tension within the pdl too resulted in significantly increased levels of mmp-1 protein after 1hr of force application which also subsequently disappeared . Mmp-2 protein was induced by pdl compression, which increased significantly in a time - dependent fashion, reaching a peak after 8 hrs after mechanical loading . Mmp-2 was significantly increased on the tension side 1 hr after force application, but gradually returned to basal levels within 8 hrs . This indicates that mmp-2 could be used as a biomarker for monitoring active tooth movement during the early stages of orthodontic treatment . The cleavages of procollagen produces procollagen type i c - terminal pro - peptide (picp) and procollagen type i n - terminal pro - peptide (pinp) and were proposed to be measured as bone formation markers . However, both picp and pinp are markers that can only indicate the formation of type i collagen and not totally bone specific . Bone morphogenetic proteins (bmps), transforming growth factor - beta (tgf-) and growth - factor- (gfs) associated internal signaling molecules are other bone - forming genes that encode proteins for gfs . Bmps bind to the surface receptors on progenitor and mature osteoblasts and subsequently trigger a signaling pathway which promotes the differentiation of osteoprogenitor cells and the up - regulation of osteoblast activity . Osteoblastic activity can also be promoted by the interaction of growth factors with specific surface receptors on osteoblasts, thereby stimulating insulin - like gf-1 . They have growth - promoting effects on bone, in addition to regulating cell growth and development . Studies have found that msx 1 and msx 2 are potential regulators of osteoblastic activity . The msx 1, protein is known as a critical modulator of bone development and remodeling, and msx 2 is an alternative regulator protein of cbfa1 expression in bone formation during otm . Msx 1 and msx 2 can be used as potential biomarkers during the development of stem cells into osteoblasts during otm . Tnf- is a pro - inflammatory cytokine that is expressed during periodontal disease and is responsible for alveolar bone resorption during periodontal breakdown . Rankl and its receptor rank, which are present on osteoblasts and precursor osteoclasts, respectively, have been recognized as the key factors that stimulate osteoclast formation . Recent studies analyzed the cytokine expression pattern in compression and tension sides of the pdl during orthodontic tooth movement in humans by means of real - time polymerase chain reaction (pcr). It was found that both the pressure and tension sides showed higher expression of all the cytokines when compared to the pdl of normal teeth . The compression side exhibited higher expression of tnf-, matrix metalloproteinase i (mmp-1) and rankl, whereas the tension side presented higher expression of type i collagen, il-10, tissue inhibitor of matrix metalloproteinase i (timp-1), osteoprotegerin (opg) and osteocalcin (ocn). The expression of tgf- was found to be similar in both pressure and tension sides . It is now clear that rankl, together with macrophage - colony stimulating factor, is required for osteoclast formation from precursor monocytes and macrophages . The natural inhibitor of rank - rankl interactions is the soluble tnf receptor - like molecule osteoprotegerin (opg), which binds to rankl and prevents its ligation, thereby preventing osteoclast differentiation and activation . Rankl protein was found to be predominant in inflammatory cells adjacent to areas of pathological bone loss in periodontal disease and is associated with the progress of periodontal disease . The local rankl gene transfer to the periodontal tissue using a hemagglutinating virus of japan (hvj) envelope vector the activation of transferred rankl gene in the periodontal tissue indicates that rankl is involved during active otm especially in periodontal area . However, the activation of the opg gene through gene transfer to periodontal tissues neutralized rankl activity and hence inhibited osteo - clastogenesis . This decrease was found to be significant during the first month of active tooth movement and started to stabilize later . Batra et al ., found that the alkaline phosphatase activity in the gcf showed an increase during canine retraction which peaked during the 14 day . It is one of the important enzymes responsible for initiating orthodontic tooth movement and then sustaining it for a two week period . Nitric oxide (no) is produced through the activity of constitutive endothelial nitric oxide synthase (enos) or inducible nitric oxide synthase (inos) and plays an important role in regulating bone response to mechanical stress . High levels of no reduce the osteoclastic activity, while the inhibition of no production increases osteoclastogenesis and osteoclastic activity . The potential of tartrate - resistant acid phosphatase (trap) as a biomarker of bone resorption has been long recognized . Refinement of the activity assay to primarily measure trap 5b at a ph level of 6.1 was suggested for investigation of osteoclastic activity and bone resorption rates . However, controlled experimental studies are required to validate the use of trap as a biomarker to monitor otm . It is expressed by highly differentiated osteoblasts and is incorporated into the bony matrix during alveolar bone remodeling . Smaller osteocalcin fragments are found in areas of bone remodeling and are actually degradation products of the bone matrix . Lactate dehydrogenase (ldh) and aspartate aminotransferase (ast) are inflammatory biomarkers found outside cells during inflammation . Increased levels of lactate dehydrogenase and aspartate aminotransferase[4143] were detected in human gcf samples obtained during otm . Myeloperoxidase (mpo) is an enzyme found in polymorphonuclear neutrophil (pmn) granules and can be used to estimate the number of pmn granules in tissues . Mean mpo activity was increased in both the gcf and saliva of orthodontic patients at 2 hrs after appliance activation . Cathepsin b is an intracellular lysosomal cysteine proteinase, capable of degrading extracellular components including collagen . It is also known to play an important role in the initiation and perpetuation of inflammatory processes . The accumulation of cathepsin b in the gingival crevicular fluid has been shown to increase on mechanical loading . Cathepsin b is present in higher concentrations around osteoclasts and plays a major role in alveolar bone remodeling . Leptin and its receptor share structural and functional similarities with members of the long - chain helical cytokines including il-6, il-11, il-12 and oncostatin m. leptin stimulates the immune system by enhancing cytokine production and phagocytosis by macrophages . Several studies have observed that the levels of gcf leptin activity may play an important role in the development of periodontal disease . Karthikeyan reported that leptin levels in the gingival crevicular fluid decreased progressively as periodontal disease progressed . Bone remodeling that occurs during orthodontic tooth movement is a biological process involving an acute inflammatory response in the periodontal tissues . Application of a mechanical stimulus in the form of orthodontic forces cause an inflammatory reaction within periodontal tissues, which in turn may trigger the biological processes associated with bone remodeling . The various neurotransmitters, growth factors, interleukins, leptins and enzymes like aspartate aminotransferase, cathepsin k and matrix metalloproteinases have the potential to serve as biological markers in order to monitor and validate orthodontic tooth movement . Orthodontic tooth movement is a biomechanical exploitation of the physiologic mechanisms for developing and maintaining optimal occlusal function.
Restrictive fluid strategy contributes to better outcomes in major surgeries while goal - directed therapy targeting volume have shown improved results also . Excessive fluid restriction with hypovolemia may cause organ dysfunction, increased postoperative morbidity, and death . Central venous pressure (cvp) as a static preload variable has been challenged in its utility to predict fluid responsiveness and a systematic review of 24 studies showed that the cvp is a poor predictor of blood volume and that changes in the cvp do not predict response to fluid administration . Goal - directed fluid management with hemodynamic targets and oxygenation indices have been associated with improved outcomes and reduction in hospital stay in patients undergoing high - risk surgeries . Variations in stroke volumes have been used as predictors of fluid responsiveness in patients who are mechanically ventilated . Mechanical ventilation induces cyclic variations in cardiac preload, which is reflected in the cyclic changes in systolic arterial pressure, arterial pulse pressure and left ventricular stroke volume . While cyclical changes associated with ventilation does not produce changes in cardiac output in inadequately filled patients it can cause significant changes with hypovolemia . Essentially, patients with wide variations in the stroke volume will be on the steep portion of the frank starling's curve that plots the effects of preload on the stroke volume . The stroke volume variation (svv) is the measure of the variations in stroke volume associated with ventilation, and the fluctuations are larger with hypovolemia . As an alternative to static variables like cvp the flotrac vigileo is a minimally invasive cardiac output monitor that calculates the arterial pulsatility as the standard deviation of the arterial pressure wave . The device calculates the resistance and compliance of the vasculature by a proprietary algorithm from which values of stroke volume, cardiac output, systemic vascular resistance (svr) and svv are calculated . In this study, we used the svv measured from the flotrac vigileo as a guide for fluid replacement versus cvp targets during major abdominal surgery . The primary outcomes were the length of intensive care unit (icu) and hospital stay postoperatively . The secondary outcomes were levels of intraoperative serum lactate, inotrope use during surgery, intraoperative fluids, postoperative ventilation and time to return of bowel function . This study was a prospective, single - blinded, randomized study . Based on the key article by donati et al . With a mean difference of 2.1 days for the hospital stay (11.3 3.8 vs. 13.4 6.1) with 95% confidence limit after approval from the hospital ethics committee and informed consent, 60 patients belonging to american society of anesthesiologists (asa) physical status i and ii, undergoing major abdominal surgery that included the whipple's procedure, low anterior resection, retroperitoneal tumor and gastrectomy were recruited for the study . Those with asa physical status iii and above, age <18 years, patients with a history of cardiac arrhythmias, body mass index of> 40 kg / m and those undergoing combined abdominal and open thoracic surgery were excluded . Patients were allotted to either control group or intervention group by a closed envelope method . In the theatre, a large bore intravenous (iv) cannula (18 g) was placed, and the pulse - oximeter and electrocardiogram were attached . A thoracic epidural catheter was placed between t8 and t12 levels and checked for intravascular or intrathecal placement by administering a test dose (3 ml of lidocaine 2% with 1 in 200,000 adrenaline). All patients were induced with a standard iv anesthesia protocol that included midazolam 0.02 - 0.04 mg / kg body weight, glycopyrrolate 0.2 mg, fentanyl 2 mcg / kg and propofol titrated to loss of verbal response . Tidal volumes of 6 - 8 ml / kg were used and the respiratory rate of the ventilator was adjusted to maintain etco2 between 30 and 40 mm hg . A positive end - expiratory pressure of 4 cm h2 o was applied uniformly to all patients . Anesthesia was maintained with isoflurane 1 - 1.5%, air and oxygen (2:1) with intermittent muscle relaxants . Central venous cannulation was performed in the right internal jugular or right subclavian vein by the seldinger technique . Continuous epidural analgesia was administered by an infusion of 0.25% bupivacaine with 2 mcg / ml fentanyl as an additive at a rate between 4 and 8 ml / h . The iv fluid therapy was guided by the cvp in the control group (group c) to maintain it between 10 and 12 mm of hg . In the intervention group (group i), the svv was measured using a flotrac vigileo third generation device and fluid replacements were administered until the svv was corrected to <10% . The arterial blood gas (abg) analysis was done every 2 h during the surgery . The type of fluids replaced in the perioperative period was at the discretion of the anesthesiologist . The crystalloids used included ringer's lactate and normal saline and all patients received 1.0 l of hydroxyethyl starch (130/0.4) prior to the administration of blood or products . Packed red blood cells were infused if blood loss exceeded allowable limits and abg values showed a decrease in hematocrit to <27% . Vasopressor (noradrenaline) was added to maintain the mean arterial pressure (map)> 65 mm hg if the hypotension could not be corrected by volume replacement and the measured value of svr was low . An inotrope (dopamine / dobutamine) was added if the blood pressure did not respond to fluid boluses and if the svr was normal . Intraoperative parameters recorded included the total volume of iv fluids (colloids / crystalloids / blood), 2 hourly abg analysis from which serum lactate was obtained . We also recorded the map, cvp, svv (intervention group), and the requirement of vasoactive agents and the total duration of surgery in both groups of patients . At the end of surgery, patients were extubated after administration of neostigmine 0.05 mg / kg and glycopyrrolate 10 mcg / kg . If the patients were hemodynamically unstable or hypothermic, they were ventilated until reassessment the next morning . All patients were shifted to a surgical icu managed by an intensivist who was blinded to the study allocation . Postoperative parameters monitored were the need for postoperative ventilation, or the requirement of ventilatory assistance (bipap) after extubation . Time to return of bowel function was assessed by time to passage of flatus or functioning of stoma . The duration of icu stay and time to discharge from the hospital were noted as primary end points . The numerical data were analyzed using mann - whitney u nonparametric test . Demographic parameters and duration of anesthesia were comparable in both groups [table 1]. The primary outcome measure, the postoperative icu stay, was significantly shorter in the intervention group as compared to the control group [table 2]. The length of hospital stay was also shorter in the intervention versus the control group however this value was not significant statistically [table 2]. Comparison of demographic data comparison of icu and hospital stay in days the mean volume of iv fluids given in the intervention group to maintain the svv <10% was significantly lesser than the volume of iv fluids administered in the control group [table 3]. The mean intraoperative blood loss in the intervention and control groups were comparable [table 4]. The lactate levels during surgery were similar between the two groups [table 3]. Comparison of secondary outcome variables comparison of intraoperative blood loss and postoperative renal function all patients had urine output> 0.5 ml / kg / h during surgery and postoperative creatinine levels were comparable [table 4]. The numbers of patients needing inotropes, postoperative ventilation and the time to return of bowel function were not different between the two groups (p> 0.05, table 3). Nine patients in the intervention and eight patients in the control group developed postoperative complications [table 5]. We found that the fluid replacement according to the svv protocol resulted in a significant reduction in the postoperative icu stay . We hypothesized that the fluid administration targeted to the svv optimized the cardiac output and tissue perfusion in this group of patients similar to several studies . In the study by lopes et al . Fluid administration based upon pulse pressure variation (ppv) resulted in a significantly shorter postoperative stay in the intervention group . Studies comparing volume replacements by specific strategies had aimed to keep the cvp between 8 and 15 mm hg . We aimed a cvp between 8 and 12 mmhg in our patients as epidural vasodilatation demanded adequate fluid replacement prior to the use of vasopressors . Inadequate fluid replacement may result in the use of inotropes that could result in tachycardia, increased myocardial oxygen demand and risk for arrhythmias . Although the length of hospital stay appeared to be lesser in the intervention group, this was not different from the control statistically . This is contrary to the study by lopes et al ., where a difference in both icu stay and hospital stay were observed . In their study, the control group did not have targets for cvp while the intervention group targeted ppv unlike our study where even the control had a targeted fluid administration based on the cvp . We believe that this fluid optimization may have improved tissue perfusion in the control group also . In our study, the intervention group (svv) received significantly less fluid than cvp group . This finding although contradictory to other studies targeting the ppv or the systolic pressure variation could be explained by the fact that we allowed a liberal fluid administration . We also think that in all patients, the epidural analgesia at a thoracic level could have caused vasodilatation and hypotension that increased fluid resuscitation . We used serum lactate levels as a surrogate for adequacy of tissue perfusion in the intraoperative period . Although the mean serum lactate values were lower in the intervention group (1.26 vs. 1.52) this was not significant . The levels of lactate in both groups were well within the normal range despite the long duration of surgery and large volume shifts suggesting the overall beneficial effects of adequate volume replacements during major abdominal surgeries . The blood loss in both the groups was comparable in our study and the use of colloids and blood products were not different between the two groups . Only 4 patients in the control and 3 in the intervention group needed a transfusion (6 units vs. 5 units). There was no statistically significant difference between the two groups in the requirement of vasoactive agents . Patients in both groups maintained urine output of> 0.5 ml / kg / h and none developed postoperative acute kidney injury as reflected by a normal and comparable creatinine values in the postoperative period . Brandstrup et al . Observed that patients managed with a restrictive intraoperative fluid regimen had fewer complications and a shorter postoperative stay in the icu . This was also reported by lobo et al . Who showed that a restrictive fluid strategy while targeting oxygenation indices in high - risk surgery reduced the complications after surgery . In our study, the patients in the svv group received significantly less fluids and also had a significantly shorter postoperative icu stay than the control, although the fluid replacements were above the volumes used in a restrictive strategy . The difference between the two groups in the time to return of bowel function was not statistically significant in our study . Wakeling et al . Demonstrated that esophageal doppler guided fluid management minimized the time to return of bowel function and postoperative hospital stay . In their the incidence of postoperative complications was not significantly different between the two groups in our study (26.7% vs. 30%, table 5). One patient in the control group and 1 patient in the intervention needed re - laparotomy for sepsis and 1 patient in the control for postoperative anastomotic leak . There were no differences in the duration of postoperative ventilation or need for respiratory assist devices . In this study, we used the flotrac vigileo, a third generation device for assessment of svv . The svv derived from this was found to have a good correlation with the svv obtained from picco plus which uses a femoral arterial line and has shown to be a reliable predictor of svv in several studies . The threshold value for svv using the flotrac was 9.6% in this study and hence we aimed to keep the values of svv <10% in the intervention group . Our study had limitations in the numbers of patients and in the fact that we had excluded higher risk patients undergoing major surgeries . A larger multicenter trial including higher asa grades could be considered in the background of these results . Fluid replacements targeting to minimize svv during major abdominal surgery resulted in a shorter postoperative icu stay and lesser intraoperative fluid requirement as compared to cvp guided fluid replacement . Implementation of fluid replacement guided by a dynamic preload variable (svv) appeared to have distinct advantages over conventional static variables (cvp) on postoperative icu stay in abdominal surgery.
Cessation of tobacco use undoubtedly benefits health . However, many studies have reported that smoking cessation may implicate some hazard effects on health . It may sometimes cause weight gain and result in obesity [613], the second important preventable risk for premature death . It may also increase diabetes mellitus risk in the short - term, presumptively owing to associated weight gain [13, 15]. But there are controversies among studies regarding smoking cessation, weight gain, and risk of incident diabetes mellitus . Baum and chou reported in their nber study that smokers were 7.8% less likely to be obese . The declining use of cigarettes was the most significantly attributing factor for the soaring prevalence of obesity in usa . The average weight gain after smoking cessation varied widely and was roughly estimated to be around 4 - 5 kg in two large - scale studies [12, 17], approximately equal to the amount different between the mean weight of smokers and nonsmokers . However, weitzman et al . Reported that environmental exposure to tobacco smoke or active smoking in american adolescents was associated with higher rate of metabolic syndrome and abdominal obesity . In williamson et al . 's national cohort study, people who never smoked and smokers weighed nearly the same at a 7 to 13 years' follow - up . Marked weight gain (i.e., greater than 13 kg) may sometimes be strongly associated with smoking cessation, but it usually occurs in a minority of smoking quitters (i.e., in williamson et al . 's study, 9.8 percent of the men and 13.4 percent of the women who quit smoking). Additionally, there are debates about the risk for incident diabetes mellitus following smoking cessation, although the association between smoking and diabetes mellitus has been well established [13, 1924]. In this did a meta - analysis on 25 prospective cohort studies including 1.2 million participants, with 45844 incidental cases of diabetes mellitus during a study follow - up period ranging from 5 to 30 years . Compared with people who never smoked, the relative risk (rr) for diabetes mellitus in smokers was pooled and adjusted to be 1.44 (95% ci = 1.311.58). The risk was highest in heavy smokers (more than 19 cigarettes a day; rr = 1.61, 95% ci = 1.431.80) and lower in former active smokers (rr = 1.23, 95% ci = 1.141.33), consistent with a dose - response phenomenon . However, several studies disclosed controversial results . In nagaya's longitudinal study, the risk for diabetes mellitus was increased by heavy smoking in obese men but decreased by light smoking in lean men . But the relative risk was stronger in men with lower body mass index (body mass index less than 24.2 kg / m versus body mass index (bmi) of 24.2 kg / m or more). Suggested that smoking cessation increases short - term risk of type 2 diabetes irrespective of weight gain . Likewise, yeh et al . 's prospective cohort study found that the hazard for incident diabetes mellitus after smoking cessation reached its peak during the first 3 years (hazard ratio = 1.91; 95% ci = 1.193.05) and then gradually decreased to 0 at 12 years . Furthermore, kamaura et al . Reported that smoking cessation only raised the rate of bmi increase briefly . Importantly, a prospective cohort study using the data from the framingham offspring study disclosed that the cardiovascular benefit of smoking cessation was weakened by the presence of diabetes mellitus . Therefore, the occurrence of diabetes mellitus rather than weight gain following smoking cessation is the critical issue for care providers to encourage their clients abstaining from smoking . Taking all the above together, it is crucial to clarify whether smoking cessation may indeed bring individuals harmful metabolic effects (i.e., sustained overweight or obesity and incident diabetes mellitus) and identify individuals vulnerable to its adverse effects . In this regard, there are fairly few studies exploring smoking cessation, incident diabetes mellitus, and weight change together . We therefore conducted this study to examine the association between smoking cessation and incident diabetes mellitus and its correlation with weight gain . This retrospective cohort study was done after being approved by chang gung memorial hospital institutional review board (document number irb 102 - 1014b). The profiles for analysis were extracted from the mass health examination performed for employees in an industrial park in middle taiwan annually from 2007 to 2013 . Self - reported smoking status (including how long they have smoked or quitted smoking), drinking habit, and medical history (including diseases such as diabetes mellitus, hypertension, dyslipidemia, and viral hepatitis and medication currently prescribed) were recorded by standardized questionnaires and were reconfirmed by a nurse - administered check - up . The female employees (455 in number) were excluded because they counted less than 5% of the total number, and all did not smoke ever . To avoid complexity, people (2125 in number) who did not have complete data, smoked just socially or resumed smoking during the study period, or were diagnosed as dm at first examination were all excluded as well . There were 8452 all the included individuals were categorized into never - smokers who had never smoked before, ex - smokers who had quitted smoking, and current smokers who had been smoking until the final health examination . The ex - smokers were further divided by the number of years they had quitted smoking . The odds ratios (ors) for newly diagnosed diabetes mellitus were then calculated with adjustment for potential confounders and compared among groups . All biochemical tests were performed with fresh samples as instructed by manufacturer (7600 clinical analyzer, hitachi high - tech, tokyo, japan) under standardized quality control in the clinical laboratory department at chang gung memorial hospital at chiayi, taiwan . The confirmation of diabetes mellitus usually needs two separated occasions of elevated plasma glucose higher than 6.9 mmol / l (125 mg / dl) or even strictly meeting the requirement of standard oral glucose tolerance test . In this retrospective study, it is hard to use these criteria . To avoid missing potential cases, people without history of diabetes mellitus but with just one occasion of fasting plasma glucose higher than 6.9 mmol / l (125 mg / dl) or newly receiving drinks occasionally . Those who drank twice a month or more frequently were regarded as man with waist circumference 90 cm or higher was regarded as abdominal obesity . Systolic blood pressure equal to or higher than 130 mmhg, or diastolic blood pressure equal to or higher than 85 mmhg, or current use of antihypertensive medicines was regarded as high blood pressure . Serum triglyceride equal to or higher than 1.7 mmol / l (150 mg / dl) was regarded as dyslipidemia . Fasting plasma glucose from 5.6 to 6.9 mmol / l (100 to 125 mg / dl) was regarded as impaired fasting glucose . Logistic regression in spss 18.0 for windows (spss inc ., chicago, il, usa) was used to estimate the ors for newly diagnosed diabetes mellitus with smoking status as the main independent variable . The analysis was performed with adjustment for potential confounders including age, status of alcohol consumption, abdominal obesity, high blood pressure, dyslipidemia and impaired fasting glucose at the first examination, and weight gain between the first and the final examination . A p value less than 0.05 and a 95% confidence interval (ci) of or not containing 1 were considered statistically significant . The study included 8452 men aged, at their first health examination, from 24 to 70 with mean age 41.4 (7.1) years . There were 4370 (51.7%) men who never smoked, 1169 (13.7%) men who had quitted smoking, and 2913 (34.5%) men who kept on smoking . In the group that had quitted smoking, there were 146 (1.7%) men within the first year of smoking cessation, 144 (1.7%) within the 2nd, 278 (3.3%) within the 3rd, and 202 (2.4%) within the 4th and 399 (4.7%) had quitted smoking longer than 4 years . The characteristics of the grouped individuals by smoking status were summarized in table 1 . During this 6-year period, 374 men (4.4%) were newly diagnosed as diabetes mellitus . Compared with never - smokers, the current smokers had higher odds to have newly diagnosed diabetes mellitus (as shown in figure 1). Additionally, the ex - smokers within their first 2 years of abstinence were also inclined to have newly diagnosed diabetes mellitus (never - smokers 3.6%, or as 1; current smokers 5.5%, or = 1.499, 95% ci = 1.1471.960, and p = 0.003; ex - smokers in the first year of abstinence 7.5%, or = 1.829, 95% ci = 0.9063.694, and p = 0.092; ex - smokers in the second year of abstinence 9.0%, or = 2.020, 95% ci = 1.0313.955, and p = 0.040; ex - smokers in the third year of abstinence 2.9%, or = 0.850, 95% ci = 0.3961.826, and p = 0.677; ex - smokers in the fourth year of abstinence 5.0%, or = 1.080, 95% ci = 0.5332.187, and p = 0.831; and the ex - smokers after 4 years of smoking cessation 4.0%, or = 0.945, 95% ci = 0.5391.658, and p = 0.845). Tobacco smoking is a well established risk factor for many diseases, including several kinds of cancer [3032] and cardiovascular and lung diseases . It raises the death rate in middle age by twofold to threefold [2, 4]. In particular, it may predispose to or is associated with type 2 diabetes mellitus [13, 1925, 27, 3336], which further contributes to the risk of cardiovascular diseases . However, there are controversies about metabolic benefits from smoking cessation . In terms of smoking cessation, weight gain, and diabetes mellitus however, this association seems to interact significantly with age . In mackay et al . 's study, never - smokers or ex - smokers aged 1624 years were not more likely to be overweight or obese than active smokers of the same age . Although smoking cessation could be accompanied with a weight gain, most of it occurs during the first 6 months . People at younger age (e.g., <55 years) and lower socioeconomic status and who used to smoke heavily (e.g., more than 25 cigarettes per day) or have history of binge eating are at risk for marked weight gain (i.e., more than 10 kg) [12, 39]. Moreover, in clair et al . 's study, weight gain following smoking cessation did not influence its cardiovascular benefit unless there was a coexisting diabetes mellitus . Even in patients with diabetes mellitus, smoking cessation may still reduce risk of premature death although it usually takes several years for effect . It then becomes crucial to determine whether smoking cessation per se would increase risk for incidental diabetes mellitus despite weight gain or not . In our study, we found that ex - smokers during their first two years of abstinence have even higher odds than current smokers for newly diagnosed diabetes mellitus, though the increased odds ratio for ex - smokers in the first year of abstinence was not statistically significant (as shown in figure 1). It seems incredulous that people should immediately face rising odds for incident diabetes mellitus when they start quitting smoking . This contradictory result may arise from the immediate withdrawal of beneficial metabolic effect of certain constituents (e.g., nicotine) in tobacco [4145] and the delayed subsiding of adverse effects or irreversible harmful effects from smoking [46, 47]. Like some diet drugs (e.g., sibutramine, phentermine, and buspirone), nicotine may suppress appetite and prevent weight gain by increasing central nervous system levels of norepinephrine, dopamine, and serotonin . It may also promote release of catecholamines and cortisol and suppress adiponectin [50, 51]. Although smoking is generally implicated with harmful effects, there are several studies that disclosed beneficial metabolic effects from certain constituents of tobacco [4245]. Complexity of all the above findings could partially explain why smokers are prone to develop insulin resistance and have higher cardiovascular risk and the controversy why smoking cessation may sometimes not only cause weight gain [9, 53] but also increase incidence of diabetes mellitus [27, 28, 54]. In particular, some of the ex - smokers might quit smoking because of great ill health . Given that smoking may reflect a clustering of risky life styles, there should be quite a few residual confounders in this study . In particular, diagnostic bias defining diabetes mellitus by only one occasion of abnormal fasting glucose, monogender, and the lack of counting the quantity of cigarette smoking all make the results of our study biased and of limitation . But it may meanwhile carry a short - term (i.e., within the first couple of years) rising risk for incident diabetes mellitus . However, rising odds are seen in the first 2 years after quitting smoking in our study . In particular, it is independent of weight gain . Therefore, we suggest that intensified modification of life style or other strategies for prevention of diabetes mellitus may be needed before and immediately after smoking cessation . At least, for smokers and ex - smokers at risk for diabetes mellitus, monitoring at shorter intervals should be considered for early detection.
Image registration is used to find the spatial correspondence between two images and plays an important role in pulmonary image analysis . In a sequence of pulmonary scans, the computed correspondences describe the motion of the lung between a pair of images at the voxel level . Registration of lung volumes across time or across modalities has been utilized to establish lung atlases, estimate regional ventilation and local lung tissue expansion [25], assess lobar slippage during respiration [6, 7], and measure pulmonary function change following radiation therapy . Lung registration methods are typically intensity - based [2, 5, 913] or feature - based [1418]. Intensity - based methods consider intensity patterns of the whole lung regions to define similarity measures . They take advantage of the strong contrast between the lung parenchyma and the chest wall, and between the parenchyma, the blood vessels and larger airways . Commonly used intensity - based methods include minimizing intensity difference [3, 10], maximizing mutual information or normalized cross correlation [5, 9], and preserving tissue volume or lung mass [12, 13]. Since intensity - based methods do not use anatomical knowledge, these methods can get stuck in local minima resulting in mismatches of important anatomical structures such as bifurcations of smaller airway and vessel branches . On the other hand, they usually utilize corresponding landmarks and local intensity patterns [14, 16, 17] and surfaces correspondences [14, 15, 18]. However, due to the sparsity of features, good alignment of features cannot guarantee satisfactory matching accuracy for all lung regions . Since registration methods using either intensity - only or feature - only registration have their limitations, it is desirable to design lung registration methods that utilize both intensity and feature information [1923]. It has been shown that hybrid registration methods that combine intensity and feature information can help improve matching accuracy . Most of these methods incorporate distributed landmark pairs selected at airway or vessel branch points, that were identified manually or semiautomatically, centerline of the airway and vessel tree structures, and lung surface information . Therefore, fast feature extractions and effective methods to utilize feature information are needed to improve registration accuracy . In this paper, we couple intensity and feature information together to match 3d lung ct images acquired during breath hold at two different levels of inflation . We propose a feature - based similarity criterion utilizing the information of vessel locations and shapes in the registration process . This vesselness preserving cost function is added to four existing intensity - based similarity costs, and comparison experiments show that this criterion helps improve the registration accuracy . Higher matching accuracy makes the postanalysis of regional tissue mechanical properties more plausible and reliable . Our preliminary work on the effectiveness of using a vesselness matching similarity term was described in [2527]. The work presented in this paper provides a complete description of our vesselness matching approach . This paper extends our previous work by describing how to choose weighting factors for the different cost terms, describes a multiresolution optimization scheme, and provides more validation . This paper studies the effect of the vessel matching when used with various intensity similarity metrics such as the sum of squared intensity difference, sum of squared tissue volume difference, mutual information, and normalized cross correlation . This paper also examines the effect of using a linear - elastic constraint to regularize the displacement fields . In this study, six pairs of volumetric ct data sets from six human subjects in the supine orientation were collected on a siemens sensation 64 multidetector ct scanner . Each image pair was acquired during breath - holds near functional residual capacity (frc) and total lung capacity (tlc) in the same scanning session . For subject 1, data were acquired at functional residual capacity (frc) with 21.8% of the vital capacity (vc) and total lung capacity (tlc) with 95.6% of the vc . For subject 2, data were acquired at frc with 30.5% of the vc and tlc with 89.6% of the vc . For subject 3, data were acquired at frc with 26.3% of the vc and tlc with 95.7% of the vc . For subject 4, data were acquired at frc with 11.0% of the vc and tlc with 68.9% of the vc . For subject 5, data were acquired at frc with 25.8% of the vc and tlc with 92.9% of the vc . For subject 6, data were acquired at frc with 26.5% of the vc and tlc with 102.0% of the vc . Each volumetric data set was acquired at a section spacing of 0.5~0.6 mm and a reconstruction matrix of 512 512 . In - plane the parenchyma regions in the frc and tlc data sets were segmented using the method described in . Figure 1 gives an illustration of pulmonary ct images with renderings of the lung segmentations . The goal of image registration is to estimate a transformation h that defines the pointwise correspondences between two images i1 and i2 . More formally, let d r define the domain of the images i1: d r and i2: d r. in this work, the transformation h: d d is assumed to be a diffeomorphism . Let denote the set of all diffeomorphisms from d to d. the optimal transformation h is estimated by minimizing a cost function that consists of a similarity cost function and a regularizing term, that is, (1)h^=argminhcsim(i1,i2,h)+creg(h). The similarity cost function describes the characteristics of two images that should agree for corresponding image points . The regularization cost function is used to enforce desired properties of the transformation such as minimum distortion . The constant is used to balance the influence of the regularization cost with respect to the similarity cost . In general, the similarity and the regularization costs can be decomposed into linear combinations of more specific cost functions . For intensity - based image registration, it is usually assumed that intensities of corresponding voxels are related to each other in some way . Many criteria to construct the intensity relationship between corresponding points have been suggested as the cost function for aligning two images . Examples of intensity - based cost functions are the mean square difference (msd), correlation coefficient, mutual information, pattern intensity, and gradient correlation [29, 30]. In this work, we investigated three commonly used intensity - based cost functions and one intensity - based cost function designed for matching lung ct images . Sum of squared difference (ssd)minimizing the intensity difference at corresponding points between two images is an intuitive method to register grayscale images . A simple and common cost function is the sum of squared difference (ssd) defined by (2)cssd=x[i2(x)i1(h(x))]2, where i1 and i2 are the template and target image intensity functions, respectively . R denotes the union of lung regions in target image and deformed template image . The underlying assumption of ssd is that the image intensity at corresponding points between two images should be similar . However, considering the change in ct intensity as air inspired and expired during the respiratory cycle, the grayscale range is different within the lung region in two ct images acquired at different inflation levels . To balance this grayscale range difference, for example, a histogram matching procedure can be used before ssd registration to modify the histogram of template image so that it is similar to that of target image . Minimizing the intensity difference at corresponding points between two images is an intuitive method to register grayscale images . A simple and common cost function is the sum of squared difference (ssd) defined by (2)cssd=x[i2(x)i1(h(x))]2, where i1 and i2 are the template and target image intensity functions, respectively . R denotes the union of lung regions in target image and deformed template image . The underlying assumption of ssd is that the image intensity at corresponding points between two images should be similar . However, considering the change in ct intensity as air inspired and expired during the respiratory cycle, the grayscale range is different within the lung region in two ct images acquired at different inflation levels . To balance this grayscale range difference, for example, a histogram matching procedure can be used before ssd registration to modify the histogram of template image so that it is similar to that of target image . Mutual information (mi)mutual information (mi) similarity cost function can accommodate intensity difference between two images and is therefore well - suited to accommodate the ct intensity change during inflation and deflation of the lung . Mutual information expresses the amount of information that one image contains about the other one . Analogous to the kullback - leibler measure, the negative mutual information cost of two images is defined as [9, 32] (3)cmi=ijp(i, j)logp(i, j)pi1h(i)pi2(j), where p(i, j) is the joint intensity distribution of transformed template image i1h and target image i2; pi1h(i) and pi2(j) are their marginal distributions, respectively . The histogram bins of i1h and i2 are indexed by i and j. the experiments of mi - driven registration use 50 50 histogram bins to estimate joint distribution . Mutual information (mi) similarity cost function can accommodate intensity difference between two images and is therefore well - suited to accommodate the ct intensity change during inflation and deflation of the lung . Mutual information expresses the amount of information that one image contains about the other one . Analogous to the kullback - leibler measure, the negative mutual information cost of two images is defined as [9, 32] (3)cmi=ijp(i, j)logp(i, j)pi1h(i)pi2(j), where p(i, j) is the joint intensity distribution of transformed template image i1h and target image i2; pi1h(i) and pi2(j) are their marginal distributions, respectively . The histogram bins of i1h and i2 are indexed by i and j. the experiments of mi - driven registration use 50 50 histogram bins to estimate joint distribution . Normalized cross correlation (ncc)normalized cross correlation can be used for multimodality registration problems since it is insensitive to a constant multiplicative factor between the images . This cost function measures the pixel - wise cross - correlation between image intensities normalized by the square root of the autocorrelation of each image . Mathematically, the negative normalized cross correlation measure is given by (4)cncc=xi2(x)i1(h(x))xi2(x)2xi1(h(x))2, where the negative sign was added so that the optimal transformation h is found by minimization . When the factor of the intensity patterns from two images is a constant, the measure equals 1 . Misalignments between the images will result in decrease of the normalized cross correlation, and thus, increase of the similarity cost cncc . Normalized cross correlation can be used for multimodality registration problems since it is insensitive to a constant multiplicative factor between the images . This cost function measures the pixel - wise cross - correlation between image intensities normalized by the square root of the autocorrelation of each image . Mathematically, the negative normalized cross correlation measure is given by (4)cncc=xi2(x)i1(h(x))xi2(x)2xi1(h(x))2, where the negative sign was added so that the optimal transformation h is found by minimization . When the factor of the intensity patterns from two images is a constant, the measure equals 1 . Misalignments between the images will result in decrease of the normalized cross correlation, and thus, increase of the similarity cost cncc . Sum of squared tissue volume difference (sstvd)the sum of squared tissue volume difference (sstvd) cost function accounts for the variation of intensity in the lung ct images during respiration . Assume that lung is a mixture of two materials: air and tissue / blood (nonair). Then the hounsfield units (hu) in lung ct images is a function of tissue and air content . From the hu of ct lung images, the regional tissue volume and air volume can be estimated following the air - tissue mixture model by hoffman and ritman . Let v(x) be the volume element at location x. then the tissue volume v(x) within the volume element can be estimated as v(x) = v(x)((hu(x) huair)/(hutissue huair)), where we assume that huair = 1000 and hutissue = 0 . The intensity similarity cost function sstvd is defined as (5)csstvd=[v2(x)v1(h(x))]2dx = [v2(x)i2(x)+10001055 v1(h(x))i1(h(x))+10001055]2dx . The jacobian of a transformation j(h) estimates the regional volume changes resulted from mapping corresponding regions . Thus, the tissue volumes in image i1 and i2 are related by v1(h(x)) = v2(x) j(h(x)). The sum of squared tissue volume difference (sstvd) cost function accounts for the variation of intensity in the lung ct images during respiration . Assume that lung is a mixture of two materials: air and tissue / blood (nonair). Then the hounsfield units (hu) in lung ct images is a function of tissue and air content . From the hu of ct lung images, the regional tissue volume and air volume can be estimated following the air - tissue mixture model by hoffman and ritman . Let v(x) be the volume element at location x. then the tissue volume v(x) within the volume element can be estimated as v(x) = v(x)((hu(x) huair)/(hutissue huair)), where we assume that huair = 1000 and hutissue = 0 . The intensity similarity cost function sstvd is defined as (5)csstvd=[v2(x)v1(h(x))]2dx = [v2(x)i2(x)+10001055 v1(h(x))i1(h(x))+10001055]2dx . The jacobian of a transformation j(h) estimates the regional volume changes resulted from mapping corresponding regions . Thus, the tissue volumes in image i1 and i2 are related by v1(h(x)) = v2(x) j(h(x)). Feature information extracted from the grayscale image is important to help guide the registration process . During the respiration cycle, therefore, the shape and spatial information of vessels can be utilized to help improve the registration accuracy . In ct images, this intensity difference between parenchyma and blood vessels can effectively help intensity - based registration . Therefore, grayscale information of the small vessels give almost no contribution to the intensity - based registration . In order to better utilize the information of blood vessel locations, we utilize the vesselness measure (vm) computed from intensity images rather than using their grayscales directly . Sum of squared vesselness measure difference (ssvmd)the vesselness measure is based on the analysis of eigenvalues of the hessian matrix of image intensity . The eigenvalues, which are geometrically interpreted as principal curvatures, can be used to indicate the shape of underlying object . In 3d lung ct images, isotropic structures such as parenchyma tissues (dark) are associated with three similar nonzero positive eigenvalues . Tubular structures such as blood vessels (bright) are associated with one negligible eigenvalue and two similar nonzero negative eigenvalues . Ordering the eigenvalues of a hessian matrix by magnitude \|1 \| \|2 \| \|3\|, the frangi's vesselness function is defined as (6)f()={(1era2/22)erb2/22(1es2/22) if 2<0 and 3<0,0 otherwise, with ra=\|2\|/\|3\|,rb=\|1\|/\|23\|,s=12+22+32, where ra distinguishes between plate - like and tubular structures, rb accounts for the deviation from a blob - like structure, and s differentiates between tubular structures and noise .,, and control the sensitivity of the vesselness measure . The experiments in this paper use = 0.5, = 0.5, and = 5 . The vesselness measure is based on the analysis of eigenvalues of the hessian matrix of image intensity . The eigenvalues, which are geometrically interpreted as principal curvatures, can be used to indicate the shape of underlying object . In 3d lung ct images, isotropic structures such as parenchyma tissues (dark) are associated with three similar nonzero positive eigenvalues . Tubular structures such as blood vessels (bright) are associated with one negligible eigenvalue and two similar nonzero negative eigenvalues . Ordering the eigenvalues of a hessian matrix by magnitude \|1 \| \|2 \| \|3\|, the frangi's vesselness function is defined as (6)f()={(1era2/22)erb2/22(1es2/22) if 2<0 and 3<0,0 otherwise, with ra=\|2\|/\|3\|,rb=\|1\|/\|23\|,s=12+22+32, where ra distinguishes between plate - like and tubular structures, rb accounts for the deviation from a blob - like structure, and s differentiates between tubular structures and noise .,, and control the sensitivity of the vesselness measure . The experiments in this paper use = 0.5, = 0.5, and = 5 . The hessian matrix is computed by convolving the intensity image with second and cross derivatives of the gaussian function . In a multiscale analysis, the response of the vesselness filter will achieve the maximum at a scale, which approximately matches the size of vessels to detect . Therefore, the vesselness measure is estimated by computing (6) for a range of scales and selecting the maximum response: f = maxminmaxf(), where is the standard deviation of the gaussian function . The vesselness image is rescaled to [0, 1] and can be considered as a probability - like estimate of vesselness features . Larger vesselness value indicates that the underlying object is more likely to be a vessel structure, as shown in figure 2 . Let f1(x) and f2(x) represent the vesselness measures of images i1 and i2 at location x, respectively . In order to match similar vesselness patterns between two images, the sum of squared vesselness measure difference (ssvmd) is proposed as (7)cssvmd=x[f2(x)f1(h(x))]2 . Continuum mechanical models such as linear elasticity [11, 37, 38] and viscous fluid [37, 39] can be used to regularize the transformation . A common way to constraint the deformation is applying a differential operator on the transformation and formulating an additive cost term in the objective cost function [11, 22, 4046]. In our registration algorithms, a linear - elastic constraint was used to regularize the displacement fields u, where u = h(x) x. this regularization term is formed as (8)creg(u)=\|\|lu(x)\|\|2dx, where l can be any nonsingular linear differential operator . Here the linear elasticity operator l is formed as l u(x) = u(x) (u(x)) + u(x), where = [/x1, /x2, /x3] and = = [/x1 + /x2 + /x3]. Using the linear elasticity differential operator can help smooth the transformation and help eliminate abrupt changes in the displacement fields . The linear elasticity operator is used in this work to help avoid the transformation from folding onto itself . However, it cannot prevent the jacobian of the transformation from going negative, that is, destroying the image topology under the transformation . Finally, the total cost is defined as a linear combination of the intensity - based costs, vesselness measure preserving cost, and regularization constraint (9)ctotal(h)=cintensity(i1,i2,h)+cssvmd(i1,i2,h)+creg(h).cintensity can be one of the four intensity - based similarity cost functions: cssd, cmi, cncc, or csstvd . Constants and are weights to adjust the significance of the three terms . The transform defines how points from the template image i1 are mapping to their corresponding points in the target image i2 . In three dimensional space, let x = (x1, x2, x3) define a voxel coordinate in the image domain of the target image i2 . The transformation h is a (3 1) vector - valued function defined on the voxel lattice of target image, and h(x) gives the corresponding location in template image to the point x. the lung is composed of nonhomogenous soft tissue . Since lung expansion is nonuniform, nonrigid transformations are required to model the lung motion across different inflation levels . To represent the locally varying geometric distortions, the transformation can be represented by various forms of basis function, such as fourier transform, thin - plate splines, and b - splines . B - splines are well suited for image registration and are able to capture the local nonrigid deformation between two images [40, 45]. Considering the computational efficiency and accuracy requirement, let i = [x(xi), y(xi), z(xi)] be the coefficients of the ith control point xi on the spline lattice g along each direction . The transformation is represented as (10)h(x)=x+igi(xxi), where (x, y, z) = (x)(y)(z) is a separable convolution kernel . (x) is the uniform cubic b - spline basis function defined as (11)(x)={(46x2 + 3\|x\|3)6, 0\|x\|<1,(2\|x\|)36, 1\|x\|<2,0, \|x\|2 . There are several existing methods in numerical analysis such as the partial differential equation (pde) solvers used to solve for elastic and fluid transformations, steepest gradient descent method, conjugate gradient method, and so forth . Similar to [9, 13], our similarity cost functions were optimized using a limited memory quasi - newton minimization method with bounds (l - bfgs - b) algorithm . The bound constraints are applied on b - spline coefficients to guarantee the local injectivity (one - to - one property) of the transformation, that is, the transformation maintains the topology of two images . According to their analysis, the displacement fields are locally injective over the domain if the b - spline coefficients satisfy the conditions that x x / k, y y / k, and z z / k, where x, y, and z are the b - spline grid sizes along each direction, and k is a constant approximately equal to 2.479772335 . Validation and evaluation of image registration accuracy is an important task to quantify the performance of registration algorithms . Due to the absence of a gold standard to judge a registration algorithm, intrasubject ct images of the lung contain identifiable landmarks such as airway - tree and vascular - tree branch points . For each pair of frc and tlc data, 100150 distinctive landmark pairs were selected as branch points of the vascular tree using a semiautomatic method . The euclidean distance between the registration - predicted landmark position and its true position is defined as landmark error . Let pk and qk be the location of landmark k on template image i1 and target image i2, respectively . Vessels in the lung keep their tubular shape and tree structures during the respiratory process . Therefore, evaluating the alignment on vessel trees is an important approach to validate the matching accuracy at the lung feature level . The registration accuracy on the vessel tree was evaluated by vessel matching distance, which is calculated as the distance between a point on the target vessel tree and its closet point on warped template vessel tree . Mathematically, this distance can be stated as the vessel positioning error (vpe) (12)vpe(x)=minyv2 d(x, h(y)) for a given point x in v1, where v1 and v2, respectively, are the set of all points in the vessel trees extracted from image i1 and i2, respectively, and d() defines the euclidean distance . Examples of the vessel tree extractions are shown as red curves in figure 3 . Lobar fissures are defined as the division between adjacent lung lobes and represent important physical boundaries within the lungs . Fissure locations are extracted from the images by segmenting the lobes using and then identifying voxels adjacent to two lobe segmentations . The fissure positioning error (fpe) is defined as the minimum distance between a point on the deformed fissure and the closest point on the corresponding target fissure (13)fpe(x)=minyf2 d(x, h(y)) for a given point x in f1, where f1 and f2, respectively, are the set of all points in the fissure in image i1 and i2, respectively . Examples of the lobe segmentations are shown in different colors in figure 3 . The lung tissue deformation pattern can be used as an index to assess lung function . In this work, the jacobian determinant of the transformation field derived by image registration is used to estimate the local tissue deformation . The jacobian determinant (often simply called the jacobian) [11, 48, 54] is a measurement to estimate the pointwise expansion and contraction during the deformation . The jacobian of the transformation j(h(x)) is defined as (14)j(h(x))=\|h1(x)x1h1(x)x2h1(x)x3h2(x)x1h2(x)x2h2(x)x3h3(x)x1h3(x)x2h3(x)x3\| . Using a lagrangian reference frame local tissue expansion corresponds to a jacobian greater than one, and local tissue contraction corresponds to a jacobian less than one . Preprocessing starts by identifying the lung regions in all images using the method described in . Images and masks are downsampled by a factor of 2 in each dimension to reduce computation time . For each pair of data sets, frc images are used as the target image, and tlc images are used as the template image . In order to evaluate how the vesselness cost function affects the registration algorithm results, we performed registration experiments using different similarity costs on parenchyma region in each pair of data sets for comparison . There were four registration methods driven by intensity - only similarity cost functions described in section 2.2.1, and the same four registration methods that included the feature - based similarity cost ssvmd . After registration, the results from each method were evaluated and compared through matching accuracy on landmarks, vessels, fissures, and underlying transformation properties . We designed experiments to discover good parameter settings on intensity - based cost, feature - based cost cssvmd, and regularization term creg in (9). Here parameter settings for registration algorithms using other intensity - based cost functions, for example, cssd, cmi, and cncc can be tuned in the same way . Table 1 and figure 4 show the results for 20 ct - to - ct registration experiments, as the weighting values (ved) and (smooth) are varied . The values of and ranged from 0 to 2 and 0 to 0.5, respectively . Experiment ct01 corresponds to unconstrained estimation, in which the transformation was estimated only according to the intensity similarity cost . This experiment produced relatively the worse registration results as evident by the large values of csstvd, cssvmd, and creg in the respective tables . Experiments ct02, ct03, ct04, and ct05 demonstrate the effect of estimating the transformations without minimizing the vesselness similarity cost while varying the weight of the linear elastic cost . Minimizing the linear elastic cost is good for optimizing the other two similarity costs csstvd and cssvmd, as we can see from figures 4(a) and 4(b). Values larger than 0.2 are not recommended since they may cause the csstvd to increase dramatically . Experiments ct06, ct11, ct16, and ct21 demonstrate the effect of using vesselness similarity cost function without enforcing the linear elasticity constraint . The cssvmd values for these experiments are much lower than the previous cases since they are being minimized . The intensity similarity costs csstvd also decreased using registration with vesselness constraint, especially when is in the range of [0.5, 1]. The remaining experiments show the effect of jointly estimating the transformations, while varying the weights on both the vesselness similarity cost and the linear elasticity constraint . These experiments show that it is possible to find a set of parameters that produce better results using both constraints than only using one or none . The optimal set of parameters should be chosen to provide a good intensity match and vesselness match, while producing less spatial distortion as measured by an acceptable level of linear elastic cost . From the experiments, we observe that = 0.5 ~ 1 and = 0.05 ~ 0.1 are good for minimizing the three costs at the same time . In this work, weighting parameters were set to = 1 and = 0.1 when using sstvd as intensity cost function . Parameters in registration using other three intensity cost functions are set in the same way . A spatial multiresolution optimization procedure from coarse to fine was used to improve speed, accuracy, and robustness of the registration . In the experiments, the spatial multiresolution strategy proceeds from low to high resolution, starts at one - eighth of the spatial resolution, and increases by a factor of two until the full resolution is reached . Meanwhile, a hierarchy of b - spline grid spacings from large to small was also used . The finest b - spline grid space used in an example multiresolution scheme design for minimizing the total cost function is listed in table 2 . The image spatial resolution and b - spline grid spacing figure 5 lists the cost values at each iteration of one registration . At 1/8 and 1/4 the optimization is stopped before reaching the maximum iterations if the total cost change is nominal between consecutive iterations . Registration at full resolution further adjusts the local structures matching . During the registration procedure, the original average landmark error is 27.40 14.37 mm with a maximum landmark error of 72.79 mm . Table 3 shows the mean and standard deviation of landmark errors through all six subjects after using different registration methods . Table 4 shows the vessel positioning errors for six subjects after using different registration methods . The average errors and standard deviations were all decreased after adding the vesselness constraint . Figure 7 shows the distance map on frc vessel tree from one subject after using eight different registration methods . These results show that large errors between the deformed source and target vessel trees are reduced after adding the ssvmd constraint . For each pair of frc and tlc images, the parenchyma regions were segmented into five lobes, and the three fissures were identified, where the lobe segmentations touched each other . The mean and standard deviation of fissure positioning error over all three fissures after using eight registration methods are shown in table 5 . The average fissure positioning error across the six subjects was 9.20 mm and decreased to around 1.50 mm and 1.00 mm without and with ssvmd cost, respectively . The distance map on fissure planes from one subject after using eight different registration methods is shown in figure 8 . Notice that adding ssvmd helped improve registration accuracy in the lung regions near the thoracic cage . Registration methods producing similar matching accuracy on the feature locations may result in different underlying parenchymal tissue functions . In order to reveal the lung tissue deformation pattern, the jacobian determinant j at a given point gives important information about the behavior of transformation h near that point . Arrows denote regions that show different deformation patterns using intensity - only registration methods, but they are more similar after adding vesselness cost function . The experiments presented in this paper were designed to evaluate the performance of the vesselness constraint when it was added to intensity - based registration algorithms . The experimental results of tuning weighting parameters in the total cost function suggest that using both vesselness and smoothing constraint can help minimize the similarity cost, as shown in figure 4 . Figure 5 shows that the multiresolution scheme is important and useful for solving complex registration problem efficiently . This is because the registration is first performed at a coarse scale, where the images have much fewer voxels, which are fast and can help eliminate local minima . Table 3 and figure 6 demonstrate that adding the ssvmd cost function reduced the mean landmark errors of the four basic registration methods . Landmarks with large errors, shown as outliers in the box plot, are aligned much better when ssvmd is used . Figure 7 reflects the fact that the ssvmd constraint helps improve matching accuracy over all four basic methods on small vessels, around lung boundaries and in the region near diaphragm . The reason for this is that blood vessels in those regions are usually small and have low intensity contrast, and thus they contribute little to conventional intensity similarity criteria . The vesselness measurement enhances blood vessel information and strengthens contribution of small vessels to registration process when using the ssvmd similarity cost . Figure 8 indicates that the ssvmd not only helps match vessel structures, but also helps improve registration accuracy in other regions, such as positions on the fissure planes near the thoracic cage . Good matching accuracy on the feature locations does not guarantee that the parenchymal tissue is correctly aligned . Rather than evaluates the alignment accuracy, it reveals how well the transformation preserves topology and measures the differential lung volume change . In figure 9, the left column shows that the jacobian maps generated by the four registration methods without ssvmd have a similar ventral to dorsal gradient as expected since the subjects were imaged in the supine orientation . However, the local tissue deformation patterns derived from these methods are different even in the methods pair ssd and sstvd, which have similar landmark errors as shown in table 3 . This is consistent with the findings that while the intermethod variability on the landmark error is small, there may be discriminating difference in the jacobian maps . The right column shows that adding the ssvmd constraint produces jacobian images that are much more similar across different registration methods and reveal more detailed deformation patterns especially near vessel locations . The four jacobian maps produced using registration methods with ssvmd are similar, which may imply that the derived local deformation patterns are more reliable . Comparing the four intensity - only registration methods, registration driven by sstvd achieved lower landmark error and lower vessel and fissure positioning errors than other three methods driven by ssd, mi, and ncc . Figures 7 and 8 reflect that sstvd - driven registration resulted in more accurate matching within the region near the thoracic cage . The reason may be that sstvd cost function contains a local jacobian factor, which can constrain incorrect large displacement and help prevent distortion near the thoracic cage . After adding ssvmd on the four intensity - based cost functions ssvmd helps improve matching accuracy in regions near the thoracic cage and near the diaphragm . In our experiments, registration method using sstvd + ssvmd resulted in the smallest matching errors . Therefore, we may consider that sstvd + ssvmd is the best similarity cost function to register lung ct images according to our evaluation . The effectiveness of the vesselness preserving metric was tested on a variety of lung ct data sets as part of the grand challenge more than 20 individual registration algorithms from different groups were applied to 30 pairs of lung ct scans in the empire10 challenge . Besides our tissue volume and vesselness preserving method, one other method called the robust treereg also combined intensity and feature information . The robust treereg algorithm performed a robust tree registration (rtr) and added correspondences between bifurcations of the vessel tree to the voxel - based mutual information driven registration . For this registration challenge, our tissue volume and vesselness preserving method had better performance than the robust treereg method . This result may imply that the vesselness measure provides more feature information than the bifurcation landmarks of the vessel tree . In addition, our tissue volume (or mass) and vesselness preserving method was shown to improve matching results compared to methods that only incorporated mass preservation . This paper presented nonrigid registration algorithms driven by commonly used intensity - based criteria for lung registration, a feature - based vesselness constraint, and a linear elastic smoothing constraint . Results were presented to show that adding the ssvmd constraint to existing similarity cost functions such as ssd, mi, ncc, and sstvd reduces landmark error and improves overlap on vascular tree and fissure planes . The purpose of adding the vesselness cost in registration process is that it can help correct the mismatches of small vessels and their surrounding lung tissues . Using the ssvmd constraint was shown to produce a more detailed expansion pattern for local tissue, especially near vessel locations . This demonstrates that using the ssvmd constraint not only helps match on feature structures, but also helps align corresponding parenchymal tissues providing a more reliable pattern of local lung tissue deformation . In this paper, registration method preserving both tissue volume and vesselness measurement performed best on matching 3d lung ct data according to our evaluation.
Bilateral common iliac artery (cia) aneurysm (ciaa) is a rare entity . In the past decade, different endovascular approaches have been adopted for patients with several comorbidities or unfit for open repair (or). Recently, the use of iliac branch stent graft has been proposed, resulting in satisfactory patency rates and decrease in morbidity . Currently, according to instruction for use, the iliac branch stent graft is to be used with aortobi - iliac stent graft conjunction . We describe a case of a successful endovascular repair of bilateral ciaas using the gore excluder iliac branch endoprosthesis (ibes) without aortobi - iliac stent graft conjunction . An 83-year - old man was admitted with abdominal pain and presence of pulsatile mass in the right and left iliac fossa . Computed tomographic (ct) angiography showed the presence of large bilateral ciaas (right cia = 66 mm; left cia = 38 mm), without concomitant thoracic or abdominal aorta aneurysm . Moreover, ct scan demonstrated the presence of bilateral lower accessory renal artery close to the aortic bifurcation . Due to the high operative risk, the patient was scheduled for endovascular repair with bilateral ibes, without the aortobi - iliac stent graft conjunction to avoid the renal ischemia as a consequence of renal arteries covering . The procedure was completed without complications and duplex ultrasound demonstrated the complete exclusion of both aneurysms without any type of endoleaks at 1 month of follow - up . Gore ibes without aortobi - iliac stent graft conjunction seem to be a feasible and effective procedure for the treatment of isolated ciaas in patients with highly selected anatomical conditions . Common iliac artery (cia) aneurysm (ciaa) is defined by a transverse diameter greater than 18.5 mm for men, and 15 mm for women . The prevalence of isolated cia aneurysm is around 0.008% to 0.03% in autopsy studies and 3% of all kind of aneurysms, and they may be associated to an abdominal aorta aneurysm up to 20% of patients . Currently, elective repair is indicated when ciaa transverse diameter is greater than 30 mm, due to an increased chance of developing symptoms or complications, including rupture with high risk of mortality (50%100%). Open repair (or), compared with endovascular procedures, is associated with more blood loss, longer operative time, and higher morbidity and mortality rate . Endovascular techniques, with the off - label use of commercially available devices, have been proposed as a safe alternative to open surgery for the treatment of ciaas patients with severe comorbidities and high risk, or unfit for or . Usually, the endovascular approaches include embolization and endoluminal stenting of one or both internal iliac artery (iia) in order to create a distal landing zone in the external iliac artery (eia). However, the iia embolization may result in erectile dysfunction, buttock or thigh claudication, paraplegia, and sphincter dysfunction . To address these issues, several endovascular strategies have been proposed to preserve at least 1 internal iliac artery, including the bell bottom technique, chimney technique, sandwich technique, and parallel endograft technique . The off - the shelf iliac branch stent graft offers the possibility to create a distal landing zone both in iia and eia, therefore maintaining the iia patency and pelvic perfusion, resulting in satisfactory patency rates and decrease in mortality . Currently, the main available devices are: the zenith iliac branched device (ibd, cook, inc ., bloomington, in); the gore excluder iliac branch endoprosthesis (ibe, w.l . Gore & associates, flagstaff, az); and the jotec e - iliac stent graft (lotzencker, hechingen, germany). The main anatomical indications for the treatment of aorto - iliac and cia aneurysms using gore ibe are resumed in table 1 . Anatomical criteria for the gore excluder iliac branch endoprosthesis device for the correct implantation . According to the manufacturer instructions for use (ifu), the gore ibe is to be used in conjunction with aortobi - iliac stent graft . Therefore, due to specific anatomical limitations, not every patient is always eligible for the standard approach . We report a treatment of bilateral ciaas by endovascular positioning of bilateral gore ibes, without the need of aortobi - iliac stent graft conjunction . The patient had clinical history of hypertension, coronary artery disease, diabetes mellitus, severe chronic pulmonary disease requiring permanent o2 therapy, and moderate renal failure (serum creatinine 1.5 mg / dl). Physical examination revealed the presence of a pulsatile mass in the right and left iliac fossa . Computed tomographic (ct) angiography demonstrated the presence of a large bilateral ciaas (right cia = 66 mm; left cia = 38 mm) (fig . There were no concomitant aneurysms of thoracic or abdominal aorta, but ct angiography showed the presence of a bilateral accessory lower renal artery, 2 cm above the aortic bifurcation (fig . The length and diameter of the right cia proximal neck were 23 and 17 mm, respectively . The total length of the right cia was 104 mm and the diameter of the right external iliac artery (eia) was 9.5 mm . The internal iliac artery diameter and length to first bifurcation were 9.3 and 27 mm, respectively . The length of the left cia proximal neck was 27 mm and its diameter ranged between 15.5 and 18 mm . The total length of the left cia was 85 mm and the diameter of the right eia was 10 mm . The left internal iliac artery diameter and length to first bifurcation were 7.6 and 23 mm, respectively . Taking in consideration the advanced age and the significant comorbidities, the patient was considered not suitable for open repair and scheduled for endovascular treatment . Because of the 2 accessories renal arteries close to the aortic bifurcation, the bilateral ciaas were treated with bilateral isolated ibe, without an aortic main body endograft support, to avoid the likely renal ischemia as a consequence of polar arteries covering by the endograft . The procedure was performed under local anesthesia and was approached from a bilateral antegrade common femoral artery puncture using sonography guidance and introducer sheaths of appropriate size . After sheath insertion, 5000 units of heparin were administered, and a ber ii diagnostic catheter (4 f, 100 cm, cordis corporation, bridgewater, nj) was advanced into the left cia with the support of a 0.035 terumo guide wire (terumo medical). The gore ibe was introduced over a 0.035 stiff guide wire and through - and - through wire, and deployed at the level of the left cia bifurcation . A 12 f crossover sheath was advanced over the through - and - through wire and placed at the level of the side branch . A 0.035 short tip stiff guide wire (amplatz, boston scientific) was introduced inside 12 f sheath, and internal iliac component was advanced over the wire and deployed in the ipsilateral internal iliac artery . The angiography confirmed the complete exclusion of left ciaa and the absence of endoleak (fig . The procedure was then performed in the right cia, where a gore excluder ibe was deployed at the level of bifurcation (fig . 2b), while an internal component was deployed in the right internal iliac artery . Also in this case, 2 proximal cuffs were deployed to achieve an adequate sealing zone . At the end of the procedure, angiography confirmed the complete exclusion of the 2 aneurysms without any signs of endoleaks (fig . 2c). Postoperative course was uneventful and the patient was discharged on the 3rd postoperative day with a stable renal function (serum creatinine 1.4 mg / dl). (a) transversal slide of preoperative ct angiography image showing bilateral common iliac artery aneurysms (right cia = 66 mm; left cia = 38 mm). (b) coronal slide of preoperative ct angiography image showing the presence of accessory renal artery close to the aortic bifurcation . (c) 3d ct angiography reconstruction image showing bilateral inferior accessory renal arteries (the left accessory renal artery was the lower). Cia = common iliac artery aneurysm, ct = computed tomography, 3d = three - dimensional . (a) procedural angiography after correct positioning of the left gore excluder ibe and patency of the liic . (b) right gore excluder ibe device at the level of the right iliac bifurcation . (c) postprocedural angiography demonstrated the complete exclusion of both iliac aneurysms without signs of endoleak and patency of the left accessory renal artery . Ibe = iliac branch endoprosthesis, licc = left internal iliac component . Patients with cia aneurysm are usually followed by means of duplex ultrasound (dus) and clinical examination before discharge, and after 1 and 6 months, to evaluate patency and endoleak occurrence . This is the first report of endovascular repair, using gore ibe device, of symptomatic bilateral ciaa, without the need for an aortobi - iliac stent graft conjunction . Endovascular management of cia aneurysms with the ibe device is safe and effective in the short - term . In a review on the endovascular treatment of iliac aneurysm with the zenith iliac branched device, karthikesalingam et al reported a technical success rates ranging between 85% and 100%, with an iliac branch device occlusion in 12% of patients . Similar results were reported with the gore ibe with a technical success of 93.3%, no 30-day mortality, and with an iia patency rate of 91.4% at 1 and 5 years of follow - up, respectively . More recently, millon et al reported their experience with the gore ibe in 10 patients with aneurysmal cia: technical success was 100%, with a complete aneurysm exclusion without type ib or type ii endoleak in all patients; 1 aortic type ia endoleak was observed in the follow - up, while branch patency was 100% at 1 month and 90% at 6 months . However, anatomical limitations exclude a large number of ciaa that could be treated with dedicated branched endovascular devices . Karthikesalingam et al and gray et al reported that only one third of patients with aorto - iliac or isolated cia aneurysms fulfilled the criteria for the use of these endovascular devices . Several endovascular strategies have been proposed to preserve at least 1 internal iliac artery . In 2001, karch et al described the bell bottom technique in the treatment of ectatic and nonaneurysmal iliac aneurysm . They used the larger diameter aortic extension cuff to achieve and adequate endograft to arterial wall apposition in patients with ectatic, nonaneurysmal cias . In 14 patients, an aortic extension cuffs were placed into 18 ectatic (> 14 mm, but <20 mm) cias . No endoleaks, ruptures, and endograft migration related to this technique were recorded at a mean follow - up of 14 months . In 2010 torsello et al reported a technical success rate of 97.8% in 89 patients with aorto - iliac artery aneurysm undergoing a bell bottom technique: after a mean follow - up of 56.5 months, 8 patients died (none aneurysm related). Meier analysis was 96.3% at 1 year, 85.5% at 3 years, and 83.1% at 5 years . Technique for the treatment of ciaas, with no recorded branch occlusion or type i endoleak from the iia or chimney graft gutters at 6-month imaging studies . In 2011, lobato described the sandwich technique for isolated common and iia aneurysms or aorto - iliac aneurysms extending to the iia . This technique consists of: insertion of an aortobi - iliac stent graft through an ipsilateral femoral approach with the iliac limb distal end positioned 1 cm above the iia origin; catheterization of the ipsilateral iia through a left brachial access with a long floppy tip guidewire; placement of a covered self - expanding stent inside the iia with a 6-cm overlap into the iliac limb, followed by positioning of an iliac limb extension 1 cm below the covered stent proximal end; modeling of the iliac limb stent grafts using a latex balloon and dilation of the covered stent with an angioplasty balloon; and deployment of the contralateral iliac limb . In 2013, lepidi et al described the this technique allows avoiding main body insertion, using 2 iliac limbs endografts (iles) that are simultaneously delivered from both femoral arteries, landing parallel into the aortic neck . A 3rd parallel covered stent graft (usually a viabahn) is delivered from a left brachial approach in order to maintain blood flow to 1 iia when needed . Three patients required a reintervention: 1 patient needed an ile extension to treat a type ib endoleak and 2 for a type ii endoleaks . No endoleaks, graft displacements, or occlusions were observed during follow - up (median: 26 months, range 1242 months). It should be noted that these approaches are often used only in the case of aorto - iliac aneurysm and considered off - label and not always feasible . The gore ibe device provides very conformable technology and accurate positioning thanks to its repositionable delivery system, and the internal iliac component is a fully supported sinusoidal stent designed for kink resistance, with proven long - term results in iliac patency preservation . Currently, according to gore ifu, ibe is to be used together with an aortobi - iliac stent graft in order to obtain a reconstruction of the healthy proximal portion of the cia, and to avoid the possible migration of the device due to the large proximal diameter of the cia . In this case report, we have demonstrated the feasibility of a bilateral ciaa repair with isolated gore ibes, preserving the patency of both iias . Due to the presence of the bilateral polar renal arteries close to the aortic bifurcation, the use of aortobi - iliac stent graft has to be avoided due to the likely renal ischemia . The main body extensions were used bilaterally in order to increase the size of the available landing zone and to avoid proximal endoleaks . The main limitation of this case report is the absence of an adequate follow - up because to patient died 3-month after intervention . Nevertheless, the aim of this case report was to assess the feasibility of the gore ibe implantation, without aortobi - iliac stent graft conjunction, for the treatment of patients with isolated bilateral ciaas . Bilateral, isolated gore excluder ibes seem to be a feasible and safe procedure in the treatment of isolated ciaa, maintaining the patency of iia and reducing the risk of pelvic complications . This case report may suggest a possible ifu extension of this device, in high selected patients, in the near future . However, a larger number of patients with a longer follow - up are needed to better define suitable anatomical characteristics and determine the effective efficacy and durability of this device.
Leishmaniasis is a parasitic disease that can manifest in three forms: 1) mucosal, 2) cutaneous, and 3) visceral (who 2010, maroli et al . 2013). Visceral leishmaniasis (vl) is the most severe form caused by the protozoan flagellate, leishmania donovani (east africa and indian subcontinent) and l. infantum (also known as l. chagasi, found in europe, north africa, and latin america). As this review focuses on elimination efforts in india, we will only refer to l. donovani in this article . Globally, the annual incidence rate is approximately 200,000400,000 cases, the majority of cases are present in bangladesh, nepal, and india . Furthermore, two - thirds of those cases occur in india where vl (also known as kala - azar; black fever) is endemic in the states of uttar pradesh, jharkhand, west bengal, and bihar (alvar et al . Reports have noted the annual incidence in india as 146,700282,800 cases with a mortality rate of at least 2.4% . However, other studies involving active searches at the village level have discovered mortality rates from 1020%, partly due to a delay in diagnosis (alvar et al . Vl carries a mortality rate over 90% when left untreated (desjeux 1996, jeronimo et al . Transmission of l. donovani occurs via the bite of a female phlebotomus argentipes sand fly . Of the nearly 50 species of sand flies present in india, p. argentipes is the only one known to transmit vl in this country (kumar et al . Once the parasite is in the human body it rapidly invades macrophages and eventually moves in this way to the liver, spleen, and lymph nodes (chappuis et al . Symptoms include: fever lasting weeks to months, splenomegaly, hepatomegaly, and anemia (desjeux 1996, guerin et al . Go on to develop a rash known as post kala - azar dermal leishmaniasis (pkdl), this can occur anywhere from several months to several years following treatment (ramesh and mukharjee 1995, rahman et al . 2010). Pkdl patients do not experience other symptoms outside of the rash, however, they are thought to serve as a reservoir for l. donovani where feeding sand flies can acquire the parasite (addy and nandy 1992, rahman et al . Are the only known reservoir for l. donovani in india, pkdl patients are another concern for vl elimination efforts . Visceral leishmaniasis is a poverty - associated disease linked to poor housing and sanitary conditions and malnutrition, these factors have led to a number of difficulties regarding treatment and elimination (cerf et al . Easy access to medical care in the rural vl - endemic regions of india is still limited, meaning patients are not routinely identified or wait until the disease has progressed to more severe symptoms before seeking treatment . Moreover, until recently treatment regimens required medication injections over the course of 2028 days leading to poor compliance rates (clem 2010, moore and lockwood 2010, stockdale and newton 2013). In addition to hurdles with medical treatment, increased sand fly density has been associated with certain types of housing, owning livestock, and nearby vegetation (ranjan et al 2005, singh et al . 2010, poch et al . 2011, poch et al . 2012, perry et al . The risk of exposure to p. argentipes is even higher for some, as many people in these poor regions of india live in close proximity to their cattle, keeping them inside their dwellings (singh et al . 2014). As a by - product of ddt spraying for malaria elimination programs in the 1950s and 1970s, however, when ddt spraying ended, sand fly numbers and cases of vl rose again, leading to several major epidemics (kishore et al . In 2005, the governments of bangladesh, nepal, and india began a concerted vl elimination effort, with a target goal of 2015 for elimination (1 case in 10,000 people). Indoor residual spraying (irs) and insecticide - treated bed nets (itn) have been the main forms of control regimens tried in india, while environmental modification (evm) and feed - through insecticides (fti) have been less studied . Despite significant efforts to control sand flies and treat infected persons as such, we sought to review the current practices regarding vector / vl control programs in india . India in combination with the following keywords: phlebotomus argentipes, sand fly, kala - azar, and/or visceral leishmaniasis . The following paper is a summation of the articles collected in hopes of highlighting what has worked, what has not, and what can be learned as we move forward towards elimination goals . Irs has been the main line of defense against vector - borne diseases in india . In the 1950s and again in the 1970s there were aggressive irs initiatives to eliminate malaria (kishore et al ., households in malaria - endemic regions, many of which were the same as vl - endemic areas, were sprayed with ddt to cull mosquitoes . Sand fly populations declined in kind, and with them, cases of vl . However, at the end of those campaigns, with no systematic irs programs, sand fly populations and cases of vl quickly rebounded (kishore et al . Since then, irs using ddt has been employed to fight vl transmission in india with mixed results . Currently, the national vector borne disease control programme of india dictates that irs should be performed on all homes and cattle sheds in endemic regions . They recommend two annual applications, the first to occur in february / march and the second in may / june (nvbdcp 2014). Applied properly, irs has been shown to dramatically impact p. argentipes density (joshi et al . 2009). In that carefully controlled study involving all homes in six clusters in each of four villages, there was a 72.4% decline in sand fly numbers . Other studies that have assessed the efficacy of irs in india have instead highlighted a number of issues limiting its success in controlling sand flies and vl . Huda et al . Monitored local irs programs for control of vl in bangladesh, nepal, and india (2011). In india, (though it was not unique for some of these issues) they found functional pumps and spare parts were lacking and 23.5% of the pumps were leaking . Spraying staff were not adequately trained and as such, proper mixing of ddt was done only 29.4% of the times observed . Additionally, proper distance and swath coverage during spraying were maintained only 49% and 58.8% of the time, respectively (huda et al . 2011). These results are echoed in a similar report that found the same issues with equipment, training, mixing, and distance from surface, in addition to storage and quality issues for the ddt (chowdhury et al . This group also noted that the ddt residue levels on the walls varied at the village level from 66%90% of the intended concentration while at the household level, the concentration varied as much as 9.1% to 330% (chowdhury et al . Furthermore, spraying must be applied to> 80% of the homes in an area for mass effect (cdc 2012). According to huda another point of concern with irs in india is resistance of p. argentipes to ddt . Resistance to ddt was documented for the district of samastipur in bihar, india as early as 1990 (mukhopadhyay et al . 1992). In general, p. argentipes remains largely susceptible to ddt with the majority of flies succumbing to the insecticide (singh et al . However, select districts are showing signs of this trend shifting (kishore et al . Documented only a 54% mortality rate after 24 h for sand flies exposed to walls sprayed with ddt while another group found up to 70% were killed when exposed to surfaces that had been sprayed 2 weeks prior (chowdhury et al . It is possible that the results obtained by huda and chowdhury et al . May have been partly due to improper storage, mixing, spraying, and/or active ingredient concentration which were all problems documented by those studies (chowdhury 2011a, huda 2011a). A controlled study involving irs in three states in india found mortality rates for p. argentipes ranging from 3189%, indicating moderate to significant levels of resistance to ddt, even with proper use (singh et al . 2012). Despite these many problems recorded by groups studying irs, in all of the aforementioned studies, post - treatment sand fly abundance was significantly reduced in the short term (chowdhury et al . In fact, joshi et al . Demonstrated a negative effect on sand fly numbers up to five months post - treatment (joshi et al . Both nepal and bangladesh employ pyrethroids for their irs regimens and have shown them to be effective (joshi et al . Some work has been conducted using different insecticides in india . In one report they found sand flies were resistant to ddt but not deltamethrin (dhiman et al . A more recent study documented that deltamethrin was effective in nearly 100% of the locations tested in india (singh et al . The government of india is currently testing the efficacy of synthetic pyrethroids on sand fly control in bihar (nvbdp 2014). A comprehensive table of susceptibility studies can be found in the review by ostyn et al . P. argentipes are peridomestic, found in cattle enclosures as well as vegetation (poch et al . Another matter that is infrequently addressed in studies regarding irs is the health effects to humans, animals, and the environment as a result of frequent and long - term irs . The stockholm convention on persistent organic pollutants has banned ddt (van den berg 2009). However, there is yet no consensus on whether ddt exposure leads to deleterious health effects among humans (sharpe and stewart 2004, beard 2006). Regardless, there are well documented studies regarding its toxicity in birds and a variety of aquatic species (blus 2003, sparling 2010, beckvar and lotufo 2011). While further work is needed to verify them, these data indicate that alternative irs compounds may be more efficacious in controlling p. argentipes abundance . Research looking at effects on human health due to long - term exposure would still need to be addressed . Ultimately, these data suggest that with proper execution, irs could be an even more effective tool against vl in india but is not sufficient as a standalone given that p. argentipes is found outdoors in significant numbers . Bed nets, in particular, itns and long - lasting insecticide nets (llins), have been suggested as alternatives and/or complements to irs for the control of sand fly populations and vl . Itn / llins have been effective against other vector - borne diseases, including cutaneous leishmaniasis (lengeler 2004, kulkarni et al . 2007, wilson et al . 2014) but there are mixed results when it comes to sand flies in india . A trial using untreated nets found that the number of female, blood - fed p. argentipes declined by 85% following the introduction of the nets (picado et al . However, the authors of that study note that they lacked concurrent controls and thus, blood - feeding rates could potentially be attributed to changes in environmental factors (temperature, humidity, precipitation) or changes in host availability (i.e. An increase in domestic animals). Even so, other groups in bangladesh and nepal have found similar results (bern et al . The report by joshi et al . Investigated the usefulness of irs, llins, and evm to mitigate sand fly density in india, nepal, and bangladesh . When llins were in place, there was a village - wide reduction in p. argentipes numbers by 43.7% in india when measured 5 months post - intervention (joshi et al . A subsequent village - wide study in india noted a 25% decline in sand fly density (picado et al . In that study, 16 clusters were enrolled, of those, 10 were used for sand fly capture studies . The reduction appeared to be at the community level and not displacement as p. argentipes abundance did not increase in cattle enclosures (picado et al . However, this observation does not exclude other environmental refuges, such as vegetation (poch 2011, poch 2012). As a part of the same study by picado et al . They also investigated rates of vl in these same villages and found no protective effect of llins on the rate of seroconversion (picado et al . Over the course of 24 months, incidence of l. donovani infection was found to be 5.4% in the intervention group and 5.5% in the control group while clinical vl rates were 0.38% and 0.40% in the intervention and control groups, respectively . One explanation for this result was that llins do not prevent outdoor transmission (picado et al . An earlier study of 48 homes and mixed dwellings (homes shared with livestock) in bihar compared two types of llins to two types of untreated nets and looked at sand fly densities . At all time - points, up to 9 weeks post - treatment, there were no differences in p. argentipes abundance between the groups (dinesh et al . It should be noted, that during the course of the aforementioned studies, unplanned irs took place, and in both cases there was no noticeable effect on the number of sand flies captured in either study (dinesh et al . Furthermore, when the statistical models used to analyze the vl seroconversion data were adjusted for irs, no changes were found (picado et al . While the number of studies has been limited, the efficacy of bed nets in india to combat vl have not shown the same promise as they have in bangladesh (chowdhury et al . The potential reasons for this are many, including: study design, environmental factors (temperature, humidity, flooding etc . ), and susceptibility to insecticides - p. argentipes in bangladesh may be more susceptible, given their history of irs campaigns is more recent compared to india (bern et al . As breeding sites are yet unknown, and sand flies have been associated with vegetation in india, these remain confounding factors when accounting for differences in intervention strategies (poch et al . Non - compliance and net quality are another concern . In the studies by picado and joshi, even so, compliance with nets can be difficult as sand flies are much smaller than mosquitoes requiring finer mesh that people find stifling to sleep under in the heat of summer (ostyn et al . While in theory, the impregnated insecticide should provide repellent activities, thus allowing for a larger mesh, efficacy in the field has not been shown (picado et al . Moreover, 93% of 1,217 people surveyed in 20092011 in vl - endemic regions reported sleeping outside at some point during the hottest months of the year (perry et al . 2013). Assuming this trend holds true throughout other vl - afflicted areas, this is a serious hurdle to be faced in regards to the efficacy of irs, itns, and evm as none mitigate transmission that occurs outside of a person s dwelling . Typical dwellings in the regions of india afflicted by vl are often made of mud and thatch, or brick / plaster . 2014). In all cases, cracks and crevices where sand flies can rest and hide are prevalent . All home types are susceptible to habitation by p. argentipes, but thatched homes in particular cater to high densities (malaviya et al . Evm is a method little studied when compared to irs and itns as a means to control vl in india . Evm has generally meant alterations to the home or surrounding environment by means of covering or filling in cracks and crevices in walls and floors . A pilot study involving 15 homes saw a reduction in sand fly numbers using a mud and lime plaster mix to seal cracks in homes and cattle enclosures (kumar et al . The same well - controlled study comparing the efficacy of irs and itns in india, nepal, and bangladesh found a 42% decline in sand fly abundance five months after the walls of homes and cattle enclosures were plastered with a mud and lime mixture . The negative effect on sand fly populations may be due to the lime ph or limiting available moisture, thus, inhibiting breeding of p. argentipes, though breeding sites for these flies have not been confirmed (kumar et al . Indeed, a study in bangladesh saw no effect on sand fly populations when crevices were filled with mud (chowdhury et al . Importantly, another study in india found mud - plastered walls themselves to be risk factor for vl (ranjan et al . However, while mud / lime mixes are effective, this method requires continual maintenance and is costly compared to irs and itns (das et al . 2008). But like irs and itn, it is only effective against the sand flies that are inside homes; populations that reside in outdoor enclosures and vegetation would remain a source of vl . A newer, relatively untested, yet promising addition to the vector - control arsenal is the use of ftis . Several compounds including ivermectin, fipronil, and imidacloprid have been tested in rodents to cull p. papatasi, a vector for cutaneous leishmaniasis (mascari et al ., the agents have been effective at controlling adult and larval sand flies when fed on blood or feces from treated animals, respectively . In regards to p. argentipes, ivermectin and fipronil as well as diflubenzuron and eprinomectin were tested as ftis in rats (ingenloff et al . Fipronil at 150 ppm was shown to result in the quickest mortality and its effects had greater longevity than the other three compounds, even when they were used at greater concentrations . Cattle and other livestock act as a major source of blood meals for female sand flies . Blood - meal analysis in one study found that 39.3% of the flies tested had fed on some form of livestock (garlapati et al . So as to target p. argentipes that are feeding off of cattle and therefore, possibly residing outdoors, a controlled study involving cattle dosed with 0.5, 1.0, 2.0, and 4.0 mg / kg body weight of fipronil was conducted (poch et al . 2013). That investigation demonstrated that fipronil as an fti is effective, even at the lowest dose, at killing both adult and larval sand flies . The majority of adult sand flies had a 100% mortality rate within four days when fed on cattle up to 21 days post - treatment for all but the lowest dose (which had a 22% mortality rate at 21 days post - treatment). Nearly all of the flies that were fed on days 1, 3, and 5 post - treatment succumbed on the same day . For larval p. argentipes, the mean - time to death when fed on feces from cattle 1 or 3 days post - treatment was 4.5 days, larva fed on feces with the lowest dose (0.5 mg / kg) on day 1 post - treatment had 100% mortality within 5.5 days while the highest dose (4.0 mg / kg) was 4.0 days . Additionally, for all doses tested, 100% mortality of larval sand flies was achieved by day 15.5 when fed on dung collected 21 days post - treatment (poch et al . 2013). Given that this method is untested under field conditions, conclusions regarding its effect on p. argentipes abundance or vl transmission cannot be drawn at this time . While no effects to cattle health were noted in the aforementioned study, future work will need to be done to assess the health of cattle treated long - term . Moreover, at the lowest dose, fipronil can remain in the animal for up to 35 days but it is at concentration below international allowable limits (poch et al . Studies regarding the efficacy of fipronil as an fti are ongoing and if they prove positive, this method would be a valued addition to irs and itns as it would better address exophilic p. argentipes, making control measures more comprehensive . Irs has been the main line of defense against vector - borne diseases in india . In the 1950s and again in the 1970s there were aggressive irs initiatives to eliminate malaria (kishore et al ., households in malaria - endemic regions, many of which were the same as vl - endemic areas, were sprayed with ddt to cull mosquitoes . Sand fly populations declined in kind, and with them, cases of vl . However, at the end of those campaigns, with no systematic irs programs, sand fly populations and cases of vl quickly rebounded (kishore et al . Since then, irs using ddt has been employed to fight vl transmission in india with mixed results . Currently, the national vector borne disease control programme of india dictates that irs should be performed on all homes and cattle sheds in endemic regions . They recommend two annual applications, the first to occur in february / march and the second in may / june (nvbdcp 2014). Applied properly, irs has been shown to dramatically impact p. argentipes density (joshi et al . 2009). In that carefully controlled study involving all homes in six clusters in each of four villages, there was a 72.4% decline in sand fly numbers . Other studies that have assessed the efficacy of irs in india have instead highlighted a number of issues limiting its success in controlling sand flies and vl . Huda et al . Monitored local irs programs for control of vl in bangladesh, nepal, and india (2011). In india, (though it was not unique for some of these issues) they found functional pumps and spare parts were lacking and 23.5% of the pumps were leaking . Spraying staff were not adequately trained and as such, proper mixing of ddt was done only 29.4% of the times observed . Additionally, proper distance and swath coverage during spraying were maintained only 49% and 58.8% of the time, respectively (huda et al . 2011). These results are echoed in a similar report that found the same issues with equipment, training, mixing, and distance from surface, in addition to storage and quality issues for the ddt (chowdhury et al . This group also noted that the ddt residue levels on the walls varied at the village level from 66%90% of the intended concentration while at the household level, the concentration varied as much as 9.1% to 330% (chowdhury et al . Furthermore, spraying must be applied to> 80% of the homes in an area for mass effect (cdc 2012). According to huda another point of concern with irs in india is resistance of p. argentipes to ddt . Resistance to ddt was documented for the district of samastipur in bihar, india as early as 1990 (mukhopadhyay et al . 1992). In general, p. argentipes remains largely susceptible to ddt with the majority of flies succumbing to the insecticide (singh et al . However, select districts are showing signs of this trend shifting (kishore et al . Documented only a 54% mortality rate after 24 h for sand flies exposed to walls sprayed with ddt while another group found up to 70% were killed when exposed to surfaces that had been sprayed 2 weeks prior (chowdhury et al . It is possible that the results obtained by huda and chowdhury et al . May have been partly due to improper storage, mixing, spraying, and/or active ingredient concentration which were all problems documented by those studies (chowdhury 2011a, huda 2011a). A controlled study involving irs in three states in india found mortality rates for p. argentipes ranging from 3189%, indicating moderate to significant levels of resistance to ddt, even with proper use (singh et al . 2012). Despite these many problems recorded by groups studying irs, in all of the aforementioned studies, post - treatment sand fly abundance was significantly reduced in the short term (chowdhury et al . In fact, joshi et al . Demonstrated a negative effect on sand fly numbers up to five months post - treatment (joshi et al . Both nepal and bangladesh employ pyrethroids for their irs regimens and have shown them to be effective (joshi et al . Some work has been conducted using different insecticides in india . In one report they found sand flies were resistant to ddt but not deltamethrin (dhiman et al . A more recent study documented that deltamethrin was effective in nearly 100% of the locations tested in india (singh et al . The government of india is currently testing the efficacy of synthetic pyrethroids on sand fly control in bihar (nvbdp 2014). A comprehensive table of susceptibility studies can be found in the review by ostyn et al . P. argentipes are peridomestic, found in cattle enclosures as well as vegetation (poch et al . Another matter that is infrequently addressed in studies regarding irs is the health effects to humans, animals, and the environment as a result of frequent and long - term irs . The stockholm convention on persistent organic pollutants has banned ddt (van den berg 2009). However, there is yet no consensus on whether ddt exposure leads to deleterious health effects among humans (sharpe and stewart 2004, beard 2006). Regardless, there are well documented studies regarding its toxicity in birds and a variety of aquatic species (blus 2003, sparling 2010, beckvar and lotufo 2011). While further work is needed to verify them, these data indicate that alternative irs compounds may be more efficacious in controlling p. argentipes abundance . Research looking at effects on human health due to long - term exposure would still need to be addressed . Ultimately, these data suggest that with proper execution, irs could be an even more effective tool against vl in india but is not sufficient as a standalone given that p. argentipes is found outdoors in significant numbers . Bed nets, in particular, itns and long - lasting insecticide nets (llins), have been suggested as alternatives and/or complements to irs for the control of sand fly populations and vl . Itn / llins have been effective against other vector - borne diseases, including cutaneous leishmaniasis (lengeler 2004, kulkarni et al . 2007, wilson et al . 2014) but there are mixed results when it comes to sand flies in india . A trial using untreated nets found that the number of female, blood - fed p. argentipes declined by 85% following the introduction of the nets (picado et al . However, the authors of that study note that they lacked concurrent controls and thus, blood - feeding rates could potentially be attributed to changes in environmental factors (temperature, humidity, precipitation) or changes in host availability (i.e. An increase in domestic animals). Even so, other groups in bangladesh and nepal have found similar results (bern et al . The report by joshi et al . Investigated the usefulness of irs, llins, and evm to mitigate sand fly density in india, nepal, and bangladesh . When llins were in place, there was a village - wide reduction in p. argentipes numbers by 43.7% in india when measured 5 months post - intervention (joshi et al . A subsequent village - wide study in india noted a 25% decline in sand fly density (picado et al ., 16 clusters were enrolled, of those, 10 were used for sand fly capture studies . The reduction appeared to be at the community level and not displacement as p. argentipes abundance did not increase in cattle enclosures (picado et al . However, this observation does not exclude other environmental refuges, such as vegetation (poch 2011, poch 2012). As a part of the same study by picado et al . They also investigated rates of vl in these same villages and found no protective effect of llins on the rate of seroconversion (picado et al . Nearly 20,000 people were enrolled in the seroconversion study . Over the course of 24 months, incidence of l. donovani infection was found to be 5.4% in the intervention group and 5.5% in the control group while clinical vl rates were 0.38% and 0.40% in the intervention and control groups, respectively . One explanation for this result was that llins do not prevent outdoor transmission (picado et al . . An earlier study of 48 homes and mixed dwellings (homes shared with livestock) in bihar compared two types of llins to two types of untreated nets and looked at sand fly densities . At all time - points, up to 9 weeks post - treatment, there were no differences in p. argentipes abundance between the groups (dinesh et al . It should be noted, that during the course of the aforementioned studies, unplanned irs took place, and in both cases there was no noticeable effect on the number of sand flies captured in either study (dinesh et al . Furthermore, when the statistical models used to analyze the vl seroconversion data were adjusted for irs, no changes were found (picado et al . While the number of studies has been limited, the efficacy of bed nets in india to combat vl have not shown the same promise as they have in bangladesh (chowdhury et al . The potential reasons for this are many, including: study design, environmental factors (temperature, humidity, flooding etc . ), and susceptibility to insecticides - p. argentipes in bangladesh may be more susceptible, given their history of irs campaigns is more recent compared to india (bern et al . As breeding sites are yet unknown, and sand flies have been associated with vegetation in india, these remain confounding factors when accounting for differences in intervention strategies (poch et al . Non - compliance and net quality are another concern . In the studies by picado and joshi, even so, compliance with nets can be difficult as sand flies are much smaller than mosquitoes requiring finer mesh that people find stifling to sleep under in the heat of summer (ostyn et al . While in theory, the impregnated insecticide should provide repellent activities, thus allowing for a larger mesh, efficacy in the field has not been shown (picado et al . Moreover, 93% of 1,217 people surveyed in 20092011 in vl - endemic regions reported sleeping outside at some point during the hottest months of the year (perry et al . 2013). Assuming this trend holds true throughout other vl - afflicted areas, this is a serious hurdle to be faced in regards to the efficacy of irs, itns, and evm as none mitigate transmission that occurs outside of a person s dwelling typical dwellings in the regions of india afflicted by vl are often made of mud and thatch, or brick / plaster . 2014). In all cases, cracks and crevices where sand flies can rest and hide are prevalent . All home types are susceptible to habitation by p. argentipes, but thatched homes in particular cater to high densities (malaviya et al . Evm is a method little studied when compared to irs and itns as a means to control vl in india . Evm has generally meant alterations to the home or surrounding environment by means of covering or filling in cracks and crevices in walls and floors . A pilot study involving 15 homes saw a reduction in sand fly numbers using a mud and lime plaster mix to seal cracks in homes and cattle enclosures (kumar et al . The same well - controlled study comparing the efficacy of irs and itns in india, nepal, and bangladesh found a 42% decline in sand fly abundance five months after the walls of homes and cattle enclosures were plastered with a mud and lime mixture . The negative effect on sand fly populations may be due to the lime ph or limiting available moisture, thus, inhibiting breeding of p. argentipes, though breeding sites for these flies have not been confirmed (kumar et al . Indeed, a study in bangladesh saw no effect on sand fly populations when crevices were filled with mud (chowdhury et al . Importantly, another study in india found mud - plastered walls themselves to be risk factor for vl (ranjan et al . However, while mud / lime mixes are effective, this method requires continual maintenance and is costly compared to irs and itns (das et al . 2008). But like irs and itn, it is only effective against the sand flies that are inside homes; populations that reside in outdoor enclosures and vegetation would remain a source of vl . A newer, relatively untested, yet promising addition to the vector - control arsenal is the use of ftis . Several compounds including ivermectin, fipronil, and imidacloprid have been tested in rodents to cull p. papatasi, a vector for cutaneous leishmaniasis (mascari et al . 2013, derbali et al . 2014). In these reports, the agents have been effective at controlling adult and larval sand flies when fed on blood or feces from treated animals, respectively . In regards to p. argentipes, ivermectin and fipronil as well as diflubenzuron and eprinomectin were tested as ftis in rats (ingenloff et al . Fipronil at 150 ppm was shown to result in the quickest mortality and its effects had greater longevity than the other three compounds, even when they were used at greater concentrations . Cattle and other livestock act as a major source of blood meals for female sand flies . Blood - meal analysis in one study found that 39.3% of the flies tested had fed on some form of livestock (garlapati et al . So as to target p. argentipes that are feeding off of cattle and therefore, possibly residing outdoors, a controlled study involving cattle dosed with 0.5, 1.0, 2.0, and 4.0 mg / kg body weight of fipronil was conducted (poch et al . That investigation demonstrated that fipronil as an fti is effective, even at the lowest dose, at killing both adult and larval sand flies . The majority of adult sand flies had a 100% mortality rate within four days when fed on cattle up to 21 days post - treatment for all but the lowest dose (which had a 22% mortality rate at 21 days post - treatment). Nearly all of the flies that were fed on days 1, 3, and 5 post - treatment succumbed on the same day . For larval p. argentipes, the mean - time to death when fed on feces from cattle 1 or 3 days post - treatment was 4.5 days, larva fed on feces with the lowest dose (0.5 mg / kg) on day 1 post - treatment had 100% mortality within 5.5 days while the highest dose (4.0 mg / kg) was 4.0 days . Additionally, for all doses tested, 100% mortality of larval sand flies was achieved by day 15.5 when fed on dung collected 21 days post - treatment (poch et al . 2013). Given that this method is untested under field conditions, conclusions regarding its effect on p. argentipes abundance or vl transmission cannot be drawn at this time . While no effects to cattle health were noted in the aforementioned study, future work will need to be done to assess the health of cattle treated long - term . Moreover, at the lowest dose, fipronil can remain in the animal for up to 35 days but it is at concentration below international allowable limits (poch et al . Studies regarding the efficacy of fipronil as an fti are ongoing and if they prove positive, this method would be a valued addition to irs and itns as it would better address exophilic p. argentipes, making control measures more comprehensive . With humans as the only known reservoir for vl in india, rapid diagnosis and treatment would go a long way in controlling epidemics . However, if pkdl patients serve as a source of l. donovani, vector - control programs will also be needed to reach the goal of elimination . In this way, surveillance and reporting programs may need to be reevaluated as under - reporting of vl remains an issue within india (mubayi et al . Taking into account the diverse feeding and living habits of p. argentipes, it is likely that a combination of control measures will be necessary to eliminate vl in india . Switching insecticides may be beneficial with mounting evidence of increased resistance of p. argentipes to ddt coupled with potential health effects on humans, animals, and the environment (blus 2003, van den berg 2009, sparling 2010, huda et al . 2015). In the end, the efficacy of irs is dependent on organized government programs that coordinate the spraying schedule and train the technicians, which can be either a benefit or hindrance depending on the resources available to these groups (joshi et al . 2011a, huda et al . 2011). Even still, irs works well to curb adult p. argentipes abundance . Efforts should also be placed on identifying breeding grounds . To this end, irs could be focused and effective at eliminating both adult and larval flies . Insecticide treated nets have shown limited success in india despite reports of their usefulness in other countries in combating sand fly densities (dinesh et al . 2008, picado et al . While there is some level of personal protection from sand flies afforded to people who use bed nets, more work would need to be done to confirm that the cost of these initiatives is validated . Itns can often be more effective control measures against disease - transmitting vectors as proper use is in the hands of the affected persons instead of an outside program . That being said, non - compliance in india could be a problem as many people report sleeping outdoors during the hot summer months (perry et al . Long - lasting insecticide nets can have repellent and insecticidal properties for years, making them a relatively cheap supplement to irs, however, given the impoverished state of most of the afflicted regions, nets may still need to be provided by government or non - profit groups . The most expensive of the three most studied intervention strategies is evm (das et al . A mud and lime mixture to seal cracks and crevices in walls and floors has shown some negative effects on sand fly abundance, yet these studies have been limited (kumar et al . Given the cost and need for continual maintenance, evm is a strategy that may be best left for use on a case - by - case basis rather than a district - wide, vector - control measure . The state of dwellings in vl - endemic villages is a by - product of the greater issue of region - wide poverty, if that issue were better addressed, evm would be a moot point . This relatively new addition to vl vector - control has been effective at killing both adult and larval p. argentipes under controlled settings (inglenoff et al . There is no known resistance by sand flies to fipronil and the tactic of dosing cattle begins to address outdoor transmission of vl . However, ftis would still rely on proper usage by individuals and like itns, would carry similar benefits and risks of proper use . Similar to irs, ftis will result in the potential for some human, animal, and environmental exposure . Although, reported residue levels of fipronil in milk is below international standards (poch et al . Work would need to be done to monitor for any adverse effects to humans, animals, and the environment, following long - term use of ftis to treat cattle . Should ftis prove effective under field conditions, they would begin to fill the gap in available treatments that target sand flies outside of homes and cattle enclosures . Similar to fti and irs is the use of natural botanicals as insecticides in place of synthetic chemicals . One study demonstrated that a 2% concentration of neem oil mixed with either coconut or mustard oil was effective at repelling sand flies from human subjects in india (sharma and dhiman 1993). While no work has been done regarding the use of potential biochemicals as insecticides in india, studies conducted in other countries on different sand fly species showed that various plant - derived compounds were effective at killing adult and larval stages of the insects (dinesh et al . Although many of the tested biochemicals only had a 50% mortality rate, this area of research is still relatively unexplored and may be a useful alternative to synthetic insecticides . With millions of homes that would require intervention, vector - control programs need to balance rapid efficacy with long - term cost to ensure that if a treatment measure is terminated the country does not experience the rapid resurgence as occurred at the end of ddt irs in the 1970s . One year of irs costs on average, $5.90 per household which is more than llins ($4.50/house / year) but less than evm ($8.70/house / year), and those costs have likely only gone up since that report was conducted (das et al . There is a real need for thorough cost - effectiveness studies using combinations of control measures and disease prevalence / sand fly population scenarios . Research documenting the direct and indirect costs, estimated expenditures for effective training and supervision of staff, as well as equipment maintenance would help to make informed decisions on the best use of resources for this endeavor . Moreover, modelling has been done to estimate what percent of the sand fly population would need to be culled in order for vl to be eliminated . In that report, if sand fly life expectancy was reduced (eg via irs, itns, or ftis), there would need to be a 67% decline in abundance in order for vl to be eliminated (stauch et al . Only the fti report and some of the irs studies meet that threshold (chowdhury et al . 2013). Further focusing of efforts and resources could be done by implementing remote sensing and gis data . Preliminary work done in brazil and india modeling both climate and land data has been used to try and identify vector habitat as well as regions that may be the focus of vl outbreaks due to various weather and geo - environmental factors (bhunia et al . While standardization of analysis methods and data acquisition are still needed, gis and remote sensing could greatly help to target high - risk areas before an outbreak occurs . Vector - control and vl - transmission studies should begin to focus on combination intervention strategies as well as enhanced public education programs . If the public are unaware of the risk, the symptoms, and treatments for vl, control programs will struggle (singh et al . 2010, malaviya et al 2013). From what is known at this point, irs is efficacious in quickly curbing adult sand fly populations, itns may have some benefit for personal protection and might also be useful if irs is not performed correctly 100% of the time . Lastly, fti is deleterious to both adult and larval p. argentipes and has the potential to disrupt populations of exophilic sand flies . What has been lacking outside of the one study by picado et al . (2010b), are studies that attempt to link a decline in sand fly abundance with a subsequent drop in cases of vl . A broader review of 84 studies in 22 countries involving all forms of human leishmaniasis found that only 35% measured leishmania infection as an outcome (stockdale and newton 2013). This is an issue afflicting many vector / disease - control studies (wilson et al . Future studies should concentrate their efforts on making this connection . While a decline in sand fly numbers should in theory correlate to a decline in vl infections, this link has yet to be demonstrated clearly by the research performed to date . Future work investigating the connection between reduced sand fly abundance and vl infection rates as well as shifts in parasite availability and sand fly feeding behaviors should be a priority . In combination, these tactics may work to bring sand fly numbers down quickly in the short - term, allowing for vl - patient identification and treatment . Currently, the state of bihar is beginning tests for use of synthetic pyrethroids in place of ddt for irs . The national roadmap laid out by the government of india emphasizes case detection and treatment as well as surveillance for pkdl . While they state the need for integrated vector management, irs remains the mainstay for vector control (nvbdp 2014). Ongoing work with sand fly breeding site identification, and implementation of more novel technologies like gis and remote sensing the global initiative to eliminate vl in all endemic regions has been strong, however a coordinated effort between groups employing the various control tactics will be vital to see the elimination goal met . Once caseloads have been brought to 1:10,000, the use of irs, itns, ftis and all of the aforementioned technologies and trainings should be evaluated to determine which would be best for continued control measures.
Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process . Also, it has been reported to be associated with the onset of various cancers . Acute inflammation is the exudation of plasma proteins and fluids and the emigration of leukocytes . Chronic inflammation is inflammation of prolonged duration in which tissue destruction, active inflammation, and attempts at repair are proceeding simultaneously . Current approaches to overcome the inflammation include the use of nonsteroidal anti - inflammatory drugs (nsaids), immune selective anti - inflammatory derivatives, selective glucocorticoid receptor agonist, resolvins / protectins, and tnf inhibitors . Although drug treatment has been improved to some extent yet, it is still a challenge for the pharmaceutical chemists to explore the more effective and potent therapeutic agents to treat inflammation and reduce the signs and symptoms of acute inflammation and chronic inflammatory diseases . Thus, it is well evident from the literature and numerous studies that there is requirement for appropriate modification of the molecules to attenuate the effective potent therapeutic agents for the treatment of inflammation and also ensure that the host immune defense against infection is not impaired . The chemistry of chalcones has generated an intensive scientific interest due to their wide spectrum of biological properties such as antibacterial, antifungal, insecticidal, anaesthetic, anti - inflammatory, analgesic, and ulcerogenic properties . Some substituted chalcones and their derivatives, including heterocyclic analogues, have been reported to possess some interesting biological properties [47] which are detrimental to the growth of microbes tubercle bacilli, malarial parasites, acrus, schistosoma, and intestinal worms . Some of the compounds are also claimed to be toxic for animals and insect and are also reported to exhibit inhibitory action on several enzymes, fungi, and herbaceous plants . 1-acetyl-3,5-diaryl-4,5-dihydro(1h)pyrazoles have previously been reported to exhibit a variety of biological activities such as inhibitors of monoamine oxidases, swine kidney oxidase and bovine serum amine oxidases [8, 9], anticancer via binding to p - glycoprotein [10, 11], anti - helicobacter pylori, antiviral, antibacterial via inhibition of fabh, and anti - inflammatory [15, 16]. In the present paper, we describe the synthesis of some substituted n - acetylpyrazoles (4a j) and their corresponding o - propargylate analogs (5a j) as potent anti - inflammatory agents . Ir spectra are recorded on perkin elmer rx1 spectrophotometer using kbr pellets and are expressed in cm . The hnmr spectra were recorded on bruker 300 mhz spectrometer in (cdcl3) using tms as an internal reference and chemical shifts were measured in ppm . The progress of the reaction was monitored by tlc using 0.2 mm thickness aluminium sheet precoated with silica gel merck 60f 254 and visualization was done using iodine / uv lamp for detection of the spots . The solvent was removed under reduced pressure using buchi rotary evaporator . Concentrated sulfuric acid (2.5 ml) was added slowly with stirring into a solution of various substituted benzaldehydes (1 mmol) and acetophenones (1 mmol) in glacial acetic acid . The stirring was continued for 48 h keeping the temperature of reaction mixture below 2025c . After the completion, the reaction mixture was poured onto ice cold water (100 ml) and extracted thrice with ethylacetate . The organic layer was pooled and concentrated under reduced pressure to get crude 1,3-diarylpropenones which was recrystallized from ethanol and further used for the next step . A mixture of various 1,3-diarylpropenones (1 mmol) in acetic acid and hydrazine monohydrate 80% (2 mmol) was refluxed for 4 hours . The mixture was then poured onto ice cold water (50 ml) to get crude pyrazole analogs (4a j), which were purified by crystallization with ethanol to afford pure title compounds . Propargyl bromide (1.5 mmol) was added to the stirred mixture of pyrazole (1 mmol) and k2co3 (1.5 mmol) in dry dmf . The stirring was continued under anhydrous condition for 24 h keeping the temperature of reaction mixture below 5c . After completion of reaction, as evident by tlc, the reaction mixture was poured onto ice cold water . Precipitates of o - propargylated - n - acetylpyrazole were then filtered out and dried . Molecular formula: c18h18n2o3; yield: 80%; ir (kbr, cm): 1665 (c = o), 1630 (c = n), 1590 (aromatic c = c), 1265 (c o), 1125 (c n); hnmr (300 mhz, cdcl3, tms = 0): 1.82.0 (d, 2h, ch2), 2.1 (s, 3h, ch3), 3.7 (s, 3h, och3), 4.7 (t, 1h, ch), 5.1 (s, 1h, oh), 6.56.7 (m, 3h, ar c nmr (300 mhz, cdcl3): 168.3, 151.2, 143, 136.5, 134, 131.2, 128.4, 120.5, 115.7, 112.4, 59.3, 56.4, 39.2, 23.0 . Molecular formula: c18h17brn2o3; yield: 75%; ir (kbr, cm): 1660 (c = o), 1630 (c = n), 1590 (aromatic c = c), 1130 (c n); hnmr (300 mhz, cdcl3, tms = 0): 1.92.1 (d, 2h, ch2), 2.1 (s, 3h, ch3), 3.6 (s, 3h, och3), 4.8 (t, 1h, ch), 5.0 (s, 1h, oh), 6.56.7 (m, 3h, ar c nmr (300 mhz, cdcl3): 168.5, 151.2, 143.4, 137.2, 133, 131.4, 125.4, 120.2, 116.3, 112.4, 59.3, 56.2, 39.4, 23.2 . Molecular formula: c18h17cln2o3; yield: 70%; ir (kbr, cm): 1665 (c = o), 1630 (c = n), 1590 (aromatic c = c), 1265 (c o), 1125 (c n); hnmr (300 mhz, cdcl3, tms = 0): 1.82.0 (d, 2h, ch2), 2.0 (s, 3h, ch3), 3.7 (s, 3h, och3), 4.8 (t, 1h, ch), 5.0 (s, 1h, oh), 6.56.7 (m, 3h, ar h), 7.37.6 (m, 4h, ar h). Molecular formula: c19h20n2o4; yield: 75%; ir (kbr, cm): 1665 (c = o), 1630 (c = n), 1590 (aromatic c = c), 1265 (c o), 1130 (c n); hnmr (300 mhz, cdcl3, tms = 0): 1.82.0 (d, 2h, ch2), 2.1 (s, 3h, ch3), 3.6 (s, 6h, och3), 4.7 (t, 1h, ch), 5.0 (s, 1h, oh), 6.56.8 (m, 5h, ar h), 7.47.6 (m, 2h, ar h). Molecular formula: c18h17n3o5; yield: 70%; ir (kbr, cm): 1660 (c = o), 1635 (c = n), 1590 (aromatic c = c), 1125 (c n); hnmr (300 mhz, cdcl3, tms = 0): 1.92.1 (d, 2h, ch2), 2.2 (s, 3h, ch3), 3.7 (s, 3h, och3), 4.8 (t, 1h, ch), 5.1 (s, 1h, oh), 6.56.7 (m, 3h, ar c nmr (300 mhz, cdcl3): 168.2, 151.4, 143.2, 140.2, 137, 130.2, 121.4, 120.5, 116.5, 112.4, 59, 56.4, 39.1, 23.2 . Molecular formula: c21h24n2o6; yield: 65%; ir (kbr, cm): 1665 (c = o), 1635 (c = n), 1590 (aromatic c = c), 1270 (c o), 1130 (c n); hnmr (300 mhz, cdcl3, tms = 0): 1.82.0 (d, 2h, ch2), 2.0 (s, 3h, ch3), 3.6 (s, 12h, och3), 4.7 (t, 1h, ch), 4.9 (s, 1h, oh), 6.56.6 (m, 5h, ar molecular formula: c17h16n2o2; yield: 65%; ir (kbr, cm): 1660 (c = o), 1635 (c = n), 1595 (aromatic c = c), 1130 (c n); hnmr (300 mhz, cdcl3, tms = 0): 1.82.0 (d, 2h, ch2), 2.1 (s, 3h, ch3), 4.8 (t, 1h, ch), 4.9 (s, 1h, oh), 6.77.3 (m, 9h, ar c nmr (300 mhz, cdcl3): 168, 160.4, 151.2, 143.0, 130.2, 128.2, 127, 126.4, 116.4, 58.1, 39.4, 23.4 . Molecular formula: c18h18n2o3; yield: 70%; ir (kbr, cm): 1665 (c = o), 1635 (c = n), 1590 (aromatic c = c), 1130 (c n); hnmr (300 mhz, cdcl3, tms = 0): 1.82.0 (d, 2h, ch2), 2.0 (s, 3h, ch3), 3.7 (s, 3h, och3), 4.9 (t, 1h, ch), 5.0 (s, 1h, oh), 6.77.3 (m, 8h, ar molecular formula: c19h20n2o4; yield: 65%; ir (kbr, cm): 1660 (c = o), 1635 (c = n), 1595 (aromatic c = c), 1270 (c o), 1130 (c n); hnmr (300 mhz, cdcl3, tms = 0): 1.82.0 (d, 2h, ch2), 2.0 (s, 3h, ch3), 3.6 (s, 6h, och3), 4.9 (t, 1h, ch), 5.0 (s, 1h, oh), 6.67.4 (m, 7h, ar molecular formula: c20h22n2o5; yield: 65%; ir (kbr, cm): 1665 (c = o), 1635 (c = n), 1590 (aromatic c = c), 1270 (c o), 1135 (c n); hnmr (300 mhz, cdcl3, tms = 0): 1.92.1 (d, 2h, ch2), 2.1 (s, 3h, ch3), 3.7 (s, 9h, och3), 4.8 (t, 1h, ch), 5.0 (s, 1h, oh), 6.46.7 (m, 4h, ar molecular formula: c21h20n2o3; yield: 75%; ir (kbr, cm): 1665 (c = o), 1630 (c = n), 1590 (aromatic c = c), 1265 (c o), 1125 (c n); hnmr (300 mhz, cdcl3, tms = 0): 1.82.0 (d, 2h, ch2), 2.1 (s, 3h, ch3), 2.5 (s, 1h, ch), 3.7 (s, 3h, och3), 4.5 (s, 2h, ch2), 4.8 (t, 1h, ch), 6.77.5 (m, 8h, ar molecular formula: c21h19brn2o3; yield: 70%; ir (kbr, cm): 1660 (c = o), 1635 (c = n), 1595 (aromatic c = c), 1265 (c o), 1130 (c n); hnmr (300 mhz, cdcl3, tms = 0): 1.92.1 (d, 2h, ch2), 2.1 (s, 3h, ch3), 2.6 (s, 1h, ch), 3.6 (s, 3h, och3), 4.5 (s, 2h, ch2), 4.8 (t, 1h, ch), 6.67.5 (m, 7h, ar molecular formula: c21h19cln2o3; yield: 60%; ir (kbr, cm): 1665 (c = o), 1635 (c = n), 1595 (aromatic c = c), 1265 (c o), 1125 (c n); hnmr (300 mhz, cdcl3, tms = 0): 1.82.0 (d, 2h, ch2), 2.0 (s, 3h, ch3), 2.5 (s, 1h, ch), 3.6 (s, 3h, och3), 4.6 (s, 2h, ch2), 4.8 (t, 1h, ch), 6.67.5 (m, 7h, ar molecular formula: c22h22n2o4; yield: 70%; ir (kbr, cm): 1660 n), 1590 (aromatic c = c), 1265 (c o), 1130 (c n); hnmr (300 mhz, cdcl3, tms = 0): 1.82.0 (d, 2h, ch2), 2.0 (s, 3h, ch3), 2.6 (s, 1h, ch), 3.7 (s, 6h, och3), 4.6 (s, 2h, ch2), 4.8 (t, 1h, ch), 6.67.5 (m, 7h, ar molecular formula: c21h19n3o5; yield: 65%; ir (kbr, cm): 1660 (c = o), 1635 (c = n), 1590 (aromatic c = c), 1125 (c n); hnmr (300 mhz, cdcl3, tms = 0): 1.82.0 (d, 2h, ch2), 2.1 (s, 3h, ch3), 2.5 (s, 1h, ch), 3.7 (s, 3h, och3), 4.6 (s, 2h, ch2), 4.9 (t, 1h, ch), 6.66.7 (m, 3h, ar c nmr (300 mhz, cdcl3): 168.2, 151.4, 150.5, 149.6, 147.2, 140.1, 136.2, 130.2, 121.3, 120.2, 115.4, 112, 78.1, 59, 57.2, 39.1, 23.1 . Molecular formula: c24h26n2o6; yield: 60%; ir (kbr, cm): 1665 (c = o), 1635 (c = n), 1590 (aromatic c = c), 1270 (c o), 1130 (c n); hnmr (300 mhz, cdcl3, tms = 0): 1.82.0 (d, 2h, ch2), 2.0 (s, 3h, ch3), 2.5 (s, 1h, ch), 3.7 (s, 12h, och3), 4.6 (s, 2h, ch2), 4.9 (t, 1h, ch), 6.66.7 (m, 5h, ar h). Molecular formula: c20h18n2o2; yield: 65%; ir (kbr, cm): 1660 (c = o), 1635 (c = n), 1595 (aromatic c = c), 1130 (c n); hnmr (300 mhz, cdcl3, tms = 0): 1.82.0 (d, 2h, ch2), 2.0 (s, 3h, ch3), 2.5 (s, 1h, ch), 4.5 (s, 2h, ch2), 4.9 (t, 1h, ch), 6.87.4 (m, 9h, ar molecular formula: c21h20n2o3; yield: 70%; ir (kbr, cm): 1665 (c = o), 1635 (c = n), 1590 (aromatic c = c), 1130 (c n); hnmr (300 mhz, cdcl3, tms = 0): 1.82.0 (d, 2h, ch2), 2.1 (s, 3h, ch3), 2.4 (s, 1h, ch), 3.7 (s, 3h, och3), 4.5 (s, 2h, ch2), 4.8 (t, 1h, ch), 6.77.5 (m, 8h, ar molecular formula: c22h22n2o4; yield: 65%; ir (kbr, cm): 1660 (c = o), 1635 (c = n), 1595 (aromatic c = c), 1270 (c o), 1130 (c n); hnmr (300 mhz, cdcl3, tms = 0): 1.82.0 (d, 2h, ch2), 2.0 (s, 3h, ch3), 2.5 (s, 1h, ch), 3.7 (s, 6h, och3), 4.6 (s, 2h, ch2), 4.9 (t, 1h, ch), 6.66.8 (m, 5h, ar h), 7.47.5 (m, 2h, ar h). Molecular formula: c23h24n2o5; yield: 60%; ir (kbr, cm): 1660 (c = o), 1635 (c = n), 1595 (aromatic c = c), 1270 (c o), 1135 (c n); hnmr (300 mhz, cdcl3, tms = 0): 1.82.0 (d, 2h, ch2), 2.0 (s, 3h, ch3), 2.6 (s, 1h, ch), 3.7 (s, 9h, och3), 4.6 (s, 2h, ch2), 4.9 (t, 1h, ch), 6.46.8 (m, 4h, ar adult wistar rats of either sex weighing within 150180 g were used throughout the work . The selected animals were kept under standard conditions of light and temperature with free access to food and water . All experimental procedures were carried out in strict accordance with the guidelines prescribed by the committee for the purpose of control and supervisions on experimentation on animals (cpcsea) and were approved by the institutional animal ethics committee of guru gobind singh college of pharmacy, yamuna nagar, haryana (regn . The paw edema was induced by subplantar injection using carrageenan and the increased foot volumes were measured in a plethysmograph by water displacement . The anti - inflammatory activity was carried out by carrageenan - induced paw edema test . Test compounds (4a i and 5a i) and standard drug indomethacin were suspended in 0.5% w / v of sodium carboxyl methylcellulose (cmc), which was used as a vehicle for the control group . The rats were dosed with test drugs orally (100 mg / kg body weight) including the reference standard (10 mg / kg body weight) with help of oral catheter . After 30 minutes of drug administration, 0.1 ml of 1% w / v carrageenan solution in saline (0.9%) was injected in the subplantar region of the left hind paw of control as well as standard and test groups . The increased volume of paw edema (in ml) was determined immediately after injection of carrageenan and 4 h later . The difference between these two values was taken as edema volume . The percentage protection against inflammation was calculated as follows: (1 vd)/vc 100, where vc is the increase in paw volume in the absence of the test compound (control) and vd is the increase of paw volume after injection of the test compound . Significant differences between the control and treated groups were obtained using dunnett's test with p value <0.05 . Claisen - schmidt condensation of substituted aryl ketones (1) and benzaldehydes (2) in glacial acetic acid and h2so4 yielded 1,3-diarylpropenones (scheme 1, 3a j; 7090%) which on further treatment with hydrazine hydrate in presence of acetic acid under reflux afforded n - acetylpyrazolines (scheme 1; 4a j; 6580%). These pyrazoles analogs were further treated with propargyl bromide and k2co3 in dry dmf affords title o - propargylated - n - acetylpyrazole (scheme 1, 5a j, 6075%) after recrystallization in ethanol (schemes 1(a) and 1(b)). The purity and structures of all the synthesized compounds have been elucidated on the basis of their spectral data including ir and hnmr . The in vivo anti - inflammatory activity was studied using carrageenan - induced rat paw edema model . The anti - inflammatory activity of all the prepared analogs along with standard drug indomethacin is depicted in table 1 . The test and standard drug produced significant inhibition of paw edema as compared to control . Out of all prepared analogs, 4e, 4 g, 5e, 4b, and 4a exhibit a comparable activity to that of standard drug indomethacin . In summary, the present investigation describes synthesis and anti - inflammatory potential of some o - propargylated - n - acetylpyrazole (5a j) along with n - acetylpyrazole analogs (4a j) derived from 1,3-diarylpropenones (3a j), which was prepared by condensation of various substituted benzaldehydes and acetophenones . The prepared compounds were characterized by suitable methods such as spectroscopic evaluation like ir, hnmr, and c nmr . The prepared analogs show remarkable reduction in inflammation, after 4 h of carrageenan administration as compared to control . Out of all prepared analogs, five compounds (4e, 4 g, 5e, 4b, and 4a) delivered by oral route exhibit a comparable anti - inflammatory activity to that shown with indomethacin, suggesting a good oral bioavailability and a potent effect on inflammation . The promising activity of these compounds related to their structures can also be useful for establishing the structure activity relationship studies . Sar study revealed that nature of substituent(s) particularly electron withdrawing groups on aromatic ring greatly affects the anti - inflammatory activity . These novel compounds will be further examined upon an oral repeated oral delivery for their toxicity to verify their potential therapeutic effect.
Neoadjuvant therapy followed by surgical resection has been attempted to improve the survival of patients with locally - advanced non - small cell lung cancer (nsclc). Proven strategies to select patients who would obtain a survival benefit from surgery after neoadjuvant therapy have not been established, except for the response evaluation criteria in solid tumors (recist) and re - staging . Besides the recist, several studies have assessed the prognostic significance of a reduction in the maximum standardized uptake value (suvmax) on f - fluorodeoxyglucose positron emission tomography (pet) in monitoring the response to treatment . In addition, we hypothesized that a reduction in the tumor volume would show a stronger correlation with the outcomes than the diameter - based recist criteria . Therefore, this study was designed to evaluate the predictive significance of the variables obtained by diagnostic imaging in patients who underwent surgical resection after neoadjuvant therapy for nsclc, and included the variables of the recist, the volume - based assessment and the suvmax - based assessment this study was conducted with the approval of the institutional review board of nagoya university hospital . Between december 2006 and june 2012, 41 consecutive patients were diagnosed with clinical stage ii and iii nsclc and underwent pulmonary resection following neoadjuvant therapy . Five patients with r2 resection, two patients who did not undergo a post - neoadjuvant treatment pet scan and one patient who died within the first 30 days after the operation were excluded from the analysis . All patients had histological or cytological confirmation of nsclc before they received neoadjuvant therapy . As neoadjuvant therapy, 24 patients received one or two cycles of platinum - based chemotherapy combined with 4064 gy of concurrent radiotherapy . Five patients received one to six cycles of platinum - based chemotherapy and four patients were given radiation of 4060 gy . Pet scans were performed with a pet - ct scanner (biography 16; siemens medical solutions, erlangen, germany). The fdg dose was 3.7 mbq / kg (body weight <60 kg) or 4.07 mbq / kg (body weight 60 kg). The maximum value in a region of interest around the pulmonary lesion (not the lymph nodes) was defined as the suvmax . Post - suvmax was defined as the post - neoadjuvant treatment suvmax . A partial response (pr) and stable disease (sd) volume response (vr) was defined as a more than 50% reduction of the tumor volume (tumor volume = a b c 0.52, using the three orthogonal crystal axes a, b and c from the data obtained by ct scans). The dfs was defined as the time from the pretreatment histological diagnosis to relapse or death due to any cause . The os was defined as the time from pretreatment histological diagnosis to death due to any cause . The seventh edition of the tumor - node - metastasis classification was applied in this cohort, and the pathological diagnosis of the tumor was made based on the world health organization classification . A pcr was defined as no evidence of residual vital tumor cells around the primary pulmonary lesion . The correlations between the variables and the pcr of the primary tumor, the dfs and the os were analyzed . A logistic regression analysis was performed to evaluate the diagnostic accuracy of the data for the imaging tests used to assess whether there was a pcr . The kaplan meier method was used to estimate the dfs and os, and the log - rank test was used to compare the survival curves . We performed the multivariate cox regression analyses to estimate hazard ratios (hrs) and 95% confidence interval (ci) for dfs and os . As the variables, we select vr (and non - vr), post - suvmax as well as the variables which were considered to be important based on the current evidence . Consequently, the following variables were included: neoadjuvant therapy, vr (and non - vr), post - suvmax and clinical stage after neoadjuvant therapy . All analyses were conducted using the jmp software program (version 8.0.1, sas institute inc ., cary, nc, usa)., 12 tumors were classified to have a pr and 21 tumors were classified as sd . In the volume - based assessment, 21 tumors showed a vr, and 12 tumors did not . In the suvmax - based assessment, 25 tumors [20 (83%) tumors with chemoradiotherapy, 2 (40%) tumors with chemotherapy and 3 (75%) tumors with radiotherapy] had a post - suvmax 7.5 and eight tumors had a post - suvmax> 7.5 . A pcr around the primary pulmonary lesion was observed in eight patients (24%). The characteristics of patients who underwent pulmonary resection after neoadjuvant therapy recist: response evaluation criteria in solid tumors; suvmax: maximum standardized uptake value; pcr: pathological complete response with a median follow - up period of 42 months, the estimated three - year dfs and os were 70% and 78%, respectively . Figure 1 shows the dfs and os curves based on the recist, vr and post - suvmax . Not the recist, but the vr and a post - suvmax were significant variables predicting the dfs . In the multivariate cox regression analysis, only the non - vr and post - suvmax> 7.5 were significant variables predicting the dfs (hr 3.9, 95% ci 1.015.8, p = 0.0422 and hr 6.1, 95% ci 1.525.5, p = 0.0127, respectively), but either was not for os . The post - suvmax as a continuous valuable had a significant correlation with a pcr (p = 0.0067), while the recist and non - vr did not . The post - suvmax of the eight nodules with a pcr ranged from 1.9 to 4.2 . The disease - free survival and overall survival curves categorized based on the recist (a, b), the tumor volume response (vr: a more than 50% reduction in the tumor volume) (c, d) and the metabolic response (with the cut - off value of the post - suvmax set at 7.5) (e, f) in this study, the results indicated that a post - suvmax> 7.5 and a non - vr following neoadjuvant therapy can be valid variables to predict the dfs . These results should be considered, especially in patients considered to be at high risk for invasive surgery . The pathological response after neoadjuvant therapy in resectable nsclc was reported to have the potential to be a surrogate endpoint for predicting the survival . Our results indicate that the post - suvmax has a significant correlation with the pcr, while volume - based assessment does not . There were a number of possible limitations associated with our retrospective study which should be recognized . First, the small number of patients and the short follow - up period should be noted . Moreover, the number and outcomes of patients who were rejected for surgery were not clear . Third, we used a suvmax of 7.5, which was an integral multiplication of the value of 2.5 widely used to discriminate between benign and malignant diseases . One study proposed the use of cut - off values for the post - treatment peak standardized uptake value of 7.0 for the clinical decision - making after chemoradiotherapy . However, the exact significance of the cut - off value for the post - suvmax remains vague due to the shortage of evidence . In conclusion, the post - treatment suvmax can be a valid alternative variable that can be used to predict the effect of neoadjuvant therapy and the survival of patients with stage ii and iii nsclc
Provisional crown and fixed partial prosthesis today represent an important element of fixed prosthetic treatment.1,2 these prosthesis are made with the aim of supporting the teeth for which preparation is being made, observing prognosis, and giving the patient function, phonation, aesthetic appearance and tissue compatibility until permanent restoration can be administered.35 in order for provisional restorations to be successful, they have to be resistant to forces inside the mouth . Many fibers of various kinds have been tested in recent years in order to increase the fracture resistance of provisional crowns . While there have been many studies showing that these fibers increase the resistance of acrylics, there are none on their impact on surface roughness . The surface roughness of dental materials is the main influence on plaque formation, discoloration, abrasion and aesthetic appearance . Many bacteria are able to adhere to hard surfaces in the oral cavity.6,7 the roughness of intra - oral hard surfaces and free energy have a significant effect on primary adhesion and oral micro - organism retention . A surface roughness of 0.3 mm can be felt by the tongue, thus having a negative impact on patient comfort.8 in vitro studies regarding surface roughness for bacterial plaque retention have shown that an average surface roughness above 0.2 mm in fixed restorations increases the level of bacterial retention.810 the aim of this study was to investigate surface roughness in provisional crown resins, after polishing, reinforced with different concentrations of glass fibers . Generally used and commercially available autopolymerizing resin was used in this study for provisional crown and fixed partial restoration (dentalon plus, heraeus, kulzer gmbh, wehrheim, germany). The manufacturers recommended powder / liquid ratio was 2 grams of powder to 1 ml of liquid . Four different glass fiber groups in different concentrations were established, according to the powder to liquid mixture; group a (no fiber), group b (0.5%), group c (1%) and group d (2%), each group containing 12 disk specimens . A teflon mold was made in order to produce disk - shaped specimens (10 mm 2 mm). The unprocessed glass fibers were then cut to a length of 3 mm and kept in a predetermined amount of monomer . The provisional crown acrylic was mixed in the light of the manufacturer s recommendations and added to the glass fiber mixture in predetermined quantities . The provisional resin paste added to glass fiber in desired concentrations was kneaded manually for 40 seconds and placed in the mold . This was then placed in a hydraulic press (rucher phi, birmingham, uk) and pressure slowly applied in such a way as to permit excess resin to escape . Pressure of 20 psi (140 kpa) was applied for 5 minutes . Once polymerization had been completed, the specimens were removed from the mold and analyzed with regard to air bubbles and size . All specimens were abraded for 10 seconds in a wet environment by means of a 300 rpm polishing device (beuhler, meta serv, dusseldorf, germany) using 600 grit sandpaper . All specimens were then polished for 15 seconds using a polishing machine with pumice mixed at a level of 2 g/2 ml . Finally, diamond polishing paste was applied using a polishing device at 15,000 rpm . All specimens were washed in distilled water and left in an ultrasonic bath for 10 minutes . An average surface roughness value (ra) from 4 randomly selected points on the surface was calculated using a profilometer (mitutoyo surf test 201, japan). A 7.5 mm field was scanned at every measurement using the profilometer with a study gap of 250 m . Forty - eight pieces of data (12 4) were obtained from each group, giving a total of 192 (table 1). In addition to profilometric analysis, photographic images, 2 from each group, were obtained using a scanning electron microscope (sem) at an original magnification of 400 in order to analyze post - polishing surface roughness . All analyses were performed using the statistical package for scientists (sigmastat) windows version 3.10b . The averages and standard deviation values obtained as a result of the surface roughness test are shown in table 1 . A significant difference was determined among the surface roughness values of provisional crown resins to which different concentrations of fiber had been added (p<.001) (table 1). Tukey s test was then used to perform paired comparisons of the data between the different groups, and a significant difference was found between group a (no fiber) (figure 1) and the other groups, between group b (0.5%) and group d (2%) and between group c (1%) and group d. on the other hand, there was no significant difference between group b and group c. in other words, no statistically significant difference was determined between the surface roughness of provisional crown resins reinforced with glass fibers at concentrations of 0.5% or 1% . In addition, compared with other groups, provisional crown resins reinforced with a 2% glass fiber concentration had a significantly greater surface roughness (figure 2). The sem images obtained clearly show the glass fiber particles in the group b, c and d provisional crown resins (figures 3, 4 and 5). The surface roughness in group a, with an average ra value of 0.309, was statistically significantly different to that of the other groups . Sem images showed that provisional crown resins with no additional fiber had the lowest surface roughness, while provisional crown resins with a 2% concentration had a very different level of fiber coverage . The absence of any significant difference between groups b and c, with 0.5% and 1% concentrations, respectively, according to measurements performed using tukey s test, was confirmed by the sem images . Surface quality is an important factor affecting the state of dental restorations in the oral cavity in a number of regards . Studies have shown that decreased roughness on intraoral surfaces reduces plaque formation.9,11,12 various fibers have been used to strengthen the polymethylmetacrylate (pmma) resins used in dentistry.8,13,14 although carbon fibers increase the abrasion, tensile and transverse strength, bending and elasticity modulus of pmma resins, they are unpleasant in color . This makes them unpopular.1517 polyethylene fibers increase pmma resins tensile and flexural strength and young s modulus . In addition, they are not dark in color in the way carbon fibers are, but it may not be practical to roughen them in the dental surgery . However, glass fibers are invisible in pmma resin . Glass fibers also have good biocompatibility, possess an appropriate capacity for bonding to the tooth structure and other resins, and are easily manipulated in the clinic or laboratory.18,19 they have thus become very popular in recent years . Although the effect of glass fibers on provisional crown resistance is known, the same cannot be said for their effect on surface roughness . Our study evaluated the effect of glass fibers added in different concentrations to provisional crown resins on surface roughness using polymetric analysis and sem . Fiber is installed in the resin in three different ways in dentistry: chopped, longitudinal and woven form.14 vallittu et al20 reported that fibers installed longitudinally in the resin changed place with the pressure applied when the mold was placed in a hydraulic press and that their parallelism was impaired . Since the woven form resembles cloth, its contact with the acrylic is problematic and problems with bonding to the acrylic arise.14,21 since the negative features observed in the other forms are not seen in the chopped form, this was employed in our study . The ra values determined as a result of the surface roughness were: group a (no fiber) 0.309 ra, group b (0.5%) 0.2 ra, group c (1%) 0.990 ra, and group d (2%) 1.73 ra . A significant difference was determined among the groups to which different concentrations of fiber were added (p<.001). No statistically significant difference was determined between the surface roughness values of provisional crown resins reinforced with different concentrations, 0.5% and 1%, of glass fiber in paired comparison tests . The reinforcement of provisional crown acrylics with 1% glass fiber increases mechanical properties more than reinforcement with 0.5% glass fiber.14,21 however, no significant difference in surface roughness in provisional crown acrylics reinforced with 0.5% and 1% concentrations of glass fiber was determined in our study . Therefore, in terms of surface roughness, we recommend the reinforcement of provisional crown acrylics with 0.5% and 1% concentrations of glass fiber . In this study, group a specimens to which no fiber was added exhibited smoother surfaces than specimens from groups b, c and d to which fiber had been added, and the sem images obtained strengthened this conclusion (figures 6,7,8 and 9). According to an in vivo study by quirynen et al,22 clinically acceptable roughness values in the oral environment after hard surfaces have been polished should not exceed 0.2 m . Wietnam and eames23 reported plaque accumulation on the surfaces of composite specimens with a surface roughness of 0.71.44 m . This study shows that 2% glass fiber added to provisional crown resins leads to a level of surface roughness that will increase plaque accumulation (ra=1.734). Glass fibers, which make a positive contribution to the physical characteristics of provisional crowns, have a negative effect on surface roughness and on plaque accumulation . The sem images obtained show glass fiber particles on the homogeneous structure of the provisional crown methacrylate resin acrylic material . The glass fiber concentration may affect distribution, acrylic arrangement or materials natural chemistry surface roughness . Borchers et al24 reported that when long - term provisional crowns are employed the importance of preventing plaque accumulation rises and far more effective polishing systems are essential for provisional restorations . It was determined within the parameters of this study that the reinforcement of methyl methacrylate with different concentrations of glass fiber increased surface roughness . The standardization of this study according to an in vitro protocol and its inability to ideally reflect clinical conditions are its main disadvantages . The polishing process in a clinical environment or in the laboratory is not as successful as the polishing process in this study . The first reason for this is that provisional crowns have concave and convex surfaces, for which reason polishing can never be performed on perfectly smooth surfaces as in this study . The second reason is that it is difficult to adjust the recommended speed and power of polishing materials in the surgery . In addition, effectiveness of polishing under clinical conditions is to a large extent dependent on the operator doing the polishing . The following conclusions emerged from within the parameters of this study: the reinforcement of provisional crown and fixed partial denture resin with glass fibers increases surface roughness . Paired comparisons between groups revealed no significant difference only between the average surface roughness values of provisional crown and fixed partial denture resin specimens reinforced with 0.5% and 1% concentrations of glass fiber.
The hunt for highly potent new drug molecules, often targeting receptors with lipophilic molecular requirements, has resulted in a general trend of increasing number of highly lipophilic, poorly water - soluble drugs . Between 75 and 90% of all new drug molecules have a solubility which is too low to allow complete dissolution of the dose in the intestinal fluid after oral administration . Therefore, the bioavailability of the drug is compromised due to low and erratic absorption . In the gastrointestinal tract lipophilic drug molecules are solubilized in mixed lipid aggregates in the intestinal fluids . In the fasted state, these are composed of bile salts and phospholipids . For lipophilic drug molecules, the interaction of the drug with the bile salts and phospholipids can have a significant effect on the partitioning into these structures and, hence, impact the solubilization and, consequently, the bioavailability . The composition of the intestinal fluid shows high interindividual variability in the fasted state, and the large variation in bile secretion and composition adds another unpredictable element to drug delivery . Unpredictable bioavailability can be critical when the drug has a limited therapeutic window; i.e., a low dose may be ineffective while a high dose results in toxicity . The two primary components in simulated human intestinal fluid are the bile salt taurocholate (tch) and the phospholipid phosphatidylcholine 1,2-dilinoleoyl - sn - glycero-3-phosphocholine (dlipc). Phospholipids have polar headgroups and long (nonpolar) acyl chains (figure 1). They therefore tend to self - assemble into ordered and extended bilayered membranes and micelles in water to minimize the exposure of their nonpolar groups to water . Bile salts are amphiphilic surfactants that easily self - assemble into small micelles in water solutions at millimolar concentrations, with the capacity to solubilize both polar and nonpolar compounds . Bile salts originate from cholesterol in the liver and contain a hydrophilic side chain attached to a large, rigid, and boat - shaped tetracyclic ring system . The ring system is characterized by a hydrophilic and a hydrophobic face, where the overall amphiphilic properties stem from the hydrophilic hydroxyl and hydrophobic methyl groups located on different sides of the ring system (see figure 1). United - atom molecular dynamics snapshots and lewis structures illustrating the deprotonated bile salt taurocholate (tch, top) and the phospholipid 1,2-dilinoleoyl - sn - glycero-3-phosphocholine (dlipc, bottom). Note the stereochemistry of the tch hydroxyl groups . Experimental characterization and drug dissolution studies show that mixtures of dlipc and tch produce different mixed lipid aggregates that may increase the solubility of certain drugs by several tenfold . The conditions of formation of these mixed lipid aggregates and their colloidal structure (micellar, uni - multilamellar vesicles) are hence of fundamental importance for the solubilization of drug molecules in the intestine . The aggregate size and the type of the structures formed in the three - component systems of water / bile salt / phospholipid differ significantly compared to two - component systems (water / bile or water / phospholipid) that typically form micelles or vesicles . In this study, we used md simulations to study drug partitioning into colloidal structures typically found in fasted intestinal fluid . As the first step, we investigated spontaneous aggregation of mixed tch and dlipc systems and simulated a range of systems with different water / tch / dlipc ratios on the united - atom (ua) level, starting from either random or ordered bilayer structures . Then, to quantify and further investigate the mixed lipid aggregates, we performed simulations of embedded tch clusters in dlipc bilayers using free energy calculations via umbrella sampling (us) simulations . Finally, we investigated the free energy barriers of the interaction of selected model drug compounds with representative dlipc bilayer membranes, either without or in the presence of embedded tch at a tch: dlipc ratio of 1:2, using us simulations on the all - atom level . Initially, several different attempts to probe the tendency for tch and dlipc molecules to spontaneously aggregate into ordered bilayers were performed on the united - atom level using the berger / gromos54a7 force fields by simulating systems having random initial configurations and different tch and dlipc compositions and sizes . These initial simulations were performed in view of the findings by marrink et al ., who showed that several phospholipids (1,2-dipalmitoyl - sn - glycero-3-phosphocholine (dppc), 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine (popc), 1,2-dioleoyl - sn - glycero-3-phosphocholine (dopc), and 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine (dope)) spontaneously aggregate into an equilibrated bilayer phase in water within a few tens of nanoseconds, using random starting configurations . In contrast to the reported systems and simulations, only a handful of the simulated dlipc and mixed dlipc / tch systems performed in this study from random starting configurations yielded well - defined lipid aggregates after more than 100 ns of simulations . As for dlipc, this can be explained by the polyunsaturated acyl chains yielding a low gel to liquid phase transition temperature (tm) of 57 c . For comparison, this is approximately 100 c lower than for the saturated 16- and 18-carbon analogues, dppc and dspc (tm = 41 and 55 c, respectively). With this in mind, it appeared that the full equilibration of the self - assembly between mixed dlipc / tch systems may require much longer time scales than those used in this study and that by marrink et al . In line with previous studies, however, the obtained lipid structures were found to be highly dependent on the properties of the periodic boundary conditions (pbc) and pressure control . For instance, 64 dlipc molecules (with or without 16 tch molecules saturated with 55 water molecules per lipid) did form a bilayered phase when semi - isotropic pressure control was used . However, cylindrical micelles were formed with isotropic pressure control . It is interesting to note that both types of structures can be found experimentally and are known to coexist under certain conditions . Given that fully equilibrated self - assemblies of the dlipc / tch lipid systems typically appeared to be outside the time scale of our simulations and likely subjected to pbc artifacts, the stability of preformed bilayer structures of dlipc with tch molecules were also investigated for six systems having 128 dlipc molecules and tch: dlipc ratios of 0:1, 1:4, 1:2, 1:1, 2:1, and 4:1, respectively, containing 55 water molecules per lipid (i.e., fully water saturated conditions). The resulting bilayer structures displayed remarkably high stability on the time scale of the simulations . Interestingly, even at a 1:1 ratio, the dlipc sustained a seemingly normal bilayer structure with the polar headgroups extending toward the aqueous phase, well beyond the tch polar side chain . This was further confirmed by the similar positions of the dlipc main functional groups (choline, phosphate, glycerol) in the system with a 1:2 tch: dlipc ratio (figure 2, center) as compared with the pure dlipc system (not shown). As for tch (figure 2, bottom), most of the sterol groups are buried deep into the dlipc acyl chains in the bilayer interior, and the polar side chains face the aqueous phase, although not beyond the dlipc polar headgroups . Interestingly, with increasing tch concentration in the simulated systems, increasing amounts of water was also found within the bilayer interior, increasing from 0 wt% to 0.2, 1, 1.7, 3.3, and 12 wt% with increasing tch: dlipc ratio . Top: density profiles of mixed tch / dlipc united - atom systems containing 0512 tch (green) and 128 dlipc (black) molecules at ratios of 0:1, 1:4; 1:2, 1:1, 2:1, and 4:1 tch to dlipc hydrated with 55 water molecules per lipid . Dashed lines show the pure dlipc system . Middle and bottom: density profiles of the 32:128 tch: dlipc system, displaying the relative positions of the molecular moieties of dlipc (middle) and tch (bottom). For clarity, add the following at the end: (abbreviations in the middle panel refer to the nitrogen group (n grp), the phosphate group (p grp) and the glycerol linker (glyc grp). The apparent stability of the mixed tch: dlipc bilayers under the given conditions might be due to the ability of the tch molecules to organize into transmembrane clusters in the normal direction within the dlipc bilayers (figure 3). At low tch: dlipc ratios, most tch molecules were found slightly below the dlipc headgroups; the nonpolar groups faced the bilayer interior, and the polar groups interacted with the dlipc headgroups and water molecules . However, with increasing tch concentrations within the bilayer, the number of transmembrane clusters increased, extending throughout the bilayer in the normal direction . This behavior can be explained by the amphiphilic nature of tch, which maximizes the hydrophilic and hydrophobic interactions of the polar and nonpolar groups simultaneously . Left: snapshot of a mixed tch: dlipc united - atom bilayer system . The equilibrated structure from a bilayered system contains 32 deprotonated tch and 128 dlipc molecules . Water molecules are omitted for clarity; charge - balancing na ions are shown in blue . Right: an isolated 3/4 tch cluster having three and four tch molecules in the top and bottom bilayer leaflet, respectively . The molecules are stabilized by hydrogen bonds between the tch molecules within the same bilayer leaflet and between tch molecules in the opposing leaflet . Note the single water molecule in the upper tch cluster; it penetrates deep into the bilayer interior due to the interaction with the buried hydrogen donors and acceptors of the tch . Pdb files with these structures are available as supporting information . To study the stability of the transmembrane tch clusters as a function of number of tch molecules per cluster, a set of simulations were performed with systems of 64 dlipc lipids containing a single isolated tch cluster . One to four tch molecules were kept in the upper leaflet and four in the lower one . The tch transmembrane clusters in systems with 1 and 2 tch in the upper leaflet were not stable during the timespan of the simulation, and the cluster disaggregated . These systems had a lower probability of transmembrane hydrogen bonding, in contrast to the systems with 3 or 4 tch upper leaflet tch molecules; hence, the tch clusters were not stabilized . With only 1 or 2 tch molecules in the upper leaflet, the initial hydrogen bonds with the tch in the opposing bilayer leaflet were disrupted, and the tch molecules positioned themselves adjacent to and below the dlipc headgroups after a few tens of nanoseconds . However, the systems with 3 or 4 tch in the upper leaflet remained virtually unchanged even after 200 ns, indicating superior stability over the smaller clusters . Hydrogen bond analysis (with the maximum bond length and angle between the hydrogen bond donor and acceptor being 0.35 nm and 30) further revealed a slightly larger number of h - bonds per tch molecule between both tch / tch and tch / dlipc for clusters with 3 tch compared to 4 tch molecules, indicating greater stability of the former type (figure 4). Hydrogen bond participation per united - atom tch in transmembrane clusters containing 3 (blue) and 4 tch (red) molecules per bilayer leaflet . The number of inter - tch hydrogen bonds (approximately 3.5) is highest in clusters with 3 tch molecules; in general, this number of tch molecules also forms more hydrogen bonds with its surroundings . The tch clusters with 3 tch molecules in the same bilayer leaflet were often slightly tilted relative the normal direction of the bilayer, with the polar oh groups in the sterol moiety facing each other . The clusters with 4 tch in the same bilayer leaflet typically had one peripheral and partly excluded tch, or one to two tchs slightly twisted around their principal axis, exposing oh groups toward the dlipc acyl chains . Analysis of the external surface area of the entire tch transmembrane clusters with a 0.14 nm probe (i.e., the equivalent of the water molecule radii) was also performed on the hydrophobic and hydrophilic parts of those clusters with atom charges less or greater than 0.2 . This analysis revealed that the 3 tch clusters had an overall 15% larger hydrophobic area and 4% larger hydrophilic area per molecule than the clusters with 4 tch . However, analysis of the external surface area of the sterol moieties only (without the polar side chain) resulted in a 12% increase and 10% decrease in the hydrophobic and hydrophilic areas, respectively . This shows that the sterol groups of the 3 tch cluster display a more optimal packing geometry when embedded in the dlipc bilayer than the corresponding 4 tch cluster, by exposing more hydrophobic and less hydrophilic atoms toward the lipid bilayer interior . Spontaneous formation of the transmembrane tch clusters was also investigated, i.e., tch cluster formation independent of the possible artifacts introduced by using ordered starting configurations . For this, we performed simulations using a pre - equilibrated micelle composed of 8 tch embedded in the center of 64 dlipc bilayer . Upon initial equilibration, predominately pairs of tch molecules interacting with their hydrophilic groups formed within 12 ns, with some orienting the headgroup toward the center of the bilayer . However, during the production run (500 ns), a 3 + 3 transmembrane tch cluster formed within a few nanoseconds with the two residual tch molecules positioned adjacent to the dlipc headgroups on each side of the bilayer . This result further indicated that tch transmembrane clusters could form naturally in bilayers of dlipc . To further investigate the tch and dlipc interactions in mixed lipid bilayers, the potential of mean force (pmf) for the tch molecule perpendicular the bilayer (z - direction) was determined using us simulations followed by the weighted histogram analysis . The overall pmf profile also reveals the free energy barriers along the chosen reaction coordinate . Figure 5 shows the pmf profiles for tch molecules in different transmembrane clusters over half the bilayer . For a tch molecule not interacting with any other tch molecules (denoted 1/0 in figure 5), the pmf profile is even positive (+ 8 kj / mol) in the center of the bilayer . This clearly shows that the interior of the bilayer is an unfavorable environment for the tch alone . However, with 4 tch molecules in the opposing bilayer leaflet (denoted as 1/4 in the figure), the corresponding value decreased to 68 kj / mol, indicating a gain in stability by formation of a transmembrane cluster . It is also notable that the minimum of the pmf profile (the optimal position of the molecule com) of the 1/4 system was more shifted toward the bilayer center and away from the dlipc headgroups than the 1/0 system . With additional tch molecules present in the same bilayer leaflet (i.e., systems with 2/4 and 3/4 tch molecules in the upper and lower bilayer leaflets, respectively), there were even deeper pmf profiles, demonstrating a greater overall stability of the tch clusters . For clusters with up to 3 tch molecules in the upper bilayer leaflet, the minimum of each pmf curve shifted away from the bilayer center due to the increasing interactions with adjacent tch . For the same systems, this trend was accompanied by an increasing free energy penetration barrier, gpen (defined analogously to eq 1), as indicated in figure 5 . However, the same trend was broken upon additional tch, suggesting that clusters with> 3 tch are less rigid due to the small energy gain achieved by adding an additional tch to the otherwise stable and rigid 3 tch - molecule clusters . Top: simulation snapshot showing the system setup for the us simulations with black arrows indicating the approximate position of the on average 40 umbrella windows used . Middle: density profiles of dlipc and the tch cluster, with five separate pmf curves for tch along the z - direction, normal to the dlipc bilayer . Note the shift in the position of the pmf minimum with a changing number of tch molecules . Bottom: density of dlipc and tch and the pmf profiles for a 3/4 tch cluster without and with additional (xtch) of surface tch . To better represent physiological conditions where free tch molecules are also present, an additional system was created by adding 64 tch to the aqueous bulk phase . After equilibration, the aqueous phase tch mostly accumulated at the dlipc / water interface, which drastically changed the pmf profile of the probe tch molecule (figure 5, bottom). Nevertheless, the minimum value and position of the pmf profile strongly resembled systems without bulk phase tch . Hence, tch was energetically favored to interact with the transmembrane tch clusters in the lipid bilayer rather than interacting with the tch - rich water phase in contact with the dlipc bilayer . Bile salts and phospholipids such as tch and dlipc solubilize drugs and alter lipid membrane properties . Therefore, additional us simulations were performed to investigate the interactions of drug molecules with the mixed lipid bilayers of dlipc and tch (figure 6). The simulations were performed at the all - atom level using the slipids and gaff force fields, as recommended by a recent benchmarking study, and to better account for specific small molecule lipid interactions . Because of the computational expense of all - atom us simulations for three - component systems, the simulations were limited to five different molecules: water, ethanol, carbamazepine, felodipine, and danazol (figure 7). These molecules were chosen to cover a wide range of lipophilicity, and the resulting log p values, as obtained from eq 1, are summarized in table 1 . Density profiles of a mixed lipid bilayer containing 48 dlipc and 24 tch molecules . Experimental log poct . Left: molecular structures of ethanol (top left), carbamazepine (top right), felodipine (middle), and danazol . Right: symmetrized pmf profiles of water (red), ethanol (green), carbamazepine (blue, denoted car), felodipine (purple, denoted fel), and danazol (black, denoted dan) over half an all - atom bilayer (centered at z = 0) containing only dlipc or (dotted line) mixed tch / dlipc (solid line). For water, there was a clear decrease in the otherwise positive pmf profile in the mixed tch / dlipc system compared to the pure dlipc bilayer when investigating deep partitioning into the membrane (> 1 nm). Inspection of the trajectories revealed in line with the results in figure 4 and the united - atom simulations occasional penetration of water molecules into the transmembrane tch clusters, facilitated by hydrogen bonding with the tch sterol hydroxyl groups . The overall free energy difference (glipid / water) of water in the mixed tch / dlipc and the dlipc reference was 18.6 2.3 and 27.6 0.6 kj / mol, respectively, well in line with previous studies . This demonstrates that the water partitioning of a dlipc bilayer increases by a factor of 30 when tch is present (calculated from glipid / water and eq 1). The pmf profiles over the mixed tch / dlipc and pure dlipc bilayer revealed only small differences in partitioning of the drug molecules and ethanol (table 1). However, intricate details governing the molecular interactions between the different compounds and tch / dlipc were found by interaction energy analysis (separating out electrostatic and van der waals interactions). For the lipophilic compound danazol (log poct of 4.53), the corresponding log plipid / water value was higher in the mixed tch / dlipc and the pure dlipc systems, respectively, than that observed for octanol . The increased partitioning of danazol in the mixed tch / dlipc bilayer and the shift of the minimum value in the pmf profile toward the center of the bilayer are explainable by the van der waals interactions with the embedded tch molecules; these were much stronger than the electrostatic interactions . This was further indicated by the higher total density of atoms in the mixed bilayer than in the pure bilayer (figure 6). Despite these findings, the simulated trajectories did not point to greater extent of binding between danazol and tch as compared to danazol and dlipc . For felodipine (log poct of 5.58), the corresponding log plipid / water values were lower in the mixed tch / dlipc than the pure dlipc system . The decrease in partitioning of felodipine in the mixed tch / dlipc bilayer resulted from both weaker van der waals and electrostatic interactions with dlipc . Unlike in the danazol system, these were not compensated for by the interactions with the embedded tch . Figure s1 of the supporting information shows the overlap of the umbrella sampling windows, as well as the effect of the symmetrization procedure on the resulting pmf . The differences in the pmf profiles for the tch / dlipc and pure dlipc bilayers were smaller for the more hydrophilic carbamazepine and ethanol (log poct values of 2.32 and 0.30, respectively). An inspection of the respective trajectories identified that these molecules interacted with the tch oh groups in the center region, which was highly accessible due to low particle density in the bilayer center . Although the differences in the pmf profiles were small, the embedded tch may accelerate drug diffusion over the lipid bilayer by lowering the gpen . The resulting log plipid / water for carbamazepine was slightly lower in the mixed tch / dlipc compared to the pure dlipc system, which was equivalent to the reported log poct value . The corresponding log plipid / water values for ethanol were, on the other hand, slightly higher in the mixed tch / dlipc compared to the pure dlipc system; i.e., there was a slightly increased partitioning of ethanol when tch was embedded . The md simulations strongly suggest that in mixtures of tch and dlipc, reflecting the composition of fasted state simulated intestinal fluid, embedded transmembrane tch clusters constitute an integral component in phospholipid bilayers composed of dlipc . Phospholipid micelle and bilayer systems by revealing the preferred number and configuration of bile salt molecules embedded in the phospholipid bilayers . Both unconstrained and us free energy united - atom simulations showed that the transmembrane tch clusters were (i) stabilized by mutual hydrogen bonds (oh3oh3) across the bilayer center and (ii) preferably consisted of 3 or 4 (3 + 1) adjacent and clustered tch molecules per bilayer leaflet . In the latter type, the additional tch constituted a more peripheral member to the overall cluster . From simulations on the all - atom level, the effect of embedded tch in dlipc bilayers on the partitioning of five model substances (water, ethanol, carbamazepine, felodipine, and danazol) showed that only water displayed a positive free energy profile over the entire bilayer regardless of embedded tch . For water this positive free energy barrier was however reduced in the presence of tch due to penetration into the transmembrane clusters . For the other hydrophilic solutes ethanol and carbamazepine the overall lipid / water partitioning into the lipid bilayer was similar to and without embedded tch . The effect of embedded tch on the overall lipid / water partitioning (i.e., from glipid / water) was most significant for danazol, demonstrating that the embedded tch may facilitate partitioning into, and hence, solubilization of lipophilic solutes in the mixed bilayer . However, this was not observed for the other highly lipophilic drug felodipine, which displayed a slightly reduced log plipid / water in the presence of embedded tch . The gromacs suite of programs was used for all simulations . Unless otherwise stated, all lipid simulations were conducted at a physiological nacl concentration of approximately 0.15 m. for the united - atom simulations (spc / gromos54a7/berger) and the all - atom simulations (tip3p / gaff / slipids) we used a universal short - range cutoff at 1.2 nm for both electrostatic and lennard - jones interactions . Long - range electrostatic interactions were treated by particle mesh ewald (pme). All covalent bond lengths were constrained using lincs, and a time step of 2 fs was used in all production simulations . Simulation systems of dlipc with and without tch were constructed to have either random starting configurations or ordered bilayers . The latter type was obtained by initially distributing the lipid monomers on a square grid and pre - equilibrating bilayered systems for> 50 ns while weakly restraining the lipid movements in the direction normal to the bilayer . To model the mixed lipid systems at the united - atom level, berger force field parameters for dlipc were adapted from a dopc topology, by enforcing a cis - conformation of the double bonds in the acyl chains . For tch, the topology and all interaction parameters were obtained from the automated topology builder server using the gromos54a7 force field . However, the partial charges were calculated from restricted electrostatic potential fitting using the pyred server as opls compatible charges (resp - o1), i.e., by gaussian09 calculations at the hf/6 - 31 g * level followed by resp . Similarly, a slipids compatible topology for dlipc was adapted from a dopc topology . This was done by removal of two hydrogen atoms and by enforcing a cis - conformation of the two neighboring double bonds and by increasing the partial charges of the ch2 atoms in the adjacent methylene group by + 0.03 (to + 0.06) to maintain charge neutrality . For the all - atom tch and drug molecules, the general amber force field (gaff) was used, having molecular topologies constructed by antechamber software through the acpype.py script (available online: http://code.google.com/p/acpype/) applied to molecular coordinates obtained from the zinc database . All regular and unconstrained md productions runs were typically preceded by (i) energy minimization using the steepest descent algorithm with a tolerance of 1000 kj mol nm, followed by (ii) a 50 ps simulation in the canonical (nvt) ensemble using position restraints on all solute particles, followed by (iii) a 5 ns volume equilibration simulation in the isothermal the latter simulations used the berendsen barostat for semi - isotropic pressure control, whereas all production runs used the parrinello the modified berendsen and velocity - rescaled thermostats were used for equilibration and production runs, coupling the non - water and water molecules to separate thermostats . By analogy, removal of center of mass (com) translation was applied to the lipids and water molecules independently; for bilayer simulations, the com of the upper and lower bilayer leaflets were controlled separately . Pure dlipc and mixed tch: dlipc us systems with either a random or an ordered bilayered initial configuration were simulated for a minimum of 100 ns, with either 64, 128, or 256 dlipc molecules and with a tch: dlipc ratio from 0 to 4:1 . To ensure full water saturation, the systems contained a minimum of 30 water molecules per lipid, in contrast to 21.5, the reported experimental value for dlipc . In the following studies we focused on free energy calculations making use of the bilayers since it is well - known that unilamellar vesicles are naturally present in fasted intestinal fluids . Free energy profiles for tch molecules over the bilayers, i.e., the potential of mean force (pmf), were computed from us simulations . To generate the starting configurations for the sampling, a single tch molecule was pulled to the aqueous bulk phase from the center of a representative tch cluster embedded in a bilayer consisting of 64 dlipc molecules . The systems contained 04 adjacent tch molecules within the same bilayer leaflet and 4 tch in the opposing bilayer leaflet . The total number of lipids in each leaflet was set constant by removal of dlipc molecules from the opposing bilayer leaflet . Each starting structure was equilibrated with 90 water molecules per lipid, after which a tch molecule was pulled by a harmonic force at a rate of 0.00025 nm / ps with the gromacs pull code . For the us simulations, more than 40 configurations were chosen from an initial set of 2500 . In these 40 configurations, the probed tch molecule was displaced approximately 0.15 nm along the reaction coordinate, i.e., in the normal direction (z - axis) to the bilayer . Each configuration was then simulated for 5 ns with a harmonic force constant of 800 kj mol nm . To ensure proper overlap of the sampled umbrella windows, each umbrella simulation was performed twice with different starting configurations . From the resulting potential of mean force (pmf) profiles an equivalent bilayer / water partitioning (log plipid / water) was calculated as1where glipid / water is the free energy of transfer from the aqueous phase to the lipid bilayer, expressed as the difference in the free energy of solubilization, gwater glipid, i.e., analogous to calculations of the traditional octanol and water partition coefficient log poct / water . The free energy profiles over the bilayers for the small molecules (danazol, felodipine, carbamazepine, water, and ethanol) were similarly computed from us simulations, this time using all - atom systems . To evaluate the effect of tch in the mixed lipid bilayers, umbrella simulations the mixed lipid bilayer was constructed with 24 tch and 48 dlipc molecules, whereas 64 dlipc molecules were used in the systems without tch . Similarly to the tch - only us simulations, representative configurations were chosen with a spacing of 0.15 nm along the pull vector . Each chosen configuration was simulated for 20 ns using a harmonic force constant of 700 kj mol nm . Unlike the tch us simulations, however, 2 (or 4 for ethanol) equally spaced probed molecules were pulled simultaneously through the bilayer, i.e., sampling both sides of the bilayer leaflet at the same time . This was possible because of the small size and neutral charge of these compounds . To avoid overlap of the inserted molecules, a slow growth protocol over 50 consecutive simulation steps was applied, after which the complete drug / solvent / tch / dlipc structure was energy minimized and equilibrated . Because of this, a lower pull rate of 0.000 05 nm / ps was used to avoid structural artifacts in the bilayer caused by the pulled drug molecules . To obtain the final pmf along the reaction coordinate, i.e., the free energy profiles, all us simulations were analyzed with the weighted histogram analysis method as implemented in the gromacs g_wham utility . Standard deviations were computed from bootstrapping over 200 runs using a tolerance of 1 10 . In the mixed tch / dlipc system, the pmf with calculated standard deviations for water was obtained from the averaged boltzmann - weighted density profiles of water in the drug molecule simulations, i.e., from unconstrained conditions and a total simulation time of approximately 2 s.
Our understanding of chronic pain has changed dramatically in the past decade or so, but researchers have yet, with the exception of triptans, to use new scientific approaches to successfully develop effective medications . The ideal analgesic for chronic pain should surpass other treatments efficacy, have few side effects, be easily available and inexpensive, modify disease, be predictive, and be preventive where possible . But current medications for most chronic pain conditions are relatively ineffective (less than 30 percent in placebo - controlled trials6) and are used because there are few alternatives . In fact, the field is beginning to re - evaluate the use of opioids for chronic pain because long - term benefits are for the most part limited, the drugs can produce chronic changes in the brain, and opioid use can result in addiction . Alternative pain treatments are either inadequate or are unproven; clinical trials for complementary and alternative medicines have for the most part not shown significant efficacy . New efforts to evaluate the placebo response in the field of pain and analgesia may prove highly useful in our approach to chronic pain patients and may have implications for treatment trials where the placebo response is a major obstacle . Harnessing the placebo response in an effective manner has enormous implications, but the focus should be on replacing inadequate or unproven treatments . By using a more aggressive translational process, researchers must discover new opportunities to treat pain and to define which therapies now in use work . Procedures with no validity need to be re - evaluated while promising treatments need to be built on so they can be replaced by more effective choices . The use of unproven techniques driven by income generation rather than scientific merit needs to end . Most chronic pain conditions produce changes in the brain that contribute to what can be termed the centralization of pain . This implies that ongoing pain produces progressive alterations in brain connections, molecular biology, chemistry, and structure, with behavioral consequences . One brain region consistently affected in chronic pain conditions is called the dorsolateral prefrontal lobe, a region in the front of our brains thought to be involved in several higher - order functions, including cognition, motor planning, and working memory . This centralization of pain involves alterations in sensory, emotional, and modulatory circuits, which normally inhibit pain . Thus chronic pain may alter cognition and emotion, leading to increased fear, anxiety, or depression . Previously healthy people, such as wounded warriors, may have an injury that results in a chronic pain syndrome, such as phantom limb pain . Conversely, a significant number of patients with primary depression and no history of pain may develop a generalized pain syndrome, suggesting changes in brain circuits that result in these clinical manifestations . Both human and animal studies show that chronic pain changes the brain functionally, structurally, and chemically.79 initial reports point to loss of or decreased volume in gray matter in the brain areas noted above, along with other regions . Chronic pain induces abnormal function in brain circuits, including circuits involved in cognition, autonomic responses, and other more complex integrative behaviors (e.g., fear, anxiety, interoception, reward and aversion). Chronic pain also alters the chemistry of brain regions, generating abnormal levels of excitatory and inhibitory chemicals called neurotransmitters . The ways different parts of the brain talk to each other also become abnormal, presumably due to changes in gray matter and its connections through fiber tracts.10 to make matters even more complex, these fiber tracts themselves show abnormal structure in chronic pain . We know less about how to undo these processes in a permanent manner, but early reports suggest that these changes may be reversible in some patients . Chronic pain may be considered a reward deficit syndrome,11 which suggests that drugs or treatments that alter the brain s reward circuitry may be valuable to treating pain.11 in addition, objective measures of brain circuits could provide a fingerprint of specific behaviors (such as sensory, emotional, and cognitive changes), serving as a potential biomarker of disease state and analgesic drug effects . Defining a useful biomarker for chronic pain would allow for objective evaluation of pain treatments, an enormous advance.12, 13 scientists are now studying chronic pain treatment from a variety of research angles, several of which i summarize below . Strategies for understanding the neurobiology of pain are front and center to all research in the field . Some topics, such as translational aspects and the molecular biology of pain, are not discussed here but are relevant as major contributors to pain neurobiology and the discovery of new treatment approaches for chronic pain (figure 2). Here we now have a better understanding of genetic risk factors for chronic pain.1416 researchers have evaluated four major categories with an immediate clinical impact: epigenetic contributions to chronic pain;17 genes that can predict the outcome of pain following injury or surgery;18 genes that provide information about the efficacy of drugs (so called pharmacogenetics and pharmacogenomics);19, 20 and genes that may define specific pain syndromes, such as migraine.21, 22 one example of the latter is pain sensitivity induced by a gene that controls the sodium channel nav1.7 . Channelopathies have provided a novel model for understanding pain pathophysiology; patients with excessive gene expression have exaggerated pain, and those with a deficit of the gene have congenital insensitivity to pain.23 new treatments may be designed around these discoveries.24, 25 chronic neuropathic pain alters neuronal structure and function along the neural axis from the peripheral nerve to the spinal cord and higher brain centers (cortical and subcortical regions). The ability to replace neurons through cytotherapeutic technologies such as embryonic stem cells (currently in animal models)26 or gene therapy27 offers exciting new opportunities to the research community . Some non - neuronal inflammatory systems play an important role in conditions such as chronic neuropathic pain,28, 29 which can produce persistent neuroinflammatory changes in the central nervous system.30 evaluating ongoing pain in the context of low - grade neuroinflammatory processes in the brain could further define exciting targets for potential therapies.31 some drugs, such as ketamine, may be beneficial thanks to anti - inflammatory effects.32, 33 several neurorehabilitative approaches may allow for the reversal of maladaptive changes in the brain . From mirror therapy and immersive virtual reality34, 35 to processes that can control prosthetic limbs36, 37 to brain stimulation techniques such as transcranial magnetic stimulation (tms),38 researchers have developed new approaches with potentially useful applications in the treatment of chronic pain . The way in which motor activity or motor cortex stimulation (through tms or direct stimulation) helps pain is unclear, but it may relate to forced interactions between the brain circuits involved in motor control and those involved in sensory processing . The notion of resetting disrupted or abnormal pain networks is clearly part of disease modification.39 our understanding of how these and other treatments may modify and improve neural circuits involved in pain is limited, and more randomized clinical trials are warranted . We now have a better understanding of genetic risk factors for chronic pain.1416 researchers have evaluated four major categories with an immediate clinical impact: epigenetic contributions to chronic pain;17 genes that can predict the outcome of pain following injury or surgery;18 genes that provide information about the efficacy of drugs (so called pharmacogenetics and pharmacogenomics);19, 20 and genes that may define specific pain syndromes, such as migraine.21, 22 one example of the latter is pain sensitivity induced by a gene that controls the sodium channel nav1.7 . Channelopathies have provided a novel model for understanding pain pathophysiology; patients with excessive gene expression have exaggerated pain, and those with a deficit of the gene have congenital insensitivity to pain.23 new treatments may be designed around these discoveries.24, 25 chronic neuropathic pain alters neuronal structure and function along the neural axis from the peripheral nerve to the spinal cord and higher brain centers (cortical and subcortical regions). The ability to replace neurons through cytotherapeutic technologies such as embryonic stem cells (currently in animal models)26 or gene therapy27 offers exciting new opportunities to the research community . Some non - neuronal inflammatory systems play an important role in conditions such as chronic neuropathic pain,28, 29 which can produce persistent neuroinflammatory changes in the central nervous system.30 evaluating ongoing pain in the context of low - grade neuroinflammatory processes in the brain could further define exciting targets for potential therapies.31 some drugs, such as ketamine, may be beneficial thanks to anti - inflammatory effects.32, 33 several neurorehabilitative approaches may allow for the reversal of maladaptive changes in the brain . From mirror therapy and immersive virtual reality34, 35 to processes that can control prosthetic limbs36, 37 to brain stimulation techniques such as transcranial magnetic stimulation (tms),38 researchers have developed new approaches with potentially useful applications in the treatment of chronic pain . The way in which motor activity or motor cortex stimulation (through tms or direct stimulation) helps pain is unclear, but it may relate to forced interactions between the brain circuits involved in motor control and those involved in sensory processing . The notion of resetting disrupted or abnormal pain networks is clearly part of disease modification.39 our understanding of how these and other treatments may modify and improve neural circuits involved in pain is limited, and more randomized clinical trials are warranted . The strategies below could form a cornerstone for integrated planning to conquer the pervasive problem . First, establishing an independent program or division of pain at the nih, or more fully integrating pain research across the nih institutes, could be a powerful engine for change . The current pain consortium at the nih goes a long way to try to integrate pain research, but it lacks essential financial backing that would make it more effective . Public - private partnerships such as the foundation for the nih or networks such as the london pain consortium, the german research network on neuropathic pain and the europain innovative medicines initiative are the kinds of approaches that we need to set up and embrace in the united states to enhance a focused, deliverable, and integrated research enterprise to conquer pain . Second, a pain equivalent to the howard hughes medical institute s support for biomedical research and science would help move the research field forward . Third, the government should provide incentives for biotechnology companies to invest more heavily in the pain field; this is a major concern at a time when large pharmaceutical companies are pulling out of drug development for brain disorders, including pain, due to perceived risk . Small funding sources are usually adept in taking risks on proof - of - principle research that can leverage applications for larger funding, as the new york based mayday fund, which supports pain research, has demonstrated . Large - scale programs, while more complex to manage and carry out efficiently, can begin to address complex processes such as biological, psychological, and social issues related to chronic pain and its treatment . More efficient, integrated, and better - designed clinical trial approaches, such as action (analgesic clinical trial innovations, opportunities, and networks, a collaboration between academia, the fda, and the pharmaceutical industry), could decrease the time needed to evaluate new chronic pain treatments . A layered approach could be a strategy for immediate, intermediate, and long - term progress . For example, significant research investment and focus on surgically induced neuropathic pain could have early benefits . Doctors in the united states perform an estimated 48 million inpatient surgeries each year; these are open laboratories in which to collect and evaluate data and implement clinical trials.40 our knowledge of the transition from acute to chronic pain is based not on scientific evidence but on an arbitrary timeline; clearly, for conditions such as surgically induced neuropathic pain, the initial surgical trauma (even while under anesthesia) is the initiating event that progresses to chronic neuropathic pain in substantial numbers of patients.4 another example where the clinical chronic pain problem looms large but is largely unaddressed in terms of effective therapies is osteoarthritis . Nerve growth factor drug have been very promising, and joint deterioration, an initial side effect, seemed to be linked with concurrent administration of nonsteroidal drugs . Determining differences in patients who respond and patients who do not respond even to current treatments would seem to be a pragmatic approach to research . More complex clinical research requiring larger cohorts such as to evaluate the genetics of pain, reconstitute neural systems with embryonic stem cells, and modify brain circuits may require longer - term processes before direct patient applications become possible . All treatments, including so - called alternative or homeopathic medications, need to be evaluated in clinical trials to filter out what does and does not work . . Currently, there are no objective measures for chronic pain or analgesic effects, and subjective responses are notoriously variable.4143 evaluation of treatments (in the clinic or in clinical trials) is fraught with difficulties because of subjective measures . Researchers need to determine measures that can be used as biomarkers for pain; their development would be a huge boost to clinical treatment and therapeutic trials . In addition, physicians must be trained to have a better understanding of chronic pain as a disease that affects the brain, using clinical insights to drive treatment plans . Integrating basic scientists into the clinic, so that researchers work with clinicians, would be a major step forward in having our brightest minds in pain neurobiology actually understand a patient and her pain - related problem . When it comes to pain treatments, particularly pharmacological treatments, some populations are neglected . This is particularly true of women, for whom the prevalence of pain is much higher than in men for the majority of chronic pain conditions, and also in children, for whom drugs tested in controlled trials in adults are for the most part simply down - dosed instead of evaluated in trials in pediatric populations . Given the cost of chronic pain to society, it would seem prudent to establish an aggressive agenda whereby private industry and academic institutions have incentives to develop new analgesics in both adults and children in proper clinical trials . Age, genetic, and sex differences related to chronic pain need to be evaluated in appropriately designed clinical trials for new drugs . Again, the establishment of clinical research centers of excellence (coes), set up in a manner similar to the howard hughes institute for medical research or the europain could have a great impact . Such coes do not have to be at a single institution but can be distributed to take advantage of talent, patient populations, and research infrastructure . Large data sets from patients and failed trials could be collected to help define issues such as clinical phenotype, genetic markers for disease states, and drug effects . These centers could be the focus for rapid deployment of clinical trials for new treatments . The nih and academics need to adapt to encourage these programs and provide due process for crediting individuals within them . Each of these centers should have an integrated pain research clinic that encourages interaction among basic scientists and research clinicians . We need to start with patients and integrate molecular, genetic, and other processes around them . In order to do this, we need to set up processes for consortia to work effectively and efficiently . Society depends on pharmaceutical and biotechnology companies, and federal regulatory agencies such as the fda, to develop and bring treatments to the public domain in a safe and efficient manner . The financial incentive to produce new and effective medications is great: pain is currently a $100 billion industry, and the successful introduction of more effective medications would be extremely competitive in this marketplace . The reformulation of old products, while financially sound, does not enhance the clinical armamentarium and should be discouraged because the reformulations generally are not more effective than the original products . Approaching chronic pain as a national emergency would allow for a better future in terms of both treatments for chronic pain, costs to society, and individual well - being . It is time to take an honest look at where we are, get rid of unnecessary and unproven treatments, and advance neuroscience to the patient in the form of better treatments . Pain neuroscience has never been so exciting or well positioned in terms of the potential it offers to change the current impasse . But it will take bold decisions to put into place a vision and plan of action.
Rna interference (rnai), the inactivation of gene expression by double - stranded (ds) rna, has become a major method of gene inactivation in the past ten years . The fact that the trigger for rnai is composed of dsrna was discovered in the nematode worm caenorhabditis elegans . This gene - inactivation method is far from being applicable to all nematodes, however, especially in the external application mode used in c. elegans . A recent paper by shannon et al . In bmc molecular biology describes its successful use in two panagrolaimus species that belong to a different nematode suborder from c. elegans . This increases the range of nematode species over which comparative functional genomics is in principle possible, and reinforces the accumulating evidence that susceptibility to rnai is widely distributed over nematode species . Rnai was first described in plants and has now been found in a variety of unicellular and multicellular eukaryotes . The mechanism of inhibition entails the cleavage of the dsrna trigger into smaller dsrnas of 2123 base pairs, called small interfering (si)rnas, which recognize the target mrna and lead to its destruction . Sensitivity to long endogenous dsrnas may be maintained by selection against the spread of transposons or viruses or against the spurious expression of other repetitive sequences, all of which are likely to be transcribed in both directions to at least some degree, and thus to produce dsrna . It is a particularly convenient feature of c. elegans that it is sensitive to external dsrnas provided very simply either by soaking the worms in a rna preparation or by feeding them with escherichia coli bacteria expressing dsrna from a plasmid, as outlined in figure 1 . Sensitivity to external dsrnas is thought to be required for repression of viruses, although this remains to be directly proven in c. elegans, for which no natural viruses are known . It requires both a mechanism for uptake of the dsrnas into intestinal cells, and the spread of the interference to other cells . In c. elegans, this phenomenon has made possible systematic gene - inactivation screens using a library of bacteria expressing dsrnas against most open reading frames . Similar gene inactivation by feeding with bacteria expressing dsrnas is also possible in species of flatworms and cnidarians . . External application of rnas by soaking or feeding bacteria requires the dsrnas to cross the intestinal barrier and the sirna signal to spread systemically . In some species octopamine internal application by injection into the syncytial female gonad ensures transmission to the next generation, regardless of systemic spreading . The efficiency of rna interference is far from general, however, even in nematodes (figure 2). Until recently, the use of bacteria expressing dsrnas in nematodes was restricted to c. elegans . Even within the caenorhabditis genus, the second most studied species, caenorhabditis briggsae, was found to be insensitive to external application of dsrnas . Interestingly, c. briggsae seems to be deficient in the uptake of dsrnas in the intestine, a deficiency that can be complemented by expression of c. elegans sid-2 (systemic rna interference defective 2), a gene that was found in a genetic screen for mutants defective in systemic rnai . The sid-2 gene encodes a putative transmembrane protein expressed in the intestine and probably involved directly in the uptake of dsrnas from the intestinal lumen . Only one other tested caenorhabditis species (c. sp . 1 sb341) and none of the close relatives of c. elegans was found to be sensitive to external rnai . However, so far all species of the genus tested have been found to be sensitive to dsrnas introduced by injection into the gonad . Susceptibility of each species to internal and external modes of rnai administration is indicated on the right . A + sign indicates that rnai has been successful, a sign that it has not . A + /- colors of species name indicate the status of genome sequencing: green, published; orange, ongoing or planned; red, not planned . * it has not been possible to implement rnai (by injection, soaking or feeding) in the two laboratory' satellite' model systems used for extensive genetic screens and comparative studies to c. elegans morpholino oligonucleotides had to be used instead, a poor and expensive alternative (figure 2). The recent article by shannon et al . However encourages the hope that other culturable nematode species may turn out to be sensitive to rnai . Panagrolaimus is a genus of free - living nematodes more distantly related to caenorhabditis than are oscheius and pristionchus, and it encompasses many different ecologies and reproductive modes . Burnell and colleagues show, using dsrna - expressing bacteria, that panagrolaimus superbus and, to a lesser degree, panagrolaimus sp . This study opens the way for functional genomic analysis in these panagrolaimus species and suggests that other culturable' free - living' nematodes may be sensitive to rnai . So far, only a few key species have been tested, and we can now expect variation in rnai efficiency even between closely related animals . Several plant - pathogenic nematodes are sensitive to external application (by soaking in dsrnas, in the presence of octopamine to induce feeding behavior) and possibly also through expression of dsrnas from a transgenic plant . The insect - pathogenic nematode heterorhabditis bacteriophora is also sensitive to dsrnas administered by soaking . Rnai has been unsuccessful or unreliable in nematodes that are vertebrate pathogens, however, including various evolutionary groups within nematodes (figure 2). In the genomes of these nematodes, some of the genes encoding upstream components of long dsrna processing seem to be lacking or unrecognizable . In summary, the use of rnai for functional genomics is so far restricted to a few nematode species . The work of burnell and colleagues suggests that the phylogenetic distribution of the dsrna effect on nematodes is capricious and that researchers who find no effect in one species should keep trying in a variety of other species . The complex phylogenetic distribution also raises questions about the evolutionary pressures, perhaps from pathogens, acting on the mechanisms of response to various forms of internal or external dsrnas . It also makes retrospectively remarkable the choice of c. elegans as the laboratory model system, 35 years before rnai was discovered . In the 1960s, sydney brenner, after his earlier work using phage genetics, chose c. elegans to study development and neurobiology of a multicellular organism . Brenner said: " thus we want a multicellular organism which has a short life cycle, can be easily cultivated, and is small enough to be handled in large numbers, like a micro - organism . It should have relatively few cells, so that exhaustive studies of lineage and patterns can be made, and should be amenable to genetic analysis . " He later added: " since a nervous system is essentially a cellular network, we had to be able to observe junctions between cells and their processes and this could only be achieved with the electron microscope, which has the necessary resolution . " (nobel lecture, 2002). After trying several other exotic organisms and isolating " nematodes from nature to find the best one ", brenner chose c. elegans for several reasons: its easy culture in large numbers on two - dimensional surfaces of agar plates with e. coli and in defined liquid media; its fast generation time (3.5 days); its mode of reproduction with self - fertile hermaphrodites and facultative males for crosses; its transparency in light microscopy and good contrast in electron microscopy; the constancy of its neuronal composition (which was known at least for other nematodes such as ascaris, though at the time not for c. elegans). Several common free - living nematode species could have met most of these criteria, for example in the oscheius, rhabditis or pristionchus genera . Within the caenorhabditis genus, brenner indeed first intended to work on c. briggsae (proposal to the medical research council, october 1963) which was the species that ellsworth dougherty, his nematode contact in berkeley, was beginning to culture axenically . However, the dougherty c. briggsae strain turned out to grow less well than a c. elegans strain isolated in bristol (n2). Brenner was surely a visionary when he turned to studies of development and behavior of c. elegans, but there are several future developments he could not possibly have foreseen when he chose c. elegans over c. briggsae and other nematode genera . The first of these is that c. elegans is so far the only hermaphroditic free - living species in which external application of dsrnas inactivates gene expression . Furthermore, the n2 strain that brenner chose as a reference is much more sensitive to rnai in the germ line than other c. elegans isolates (such as the cb4856 strain commonly used for single nucleotide polymorphism (snp)-based mutant mapping;). Second, easy transgenesis of c. elegans is possible because of its syncytial germ line: any form of injected dna recombines and forms an additional chromosome that is frequently passed onto later generations . Transgenesis by injection has so far proved impossible in oscheius tipulae, pristionchus pacificus and panagrolaimus species and is much more difficult in c. briggsae (the efficiency of establishing lines is lower and there is more silencing and mosaicism). Third, c. elegans can be frozen, as was successfully achieved by john sulston in 1969 . Freezing pristionchus pacificus, some rhabditis species or other caenorhabditis species such as c. sp . Finally, the efficiency of chemical mutagenesis by ethane methyl sulfonate is a balance between toxicity and mutagenic effect; mutagenesis is less efficient in oscheius tipulae, for example, than in c. elegans . In the history of science, a model organism may remain successful because of unexpected turns of chance, and c. elegans might not have become so popular had some of the features mentioned above been lacking . A further retrospective bias is introduced by the fact that c. elegans has been studied more than other species . N2: for example, culture conditions have to be changed for optimal culture of other c. elegans or c. briggsae strains (higher agar concentration because of worm burrowing); immunostaining has been optimized for c. elegans; and so on . On the other hand, other discoveries might have been made if another species had been chosen . The recent finding of caenorhabditis sp . 9, a male - female species that can hybridize with the hermaphroditic c. briggsae (maf, unpublished data) may make c. briggsae a popular species for some evolutionary studies . In contrast, a close relative of c. elegans that can hybridize with it is still missing . In addition to these methodological advantages, it is starting to become clear that the choice of strain and species has also influenced the biological results . Taking the example of vulva development, the effect of gonad ablation is clear cut in c. elegans: vulval tissue does not form at all, because of the lack of induction by the gonadal anchor cell . Yet in panagrolaimus sp . Ps1159, the outcome of the same ablation is much less clear: some vulval tissue forms in a variable manner depending on the individual . At a smaller evolutionary scale, wnt pathway mutations were not found in the original screens for vulva mutants on the n2 strain, but have a stronger effect in the genetic background of other c. elegans wild isolates (j. milloz, i. nuez and maf, unpublished data). Brenner's choice is therefore also important for the textbook picture that emerges from studying a model organism . Even given the overall universality of biological mechanisms and molecular pathways, the rapid evolution of partial redundancy between processes and molecular pathways means that key results of experimental manipulations (such as cell ablation and gene inactivation) are highly dependent on the species and strain chosen as the model organism . C. elegans was the first multicellular organism to have its genome fully sequenced . With the increase in sequencing capabilities, genome sequencing of various nematode species is under way, regardless of the possibilities for functional studies . The successful use of rnai in panagrolaimus superbus makes it a' superb' candidate for genome sequencing . In the caenorhabditis genus, the genome sequences of five species are now available, with the best assembly being that for c. briggsae . Molecular divergence is high, which makes these genomes useful for the annotation of conserved noncoding regions of the c. elegans genome . Most other nematode genome and expressed sequence tag sequences (completed or planned) are for animal- or plant - parasitic nematodes, because of their medical or economic importance . Annotation of sequences from these parasites can make use of the good annotation of the c. elegans genome . The draft assembly of the genome of brugia malayi (the cause of filariasis) suggests conservation of large - scale, but not small - scale, synteny and of many operons . Functional characterization of gene function may be possible for plant parasites using rnai interference, but direct characterization of gene function in the vertebrate parasites will be difficult because of the present lack of gene - inactivation techniques . Alternative methods for delivery of dsrnas or sirnas will be needed . Besides their use for the development of nematicides the nematodes form an abundant and highly diverse group of animals . Perhaps because of the short generation time of many of them, genome evolution is rapid in nematodes . Large sequencing projects will provide tools to study genome evolution at different evolutionary scales: from intraspecific evolution to large - scale divergence within nematodes . I thank past and present members of my lab and other colleagues who shared their experience with various difficult nematode strains . I acknowledge funding by the centre national de la recherche scientifique (cnrs) and grants from the association pour la recherche sur le cancer and the agence nationale de la recherche.
The incidence of tuberculosis is decreasing with proper medication and the improved economic and environmental status of the republic of korea . Skeletal tuberculosis is not frequent and chest wall tuberculosis is very rare . The incidence of extrathoracic tuberculosis is about 15% to 20% of all tuberculosis, and in bone and joint involvement, below 2% . Chest wall tuberculosis has been reported to be 1% to 10% of skeletal tuberculosis cases . Most patients with a tuberculous abscess of the chest wall do not complain of specific signs or symptoms, and it is difficult to diagnose and often misdiagnosed as another benign or malignant disease of the thoracic cage . Also, given that this type of abscess often fails to respond to anti - tuberculosis medication, a proper combination of medical and surgical management is very important to handle it . However, few reports have been published regarding tuberculous abscesses of the chest wall, and most reports that provide a clinical presentation and optimal treatment strategy for this disease have enrolled only a small number of patients [3 - 5]. The purpose of this study was to evaluate the characteristics of this disease and determine the role of surgery in the management of tuberculous abscesses of the chest wall . We reviewed 68 patients who underwent surgical treatment of tuberculous abscess of the chest wall and analyzed the characteristics of the patients, method of surgery, results, operative complications, and postoperative treatment . From october 1981 to december 2009, 68 patients with tuberculous abscess of the chest wall were surgically treated in our institution . Retrospective analysis focusing on their clinical features, diagnostic methods, operative methods, and postoperative management was performed . Preoperative diagnosis of the disease was made upon the history taking, physical examination, laboratory examination, and radiologic findings including computed tomography (ct) of the chest and a fistulogram in selected patients . Preoperative bacteriologic studies using acid fast bacilli staining, polymerase chain reaction, or culture were not performed routinely . Confirmation of diagnosis was made by a pathologic examination showing caseous granulomatous necrosis, langhan's giant cells, or tuberculous bacilli on the operative specimens . The study protocol was approved by our institutional review board, and all data were collected through patient records . The study included 33 men and 35 women, with ages ranging from 17 to 74 years (mean, 39.984.09). Thirty - one patients (45.6%) had a past or current history of tuberculosis . Among these patients, 10 had a past history of tuberculous pleurisy, 1 of renal tuberculosis, 1 of meningeal tuberculosis, and 3 of spinal tuberculosis . One patient had a history of tuberculous abscess of the chest wall 20 years earlier; at that time, excision of the cavity and partial rib resection was performed at another institution . The chief complaints of the patients were pain or tenderness at the lesion site in 40, a palpable mass in 24 patients, pus discharge in 3, and coughing with blood - tinged sputum in 1 . The lesion site was the right chest wall in 40 (58.8%), the left in 26 (38.3%), and the median in 2 (2.9%). In 64 patients, the lesion was single, and multiple lesions (triple in 1 patient and double in 3) were found in 4 patients (table 1). Preoperative chest ct was performed for 53 patients and a preoperative fistulogram was performed for 4 patients (fig . Preoperative bacteriologic diagnosis was performed in 12 patients using acid fast bacilli fluorochrome staining, polymerase chain reaction, or a culture of pus for tuberculosis bacilli . In 31 patients, preoperative anti - tuberculous medication was performed, with the medication period ranging from 2 to 32 weeks . When the patients had no history of antituberculous medication for tuberculosis and a tuberculous abscess was highly suspected, anti - tuberculosis medication was administered for 2 weeks . In cases of a rupture or impending rupture of the cavity the main goal of the surgery was complete excision of the abscess cavity and surrounding tissue . 1) involvement of the periosteum or perichondrium such as bony destruction or periosteal granulation tissue formation was suspected . 2) the abscess cavity in the chest wall made a fistulous track to the pleural cavity forming a loculated pleural abscess . 3) the subpleural abscess cavity was large, thus requiring the complete obliteration of the pleural space . In total after excision of the abscess, massive irrigation of the wound and meticulous obliteration of dead space using adjacent muscle mobilization or a pedicled flap was performed in every operation . Primary closure of the wound was performed in every case, and the chest wound was closed with a penrose drain in the chest wall or a chest tube drain in the pleural cavity . The surgical methods were as follows: abscess cavity excision in 54 (79%), abscess cavity excision with concomitant partial rib resection in 12 (17.6%), wedge resection of the lung parenchyme with abscess excision and rib resection in 1 (1.5%), and clavicular and sternal partial resection with abscess cavity excision in 1 (1.5%). Postoperative wound infection was noted in 16 patients (11.24%) and managed with conservative dressing and delayed wound closure . However, in 1 patient, after abscess cavity excision only, after 3 months of conservative management, reoperation for fistulectomy and partial rib excision was done . Respiratory complications such as pneumonia or atelectasis were noted in 4 patients . The pathologic examination confirmed the disease in all cases . Postoperative anti - tuberculsis medication was given for 6 to 19 months including the preoperative medication periods . The regimen was based on triple (isoniazid, rifampin, ethambutol) or quadruple (triple+pyrazinamide) therapy . The disease recurred in 5 patients (7.35%) between 6 months and 38 months postoperatively, and all these 5 patients underwent a second operation . The surgical method of the second operation in all of the recurred patients was abscess cavity excision and partial rib resection . Four patients had single site recurrence and 1 had recurrence at 2 sites . In 4 patients, the abscess cavity excision was performed at the first operation, while concomitant rib resection was performed in 1 patient at the first operation . In 3 patients, a postoperative wound problem occurred at the first operation, but in all of the patients the abscess recurred in a location other than the previous site . Only in the 1 patient with multiple lesions at the first operation did one abscess recur at a previous site and one at a newly formed site (table 3). Tuberculous abscesses of the chest wall are not frequently encountered and constitute less than 10% of skeletal tuberculosis . Sometimes abscesses make a fistulous tract to the pleural cavity and destroy underlying bone or cartilage . There are three mechanisms in the pathogenesis of a tuberculous abscess of the chest wall: 1) direct extension from the underlying pleural or pulmonary tuberculosis, 2) direct extension from lymphadenitis of the chest wall, and 3) hematogeneous dissemination from dormant tuberculous lesions . In the second case, mycobacterium tuberculosis in the pulmonary parenchyma or visceral pleura invades the pleural space forming pleuritis . Lymphatics in the pleural space develop by inflammation and finally communicate to the lymphatics of the chest wall, forming caseous necrosis or a fistulous tract . The third case can result from iatrogenic spread of tuberculosis to the chest wall during aspiration or drainage of tuberculous pleurisy or empyema . In our study, 22 (32.4%) patients had a history of tuberculosis or current tuberculosis . In general, retrospective studies based on medical records have the limitation that it is difficult to determine the exact mechanism of the tuberculous abscess of the chest wall . These lesions are generally solitary, but in some patients multiple lesions were found in two or more sites . Faure et al . Reported 16 single - lesion patients of 18 patients . In our study, 60 of 64 patients had a single location . The diagnosis of tuberculous abscess of the chest wall is difficult and often diagnosed by postoperative pathologic examination . Preoperatively, it can be confirmed by detecting acid - fast bacilli, polymerase chain reaction, or culture of pus in the abscess . Many authors recommend needle aspiration or incisional biopsy to confirm the tuberculous disease or to exclude other inflammatory disease [2,4 - 6]. Faure et al . Reported a diagnostic rate of only 36.3% by needle aspiration . Reported that 9 (62.29%) of 13 patients had positive acid - fast bacilli tests, 4 (28.57%) had positive polymerase chain reaction, and 6 (42.86%) had positive cultures from preoperative specimens . However, needle aspiration diagnosis of tuberculosis is not as reliable and some authors recommend surgical biopsy . Kim et al . Reported that only 3 of 80 patients had a preoperative bacteriologic diagnosis, and a tuberculous cold abscess was confirmed by postoperative pathologic examination in all cases . With this result, they recommended that the diagnosis can be made without a preoperative bacteriologic examination in geographic areas where the prevalence of tuberculosis remains high; instead, it can be clinically diagnosed by the clinical features of the chest wall abscess, history of pulmonary tuberculosis, and typical ct findings . Cho et al . Recommended needle aspiration or biopsy preoperatively when the patient has no past history of tuberculosis and no concomitant active pulmonary lesions, when ct findings show poorly demarcated mass lesions . In this study, only 12 patients were diagnosed preoperatively with acid - fast bacilli staining, polymerase chain reaction, or culture of bacilli, and the other cases were diagnosed with clinical information and ct findings, and confirmed with postoperative pathologic examination . A chest ct should be performed for every patient to evaluate the extent of abscess cavities and the status of the ribs, sternum, cartilage, and pleural space . The ct also provides information on the functional state of the lung parenchyma and thoracic cavities, and concomitant diseases, and excludes other inflammatory or tumor lesions . Brown noted that tuberculous abscess of chest wall can be easily diagnosed in endemic geographic areas in cases with a chest wall abscess with rib destruction and an extrapleural soft tissue mass lesion on chest ct . Cho et al . Classified the ct findings of tuberculous abscess of the chest wall as follows: 1) a lesion confined to the chest wall, 2) a lesion confined to the inner chest wall beneath the ribs and also protruding into the pleural space, 3) a lesion involving most of the chest wall and also protruding into the pleural space . With this classification, they found that more a superficial location of the abscess did not exclude the risk of rib involvement . In our study, chest ct films before the mid-1990s' were not available due to storage problems, and radiologic reports of chest ct were not sufficient to classify the type of the disease . A fistulogram with contrast media was taken in some patients, but was no longer done once the ct scan became popular in evaluating these patients . A few studies have reported successful treatment with anti - tuberculosis chemotherapy alone, but many authors have reported frequent recurrence after medication only . Hence, medical treatment alone is not suitable for this disease; many authors recommend a combination of medical and surgical management to reduce the recurrence . On the other hand, faure et al . Strongly suggested chemotherapy when a tuberculous infection is confirmed or highly suspected, but fails to improve with 1 to 3 months medication, and when it worsens, surgical intervention is indicated . In our study, 31 patients were treated with anti - tuberculosis medication preoperatively with a medication period ranging from 2 to 32 weeks, but no patients were treated with medication only . Among 5 recurred patients, 2 were preoperatively medicated, and 3 were not medicated . Weissberg mentioned in his commentary that this disease is best treated with antimicrobials and drainage, with debridement and excision reserved for the most extensive cases . The optimal surgical approach consists of excising the abscess and primary closure of the wounds, but the extent of resection is not defined . However, complete excision of the abscess including the abscess wall, fistulous tract, enlarged lymph node, and adjacent bony structure avoiding unnecessary extensive chest wall resection would be important to reduce complications and recurrence [13 - 15]. If there is any bony destruction or suspicious periosteal granulation tissue exists, if a fistulous tract overlays the rib or sternum, or for complete removal of underlying pleural abscess, the rib should be partially removed . If any dead space remains, a postoperative wound infection or chronic sinus formation can occur . In our study, we noticed a high incidence of postoperative wound infection (11.24%), which may be due to inadequate control of dead space . In 1 patient, reoperation including fistulectomy and partial rib resection was performed after 3 months of conservative management . Adequate muscle mobilization or a flap and small indwelling catheter would decrease the incidence of wound infection . Three other patients recurred among 16 postoperative wound infection patients, but at new sites . Classified patients into a debridement and drainage group and a complete resection group, and they found a higher recurrence rate in the debridement and drainage group (40%) than the complete resection group (9.2%). Reported a 16% recurrence rate in abscess excision patients, but only a 1.6% recurrence rate in the rib resection group . The duration of anti - tuberculosis therapy is controversial, but, a minimum of 6 to 9 months of isoniazide- and rifampin - based therapy would be needed . . Suggested medication for a minimum of 12 months especially in endemic geographical areas . If periosteal involvement of the abscess is suspected, excision of the skeletal structure should be performed . To decrease postoperative morbidity, complete removal of the abscess and obliteration of any dead space
Diabetes represents a major worldwide epidemic that poses a great social, economic, and medical burden on both developed and underdeveloped countries . An analysis of health examination surveys and epidemiological data involving 2.7 million participants performed in 2011 by danaei et al . Showed that the number of people with diabetes doubled between 1980 and 2008, increasing from 153 million to 347 million . According to the international diabetes federation these figures further increased to 415 million in 2015 and are estimated to reach 642 million in 2040 . Furthermore, it is estimated that 192.8 million people with diabetes have not been diagnosed and have an increased risk of developing complications, thus posing an additional burden on society . The estimated global health spending for treatment of diabetes and its complications for 2015 ranged between 673 and 1,197 billion usd . An increase in the number of patients with diabetes has been also reported for romania; the number of patients increased from 482,250 in 2005 to 803,489 in 2011 [3, 4]. The most recent epidemiological study performed in romania between december 2012 and february 2014 showed that in adults 20 to 79 years of age the overall prevalence of diabetes adjusted for age and gender was 11.6% however, except for the analysis of the trends in the diabetes - related lower extremities, little information is available on the prevalence of its chronic complications or patient attitudes toward diabetes in romania . The quality of life in patients with diabetic neuropathy in romania was a cross - sectional, noninterventional, multicenter survey performed with the involvement of 181 healthcare professionals and aimed to capture undiagnosed neuropathy in patients with self - reported diabetes in romania by using the norfolk quality of life (norfolk - qol - dn) questionnaire as a screening tool . This survey revealed a high prevalence of undisclosed neuropathy (50%), as well as a high prevalence of foot ulcers (14.85%) and amputations (3.60%) in this population . Here we present a post hoc analysis of data collected in this survey aiming to assess diabetes chronic complications (i.e., neuropathy, foot ulcers, gangrene, and amputations) as a function of time between the onset of symptoms of diabetes or its complications and the physician visit for those symptoms . At this time, no studies in romania have assessed this association in patients with neuropathic symptoms . These results may help to fill the knowledge gap on patients' health beliefs and provide invaluable support for developing future educational programs aimed at preventing diabetes complications . The methodology of this cross - sectional survey performed between january and december 2012 was previously described elsewhere . Briefly, self - administered questionnaires were distributed by physician specialists in diabetes, neurologists, general practitioners, and nurses from all regions in romania to their patients with diabetes . Data were collected using the romanian version of the norfolk qol - dn questionnaire . Of the 25,000 questionnaires distributed, the questionnaire used comprises 35 scored items reflecting patients' health perception and used to calculate the total norfolk qol - dn score and 5 subdomain scores for symptoms, activities of daily living (adls), autonomic neuropathy, physical functioning / large fiber neuropathy, and small fiber neuropathy . Additionally, the questionnaire has items which are not scored and were used to collect demographic (age, gender) and medical history information . Do you have neuropathy? Have you ever had an ulcer on your feet? Have you ever had gangrene? How soon after the onset of the first symptoms of diabetes / its complications did you make an appointment for a physician visit and see the physician? The patients were asked to respond with yes or no to all these questions, except for the last one in which the patients were asked to choose among the 6 possible responses: less than 1 month, between 1 and 6 months, between 6 and 12 months, between 1 and 2 years, over 2 years, i do not know / i do not remember.before completion of questionnaires, the patients were informed that their personal data would be collected as part of this survey and consented for their data to be analyzed . The survey was approved by the national supervisory authority for personal data processing under the number 0006753.22 - 03 - 2012 . Between 1 and 6 months, between 6 and 12 months, between 1 and 2 years, i do not know / i do not remember . The main objective of the analysis presented here was to assess the association between the presence of self - reported neuropathy, foot ulcers, gangrene and amputations and the time interval between the onset of symptoms of diabetes or its complications and the physician visit for those symptoms . Yes as an answer to the question do you have diabetes? Of these, only those with an answer other than i do not know / i do not remember to the question how soon after the onset of the first symptoms of diabetes / its complications did you make an appointment for a physician visit and see the physician? Were included in the analysis . Descriptive statistics (mean, standard deviation, and standard error, minimum, and maximum) were calculated for continuous variables . The age and total norfolk qol - dn and subdomain scores were compared between categories of responses to the questions how soon after the onset of the first symptoms of diabetes / its complications did you make an appointment for a physician visit and see the physician? By student t - test and kruskal wallis test . Gender, frequency of self - reported neuropathy, foot ulcers, gangrene, and amputations were compared by chi - square test . The association of the time interval between patient symptoms onset and physician visit for those symptoms with self - declaring the presence of neuropathy and the probability a history of foot ulcers, gangrene, or amputations was tested by logistic regression while controlling for age and sex . Additionally, the models with the history of foot ulcers, gangrene, or amputations as dependent variables were adjusted for the presence of neuropathy . Two - way bifactorial anova with age and gender as covariates was used to test the influence of time interval between symptoms onset and physician visit on the total norfolk qol - dn and the presence of self - reported neuropathy . The age and sex were used as covariates due to significant differences between groups in terms of age observed in the current analysis and the differences in the total norfolk qol - dn score in men and women previously observed in our sample . The estimated marginal means of the norfolk qol - dn total score adjusted for age and sex calculated with this model were further compared with the cut - off previously used as suggestive for the presence of diabetic neuropathy . All statistical analyses were performed using ibm spss statistics for windows, version 15.0 (armonk, ny: ibm corp). 21,756 had age and gender provided and were considered valid . After removing those with a missing answer or (495 questionnaires; 2.3% of the valid questionnaires) and those with a missing answer or i do not know / i do not remember as an answer to the question how soon after the onset of the first symptoms of diabetes / its complications did you make an appointment for a physician visit and see the physician? (3,731 questionnaires; 17.1%), 17,530 questionnaires were included in the analysis presented here (figure 1). The majority of patients reported having sought medical care within 6 months after the onset of symptoms of diabetes / its complications: 4,401 of 17,530 (25.1%) reported having sought medical care within 1 month after the symptoms of diabetes / its complications onset and 7,023 of 17,530 (40.1%) between 1 and 6 months after the onset of symptoms . 1,558 of the 17,530 included in the analysis (8.9%) and 1,239 of the 17,530 included in the analysis (7.1%) reported having sought medical care only after 1 to 2 years and more than 2 years after the symptoms of diabetes / its complications onset, respectively (figure 2). No difference was observed in the time interval between symptoms of diabetes / its complications onset and physician visit in terms of gender (table 1). Persons who sought medical care after more than 1 year following symptom onset were significantly older than those who sought medical care less than 1 month from symptom onset (p <0.001). The percentage of patients with a history of foot ulcers, gangrene, and amputations increased with the time interval between the symptoms of diabetes / its complications onset and the physician visit for those symptoms . The percentage of patients with a history of foot ulcers increased from 8.8% in the groups who sought medical care <1 month from symptom onset to 12.4% in those who sought medical care between 1 to 6 months, 16.0% in those who sought medical care between 6 and 12 months from symptom onset, 20% in those who sought medical care between 1 and 2 years, and 27.0% in those sought medical care after 2 years . For gangrene and amputations, the percentage increased from 3.1% and 2.5%, respectively, in those who sought medical care within 1 month to 8.6% and 6.1%, respectively, in those who sought medical care after more than 2 years . A similar trend was also observed for self - reported neuropathy on the prevalence of foot complications (table 1). The odds of self - reporting the presence of neuropathy were significantly higher in those who sought medical care later than 1 month from symptoms of diabetes / its complications onset as compared to those who sought medical care within 1 month from symptoms onset . Odds ratios (ors) for reporting neuropathy increased from 1.16 (95% confidence interval [ci]: 1.071.25) in those who sought medical care between 1 and 6 months from symptoms of diabetes / its complications onset to 2.28 and 2.27 in those who sought medical care in 1 to 2 years or in more than 2 years after symptoms onset . The ors for having a history of foot ulcers were also significantly higher in those who sought medical care after 1 month from symptoms of diabetes / its complications onset: 1.43 (95% ci: 1.261.63) in those who sought medical care in 1 to 6 months, 1.78 (95% ci: 1.542.06) in those who sought medical care in 6 to 12 months, 2.18 (95% ci: 1.842.58) in those who sought medical care in 1 to 2 years, and 3.08 (95% ci: 2.593.66) in those who sought medical care after more than 2 years from symptom onset . The ors for having a history of gangrene were significantly higher as compared to the reference category (those who sought medical care in less than 1 month from symptom of diabetes / its complications onset) only in patients who sought medical care more than 6 months from symptom onset . The ors for having a history of amputations were significantly higher as compared to the reference category only in patients who sought medical care more than 1 year from symptom of diabetes / its complications onset . The highest ors for a history of gangrene and amputations were observed in those who sought medical care after more than 2 years following symptom onset: 2.49 (95% ci: 1.903.26) for gangrene and 2.18 (95% ci: 1.602.97) for amputations (figure 3). The mean scores for total norfolk qol - dn and all subdomain scores increased significantly and in parallel with the time interval between the symptoms onset and the physician visit for those symptoms (figure 4, p <0.001 for all). For all these comparisons, the lower scores, which represent better qol, were observed in those who sought medical care within 1 month from symptoms of diabetes / its complications onset: 22.72 for total norfolk qol - dn score; 12.53 for physical functioning / large fiber neuropathy; 5.27 for symptoms; 2.05 for adls; 1.34 for autonomic neuropathy; and 1.53 for small fiber subdomain . The maximum score, indicating poorer qol, was observed in those who sought medical care more than 2 years after the symptom onset: 40.96 for norfolk qol - dn score; 21.72 for physical functioning / large fiber neuropathy; 8.31 for symptoms; 4.59 for adls; 2.59 for autonomic neuropathy; and 3.75 for small fiber subdomain . The anova analysis confirmed that the time between symptoms of diabetes / its complications onset and physician visit for those symptoms had a significant impact on the total norfolk qol - dn score and that this association was independent of age and gender . A significant interaction was also observed in this model between the norfolk qol - dn and self - reported neuropathy (figure 5). The estimated marginal means of norfolk qol - dn, adjusted for age and sex, in those with self - reported neuropathy were 30.54 for those who sought medical care within 1 month after symptoms of diabetes / its complications onset and increased to 47.09 in those who sought medical care after more than 2 years from symptoms onset . The estimated marginal means of norfolk qol - dn, adjusted for age and sex, increased in parallel with time interval between symptoms of diabetes / its complications onset and a physician visit (ranging from 10.56 in those who sought medical care in less than 1 month after symptoms onset and 19.35 in those who sought medical care between 1 and 2 years after the symptoms onset). All estimated marginal means of norfolk qol - dn were higher than the cut - off previously used suggestive for the presence of neuropathy . The results of this analysis of the data collected in the screening for neuropathy survey using the qol - dn norfolk tool in patients with diabetes in romania showed that the majority of patients sought medical care after only 1 month from the onset of symptoms of diabetes or its complications, while 33% waited for more than 6 months before addressing a physician and 16% sought medical care only after 1 year . We found a significant association between the time between the occurrence of symptoms of diabetes / its complications and the consult with a physician and the occurrence of self - reported neuropathy, foot ulcers, gangrene, and amputations, confirming previously observed results . The odds of self - reported neuropathy and foot ulcers were significantly higher in those who delayed seeking medical care for more than 1 month after the onset of symptoms of diabetes or its complications as compared to those presenting within 1 month from symptoms onset and increased in parallel with the time between the symptoms onset and the physician visit . Notably, the risk of more serious complications, such as gangrene and amputations, became significantly higher in those who sought medical care after 1 year from symptoms onset and more than doubled in those who sought medical care after 2 years from symptoms onset . Previously we found a high prevalence (52.5%) of undisclosed diabetic neuropathy in this population . The results of the analysis presented here show that the high prevalence of the undisclosed neuropathy is partially attributable to patients who do not report their symptoms to the healthcare providers or report them late after the symptoms onset, thus showing the limited efficacy of existing educational programs in diabetes in romania . This is in line with results of previous studies and surveys which showed that diabetes is often perceived by patients as a nonserious disease until complications occur [911]. According to the health belief model, a person will probably take action toward a disease if he / she perceives themselves to be at risk for the disease, is aware of the severity of a condition and of the benefits of certain actions, and is given cues for actions . Previous studies in patients with diabetes showed that a patient - centered multidisciplinary educational approach adapted to their level of understanding is effective in increasing the compliance and adherence to diabetes management and preventive care of chronic complications [1417] and has a positive impact on reducing the diabetes - associated complications and costs [1824]. Here we show that the answer to a single question on a positive response to the development of symptoms of diabetes / its complications should alert the individual to seek care forthwith . Our results clearly demonstrate that delaying more than one month can have a disastrous outcome on the foot complications of diabetes and education programs should impart this simple message . The type of symptoms experienced by patients may be another cause of delay between onset of symptoms and seeking physician intervention . In a previous analysis of this survey (gavan et al ., symptom characteristics that alert patients to the diagnosis of diabetic neuropathy, 2015, submitted), we identified a group of patients who self - reported neuropathy without being diagnosed by a physician . This group reported more frequently symptoms in hands and arms than the group with a previous diagnosis of neuropathy who reported more frequently symptoms in legs and feet . A higher likelihood of the patients to self - report neuropathy although they had not been told they had this diagnosis was also associated with autonomic neuropathy, large fiber neuropathy, and adls subdomain scores . The presence of symptoms in feet and legs usually alerts patient to their neuropathy and probably these patients sought for medical care soon after the symptoms onset . The group of patients with symptoms in hands and arms, autonomic neuropathy, or symptoms attributed to small fiber neuropathy may have failed to recognize the defined symptoms or, if recognized, did not report them to their physician . The mean total norfolk qol - dn score in those who sought medical care within 1 month from symptoms of diabetes / its complications onset was similar to those previously reported in patients with neuropathy without symptoms (22.72) and the mean total norfolk qol - dn score in those who sought medical care after more than 2 years after symptoms onset (40.96) was similar to the ones of a group with symptomatic neuropathy in a german population . These results confirmed once gain the deleterious impact that delays in diagnosis and treatment have on the progression of diabetic neuropathy . Another cause for the delayed appointment to a physician after the symptoms of diabetes / its complications onset may be the limited access to foot examination in the diabetic population . The foot examination in primary care is limited in both romania and elsewhere [2628]. Although as per romanian medical system organization each patient with diabetes should be seen by their primary care physician or a physician specialist in diabetes every 3 months, we have reasons to believe that the majority of them do not have a foot examination performed at least once a year . This may also have a negative impact on the patient's perception on the importance of seeking medical care when symptoms of diabetes or its complications occur . A study performed as part of the quality of care and outcomes in type 2 diabetes project aiming to assess the physician's attitude towards foot care education and foot exam in patients with type 2 diabetes showed that foot examination was performed less frequently by general practitioners as compared to physician specialists in diabetes and more frequently in those with foot complications (not diabetic neuropathy). Additionally, it was shown that patients who had a foot exam were more likely to check their feet regularly . Educational programs targeting physicians have been shown to increase the performance of screening activities and thus may have a positive impact on reducing the time between the onset of symptoms of diabetes or its complications and medical care . Notwithstanding that our survey has strengths derived from the large number of patients included and which makes its results representative for romanian patients with diabetes, it also has limitations . Although it would have provided more detailed information, we did not ask for separate answers for the time between the diabetes symptoms onset and the physician visit for those symptoms and between the time of symptoms of complications onset and the physician visit for those symptoms . We have not asked either specific question to assess which diabetes complication the patients referred to when answering the following question: how soon after the onset of the first symptoms of diabetes / its complications did you make an appointment for a physician visit and see the physician? We chose this approach to assess patients' beliefs, education, and knowledge and the overall attitude toward the health status and the primary and secondary prevention of diabetes and its complications . Therefore, we do not know if the patient answers referred to the time between the onset of symptoms of diabetes alone or its complications and the moment of seeking medical care for these symptoms . As we previously mentioned the norfolk qol - dn questionnaire was specifically designed for the evaluation of the impact of diabetic neuropathy on qol and showed good sensitivity, specificity, and positive and negative predictive values for the severity of neuropathy [25, 29, 30]. In addition, this tool has been used in romania to screen for diabetic neuropathy . Here we used an additional question to define the onset of symptoms of diabetes / complications which did not correspond with the items of the norfolk qol - dn . It remains to be ascertained more specifically what the content of the question on diabetes and its complications would be the most useful in proposing an education program to reduce the latency of patients' decision to visit a physician . However, we based our analysis on the questions asked of patients and their self - reported responses and not on reviewing medical charts or objective measures of neuropathy and its complications recall bias cannot be excluded and more specifically must depend on the nature of the question itself . In conclusion, we showed that waiting for more than 1 month after symptom onset of diabetes / neuropathy dramatically increases the risk of neuropathy, foot ulcers, gangrene, and amputations . Seventy - five percent of the patients who completed the questionnaires waited for more than 1 month after the symptom onset to seek medical attention, and 16% sought medical attention after 1 year following the symptom onset . These results are alarming and support the need to implement easily accessible educational programs on diabetes and its chronic complications.
Non - hodgkin s lymphomas are neoplastic diseases of the lymphatic system, usually involving the lymph nodes, but almost 2540% of the cases have extranodal onset . Orbital lymphomas represent about 2% of all lymphomas, 5- 15% of extranodal lymphomas and approximately 50% of all primary malignant tumors of the orbit . An association between chlamydia psittaci infection and orbital adnexal lymphoma has been described, and also, thyroid eye disease is considered to be a predisposing factor . Clinical signs and symptoms are nonspecific, often delaying the diagnosis . In 25% of the cases the conjunctiva is involved and the patients present with salmon red patches or swollen conjunctiva . In the rest of the patients, presentation is with orbital mass . Common signs at presentation are: palpable mass, pain, exophtalmia, ptosis, dyplopia, decreased vision and abnormal ocular movement . In approximately 1017% of cases the lymphoma appears bilaterally, simultaneously in both orbits in 80% of cases and subsequently in 20% of cases . Between 2040% of the patients have extraorbital lymphoma at onset, especially those with aggressive histology . The majority of orbital lymphomas are of low - grade histology (84%), the minority (16%) being aggressive lymphomas . Other histological types are: follicular, lymphocytic, mantle cell and diffuse large b - cell lymphomas . Diagnosis is based on biopsy and staging procedures should include imaging investigations: computed tomography (ct), or magnetic resonance imaging (mri) to evaluate local extension but also systemic lymphomatous involvement . Differential diagnosis should include other malignant tumors and inflammatory pseudo - tumors of the orbit and thyroid associated orbit disease . The prognosis of ocular lymphoma depends on the histological type, age, localization and stage of disease . Favorable prognostic factors are: low - grade histology, younger age, conjunctival localization and early stage at presentation . Treatment depends on the stage: in localized orbital lymphoma, radiotherapy is highly effective, while in patients with high grade histology or disseminated lymphoma, systemic chemotherapy should be used . In some localized cases, antibiotic therapy against chlamydia results in complete remission . A case of bilateral orbital lymphoma is presented below, having the approval of the ethical committee of the hospital and the written consent of the patient for publishing his pictures . A 50 years old male patient referred first to the ophtalmology clinic in april 2012 for bilateral exophtalmos and palpebral ptosis . Biopsy was refused by the patient . In november 2012, he was admitted at the maxillofacial surgery clinic for bleeding from the periorbital tumors . A biopsy was performed and diffuse large b - cell lymphoma was diagnosed . On admission to the hematology clinic the patient presented bilateral periorbital tumors (12 cm in the right and 8 cm in the left side), involving the eyelids and incorporating the eye globes (fig . 1 and 2). The patient also complained of complete loss of vision and constitutional symptoms (night sweats and weight loss). Laboratory tests revealed leucocytosis (13000/microliter) with 55% atypical lymphocytes in the peripheral blood smear, anemia (hemoglobin=10g / dl), elevated erythrocyte sedimentation rate and decreased level of igg and iga . Serological tests for chlamydia, hiv and hepatitis viruses (b and c) were negative . Cranio - orbital ct revealed bilateral orbital tumors (14/8/11 cm in the right with 2.7 cm ptosis of the right eye and 7.8/5.6/6.5 cm in the left with 2 cm ptosis of the left eye), without invasion of the eye globes, bones, paranasal sinuses or of the cerebral parenchyma (fig . 3 and 4). Cervical and thoraco - abdominal ct showed enlarged latero - cervical and retroperitoneal lymph nodes . The patient was diagnosed with stage ivb diffuse large b - cell primary orbital lymphoma and r - chop (rituximab + cyclophosphamide, adriblastin, vincristin and prednison) chemotherapy was started, which produced a rapid response . R - ice (rituximab + iphosphamide, carboplatin and etoposide) second - line chemotherapy was started and complete remission was obtained after 4 cycles (fig . 5). He did not return for completion of the chemotherapy cycles, nor for follow - up . Primary orbital lymphoma is a rare disease, diagnosis being difficult due to nonspecific symptoms at presentation . Patients usually present with unilateral orbital tumors, only 1017% of the cases having bilateral orbital involvement at onset . Ocular lymphomas have low - grade histology in the majority of the cases and prognosis is good if diagnosis is made in early stages . However, permanent loss of vision can be seen due to optical nerve atrophy, even in patients with a complete remission of the lymphoma . Treatment consists of radiotherapy in localized disease but if systemic involvement is present, chemotherapy should be used . The particularity of this case is the presence of massive orbital tumors, due to delayed diagnosis . Long - term prognosis of this patient is unfavorable, due to the advanced stage of the disease at presentation and early relapse after an initial good response to chemotherapy.
Several risk factors such as tobacco, human papilloma virus, areca nut, alcohol have been described in the literature as causative agents for head and neck squamous cell carcinoma (hnscc). In recent years, areca nut consumption has been linked to oral cancer in studies from south east asian countries. [15] betel nut has a long history of use in south east asian natives and is part of their culture and religion . The acute effects are worsening of asthma, low blood pressure, and rapid heart beats whereas chronic effects are oral submucous fibrosis (osf), precancerous oral lesions, and squamous cell carcinoma . We report a series of female patients with head and neck cancer who had the habit of chewing only areca nut without any history of tobacco usage in any form . We present their clinical parameters, socioeconomic status, education level, duration, and frequency of areca nut chewing along with review of the literature . We also intend to highlight the association of hypopharynx carcinoma with areca nut chewing habit, which has not been studied in the literature so far . These are prospectively collected data of ten patients who presented to us with biopsy proven hnscc and a history of areca nut chewing without any history of tobacco usage . We collected information on clinical parameters, duration, and frequency of areca nut chewing, socioeconomic status, and education level of the patients . There were ten female patients [table 1] with the mean age of 49 (ranging from 31 to 59 years). Two patients presented with hypopharynx cancer and eight had the lesions in oral cavity (four tongue, three buccal mucosa, and one floor of mouth). Amongst those with oral cancer, the most common complaint was that of a nonhealing ulcer in the oral cavity along with features of submucus fibrosis (trismus, white, and sensitive mucosa). Osf was present in all these cases, involving bilateral buccal mucosa, palate, and tongue . All patients complained of progressive inability to open mouth and sensitive mucosa (to hot and spicy foods) of varying degree [figures 1 and 2]. The mean duration of areca nuts chewing was 15.1 years (ranging from 6 to 50 years). Areca nut chewing habit had a wide variation in these cases, in terms of duration, frequency, socioeconomic status, and employment status . Most of the patients had the habit of areca nut chewing with an average of three to six times a day . The commonest form of areca nut chewed was the natural dried form (7/10 cases) and the rest three cases admitted to chew commercially prepared areca nut products [table 1]. The patients belonged to the upper lower socioeconomic category [table 2] and were housewives . Amongst the eight oral cancer cases, tongue (4) was most frequently involved subsite followed by buccal mucosa (3) and floor of mouth (1). Tongue followed by buccal mucosa were the primary sites of oral carcinoma in chewers of commercially available areca nut products whereas hypopharynx was primary site in chewers of natural dried areca nut, in addition to buccal mucosa and tongue . There were lot of soft deposits, plaque, and calculus and the typical stain due to tobacco and betel quid (brownish black or yellowish brown) was not seen in these cases . Also, the typical incrustations on the soft tissue especially seen in gutka chewers and chewers of pan with tobacco were absent on clinical examination in these cases . This suggested their only areca nut chewing habit . There was gross generalized attrition of teeth with exposed roots, wear facets, and root stumps, contributing to poor oral hygiene . There were two cases of scc involving pyriform sinus (subsite of hypopharyx) that presented with odynophagia with an average duration of 45 days . One of the patients underwent total laryngectomy followed by radiation therapy, and one patient required only palliative care . The submucous fibrosis was seen to involve the entire upper aerodigestive tract in these cases . Intraoperative finding in patient undergoing total laryngectomy and laryngoscopic examination in the other patient showed extensive submucous fibrosis in the hypopharyngeal and laryngeal mucosa . In majority of the cases (6/10), the lesions were in an advanced stage requiring extensive surgical resection and reconstructions followed by aggressive adjuvant therapies . In two patients curative treatment was not possible and palliative chemotherapy was advised . Age, clinical details, site and treatment details of patients with head and neck cancer clinical presentation with variable degree of restricted mouth opening and ulcerative lesion clinical presentation with ulcerations, stains on teeth with attrition and poor oral hygiene reason for areca nut chewing and socioeconomic status as per the kuppuswamy scale in contrast to the western literature wherein smoking, alcohol and human papilloma virus have been described as major risk factors for head and neck scc, smokeless tobacco, and areca nut chewing are predominant risk factors in india . Areca nut is a highly addictive substance with the score for mean severity of dependence as 7.3 (in a range of 112) similar to the problematic use of amphetamines . There have been reports of usage of these products in asian migrants to the united states, the united kingdom, singapore, australia, germany, south africa etc. [1019] this habit is an important public health problem in taiwan . Although, chewing areca nut has been found to be associated with oral cancer and recently with development of primary hepatocellular and esophageal carcinoma, this was the first time we encountered a series of carcinoma of hypopharynx in areca nut chewers . Unlike tobacco, areca nut is portrayed as a safe mouth freshener; therefore, it is freely available and widely consumed . There are reports of areca nut addiction amongst children of south east asian countries which results in oral cancer in the younger age . A study in pakistan reported that school children at primary school level are areca nut addicts and 74% children used areca nut, 35% of them chewed betel quid daily . The cases described in our study were middle - aged housewives with the chronic betel nut chewing habit . Due to the small sample size the reasons for areca nut consumption in these cases were its psycho - stimulating effects causing a euphoric feeling, sweetening breath, as a digestive or as a cultural practice . The patients had a lower level of education and not aware of the deleterious effects associated with areca nut chewing habit . Psychopharmacological effects of areca nut have been described as a popular pleasure giving substance in south asia and include increase in concentration, mild mood elevation, enhanced satisfaction after eating and relaxation . Habituation and addiction to daily chewing of the quid has also been reported amongst the people hailing from the gujarat state in india . Majority of cases in our study were of tongue and buccal mucosa, in accordance with a study from south africa . The cause of submucous fibrosis is the excessive synthesis of collagen in the submucous tissues . In vitro studies with cultured fibroblasts have shown that areca nut alkaloids such as arecoline and its hydrolyzed product arecaidine stimulate proliferation and collagen synthesis in a dose - dependent manner, higher concentrations being cytotoxic . Flavonoids, catechins, and tannins in areca nuts cause collagen fibers to crosslink, making them less susceptible to collagenase . Furthermore, the extracts of areca nut and its components have been shown to be crucial in the pathogenesis of osf and oral cancer by differentially inducing the dysregulation of cell cycle control mitochondrial membrane potential, depletion of cellular glutathione, and intracellular h2o2 production . It has been estimated that people with osf are 19.1 times more likely to develop oral cancer than those without it, after adjusting for other risk factors and another study describe a 7.6% rate of malignant transformation of osf to frank carcinoma over a 10-year period . We did not come across any other published report where areca nut has been linked to the hypopharynx cancer . The finding of the features of submucous fibrosis in the hypopharynx and larynx mucosa is a sparsely reported feature in chronic areca nut chewers . Similarly, association of hypopharynx cancer with submucous fibrosis of oral cavity is an uncommon problem that poses challenge during evaluation and treatment of these cancers . Areca nut chewing is an important risk factor in the genesis of oral potentially malignant and malignant lesions in indian women . Although there is a need to establish the carcinogenic effects of areca nut at a genetic or molecular level in these cases, wide spread health education programs are necessary to reach both urban- and rural - based populations in education and motivation against the habit of chewing areca nuts.
All procedures were reviewed and approved by the institutional animal care and use committee of the school of veterinary medicine, rakuno gakuen university (japan). Fifteen eastern grey kangaroos (macropus giganteus) with ljd aged (mean sd) 3.5 2.2 years old and with a body weight of 15.5 6.3 kg were examined in this study . The definitive diagnosis of ljd was made based on clinical findings, such as facial swelling, weight loss, excessive salivation and flicking of the tongue . Twelve eastern grey kangaroos aged 3.6 2.3 years old and with a body weight of 20.5 11.7 kg were used as the control group . The health status of the control animals was determined on the basis of a physical examination and serum biochemical analysis by zoo veterinarians . All animals were kept at hibiki animal world (fukuoka, japan) and consumed concentrated pellets (zc pellets, oriental yeast co., ltd ., tokyo, japan) for herbivores in accordance with the manufacturer s guidelines and had ad libitum access to hay (timothy and alfalfa), vegetables (including carrots, cabbage and potatoes), apples and water . Four milliliters of whole blood was collected via jugular venipuncture into heparinized tubes for the endotoxin analysis and then centrifuged for 10 min at 3,000 g at room temperature within 1 hr of collection . Approximately 1.8 ml of plasma was harvested and stored in sampling tubes (cryotubetm vials, nunc, roskilde, denmark) at 30c for later analyses . Immediately prior to testing, plasma samples were diluted 20-fold in endotoxin - free water (otsuka distilled water, otsuka pharmaceutical co., ltd ., specimens were then heated for 10 min at 80c in order to inactivate interfering substances, such as protease . The usp endotoxin reference standard (rse, usp endotoxin reference standard lot g, the united states pharmacopeial convention, inc ., rockville, md, u.s.a . ), which contained 10,000 endotoxin units (eu) per vial, was used as the positive control . The lal reagent for the lal kt (endosafe kta, charles river) assay was reconstituted with endotoxin - specific buffer solution (charles river) in order to eliminate any interference from -glucans . The traditional lal - based assay was performed on a 96-well microplate (endosafe 96-well, flat bottom microplate m9001, charles river), and endotoxin activity was determined using a microplate reader (sunrise, tecan group ltd ., mnnedorf, switzerland) and endoscan - v endotoxin - measuring software (charles river). The range covered by the standard curve (0.003 to 3.0 eu / ml) was established according to the package insert of the lal product . The lower limit of quantitation for this assay was 0.027 eu / ml . All samples tested with the pts system used 10.001 eu / ml sensitivity cartridges (fig . 2fig.2.the limulus amebocyte lysate (lal) spectrophotometer (upper) and reagent cartridge (bottom) for the endosafe pts system . ). The pts system, which comprised a spectrophotometer, reader and lal reagent cartridge, was used in the present study . The reagent cartridges (lot #3183249) were prototypes that did not react with -glucan, and were provided by charles river laboratories . Precise amounts of lal reagents, buffer components and oligosaccharides as a -glucan blocker, chromogenic substrates and control standard endotoxin were dried on the channels of the cartridges . The reagent cartridges were potency tested, spike recovery was performed, and the calibration code was then determined . The calibration code (cal #419065608093) contained the reagent cartridge test parameters that were determined during potency testing, as well as the archived curve for that batch of cartridges . The analyst loaded 25-l samples into the cartridge sample reservoirs, and the reader drew, mixed and incubated the samples at different time intervals after the assay was started . The product endotoxin concentration (endotoxin activity), product positive control with a known endotoxin concentration, percentage sample coefficient of variation, percentage endotoxin spike coefficient of variation and percentage recovery of the product positive control were automatically calculated using software for research use involving an extrapolation function . In the present study, 20-fold diluted plasma, which was heated for 10 min at 80c, was used to measure endotoxin activity . The lower limit of quantitation for this assay was 0.050 eu / ml . A detailed description of pts is provided elsewhere [6, 18]. The limulus amebocyte lysate (lal) spectrophotometer (upper) and reagent cartridge (bottom) for the endosafe pts system . Statistical analysis: a test result was considered valid based on the percentage spike recovery and percentage coefficient of variation (cv) parameters falling within the acceptance criteria (25%) established by the pts system and kt assay . Spike recovery values were considered valid, if the results were between 50% and 200%, according to the bacterial endotoxin test in the us pharmacopeia . The absolute value of the correlation coefficient of the standard curve generated using reference standard endotoxin was greater than or equal to 0.980 for the range of endotoxin concentrations established, according to the bacterial endotoxin test in the us pharmacopeia . The results of the pts and kt assay were compared using the friedman test, which is a non - parametric statistical test used to detect differences across multiple test attempts . Pearson s product moment correlation coefficients were calculated to evaluate relationships between any two continuous variables . A linear regression model analysis was also performed in order to obtain the equation . The median values for endotoxin activity obtained from the pts method were compared with the healthy controls, and the mann - whitney u test was employed for comparisons between groups . Roc curves were used to characterize the sensitivity and specificity of each parameter with respect to changes associated with ljd . The optimal cut - off point for a test is defined as the maximum vertical distance between the roc curve and diagonal or chance line and is calculated as j = maximum [sensitivity+specificity1]. The cut - off point on the roc curve that corresponds to j was regarded as the optimal cut - off point . The kt test effectively recovered endotoxin from plasma (82.4%, range 73.2% 88.5%) over the range of concentrations tested . The linearity of the standard curve was also satisfactory for the kt assay (r=0.984) over the range of concentrations tested . Cv, a parameter in the kt assay test for endotoxin activity, was 10.9% (range, 0.2% cv, a parameter in the pts for endotoxin activity, was 11.1% (range, each of the assays (kt and the pts) effectively recovered endotoxin from the plasma of kangaroos with or without ljd . The median ranges of endotoxin activity detected by each of the tests, kt and the pts, were 0.206 (min to max, 0.0271.06) and 0.222 (min to max, 0.0501.43) eu / ml, respectively . 3.relationship between endotoxin activity in plasma between the portable test system (pts) and traditional limulus amebocyte lysate kinetic turbidimetric (kt) analysis . Endotoxin activities detected in plasma samples using the pts positively correlated with those using the kt assay by pearson s product - moment correlation coefficient ., the results obtained from the pts correlated well with those from the kt assay (r=0.915, p<0.001). Based on the results of the friedman test, the ability of the pts to recover endotoxin from plasma was not significantly different from that of the kt assay (p>0.05). Relationship between endotoxin activity in plasma between the portable test system (pts) and traditional limulus amebocyte lysate kinetic turbidimetric (kt) analysis . Endotoxin activities detected in plasma samples using the pts positively correlated with those using the kt assay by pearson s product - moment correlation coefficient . Plasma endotoxin activities were higher in kangaroos with ljd (0.326 eu / ml; min to max, 0.05 to 1.56 eu / ml) than in those without ljd (0.100 eu / ml; min to max, 0.05 to 0.74 eu / ml, p<0.05). The area under the roc curve for plasma endotoxin activity was 0.793 (fig . 5fig . 5.roc curve for plasma endotoxin activity in order to detect kangaroos with lumpy jaw disease (ljd). The optimal cut - off point for the test was calculated by the youden index . P<0.05). The proposed diagnostic cut - off point for plasma endotoxin activity in order to identify kangaroos with ljd based on analyses of roc curves was set at> 0.22 eu / ml . The sensitivity and specificity of the proposed diagnostic cut - offs for plasma endotoxin activity were 80.0% and 80.0%, respectively . Medians of plasma endotoxin activity in kangaroos with lumpy jaw disease (ljd). The horizontal line in each box represents the median value . Roc curve for plasma endotoxin activity in order to detect kangaroos with lumpy jaw disease (ljd). The optimal cut - off point for the test was calculated by the youden index . We investigated the relationship between oral necrobacillosis, commonly referred to as ljd, in captive kangaroos and plasma endotoxin activity using an automated handheld endotoxin testing system named the pts . In the present study, positive control recoveries in the traditional kt assay and pts were rarely outside the acceptable range . The pts effectively detects plasma endotoxin activity in the kangaroo and is practical for simple and easy use to assess endotoxin activity in plasma . It offers several advantages over the microplate kinetic lal assays currently in use by diagnostic laboratories, namely, it is small and portable, requires only small quantities of specimens, and rapidly provides results [6, 18]. The activity of endotoxin in the plasma was higher in kangaroos with than in those without ljd . Furthermore, plasma endotoxin activity was significantly higher in kangaroos with ljd than in the healthy control group . Therefore, the proposed diagnostic cut - off for plasma endotoxin activity based on the roc curve analysis to detect ljd was set at> 0.22 eu / ml . The sensitivity and specificity of the proposed diagnostic cut - off for plasma endotoxin activity were 80.0% and 80.0%, respectively . Usp chapter 85, which addresses photometric bacterial endotoxin test methods, allows for a wide recovery range for the positive control, between 50% and 200%, because small discrepancies in test conditions and cartridge flaws contribute to variable recovery values for the positive control [6, 12, 13, 18]. An out - of - specification percentage recovery for the positive control was previously associated with a calculated product endotoxin concentration that expressed any interference, such as inhibition and enhancement . When any criteria, mainly percentage recovery of the positive control, were not within the acceptable range, the test was not considered to be valid . In the present study, positive control recoveries in the traditional kt assay and pts the photometric pts represents a rapid, simple and accurate technique using the quantitative kinetic chromogenic lal method to assess plasma endotoxin activity in kangaroos and meets all the requirements for endotoxin activity including the percentage of cv and recovery of the positive control . Furthermore, the results of the pts, which used plasma diluted 1:20 in endotoxin - free water and heated to 80c for 10 min, correlated with those obtained by the traditional kt assay . Therefore, the results of the present study confirmed that the pts is practical for simple and easy use in order to assess endotoxin activity in plasma . Our results showed that average plasma endotoxin activity was higher in kangaroos with ljd than in controls . Based on roc curves, we proposed a diagnostic cut - off point for endotoxin activity of> 0.22 eu / ml for the identification of ljd . Endotoxin, which is released from bacteria including f. necrophorum at the time of cell death and initiates an inflammatory response, refers to the lipopolysaccharide protein of the gram - negative bacterial wall and is the primary virulence factor of gram - negative bacteria responsible for damage to the kangaroo . Endotoxin is known to be responsible for many of the pathophysiological signs observed during gram - negative bacterial infections in mammalians, such as fever, leukopenia, complement activation, the activation of macrophages and changes in the plasma levels of metabolites, minerals, acute phase reactants and hormones . In conclusion, we herein investigated the diagnostic value of plasma endotoxin activity in kangaroos with systemic inflammation caused by oral necrobacillosis and identified plasma endotoxin activity as a sensitive marker of systemic inflammation in kangaroos with ljd . Based on roc curves, we proposed a diagnostic cut - off point for endotoxin activity of> 0.22 eu / ml for the identification of ljd . Our results indicate that the assessment of plasma endotoxin activity is a promising diagnostic tool for the outcome of ljd in captive macropods . In addition, the photometric pts represents a rapid, simple and accurate technique, which uses a quantitative kinetic chromogenic lal method for the assessment of plasma endotoxin activity in kangaroos . Therefore, the results of the present study confirmed that the pts is appropriate for assessing endotoxin activity in plasma.
Written informed consent was obtained from the patient for publication of this case report and accompanying images . A copy of the written consent is available for review by the editor - in - chief of this journal on request . Spencer r. anderson: responsible for research, organization, and composition of this case report . Charles s. scarborough, md: responsible for the interview, exam, and management of the patient.
Administration of antiepileptic drugs (aed) in the setting of aneurysmal subarachnoid hemorrhage (asah) has become routine in the hope of achieving better outcomes for patients . Previous studies have reported a high incidence of seizures in the setting of asah (12.5 - 22%).4)23) aed use is standard practice for prevention of seizure episodes, which can cause hypoxic brain injury or subsequent electrical field suppression, possibly delaying recovery or adversely influencing the natural course of the disease . However, prophylactic aeds have been associated with side effects including behavioral and cognitive impairments,1)16) and complicated brain hemorrhage . Therefore, more sophisticated guidelines for treatment or prevention of seizure due to sah are needed . According to the current guidelines,8)22) prophylactic aed may be considered for patients with known risk factors for seizure, such as poor clinical grade (hunt - hess [hh] grade 4 or 5), intraparenchymal hematoma, microsurgical clipping, postoperative hematoma, aneurysms of the middle cerebral artery, and cerebral ischemia.5)11)13)14)19) among these risk factors, the most comprehensive and predictive entity is poor clinical grade such as in cases of middle cerebral arterial aneurysm with intraparenchymal hematoma requiring microsurgical clipping combined with decompressive surgery . However, the use of prophylactic aed in patients with a good clinical grade is not recommended by the guidelines but used currently in our clinical practice . Thus, we retrospectively evaluated the effects of prophylactic aed on clinical outcomes in patients with a good clinical grade suffering from asah . This retrospective study was approved by our institutional review board, and the requirement for informed consent was waived . A total of 84 patients were enrolled from a database of 133 patients with asah who underwent either microsurgery or endovascular surgery between september 2012 and december 2014 . All included patients met the following criteria: (1) presence of a ruptured intracranial aneurysm on computed tomography angiography or magnetic resonance angiography; (2) hh grade 1, 2, or 3 on admission; and (3) without seizure presentation . The remaining 49 patients from the database were excluded for the following reasons: (1) onset seizures (n = 4); (2) hh grade 4 or 5 on admission (n = 38); (3) history of previous seizure taking aed (n = 2); and (4) loss to follow - up (n = 5). Included patients were divided into two groups according to the usage of prophylactic aed; the aed group (n = 44) and the no aed group (n = 40). Patients were prescribed 1000 mg of levetiracetam or 900 mg valproic acid daily for at least 6 months (6 to 12 months). Clinical data, including age, gender, initial hh grade, fisher grade, hypertension, diabetes, dyslipidemia, smoking, treatment modality, hydrocephalus, external ventricular drainage, shunt, symptomatic vasospasm, procedure related complications, systemic complications and aneurysm characteristics (location, shape, size, and neck diameter), were reviewed and compared between the two groups . Procedure related complications were defined as new neurological deterioration related to treatment modality (microsurgery or endovascular surgery) or complications requiring additional procedures . Systemic complications were defined as major medical complications, such as infectious conditions (pneumonia or urinary tract infection), hepatic dysfunction, renal dysfunction, pulmonary complications (atelectasis, pulmonary effusion, or pulmonary edema), cardiology complications (myocardial infarction, arrhythmia, or heart failure), and electrolyte imbalance (hyponatremia or hypernatremia), affecting clinical outcomes . The patients' clinical outcomes were evaluated at discharge and after six months of follow - up using the modified rankin scale (mrs). A favorable outcome was defined as mrs of 0 to 2 and a poor outcome as mrs of 3 to 6 . No difference in overall medical management and general care in the acute period was observed between the two groups . All statistical analyses were consulted to a biostatistician and performed using spss 22.0 (spss inc ., variables are expressed as the mean sd, or the number of patients (%), as appropriate . Student's t - tests were used for numeric variables, and fisher's exact tests for nominal variables . Logistic regression analysis was performed on variables with an unadjusted effect and a p - value <0.10 in univariate analysis to determine risk factors associated with poor outcomes in the aed group . A p - value <0.05 for a 95% confidence interval (ci) was considered statistically significant . The clinical characteristics and outcomes of the 84 patients according to the use of prophylactic aed are summarized in table 1 . The mean age was 52.6 13.3 years (range: 25 - 86 years). Age, gender, underlying disease, and aneurysm profiles were not statistically different between the two groups . There was no occurrence of seizure episodes in any of the included patients during admission or follow - up . Treatment modality differed significantly between the two groups; prophylactic aed was used more frequently in patients who underwent microsurgery (84.1%) compared to those who underwent endovascular surgery (15.9%, p <0.001). Aneurysm shape was also found to differ between the two groups (p = 0.032) because all dissecting aneurysms were treated by endovascular surgery . A total of nine systemic complications occurred with higher frequency (20.5%) in the aed group compared to the no aed group (5.0%). No identifiable psychiatric problem, delirium, or hepatic dysfunction was observed even in patients treated with aed . Regardless of prophylactic aed use, there was no occurrence of seizure episodes in any patients (hh grade 1, 2, or 3) with a sah during six months of follow - up . No statistical difference in clinical outcomes at discharge and after six months of follow - up was observed between the two groups (p = 0.607 and 0.178, respectively). At discharge, 38 (86.4%) of 44 patients in the aed group showed a favorable outcome with one death due to pneumonia sepsis whereas 36 (90.0%) of 40 patients in the no aed group showed favorable outcomes without mortality (fig . 1). After six months of follow - up, patients in the aed group did not show improved clinical outcomes . However, favorable outcomes of patients in the no aed group showed a slight increase to 95.0% and poor outcomes decreased to 5.0% . Analysis of risk factors associated with poor outcomes in the aed group is shown in table 3 . Logistic regression analysis showed that hydrocephalus (odds ratio [or] = 14.286; 95% ci, 1.277 to 166.67; p = 0.031) and symptomatic vasospasm (or = 9.615, 95% ci, 1.088 to 83.333; p = 0.042) were independently associated with poor outcomes in the aed group . In the current study, seizure did not occur in asah patients with a good clinical grade during admission or follow - up regardless of prophylactic aed use . At our institution, we tended to use prophylactic aed for patients who underwent microsurgery and patients with ruptured aneurysms of the anterior cerebral artery . However, our findings indicate that treatment with aed had no effect on seizure prophylaxis . A number of retrospective series similar to ours have demonstrated no significant difference in terms of seizure outcome between groups with or without prophylaxis.3)6)9)15)18) in an analysis of four large trials, patients who received aed had an odds ratio of 1.56 for worse outcomes after three months of follow - up, as well as increased risk for vasospasm, neurological deterioration, cerebral infarction, and elevated temperature during hospitalization.18) in the current study, the clinical outcomes at discharge and after six months of follow - up were not statistically different between the two groups (p = 0.607 and 0.178, respectively). At discharge, 86.4% of patients in the aed group showed a favorable outcome, but there was one death due to pneumonia . In the no aed group, 90.0% of patients showed favorable outcomes without mortality . However, after six months of follow - up, favorable outcomes of patients in the no aed group showed a slight increase to 95.0% and poor outcomes decreased to 5.0% . In clinical practice, aed usage for patients with asah is generally accepted for prevention of seizure episodes . According to the current guidelines,8)22) however, prophylactic aed may be considered for patients with known risk factors for seizure, one of which is poor clinical grade (hh grade 4 or 5) on initial presentation . Unlike asah with a poor clinical grade, patients with a good clinical grade (hh grade 1, 2, or 3) had relatively lower risk of developing seizure due to aneurysm rupture . In addition, routine use of aed in those patients has recently come under question.21) as reported in a systematic review, the rate of early postoperative seizure was 2.3% and the rate of late postoperative seizure was 5.5%.20) the average time to late seizure was 7.45 months . Patients who experienced a late seizure were more likely to undergo repair with microsurgery than endovascular surgery . They concluded that routine perioperative aed usage did not seem to prevent seizures after asah . However, it should be noted that seizures might occur years after asah and careful monitoring for late complications remains important . In the current study, we realized that we tended to use prophylactic aed for patients who underwent microsurgery . In the international subarachnoid aneurysm trial (isat), patients treated with microsurgery had a significantly higher risk of late seizure than those treated with endovascular surgery.17) in a survey conducted by the american association of neurological surgeons, 24% of neurosurgeons surveyed routinely prescribed aed for three months after asah regardless of whether seizures occurred at presentation, in the hospital, or not at all.2)6)10)12) however, in a systematic review there was no significant difference in the incidence of early and late seizure between patients treated with aed (3.0% and 5.9%, respectively) and those not treated with aed (2.2% and 6.3%, respectively, p> 0.99).20) from the risk factor analysis for poor outcomes in the aed group, hydrocephalus and symptomatic vasospasm were found to be were well - known and understandable risk factors . Although the adverse effects of aeds have not been well characterized in patients with asah, adverse effects due to phenytoin were reported in 21.4% of patients.20) we used levetriacetam or valproic acid, and fortunately did not experience adverse effects . First, it was a retrospective study based on our prospectively collected database with a small number of enrolled patients second, we did not evaluate electroencephalograms (eeg), thus we could not determine the occurrence of non - convulsive seizures . A previous study indicated that the incidence of non - convulsive seizure from intracranial hemorrhage is approximately 38%.7) however, as we enrolled only conscious patients with a good clinical grade, non - convulsive seizures would likely have been detected clinically . Third, clinical outcomes of both groups might be affected by treatment modality, which determined aed administration in our practice . The use of anticonvulsants could decrease intelligence . In patients with asah, however, evaluation of cognitive function might be incomplete, time - consuming, and lack specificity . No difference in clinical outcomes and systemic complications at discharge and after six months of follow - up was observed between the two groups . However, favorable outcomes in the no aed group showed a slight increase after six months . Our findings suggest that discontinuation of the current practice of prophylactic aed use might be recommended in patients with a good clinical grade.
A 20 year old boy presented to our department with decreased vision, redness and watering of both eyes following electrocution with overhead transmission wires six days ago . He had multiple eschars on the back, scalp and arms along with facial erythema [fig his best - corrected visual acuity was light perception in the right eye and counting fingers in the left eye with normal intraocular pressures . Anterior segment examination showed bilateral circumcorneal congestion, sluggish pupillary reactions, superficial punctate keratitis and cataract in the left eye . Fundoscopy revealed an area of peripapillary retinal opacification and a dull foveal reflex in both eyes [figs . 2 and 3]. Fundus fluorescein angiogram showed a normal retinal circulation with late hyperfluorescence corresponding to the area of opacification . Optical coherence tomography (oct) showed retinal thickening with few cystoid spaces in the right eye [fig . Similar changes were seen the left eye (not shown). Patient was started on oral corticosteroids (prednisolone 30 mg / day) and acetazolamide (750 mg / day in three divided doses). Steroids were given to control inflammation arising from the injury, if any, and oral carbonic anhydrase inhibitor were added to reduce cystic fluid collection by possible stimulation of the retinal pigment epithelium (rpe) pump . Acetazolamide was withdrawn after 1 month and oral steroids were discontinued after another month by gradual tapering . One month later, his best corrected visual acuity was counting fingers 1 meter in the both eyes with posterior subcapsular cataract and peripapillary retinal atrophy in both the eyes [fig . 5]. Oct of both the eyes showed retinal atrophy with disorganization of all retinal layers and the rpe [fig . Patient underwent cataract surgery after 6 months from the time of injury in both the eyes with no improvement in the visual acuity . Photograph of the face of the patient in the study following electrical burn showing eschars, erythema and facial erythem fundus photograph of the right eye of the patient in the study at presentation showing areas of peripapillary retinal opacification with a dull foveal reflex fundus photograph of the left eye of the patient in the study at presentation showing areas of peripapillary retinal opacification with a dull foveal reflex optical coherence tomography of right eye of the patient in the study at presentation showing presence of retinal thickening and few cystic spaces . The retinal thickening is predominantly nasal with disruption of the inner segment /outer segment (is / os) junction and retinal pigment epithelium (rpe). Localized areas of posterior vitreous detachment can be observed fundus photograph of both eyesof the patient in the study taken at 1 month following injury showing almost complete resolution of retinal opacification with appearance of retinal atrophy in both eyes . The area of retinochoroidal atrophy can be seen as patches of pigmented and depigmented retina . Right eye shows pallor of the optic disc and attenuation of inferior peripapillary retinal vessels is noted optical coherence tomography of right eye of the patient in the study at one month following injury showing retinal atrophy with disorganization of retinal layers and clumps of photoreceptor cells (left eye not shown) optic nerve is very good conductor of electricity, with the nerve tissue conducting electricity as any metal rod . The retina usually suffers thethermal effects of injuries in the form of immediate coagulation of proteins and cells, and thus the initial whitening / opacification of the retina can be explained . The absorption of energy by the rpe results in retinal damage which was evident in the peripapillary and macular area in our patient . A normal fluorescein angiogram indicates that the retinal opacification was not as a result of ischemia, but was due to the thermal coagulation of the retinal tissue . According to a recently published report, peripapillary meridional changes in the eye have been noted in a patient suffering from electric injury . Our case illustrates that initial acute response following electric shock injury is the retinal opacification with increased retinal thickness, that subsequently progresses to retinal atrophy.
Graphene electrochemistry has gained paramount interest owing to its unique role in energy conversion and storage devices such as fuel cells, water splitting cells, and supercapacitors . One of the most extensively studied electrocatalytic applications of graphene is the oxygen reduction reaction (orr) occurring at the cathode of fuel cells and metal - air batteries, in which advanced metal - free graphene - based electrocatalysts are considered as promising alternatives to the state - of - the - art precious pt catalysts due to their low cost, fuel tolerance, and long - term durability . Tremendous efforts have been undertaken, both on the experimental and theoretical level, to improve the orr performance of graphene - based materials by tuning their electronic properties through doping nonmetallic heteroatoms into a graphene matrix . However, the key orr activity properties (e.g., exchange current density, on - set potential, 4e pathway selectivity, and kinetic current density) of metal - free materials are still incomparable with those of pt - based catalysts, which stimulates the ongoing debate on whether graphene is indeed an efficient catalyst for orr . This issue is unresolved, largely due to the lack of knowledge on the origin of graphene activity toward orr and the effect of the doping of nonmetallic heteroatoms on the graphene s (electro)chemical properties . In other words, understanding the nature of the orr process on the surface of doped graphene and recognizing the origin of its electrocatalytic activity open a new era in the design of graphene - based materials with superior electrocatalytic activity toward orr . In general, monitoring the reaction intermediates formed on the surface of catalysts represents a possible platform for understanding the pathway and mechanism of orr electrocatalysis; however, it is difficult to observe in situ these adsorbed species due to their extremely short lifetimes . Importantly, the development of computational quantum chemistry provides a feasible methodology to predict the possible intermediates formed during the orr process and to evaluate their stabilities on the surface of catalysts in terms of the adsorption free energies . For example, a theoretical methodology has been developed for studying orr on a wide variety of metal catalyst surfaces, and the free energy diagram for this reaction was constructed together with a volcano - shaped plot that correlates the apparent electrocatalytic orr activity with inherent oxygen adsorption strength . Consequently, various pt and nonprecious metal alloys with superior experimentally measured orr activities comparable to that of a pure pt catalyst were designed and developed guided by the prediction of this theoretical methodology . However, such advanced methodology has not been applied to metal - free catalysts such as the most popular graphene - based catalysts; whether these metal - free counterparts can possess similar catalytic behavior or be even more active than metallic electrocatalysts is unknown from both theoretical and experimental viewpoints . Here, we extend for the first time the aforementioned methodology used for metal catalysts to metal - free systems by exploring the relationship between the experimentally measured electrocatalytic orr performance and the theoretically predicted free energy of reaction intermediates for a series of graphenes doped with nonmetal elements . The starting point of this methodology is the construction of the orr free energy diagrams for various doped graphene models by density functional theory (dft) calculations . Next, the orr exchange current density, the on - set potential, and the reaction pathway selectivity are theoretically predicted on the basis of the free energy diagrams for each model catalyst . Also, all these descriptors of the orr activity are obtained on the basis of the electrochemical voltammograms measured for the synthesized catalysts that correspond to the models studied . The origin of the oxygen reduction activity of the graphene - based catalysts studied is explained in - depth in terms of the molecular orbital theory . By combining experimental and theoretical data, it was possible to predict for the first time the electrocatalytic orr performance of an ideal graphene - based catalyst (x - graphene) which possesses a 2.1 10 a / cm orr exchange current density, a 0.33 v on - set potential (vs normal hydrogen electrode, nhe), and a nearly 100% 4e pathway selectivity; these values are comparable with or even better than those of the state - of - the - art pt catalyst . This combined computational and experimental study reveals the nature of orr electrocatalysis for graphene - based materials and paves the way to the molecular design of more highly efficient metal - free electrocatalysts for applications beyond orr . To study the effect of different dopants on the orr activity of graphene, we designed and synthesized nitrogen (n)-, boron (b)-, oxygen (o)-, sulfur (s)-, and phosphorus (p)-doped graphenes, respectively, as well as undoped graphene . All graphene - based samples were synthesized from chemically exfoliated graphene oxide (go) prepared by a slightly modified hummer s method from graphite . After dialysis for one week, the graphite oxide dispersion was diluted, exfoliated, and centrifuged with the resulting go concentration of 0.5 mg / ml . Graphite powder was considered as a representative of pure graphene (g) without any heteroatom doping . Oxygen - doped graphene (o - graphene), which can be considered as reduced go, was synthesized by thermally reducing initial go powder at 900 c in flowing ar for 3 h without any additional precursor . Due to the strong oxidation process preceding chemical exfoliation of graphite oxide, the latter possesses many oxygen - containing functional groups . During high - temperature thermal reduction (900 c in ar atmosphere), most of the oxygen species are removed (oxygen concentration decreases from 50 atomic% in pristine go to 5 atomic% in o - graphene); only four relatively stable species are retained . Nitrogen-, boron-, sulfur-, phosphorus - doped graphenes (n - graphene, b - graphene, s - graphene, p - graphene) were produced by annealing go powder with various heteroatom - containing precursors (the mass ratio of the initial go to the respective precursor = 1:10) at 900 c in flowing ar for 3 h. melamine (c3h6n6), boron oxide (b2o3), benzyl disulfide (c6h5ch2ssch2c6h5), and triphenylphosphine ((c6h5)3p) were used as the sources of n, b, s and p elements, respectively . The x - ray photoelectron spectra (xps) were measured on a kratos axis ultra x - ray photoelectron spectrometer equipped with a 165 mm hemispherical electron energy analyzer . The incident radiation was monochromatic al k x - rays (1486.6 ev) at 225 w (15 kv, 15 ma). The survey scans were taken at the analyzer pass energy of 160 ev, and the multiple high - resolution scans were recorded at 20 ev . Transmission electron microscope (tem) imaging was conducted on a fei tecnai g2 spirit tem at a voltage of 120 kv . Nitrogen - sorption isotherms were collected on a tristar ii, micromeritics nitrogen sorption analyzer at 77 k. prior to each measurement, the samples were degassed at 150 c for at least 10 h. the brunauer teller (bet) specific surface area was calculated using adsorption data at a relative pressure range of p / p0 = 0.050.25 (figure s1, supporting information [si]). Raman spectra were recorded on a horiba labram with 514.3 nm ar laser (figure s2, si). All electrochemical measurements were performed using the same mass of catalyst (0.2 mg / cm). Linear sweep voltammograms (lsv) were recorded using a glassy carbon rotating disk electrode (rde, 0.196 cm, pine research instrumentation, u.s.a .) With a scan rate of 5 mv / s or a rotating ring - disk electrode (rrde, 0.283 cm, pine research instrumentation, u.s.a .) With a scan rate of 2 mv / s . The data were recorded using a chi 760 d potentiostat (ch instruments, inc . The reference electrode was an ag / agcl in 4 m kcl solution (all potentials were referenced to nhe by adding a value of 0.205 v) and the counterelectrode was platinum wire . The kinetic current for orr occurring on the electrode can be calculated from the intercept of koutecky levich plot using the following equation (figures s3 and s4, si):1where jk is the kinetic current density at a constant potential, jd is the measured current density on rde, is the electrode rotating speed in rpm, and b, the reciprocal of the slope, could be determined from the slope of koutecky n is the number of electrons transferred per oxygen molecule, f is the faraday constant, do2 is the diffusion coefficient of o2 in 0.1 m koh, v is the kinetic viscosity, and co2 is the bulk concentration of o2 . The constant 0.2 is adopted when the rotating speed is expressed in rpm . The overall electron transfer numbers per oxygen molecule involved in a typical orr process were determined on the basis of rrde voltammograms recorded by using a rrde configuration with a 320 m gap pt ring electrode . The disk electrode was scanned cathodically at a rate of 2 mv / s, and the ring potential was constant at + 0.5 v for oxidizing any ooh intermediate . The electron transfer number (n) and ooh intermediate production percentage (% ooh, which serves as 2e pathway selectivity) were determined as follows (figure s5, si):34where i d is the disk current, ir is the ring current, and n is the current collection efficiency of the pt ring, which was determined to be 0.37 . The tafel analysis of the orr polarization was evaluated by using the relation between the kinetic current density (jk) and overpotential (= unhe 0.455) as:5where a (v) is the tafel constant related to the exchange current density (j0) and b (v / dec) is the tafel slope . A more complete version of this equation can be derived by simplifying the butler volmer equation as:6where r is the ideal gas constant, is the transfer coefficient, n is the number of electrons transferred, and j0 is the exchange current density . Combination of eq 6 with eq 5 gives the following expression for calculation of the experimental j0 (table s1, si):7 the electrocatalytic active sites and the pathways of orr on the surface of various electrocatalysts were examined on the basis of hybrid dft calculations performed by using gaussian 09 program . All the calculations were carried out using ub3lyp/6 - 31g(d, p) level of theory with all atoms fully relaxed . The solvent (water) effect was considered by the polarizable continuum model (pcm). Only intermediate states in the orr process, as well as the reactant and product states, were proposed and evaluated; extra energy barriers might exist but were not considered due to the unbalanced electron numbers borne by different states . The calculation of the free energy diagrams was performed by setting up the reference level as that of the reaction product (table s2, si), and the reference electrode was set up to be the nhe; at ph = 0 and 0 v vs nhe, reaction (h + e) 1/2 h2 is at the equilibrium under standard conditions . The free energies of reactants and each intermediate state at an applied electrode potential u were calculated as follows: g(u) = g neu, where n is the electron number of such state and g is the free energy obtained by frequency calculations at room temperature (298.15 k) after geometry optimization . Hence, the equilibrium potential u for orr (eq 8) at ph = 13 was determined to be 0.455 v vs nhe where the reactant and product are at the same energy level . The free energy of h2o(l) was derived as gh2o(l) = gh2o(g) + rt ln(p / p0) since only gh2o(g) can be directly obtained by dft calculations, where r is the ideal gas constant, t = 298.15k, p = 0.035 bar, and p0 = 1 bar . The free energy of o2(g) was derived as go2(g) = 2gh2o(l) 2gh2 4.92 ev since the high - spin ground state of oxygen molecule is notoriously poorly described in dft calculations . The free energy of oh was derived as goh = gh2o(l) gh+, where gh+ = 1/2gh2 kbtln 10 ph (kb is boltzmann s constant). The overall reaction scheme of o2 reduction to oh in alkaline environment is:8with three possible reaction pathways (one dissociative and two associative) as demonstrated in scheme s1, si . Specifically, since the surface of a doped graphene features the relatively high energy barrier (> 1.2 ev) in the dissociative pathway (figure s7, si), the following associative mechanism is dominant and considered in our calculations:9a9b9c9dwhere * refers to a given atom in the specific graphene model (i.e., possible active site). Calculation of the current density at low overpotentials includes the following assumptions: (1) the amount of active sites on different graphene surfaces is the same; (2) the rate constant represents the upper bound of the overall reaction rate; if there are additional barriers to ooh * formation / oh * desorption, the overall reaction rate should be lower . According to reference (36), the exchange current density for a certain electrocatalytic process can be theoretically calculated as follows:10where n is the electron transfer number, f is faraday s constant, k is the standard rate constant, ctotal is the total number of active sites, is the transfer coefficient (a measure of the symmetry of the potential energy surface, ranging from 0 to 1), and is a quantity related to the highest free energy change of the whole reaction (see si eqs s59, figure s8 for a detailed derivation of this equation and parameter fitting). We selected five nonmetallic elements (b, n, p, o, s) with various electron negativities to dope graphenes to obtain single - doped graphene materials . All the doped graphene electrocatalysts were chemically prepared from go by using appropriate doping procedures (see the methods section). In all five resultant samples (b - graphene, n - graphene, p - graphene, o - graphene, s - graphene) the nanosheet morphology of the pristine go was well preserved without noticeable residues of the solid precursors, as shown in the transmission electron microscopy (tem) images (figure 1). Note that the incorporation of different heteroatoms could not change the physicochemical properties of doped graphene samples such as morphology, surface area, and defects . Nitrogen - adsorption studies indicate that all samples show similar surface areas in a range between 100 and 150 m / g, which also assures similar concentrations of the orr active sites present on the samples studied (considering that the concentrations of all doped heteroatoms are also similar, between 3 to 5 atomic% based on the xps survey results; not shown here). The raman results indicate that all samples have similar concentrations of defects with the id / ig values in a range of 1.01.2, which are closely related to the electrical conductivity of these samples . Therefore, it is presumed that the differences in the electrocatalytic orr activity of various doped graphene samples do not originate from their physicochemical properties but only from the nature of the dopant affecting their orr activities . The nature of doping was investigated by analyzing high - resolution xps spectra shown in figure 2 . Peak deconvolutions were conducted as in the case of the reported respective single - doped graphenes . The five heteroatoms chemically substituted edge or central carbon atoms in the graphene matrix, yielding 13 different species in- or out - of the graphene basal plane . Specifically, natural graphite was considered as a defect - free (nondoped) graphene with an intensive typical sp - hybridized carbon (figure 2a). B - graphene has two boron containing species as central b-3c and edge b-2c o species (excluding b2o3 precursor residue) (figure 2b). N - graphene has three nitrogen species as central graphitic nitrogen and edge pyridinic and pyrrolic n in the graphene plane (figure 2c). O - graphene has two kinds of oxygen species as in - plane central pyran - type oxygen, edge carbonyl and hydroxyl oxygens, and out - of - plane epoxy oxygen (figure 2d). P - graphene and s - graphene both have one heteroatom doping configuration as p3c(o)-type phosphorus (excluding ph3p precursor residue) (figure 2e) and edge c s c sulfur (figure 2f), respectively . The inset values represent the id / ig ratios and the specific surface areas obtained from raman spectra and nitrogen adsorption isotherms, respectively for each sample (see figures s1 and s2, si for details). High - resolution xps spectra of different heteroatoms in doped graphenes: (a) graphite; (b) b - graphene; (c) n - graphene; (d) o - graphene; (e) p - graphene; (f) s - graphene . We constructed the cluster models (figure s6, si) for the aforementioned doped graphenes according to each heteroatom s chemical environments obtained from the xps spectra discussed above . H bonds, which have been previously adopted for investigation of orr on graphene doped with nitrogen or boron . Five heteroatoms could induce 13 different doping configurations in graphene clusters with very different electronic properties; concomitantly, 32 possible orr active sites, either heteroatoms themselves or adjacent carbon atoms, were theoretically studied to build the orr free energy diagram for each catalyst by obtaining the free energy for each reaction step . According to eq 9, the associative 4e reaction pathways for each doped graphene at the equilibrium potential u = 0.455 v vs nhe can be illustrated on a free energy diagram as presented in figure 3a . From such diagrams, nitrogen- and boron - doped graphene models (gn - g and gb - g) exhibit the lowest overall reaction free energy change at u, suggesting their orr performance is the best from the theoretical viewpoint, which has been confirmed by previous reports and the experiments reported in this work (figure s3, si). Taking the gn - g cluster model as an example (black line), the diagram indicates that the first electron transfer step to form ooh * (eq 9a) is an endothermic reaction with a free energy change geq 9a(u) = 0.70 ev, while the second electron transfer step (eq 9b) to form chemisorbed o * and the third electron transfer step (eq 9c) to form oh * are all exothermic with geq 9b(u) = 0.25 ev and geq 9c(u) = 0.54 ev, respectively . The last electron transfer step reflecting oh * desorption (eq 9d) possesses an easily surmountable free energy difference of geq 9d(u) = 0.09 ev . Therefore, geq 9a(u) is the largest one among the free energy changes of all four reaction steps, which indicates that this step is the most sluggish one and represents the highest resistance for the whole orr . Such trend is true for all other heteroatom - doped graphene models, indicating that the orr rate - determining step for these catalysts is the same . Note that each graphene doped by one heteroatom can result in several different cluster configurations, which possess their own reaction pathways . The lines in figure 3a only represent the model with best performance (i.e., the lowest overall reaction free energy change) among all the investigated cluster models for each dopant . The free energy diagrams for orr on all possible active sites of each model are presented in figures s9s14, si . (a) free energy diagram of different heteroatom - doped graphenes at the equilibrium potential u. (b) the adsorption free energies of intermediates ooh * (gooh ) and oh (goh ) on the investigated sites of different doped graphene models . Blue points were not considered in the linear fitting because normal ooh chemisorption did not occur on the corresponding graphene clusters . The specific values of gooh * and goh * for different models are provided in table s3, si . (c) calculated free energy diagram of the predicted x - graphene at the equilibrium potential; data for gn - g model are also included for the purpose of comparison . Reaction intermediates for each step are shown as inset animation . According to the developed theories for metal surfaces, the orr electrocatalytic activity descriptors (specifically, the exchange current density, on - set potential, 4e pathway selectivity, and kinetic current density) of a given catalyst are governed by the adsorption free energies of orr intermediates including ooh , o , and oh , which are represented by gooh , go , and goh , respectively (equation s1, si). As shown in figure 3b, gooh * for a wide variety of the doped graphene surfaces studied scales roughly with goh , similarly as in the case of metal surfaces . Additionally, the o chemisorption is more complicated than that of oh * and ooh , since it could either form single bonding with the adsorption center on graphene or form epoxy - type bonding; within each bonding type, a linear relationship is also observed (figure s15, si). On the basis of these linear relationships (gooh vs goh * or go ), we can predict the orr free energy diagram of an optimal x - graphene, which is the basis to obtain its corresponding electrocatalytic properties at the macroscopic level . The free energy diagram of x - graphene can be obtained on the basis of the sabatier principle that an ideal catalyst should bind the reaction intermediates not too strongly nor too weakly; therefore, at equilibrium potential u, the optimal overall reaction pathway on an ideal x - graphene should follow the relationship of geq9a(u) = geq9d(u). At the same time, geq9a(u) and geq9d(u) are associated to each other via the linear relationship of gooh and goh * as shown in figure 3b; therefore by obeying these requirements, geq9a(u) for x - graphene is determined to be 0.35 ev, representing the largest free energy change on the overall pathway as shown in figure 3c (red line, see eq s4, si for free energy changes of other steps). This predicted free energy difference is lower than those for all other investigated doped graphene models (for example, gn - g denoted by black line) and is close to that calculated for pt by dft . (a) experimentally determined tafel plots for different catalysts from orr polarization curves shown in figure s3, si, data were collected at rde = 1600 rpm . (b) volcano plot between j0 and gooh * with charge - transfer coefficient = 0.5 (red dashed line). Blue hollow squares are j0 obtained from tafel plots and dft - derived gooh * for each doped graphene catalyst . Gooh * for x - graphene (blue solid square) was obtained from eq s4e, si and its j0 was obtained from eq 10 . The j0 value for pt was also shown by the blue dashed line as a reference . For a given catalyst j0 is defined as the current density at the equilibrium potential in one direction for a given reaction, which reflects the intrinsic catalytic activity of the catalyst . The measured exchange current densities, j0, for each synthesized graphene surface can be obtained from the respective tafel plot as shown in figure 4a (specific values could be found in table s1, si). Simultaneously, knowing the free energy diagram for each graphene model, the theoretical exchange current density, j0, can be calculated by using a microkinetic model with one prefactor fitted from j0 by eq 10 . The predicted j0 values for various graphene models form a volcano - shaped plot versus gooh * (figure 4b red line), while j0 for each synthesized sample perfectly follows the trend of this plot (blue squares), similarly as in the case of metal surfaces . Additionally, due to the weak binding of ooh * on graphene - based surfaces, the calculated points are all on the right branch of the volcano plot, while an optimal catalyst should possess a higher j0 induced by a gooh * closer to the volcano center . Following such trends, the j0 value for an ideal x - graphene should be located at the summit of the volcano, with a calculated value of 2.12 10 a / cm, which is even 5 times higher than that of pt / c catalyst at same testing conditions (figure s4c, si). Due to the existence of a high free energy difference for the first electron transfer step geq (9a)(u) as shown in figure 3a, the initialization of oxygen reduction requires a potential bias = u u to reduce the free energy difference to geq9a(u) that could be overcame at room temperature . Taking chemically synthesized n - graphene as an example, the measured on - set potential un = 0.029 v vs nhe (from the polarization curve shown in figure 5a). From the theoretical viewpoint (gn - g model), under this potential un, the free energies of the reactant and intermediates states (figure 3c black line) shift upward as compared to those under the equilibrium potential (figure 5b red line); as a result, geq9a(un) reduces to an easily surmountable value of 0.26 ev . In the zone of u> un, geq9a(u) keeps decreasing with increasing u, which indicates the first electron transfer step is no longer the rate - limiting step for the overall reaction, i.e. The whole orr process is activated. A similar trend was also observed for other graphene models as demonstrated in figures s9s14, si . (a) enlarged lsvs plots at the orr initial region for different catalysts on rde at 1600 rpm in an o2-saturated 0.1 m solution of koh . Inset illustrates the first electron transfer step that is o2 to adsorbed ooh. (b) potential corrected free energy diagram for gn - g at experimentally observed on - set potential un (red) and theoretically predicted un and un which meet geq9a(un) = 0.43 ev and geq9a(un) = 0.22 ev, respectively (blue). (c) experimentally derived on - set potentials of doped graphenes (red squares), and the predicted values (blue bars). Theoretically, the on - set potential for a given graphene model can be thermodynamically predicted from its free energy diagram at equilibrium potential by eqs s23, si . First, a range for geq9a is defined as [0.22 and 0.43] ev on the basis of classical reaction thermodynamics (table s4, si). It is assumed that, if geq9a at a given electrode potential possesses a value within this range, the free energy difference can be overcome . The obtained theoretical on - set potentials that satisfy 0.22 ev geq9a 0.43 ev as well as the experimentally determined ones for all chemically synthesized graphenes are shown in figure 5c . Most of the experimentally obtained values (red squares) are in the theoretically predicted range (blue bars), except for s - graphene and pure graphene samples (slightly above the theoretical range), which could be attributed to the influence of nafion and glassy carbon working electrodes on the overall current density . Furthermore, based on the same criterion, the predicted on - set potential for x - graphene that corresponds to geq9a = 0.43 ev was 0.33 v, which is closer to the orr equilibrium potential than the experimental value obtained for pt catalyst . Theoretically, as demonstrated in scheme 1 in si, orr can proceed either by two sequential two - electron reactions (2e) with formation of ooh intermediate or a more efficient direct four - electron reaction (4e). Therefore understanding the nature of the 2e pathway is essential to design more appropriate catalysts for orr that possesses high 4e pathway selectivity to enhance the electrocatalytic efficiency . In alkaline solution, the mechanism of 2e pathway is:11a11b according to previous studies, 2e and 4e pathway selectivity is associated with the desorption of ooh and oh * on a metal surface, respectively; therefore, at a given potential u, the probability of 2e reaction pathway depends on the value of geq 11b(u) in comparison to geq 9d(u). Taking gn - g model as an example (figure 6a), at equilibrium electrode potential for 2e pathway u2e (0.08 v vs nhe at ph = 13), the desorption of ooh * (eq 11b) is exothermal with an energy difference of geq 11b(u2e) = 0.16 ev, which means 2e pathway is unavoidable to compensate the 4e pathway on gn - g model . Experimentally, we used the rotating ring - disk electrode (rrde) technique to verify this theoretically derived pathway selectivity prediction by monitoring the formation of intermediate peroxide species (e.g., ooh in the alkaline solution) under different electrode potential as shown in figure 6b . The measured electron - transfer number at u2e was 3.35 for chemically synthesized n - graphene; it corresponds to a mixed 33% 2e pathway selectivity and 67% 4e pathway selectivity as shown in figure 6c . Also note that at more negative electrode potentials, 2e pathway is always unavoidable due to the negative geq 11b, which is consistent with the rrde observation (figure 6c). Additionally, the same property (mixed 2e and 4e pathways for orr) has been observed for all other chemically synthesized graphene catalysts . To fundamentally eliminate the 2e pathway, a catalyst surface that binds ooh * strongly enough to induce an endothermic ooh * desorption process this criterion is met by the ideal x - graphene model, as shown by its free energy diagram at u2e in figure 6a (red line): at the equilibrium electrode potential for 2e pathway u2e, ooh * desorption on x - graphene is endothermic while oh * desorption is exothermic, which therefore avoids the 2e reduction pathway to present a nearly 100% 4e pathway selectivity, similar as in the case of pt catalyst . Jk is the cathodic oxygen reduction current under the kinetic limitation zone when the reactant mass transfer is efficient enough to keep the concentration of o2 at the electrode surface equal to the bulk value, which represents the orr kinetic electrochemical property at a given electrode potential (always more negative than orr on - set potential). Experimentally, for n - graphene (figures 7a, b) and other chemically synthesized graphenes (figure s3, si), jk was measured by rotating disk electrode (rde) voltammogram with different rotating speeds followed by performing koutecky levich plot as presented in eqs 1 and 2 (see the methods section). (a) free energy diagrams of 2e orr pathway for gn - g model (black) and x - graphene (red) at equilibrium electrode potential for 2e pathway u2e = 0.08 v vs nhe . Under this potential, the rate - limiting step of orr on gn - g is the activation of o2 to ooh , the atomic configuration of which is shown in figure 5a inset, whereas that for x - graphene is the reduction of ooh to ooh, which atomic configuration is shown in the inset of this figure . (b) experimental ring and disk currents for the synthesized n - graphene catalyst on rrde at 1600 rpm in an o2-saturated 0.1 m solution of koh . (c) rrde measured electron transfer numbers (black) and corresponding 2e pathway selectivity (blue) for n - graphene catalyst according to eqs 3 and 4 (a) electrochemically measured lsv of n - graphene catalyst at different rotating speeds in an o2-saturated 0.1 m solution of koh . Inset illustrates the last electron transfer step: oh * desorption to generate oh . (b) koutecky levich plots for n - graphene at 0.3 v, 0.2 v, and 0.1 v vs nhe, data were collected from panel a. (c) potential corrected free energy diagram for gn - g and x - graphene models at u = 0 v (u = 0.77 v). (d) the relationship between goh * and jk for various synthesized graphene catalysts under different potentials (open symbols), data were collected from figure s3, si . The values in parentheses of the legend are jk values for x - graphene, which are predicted by extending the fitted lines to goh * = 0.10 ev, shown as closed symbols . From the theoretical perspective, the above electrochemical measured jk values can be related to the free energy change of the last electron transfer step (goh ) by a linear relationship shown in figure 7d . The microkinetic scenario of this relationship might be due to the fact that the last step in the reaction pathway, oh desorption as shown in the inset of figure 7a, now becomes the rate - determining step of the whole orr as shown in figure 7c . On the basis of this trend, the predicted jk for x - graphene can be obtained from the value of the extended fitted lines at goh * = 0.10 ev (obtained by eq s4, si), as shown in figure 7d closed symbols, which is 6.01 ma / cm at 0.3 v, comparable to pt catalyst s value (7.35 ma / cm at 0.3 v, calculated from pt s lsv plots in figure s4c, si). It should be noted that these predicted jk values for x - graphene were obtained on the basis of a mixture of 2e and 4e orr pathways (since the experimentally observed jk for line fitting all contain 2e pathway as shown in figure 6c and figure s5, si), which could be further enhanced if only 4e pathway exists like in the case of pt catalyst . On the basis of the former theoretical and experimental observations of j0, u, 4e pathway selectivity, and jk for various graphene - based catalysts, we found that all these electrochemical quantities relate well to the binding strength of intrinsic oxygen - containing intermediates (adsorbed species) on the catalyst surface . Inspired by the success of the d - band center theory that the energy level of a metal atom s d - band center serves as the activity descriptor for metal surfaces, we searched for a simple activity descriptor suitable for metal - free catalysts that could accomplish a similar correlation between the binding strength and each orr active atom s molecular valence orbital levels . We first investigated the origin of the binding strength for different graphene cluster models via natural bond order (nbo) analysis to explore the orbital information of each active site . Since the valence orbital of each active center participates in the bond formation with an oxygen - containing intermediate on the graphene surface (e.g., oh ), the valence orbital level should greatly influence its adsorption energy goh. Therefore, we introduced a descriptor ediff which is defined as the difference between lowest valence orbital energy of the active center and the highest valence orbital energy of the entire graphene cluster (fermi energy level in the form of natural atomic orbitals) to quantitively represent the valence orbital level . As shown in figure 8a, goh * data plotted against ediff formed a linear relationship for a wide variety of graphene active sites . The principle that underlies this linear relationship is that the valence band (v) of the active sites hybridizes with the bonding () orbital of the adsorbed species to form bonding (v-) and antibonding (v-) * states, as illustrated in figure 8b . For the investigated graphene models, the (v-) state is full, while the filling of (v-) * state depends on the valence orbital levels of the active atom on the graphene surface . An increased filling of the antibonding (v-) * state, induced by a lower valence band, could lead to destabilization of the graphene adsorbate interaction and hence diminish the binding between them; on the other hand, a decreased filling of (v-) * state corresponds to an enhanced binding between orr intermediates and the graphene surface . As a result, a better graphene - based orr catalyst such as x - graphene should possess higher valence orbital energies of the orr active atom to induce a smaller ediff and lead to stronger adsorption of ooh * and oh * intermediates, as marked in figure 8a, resulting in better orr activity . Such an x - graphene could in principle be realized by doping with multiple elements, introducing structural defects, or any possible combination . (a) the relationship between goh * and ediff; data were collected for the most active site of various doped graphene models and labeled according to the corresponding molecular configurations shown in figure s6, si . (b) scheme of orbital hybridization of valence band from active sites and adsorbates bonding orbital . In summary, by combining experimental data and dft calculations, we systematically investigated the nature and origin of orr activity of a series of heteroatom - doped graphene catalysts . Although the stability of each model, the electric double layer effect, and hydrogen bonding were not considered in the quantum chemistry study, our models are in good accordance with the experimental observations, from the viewpoint of the orr exchange current density, on - set potential, pathway selectivity, and the kinetic current density . This agreement also validates the predictive capability of the powerful dft model employed beyond traditional metallic catalysts . Our study shows further that graphene - based metal - free catalysts possess the potential to surpass the orr performance of the state - of - the - art pt catalyst . Using orr as a probe reaction, the explored methodology should be applicable to other energy - related electrocatalysis processes such as oxygen evolution and hydrogen evolution reactions.
Advances in modern imaging technology and the widespread implementation of conventional imaging modalities, such as ultrasonography (usg), computed tomography (ct), and magnetic resonance imaging (mri), have led to an increase in the detection of incidental renal neoplasms (1, 2). Consequently, the incidence of benign renal masses has increased along with the increasing incidence of renal cell carcinoma (rcc) because current imaging and biopsy techniques cannot predict the histological features of renal tumors with complete accuracy (1, 3, 4). In contemporary practice, a number of western studies have reported a 13%-25% incidence of benign renal lesions at nephrectomy (3, 5 - 11), although data on asian patients are scarce . The few studies conducted with patients in asian countries have reported a lower incidence, 10%-11%, than that reported by western studies (12, 13). However, these studies were limited in that the data were based on relatively small study cohorts, and most studies excluded large masses . The objectives of this study were to determine the incidence of benign renal lesions in a large cohort of korean patients undergoing nephrectomies for image diagnosis of rcc and to investigate the clinical predictors of benign renal tumors . We excluded 451 patients whose operations were recorded as simple nephrectomy, and 220 patients with recurred renal cancer, bilateral disease, metastatic disease, or familial disease . Hence, the medical records of 1,598 eligible patients who underwent consecutive radical or partial nephrectomies for presumed rcc were retrospectively reviewed and analyzed . In the 23 patients with multiple tumors in the same kidney, all patients underwent preoperative contrast - enhanced ct scans, mr imaging, or both for clinical diagnosis and staging, and all lesions included in this analysis were deemed sufficiently suspicious for rcc on preoperative imaging . Since data were collected at a single tertiary referral center, many patients were transferred from primary or secondary care centers after initial ct imaging was conducted . Experienced genitourinary radiologists at our institution reviewed all imaging studies that were performed at outside hospitals . If an image was obscure or of poor quality, we performed an additional imaging study, such as ct, mri, or contrast - enhanced renal usg . When the results of additional imaging revealed a greater chance of a benign lesion, the lesions were closely monitored in most cases . If we performed nephrectomies for such lesions, the operations were recorded as simple nephrectomies and the patients were excluded . Renal lesions were classified into the following three categories on the basis of the pathologic reports: benign lesion, rcc, or other malignancy . Bosniak category iii lesions were defined as indeterminate cystic masses with thickened irregular walls or septa in which enhancement could be seen, and bosniak category iv lesions were defined as clearly malignant cystic lesions that contained enhancing soft - tissue components . Renal carcinomas were classified according to the recommendations of the 1997 american joint committee on cancer report of rcc classification and the heidelberg classification scheme (15, 16). The data were analyzed as continuous variables and as categorical variables, stratifying tumors 2 cm or smaller, 2 - 4 cm, 4 - 7 cm, or larger than 7 cm . Statistical analyses were performed with spss 17.0 software (spss, chicago, il, usa). Categorical variables were compared using the chi - square or fisher's exact test, and continuous variables were compared using the mann - whitney test with or without bonferroni's correction . All p values were two - sided, and differences were considered statistically significant at p <0.05 . This study protocol was approved by the institutional review board (irb) of the samsung medical center (no . This study protocol was approved by the institutional review board (irb) of the samsung medical center (no . Table 1 summarizes the clinicopathological parameters that were assessed in this study . Among the 1,598 patients, 1,081 patients (67.6%) the mean age was 54.3 yr (range, 10 to 86), and the mean tumor size was 4.8 cm (range, 0.6 to 24 cm). Of the 1,598 renal masses, 1,468 (91.9%) were rcc and 16 (1.0%) were other malignancies . Benign renal lesions were found in 114 (7.1%) patients, including angiomyolipoma (aml) in 47 (2.9%), oncocytoma in 23 (1.4%), complicated cyst in 18 (1.1%), and metanephric adenoma in 6 (0.4%) patients (table 2). Aml constituted 41.2% of all benign lesions; when a tumor was 4 cm or smaller, aml was the most common benign renal tumor, whereas oncocytoma was most prevalent if the tumor was larger than 4 cm (table 2). The proportion of benign lesions was significantly higher in females than in males (13.5% vs 4.1%, p <0.001). The most common presenting symptom was gross hematuria (9.4%), followed by ipsilateral flank pain (8.0%), systemic symptoms including weight loss or severe fatigue (3.4%), and palpable abdominal masses (1.3%). Gross hematuria was significantly more common in patients with malignant lesions (p = 0.026). When renal lesions were stratified according to tumor size, the incidence of benign as opposed to malignant lesions decreased significantly as tumor size increased (p = 0.004) (table 1). In patients with renal masses 2 cm or smaller, 2 - 4 cm, 4 - 7 cm, and larger than 7.0 cm, the incidences of benign lesions were 11.7%, 7.5%, 6.1%, and 3.7%, respectively, showing a significant linear - by - linear association on cochran - armitage trend test (p <0.001). Benign lesions were more common in cystic lesions than in solid lesions (13.6% vs 6.5%, p = 0.002). On multiple logistic regression analysis, female gender, smaller tumor size, and cystic renal lesions were independent predictors of benign histological features (table 3). When 132 cystic renal lesions were classified according to their pathologic results, bosniak category iii lesions had a 31.8% chance of being benign, while bosniak category iv lesions had only a 4.5% chance of being benign (table 4). To our knowledge, this study is the largest contemporary series to evaluate the incidence of benign renal lesions in asian patients . The overall incidence of benign pathological findings in korean patients at surgery was 7.1%, which is lower than the incidences reported in recent studies from western countries (3, 6 - 11). In this respect, it has already been suggested that the low incidence of oncocytomas in asian patients is one reason why asian patients have a lower incidence of benign renal lesions compared to western patients (12). In the present study, aml (2.9%) was the most common benign finding, followed by oncocytoma (1.4%), which was approximately half as common as aml . These results were consistent with the findings of two previous studies showing that oncocytoma was less frequent in asian countries in comparison with a 6%-13% incidence in western countries (12, 13). Notably, the overall incidence of benign lesions in our data was slightly lower than the incidence of benign lesions reported in studies from japan and china (12, 13) we included renal masses larger than 7 cm and excluded a large number of benign cases through multimodal preoperative imaging studies . Studies have shown that patients with larger tumors have a lower incidence of benign lesions (17). In addition, when there was an obscure lesion on a ct image, we did not hesitate to perform additional imaging studies, including another round of ct, mri, or additional contrast - enhanced renal usg, which could improve the characterization of renal tumors (18 - 21). Therefore, a number of benign lesions, such as amls or benign cystic lesions, were excluded . This may explain why we observed a lower incidence of benign lesions compared with those in other east asian series . Many studies have demonstrated that the incidence of benign renal lesions at surgery is higher in female patients (11 - 13). In this respect, the incidence of benign lesions in women was about four times higher than that in men (odds ratio [or] 3.899, 95% confidence interval [ci] 2.62 - 5.81, p <0.001), which could be attributed to the higher incidence of aml in women than in men (6.2% vs 1.4%, p <0.001). Unlike the clear association with sex, however, there is controversy about the relationship between tumor size and pathological results . Studies have tended to show an inverse relationship between tumor size and the incidence of benign lesions on univariate analysis (22), although studies that excluded larger renal lesions did not show a relationship in multivariate analysis (11, 12, 23). By limiting the inclusion criteria to small lesions, the significant relationship between tumor size and pathological outcome disappeared . In contrast, studies that included all renal masses without size limitations exhibited a significant relationship between tumor size and pathological outcome (10). In this study, the incidence of benign lesions was significantly related to tumor size, similar to results reported by frank et al . The incidence of benign lesions was about two times higher in cystic lesions than in solid lesions (or 2.177, 95% when cystic renal lesions were classified according to bosniak classification, 31.8% of category iii lesions and 4.5% of category iv lesions were benign . The incidences of benign lesions were slightly lower than those of previous studies (24, 25). In a comprehensive literature review, warren et al . (26) reported incidences of benign lesions in category iii and iv lesions of 67% and 7.5%, respectively . However, this might also be attributed to more active implementation of additional imaging studies, such as contrast - enhanced usg, at our institution (27). (28) reported that contrast - enhanced usg was better than ct for the diagnosis of malignancy in complex cystic renal masses . In addition, the differences in the definitions of bosniak classifications among centers might influence the results . To our knowledge, our study is the first to report the incidence of benign lesions for presumed rcc in light of specific presenting symptoms . Interestingly, we found no difference between benign and malignant lesions with respect to the following presenting symptoms: ipsilateral flank pain, mass palpability, or systemic symptoms such as weight loss or severe fatigue . Although gross hematuria was more common in malignancy, the significance of this difference disappeared on multivariate analysis . First, its retrospective nature might have skewed our data, although we included a large cohort of patients . Second, as a single - center study, there may be undetected regional and demographic trends that might have influenced the types of patients who came to our institution . Third, we used the final pathological tumor diameter instead of the radiological tumor diameter . A study conducted by schlomer et al . (29) reported that preoperative ct imaging may slightly overestimate the pathological sizes of renal tumors with diameters of 1 - 5 cm . Therefore, to determine the clinical predictors of benign lesions, tumor sizes measured through imaging studies would be more reasonable . However, it is also well known that, for renal tumors, clinical size and pathological size are highly correlated with each other (30). In addition, more than one half of all of the study patients were transferred to our institution after ct or mr imaging was performed at a previous institution . Consequently, there were differences in imaging protocols that might have caused differences in measurements . Furthermore, a considerable time gap intervened between ct scanning and nephrectomy, during which time tumor growth might have occurred . For this reason, it was difficult to use radiological tumor sizes in this study . Finally, the number of partial nephrectomies was significantly higher for benign lesions . However, the reason seems to be that benign lesions were smaller . Despite these limitations, the results of this study may provide valuable information for clinicians counseling patients with renal lesions and may help clinicians decide the most appropriate therapeutic modality, including renal biopsies and close monitoring . In conclusion, a large cohort from a single institution in korea showed a lower incidence (7.1%) of benign renal lesions than those found in western studies . The most common benign lesion was aml, which accounted for 41.2% of all benign lesions . When a tumor was 4 cm or smaller, aml was the most common benign renal tumor, while oncocytoma was the most prevalent when the tumor was larger than 4 cm . Female gender, cystic renal lesions, and smaller tumor size were independently associated with the incidence of benign histological features . Incidence and predictive factors of benign renal lesions in korean patients with preoperative imaging diagnoses of renal cell carcinoma seo yong park, seong soo jeon, seo yeon lee, byong chang jeong, seong il seo, hyun moo lee, and han yong choi the incidence of benign renal masses has increased along with the increasing incidence of renal cell carcinoma because current imaging and biopsy techniques cannot predict the histological features of renal tumors with complete accuracy . In contemporary practice, a number of western studies have reported a 13%-25% incidence of benign renal lesions at nephrectomy, although data on asian patients are scarce . Hence, the present study was performed to determine the incidence and predictive factors of benign renal lesions in korean patients undergoing nephrectomy for presumed renal cell carcinoma on preoperative imaging . The overall incidence of benign pathological findings in korean patients at surgery was 7.1%, which is lower than the incidences reported in recent studies from western countries . Female gender, smaller tumor size, and cystic lesions were significantly associated with benign histological features.

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