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267101528	pes2o/s2orc	v3-fos-license	Systemic Antibiotic Use in Acute Irreversible Pulpitis: Evaluating Clinical Practices and Molecular Insights This scoping review systematically evaluates the use of systemic antibiotics in treating acute irreversible pulpitis, integrating clinical practice patterns with recent molecular insights. We analyzed clinical evidence on antibiotic prescription trends among dental professionals and examined molecular research advancements in relation to pulpitis. This review is intended to bridge the gap between clinical practice and molecular research, guiding more evidence-based approaches to treating acute irreversible pulpitis. Electronic databases were searched for relevant articles published in English based on the objective of the review. A second search using all identified keywords and index terms was undertaken across all the included databases. In addition, a reference list of identified articles was searched. Studies including original research, systematic reviews, meta-analyses, clinical trials, and observational and retrospective studies, all written in English and published from 2010 onwards, were included, and an analysis of the text words contained in the titles and abstracts of the retrieved papers and of the index terms used to describe the articles was performed. A total of N = 53 articles were selected. Altogether, N = 43 (76.79%) articles were cross-sectional studies, N = 4 (11.11%) were systematic reviews, and N = 3 (5.36%) were guidelines. The most frequent level of evidence was level VI (N = 43 (76.79%). The mean percentage of dentists who prescribed antibiotics to treat acute irreversible pulpitis was 23.89 ± 23.74% (range: 0.05–75.7). Similarly, for specialists, it was 22.41 ± 15.64 (range 2.2–50.4), and the percentage for undergraduates was 17.52 ± 20.59 (range 0–62.6). The significant developments in research models for pulpitis research and the characterisation of biomarkers have led to better management strategies. Concurrently, significant advancements in molecular research provide new understandings of pulpitis, suggesting alternative therapeutic approaches. Although there are guidelines available, increased rates of antibiotic prescription are still prevalent around the globe. Introduction The most common sequela of dental caries is 'Pulpitis'.This term is used to describe the inflammation of the pulp.According to the WHO, 60-90% of school children and 98.9% of adults have cavities, leading to pain and discomfort, and dentists come across this problem routinely in dental practice [1].Pulpitis occurs due to various etiological factors or irritants that trigger the inflammatory response in the pulp, resulting in pain.One of the methods employed in emergency care to manage pain due to pulpitis is the prescription of antibiotics.This may provide a temporary solution, but the results are poor.This practice has increased the exploitation of antibiotic prescriptions in dentistry [2]. The prevalence values of pulpitis in India estimated in one study were 60.7%, 68.2%, and 43.8% in molars, premolars, and anterior teeth, respectively [3].In the USA, a total of 403,149 hospital-based Emergency Dental (ED) visits were attributed primarily to pulp in 2006.A periapical abscess without sinus involvement was the most common diagnosis for ED visits (accounting for 79.97% of all ED visits) [4]. In this review, we seek to comprehensively examine the current landscape of systemic antibiotic usage in the management of pulpitis.By exploring the extent of the existing literature, evaluating the evidence for antibiotic efficacy, determining the impact of diverse etiological factors, and identifying recommended diagnostic and treatment approaches, we aim to contribute to a more informed and evidence-based decision-making process for the management of pulpitis. Given the variability existing in the available literature and the controversial nature of systemic antibiotic use in dentistry, we hypothesize that this scoping review will reveal a diverse range of opinions and findings regarding the efficacy of systemic antibiotics in the treatment of pulpitis.Moreover, we anticipate that this review will highlight the influence of etiological factors on the management approach, suggesting that tailored strategies based on clinical presentation and thorough investigation are recommended for optimal diagnosis and treatment. Research Questions: What is the extent of the literature available on the use of systemic antibiotics for pulpitis management? 2. What evidence supports or questions the efficacy of systemic antibiotics in treating pulpitis?3. How is the management of pulpitis influenced by different etiological factors, such as carious and non-carious sources? 4. What approaches are suggested for the optimal diagnosis and treatment of pulpitis considering clinical presentation and appropriate investigation? Search Strategy The search strategy for this scoping review was designed to be as comprehensive as possible, considering time and resource limitations.A three-step approach was followed.A literature search was conducted across various electronic databases, including PubMed, MEDLINE, Cochrane Library, and EMBASE.Keywords used were "pulpitis", "antibiotics", and related terms.The following search strategy was used: ("irreversible pulpitis") OR (pulpitis)) AND (antibiotic).This was followed by an analysis of titles, abstracts, and index terms in retrieved papers.Later, a search using all identified keywords and index terms across all selected databases was conducted.Lastly, the reference lists of identified reports and articles were searched for additional sources.The review includes sources in the English language due to a lack of skill in other languages.A single search strategy was used to search for all types of evidence sources simultaneously, enhancing sensitivity.As the review progressed, additional keywords and sources were identified and incorporated into the search strategy, ensuring transparency and auditability.Collaboration with a research librarian or information scientist was instrumental in designing and refining the search. Inclusion and Exclusion Criteria 2.2.1. Inclusion Criteria The inclusion criteria were as follows: original research articles, systematic reviews, meta-analyses, clinical trials, observational studies, and retrospective studies published in English from the year 2010 to the present.The target population was human participants diagnosed with pulpitis.The included studies had to have investigated the use of systemic antibiotics as part of pulpitis management.The reported relevant outcome for original studies was the percentage of dental professionals prescribing antibiotics to treat irreversible pulpitis.Exclusion Criteria: Studies published before the year 2010, editorials, letters, conference abstracts, case reports, animal studies, in vitro studies, and studies in-volving participants without a confirmed pulpitis diagnosis were excluded.Additionally, studies not focusing on the use of systemic antibiotics for pulpitis management, those reporting irrelevant outcomes, and those published in languages other than English were excluded.The language restriction was applied due to a lack of required interpretation skills.Duplicate publications reporting on the same study, non-peer-reviewed sources, and studies with inadequate data, or an unclear methodology were also excluded from this scoping review. Study Selection Two independent reviewers performed initial title and abstract screening to identify potentially relevant articles.A full-text evaluation followed, adhering to the predefined inclusion criteria.Discrepancies were resolved through discussion and consensus.Studies that aligned with the scope of this review were included for data extraction. Data Extraction A standardized data extraction form was created and piloted to ensure consistency.The information extracted included study characteristics, methodologies, participant demographics, antibiotic interventions, outcomes, and conclusions regarding antibiotic efficacy.Data extraction was performed independently by two reviewers, with any disagreements being resolved through discussion. Data Analysis A narrative synthesis approach was employed to analyze and summarize the extracted data.Themes related to the extent of the literature, evidence supporting or questioning antibiotic effectiveness, the influence of etiological factors on management, and recommended diagnostic and treatment approaches were identified and discussed. Rating the Level of Evidence The rating of evidence in this study was conducted based on a hierarchical approach derived from different types of published papers.This hierarchical approach was designed to assess the strength and reliability of the evidence.The highest level, Level I, was assigned to evidence derived from systematic reviews or meta-analyses that encompassed all relevant randomized controlled trials (RCTs).Level II evidence was drawn from well-designed RCTs, known for their robustness and minimal bias.Level III was assigned to evidence obtained from well-designed controlled trials without randomization, providing valuable data on interventions or outcomes.Level IV encompassed evidence from well-designed casecontrol and cohort studies, contributing to our understanding of causation and associations.Level V was associated with evidence drawn from systematic reviews of descriptive and qualitative studies, offering insights into complex phenomena.Level VI involved evidence from single descriptive or qualitative studies, serving as initial exploratory sources.Finally, Level VII was applied to evidence derived from the opinions of authorities and reports made by expert committees, recognizing the significance of expert judgment in shaping clinical understanding and decision making.This structured approach to rating evidence enabled a comprehensive evaluation of the quality and applicability of the sources included in this study [5]. Levels of Evidence in the Literature Level VI evidence was predominant, accounting for a substantial number of 43 articles, representing approximately 76.79% of the total.Level I evidence, characterized by the highest quality and reliability, was assigned to seven articles, making up 12.50% of the total.Lastly, Level VII evidence, considered to have the lowest level of reliability and quality, was observed in six articles, making up 10.71% of the total.These findings emphasize the prevalence of different types of evidence, highlighting the need for more high-quality research to strengthen the body of evidence in this field (Supplementary Table S2).There were no contributions from Level II, i.e., evidence obtained from welldesigned RCTs, or Level III, that is, evidence obtained from well-designed controlled trials without randomization. Demographics The dataset (cross-sectional surveys) consists of 44 articles, and the total sample size from the dataset is 45,240 individuals.The average response rate across the studies was approximately 61.27%.There was a wide range in the number of responses, i.e., N = 11,616 responses from dentists, N = 4283 responses from specialists, and N = 893 responses from another group. Notably, the response rates exhibit variability, with a mean response rate of approximately 61.27% and responses ranging from as low as 1.8% to 100% (Supplementary Table S3).The mean response rates from specific groups include 60.29% for dentists; N = 59.75% (95% CI 35.57% to 83.93%) for specialists; and N = 57.83%for others (95% CI 28.49% to 87.16%).There were eight studies written by undergraduate dental students. Prescription Rates among Dental Professionals The dental professionals were categorized into dentists (who have bachelor's degrees in dentistry), specialists (who have specialist degrees and training), undergraduates (who did not qualify as dentists and are/were engaged in their initial training or final years of their programs), and others (possessing medical qualifications), with dentists representing the largest group (31 individuals).Dentists had a mean antibiotic prescription rate of approximately 24.6%, while specialists, a smaller group of nine individuals, exhibited a mean rate of 22.41%.The "Others" category, comprising various dental professionals (four individuals), demonstrated the highest mean antibiotic prescription rate, amounting to 41.77%.Lastly, the analysis included eight undergraduate dental students, with a mean antibiotic prescription rate of 17.52%.These findings underscore the diversity of antibiotic prescription practices among different dental professionals, with engagement levels varying across the sampled groups (Supplementary Table S3). Literature Mapping A total of N = 53 articles examining the prescription of antibiotics for treating irreversible pulpitis were selected (Figure 1).The United States (USA) contributed significantly, accounting for 10 studies, representing approximately 20.63% of the total.Spain followed closely with seven studies, making up 11.11% of the corpus.India was also a notable contributor, accounting for three studies, or 6.35%.Furthermore, Saudi Arabia also presented four studies, reflecting a similar percentage.Italy and Australia both offered four studies each, collectively constituting 12.7% of the analyzed research.The rest of the countries, each contributing one to three studies, collectively make up the remaining part of the dataset. The journals with the highest representation were the International Endodontic Journal (12.96%), with seven articles, and Antibiotics (5.56%), with three articles.Most studies are classified as cross-sectional studies, accounting for 42 instances, corresponding to approximately 77.78% of the total.Systematic reviews also featured prominently, accounting for four articles (7.41%).Review articles followed this group, accounting for four studies, representing 6.35% of the corpus.Guidelines accounted for three (5.56%)articles.There were also two separate instances of in vitro studies, contributing 1.85% to the total (Table 1 and Table S1). Molecular Developments in the Area of Irreversible Pulpitis The molecular research with respect to irreversible pulpitis has witnessed significant developments.Researchers have published studies on various aspects, including the microbiota within teeth affected by irreversible pulpitis, the roles of autophagy and ceRNA networks in this condition, the impact of preoperative treatments, gene expression analysis, and the potential of various markers like substance P and IL-8 regarding assessing inflammation.Additionally, the analyzed studies explore the molecular and genetic underpinnings of pulpitis, including via the identification of specific bacteria and immune-related regulatory networks.The research also investigated stem cell potential, cytokines, and the diagnostic utility of dentinal fluid biomarkers.These studies collectively aim to advance our understanding of the molecular mechanisms, diagnostics, and potential treatments regarding irreversible pulpitis.Level I-Evidence from a systematic review or meta-analysis of all relevant randomized controlled trials (RCTs); Level II-evidence obtained from well-designed RCTs; Level III-evidence obtained from well-designed controlled trials without randomization; Level IV-evidence from well-designed case-control and cohort studies; Level V-evidence from systematic reviews of descriptive and qualitative studies; Level VI-evidence from single descriptive or qualitative studies; Level VII-evidence from the opinions of authorities and/or reports made by expert committees [5]. Importance of the Review The effects of the most-prescribed antibiotics for treating irreversible pulpitis are largely limited, with insufficient proof to support their significance.The misunderstanding of the pathogenesis of the pulp may have led to the observed increase in antibiotic prescription for treating pulpal diseases.In a study carried out in the USA, it was reported that 16.7% of endodontists prescribed antibiotics for the treatment of irreversible pulpitis [57].Another study organized in The Netherlands reported that only a small proportion, i.e., 4.3%, of dentists continued to advise the use of antibiotics for irreversible pulpitis [31].Similarly, a multistage sampling study in India confirmed that 71.6% of dentists over-prescribed antibiotics, mainly for treating irreversible pulpitis and acute apical periodontitis [52].The over-usage of antibiotics is highly likely to lead to the growth in resistant strains of micro-organisms.There is no convincing evidence proving that penicillin-like antibiotics relieve pain and sensitivity, but many dentists continue to prescribe antibiotics.Similar concerns are discussed in other published articles [58][59][60][61][62][63][64][65][66].Therefore, this review helps to reveal the effectiveness of antibiotics in the management of pulpitis and identify the available evidence regarding the management of pulpitis. Pathogenesis of Pulpitis It is very important to understand how pulpitis occurs because such knowledge can aid the management of patients for better outcomes.Pulpitis may occur due to a microbial insult, a chemical insult, or traumatic or iatrogenic factors.Caries and periodontal diseases are microbial in nature, while crown/root fractures and injuries are traumatic.An iatrogenic factor involves marginal leakage, dental material toxicity, or trauma caused by dental procedures.Hence, the type of management employed differs depending on the type of case and cause.The dental pulp is securely protected by dentin, cementum, and enamel, providing strong mechanical support.But when the degradation of the outer enamel or cementum layer occurs, the connective tissue of the dental pulp is rendered vulnerable to the ingress of toxins due to the exposed dentinal tubules.This allows the noxious components of the oral cavity to enter the pulp and cause pulpitis [67]. The presence of bacteria in the pulp initiates an inflammatory reaction and results in pulpal necrosis.The endotoxins and bacterial waste products that are produced by proteolytic bacteria exit through the apical foramen and accumulate in the peri-apical region or apex of the tooth.Accordingly, the immune system is triggered, and defense cells will not be able to enter the root canal, accumulating and resulting in bone loss.This apical region is free from bacteria; bacteria are only present in articles from sinus formation, actinomyces, etc. [68]. Permeability of Dentine to Bacterial Toxins Physiologically and anatomically, dentine is a complex mineralized tissue in the tooth.The dentinal tubules consist of nerves, vessels, and dentinal fluid [69].Bacterial plaque accumulation leads to a microbial insult afflicting the dentinal tubules, and dentin does not act as an effective barrier against the diffusion of bacterial components, as shown in some research [70].There is evidence that bacterial components are carried to the pulp through dentine, wherein an inflammatory process is induced.Some articles report that bacterial toxins penetrate over short distances, and initial reactions begin to occur through the initiation of some host defense mechanism present in dentinal fluids.This shows that the inflammatory process arises either due to bacterial toxins or exposed dentinal tubules and/or the activation of signal substances arising from dentinal fluids. Koch's Postulate Robert Koch tried to identify the specific organisms that caused specific diseases.Hence, he conceived of criteria that later came to be known as Koch's postulate.These criteria outline the following: • The microbes present are associated with the disease and its causative lesion; • Upon isolation from the contaminated site and later transferal, the microbes should be grown on culture media; • The microbes should induce a similar disease when a pure culture of the organism is introduced into a heathy host; • The microbes should be able to re-isolatable from an experimentally infected host. Taxa information (concerning the group to which an organism belongs) gives an understanding regarding the disease process that causes pulpitis.It helps to provide the reason behind "When the disease becomes acute and why".This will aid our understanding of the role of antimicrobials in preventing infection.The acute form of acute periodontitis or other opportunistic infections cannot be explained by Koch's Postulate [71].According to a study, Prevotella melaninogenica was noticed in all acute and pus and/or tenderness articles but not in chronic articles.Other organisms included Peptostreptococcus spp., Eubacterium spp., and Campylobacter sputorum.These organisms were cultivated from a sample obtained from an intact pulp chamber of traumatized teeth.It was found that the composition of the microbiota in the root canal drives the course of the disease.Some black-pigmented bacteroid species contain certain taxa that induce acute inflammation, and other taxa do not contain these.Enteroccocus species were found to relate to periapical health rather than a disease.The organisms found after root canal treatment were from extra-oral sources or from food rather than stemming from therapy-resistant entities. Pulpitis The carious process that results in pulpitis and endodontic infections is predominantly governed by anaerobic organisms, predominantly Gram-negative bacteria.As a result, inflammation of the pulp occurs, which ranges from minimal inflammation to marked inflammation [72].Pulpal pathosis is diagnosed based on the progress of the disease, corresponding to reversible pulpitis, irreversible pulpitis (asymptomatic), irreversible pulpitis (symptomatic), and pulp necrosis [73].When dental caries reaches the pulp, reversible pulpitis occurs, and it is usually associated with mild inflammation of the pulp and mild intermittent pain.Thermal changes, especially those induced by cold drinks, will elicit this pain. Why Is There a Need for Antibiotics? Antibiotics are usually prescribed as a strategy for preventing infection and postoperative complications and for prophylaxis.These are prescribed by general dental practitioners and oral maxillofacial surgeons, and they are sometimes prescribed at the request of the patient if the dentist has not prescribed them.A recent study confirmed that more than two-thirds of 120 patients who were included in the study responded that they expected to receive antibiotics after a routine tooth extraction, and 70% of this group indicated that they would request them if not prescribed.These findings were surprising because the patients included were educated, i.e., they had at least an initial college or college degree [44,[74][75][76][77].There is a myth among healthcare professionals and patients that antibiotics play an important role in the prevention of disease (Table 2).Instilling proper education and awareness among these two groups will help to eliminate these myths related to antibiotics prescription. Myths about Antibiotics The choice between bactericidal and bacteriostatic agents is contingent upon various factors.Bactericidal agents, which swiftly eliminate bacteria, are considered indispensable for patients with compromised immune defenses.This holds particular significance in severe infections or conditions like sepsis, wherein prompt bacterial elimination is imperative.Conversely, when a patient's natural defenses are unimpaired, bacteriostatic agents, impeding bacterial growth without immediate destruction, are often deemed satisfactory.Moreover, post-antibiotic effects (PAEs), referring to the prolonged suppression of bacterial growth after antibiotic exposure, are more enduring and reliable when administering bacteriostatic agents such as erythromycin and clindamycin than when administering bactericidal agents like betalactamase.The clinical efficacy of bacteriostatic agents appears to be less dose-dependent, contributing to their consistent post-antibiotic effects compared to bactericidal agents.The assertion that bactericidal agents are superior is supported by their rapid action, potential to prevent resistance, effectiveness in critical infections, and synergy with the host's immune response, especially in compromised immune states (Table 2). In addition, the idea that bacterial infections require a "complete course" of antibiotic therapy is a prominent myth regarding the management of irreversible pulpitis.It is crucial to dispel the notion of a "complete course" of treatment, as the duration of antibiotic therapy is contingent upon the clinical improvement of the patient.Contrary to a common misconception, sustained antibiotic use beyond the point of clinical remission is not universally necessary to prevent "rebound" infections.Specifically in orofacial infections, the idea of rebound has been debunked, provided the infection's source is effectively eliminated.Orofacial infections typically endure for a brief period, often ranging from two to seven days.For patients undergoing antibiotic therapy for orofacial infections, daily clinical assessments are imperative.Ceasing antibiotic therapy becomes appropriate when substantial clinical evidence indicates the restoration of the patient's host defenses, signaling control over the infection and its resolution.Thus, the effectiveness of an antibiotic treatment is intricately linked to ongoing clinical evaluation rather than a predetermined course of medication. When to Prescribe Antibiotics Basically, antibiotics are prescribed when there is systemic involvement due to infection.Antimicrobials are also prescribed in the following instances: • As an adjunct to the management of a acute or chronic infection; • In the management of active disease, e.g., acute necrotizing ulcerative gingivitis; • When drainage cannot be established during the treatment of an uncooperative patient who requires hospitalization and must be operated on under general anesthesia; • For a patient that needs to be treated in a hospital environment due to comorbidities [58] (Table 3).Before prescribing antimicrobials, a comprehensive case history should be acquired.Patients should be examined carefully, and any signs of systemic involvement, for example, fever, lymph node involvement (lymphadenopathy), and swelling, should be searched for.This helps to rule out if a patient can be managed in a private dental setting or needs to be referred to a hospital [58].In the Indian context, in September 2015, the chief scientific advisor for the WHO Regional Director for South-East Asia in New Delhi confirmed that guidelines will be published on the usage of antibiotics to reduce the over-prescription and tackle antibiotic resistance.The panel advised hospitals and related facilities in the country to develop their own protocols as a best practice to tackle the problem [61]. The body's defense mechanism plays an important role in preventing the spread of infection, except in articles of immuno-compromised patients.According to the literature, 60% of an infection is removed by the host's own defense mechanisms if the underlying cause is removed.Antibiotics only help in maintaining the balance between host defense and invasive agents.The most important factor that indicates whether antibiotics should be prescribed is the need for antibiotics rather than which one to prescribe.Asymptomatic articles like apical periodontitis of pulpal origin and chronic apical abscesses of endodontic origin do not require antimicrobial therapy for healing.Proper root canal cleaning with effective irrigating solutions will resolve such issues.For articles like acute apical abscesses with spontaneous pain and swelling that is localized intra-orally, proper root cleaning and irrigation shaping of the canal will help solve the problem.If the case involves cellulitis or an acute apical abscess with systemic involvement, then debridement, surgical incision, and an aptly chosen antimicrobial should be considered [44,46,58,75]. The indications for antibiotic prescription in cases of acute pulpitis extend beyond molecular and clinical aspects, encompassing specific medical conditions where antimicrobial therapy is crucial.These conditions include patients with heart valve replacements, whether mechanical or biological, especially those who have undergone surgery due to microbial endocarditis.Additionally, individuals with congenital complex heart defects, surgically corrected heart defects within the initial 6 postoperative months, or residual findings after correction fall within the scope of antibiotic indication.Patients with Grade V renal insufficiency requiring dialysis, those who have undergone organ transplantation, and individuals with hip joint prostheses or total knee arthroplasties in the first two years after surgery also necessitate antibiotic consideration.Moreover, antibiotic prescription is warranted for individuals who have undergone radiotherapy and require treatment of the irradiated jaw area, those on high-risk bisphosphonates with intravenous administration over an extended period, and HIV patients with granulocyte counts below 500/µL. Solution to the Problem The possible solution to the problem is education.One method of education is to teach from errors rather than principles.Special consideration is taken when it comes to prescribing antibiotics to patients suffering from infective endocarditis (IE).Patients visiting a dental practice for their appointment very rarely have taken their antibiotics.It is good practice for a dentist to select a different class of antibiotics if the patient is already on antibiotics for endocarditis prophylaxis.If possible, one should delay a dental procedure until at least 10 days after the completion of a course of antibiotics.This will allow for the usual oral flora to be reestablished.If an individual receiving long-term parenteral antibiotic therapy for IE requires dental treatment, the treatment should be timed to occur 30 to 60 min after the parenteral antibiotic therapy has been delivered.If the dosage of an antibiotic is inadvertently not administered before the procedure, the dosage may be administered up to 2 h after the procedure.However, administration of the dosage after the procedure should be considered only when the patient has not received the pre-procedure dose.Individuals with permanent kidney dialysis shunts should be administered a course of prophylactic antibiotics using the same protocol applied for IE 33. Antibiotic Resistance Several studies [78][79][80][81][82][83] have been carried out to determine the prevalence of antimicrobial resistance in India.A recent study revealed that, generally, resistance was observed for nalidixic acid (79%), followed by Co trimoxazole (75%) and ampicillin (72%).Moderate susceptibility was seen with fluoroquinolones, and good susceptibility was seen with Imipenem (15%) and cephalosporins.Antibiotic resistance induced by antibiotic prophylaxis has been reported recently, and the factors causing these problems need to be considered.A recent meta-analysis confirmed that between 38.7% and 50.9% of pathogens causing surgical site infections and 26.8% of pathogens causing infections after chemotherapy are resistant to standard prophylactic antibiotics in the USA [84]. Management The following is a list of actions to be taken in the case of an acute dento-alveolar infection [58]: In Case of Chronic Dento-Alveolar Infections [58] Chronic dento-alveolar infections are long-standing infections in the root canal system that result in the induction of a peri-apical infection.This can arise in decayed or root-filled teeth.The infection presents as a minor localized abscess and, in some articles, occurs in the sinus and rarely requires antimicrobial therapy unless there are signs of systemic involvement (fever, lymphadenopathy, and swelling). Clinical Approaches Precise diagnosis along with localization of the afflicted tooth should be given priority.The required testing and history documentation should be performed to achieve this.To make sure that the afflicted tooth is correctly identified, it is essential to diagnose the patient's symptoms.A precise treatment plan is devised once the initial radiographs are analyzed thoroughly to determine the anatomical complexity of the tooth. The application of a restorative or temporary sedative dressing is usually performed to treat reversible pulpitis. Root canal therapy or extraction can be performed to treat irreversible pulpitis.An antibiotic can be employed if necessary, depending on the severity of the infection and the type of causative bacteria. Overview of the Literature on the Effectiveness of Antibiotics in Treating Irreversible Pulpitis Endodontic emergencies, occurring before, during, or after treatment, result from diverse pulp and root canal conditions.In this review, we aim to outline these emergencies, emphasizing the need for timely and comprehensive management.The 3D principle-diagnosis, definitive dental treatment, and drugs-guides this process.Diagnosis, the cornerstone, requires understanding various emergency-causing conditions, aided by a comprehensive classification.Treatment varies per diagnosis and includes root canal retreatment or conservative approaches.Drugs complement treatment, which is tailored to the corresponding diagnosis.Addressing inflammation and infection distinctions is crucial for achieving effective pain relief and symptom resolution (Abbott PV 2022) [12].4.15.Regional Variations 4.15.1.USA Carlsen (2021) [14] found that while many treatments aligned with ADA guidelines, extended antibiotic courses were common, highlighting the need for guideline adherence.Vasudavan (2019) [30] noted low adherence, especially in cases of tooth pain and localized abscesses.Agnihotry (2019) [31] highlighted inappropriate antibiotic use for irreversible pulpitis, noting that better practices were adhered to by educated dentists.Lockhart et al. (2019) [32] reported limited benefits and potential harm regarding antibiotic use.Tampi et al. (2019) [34] observed mixed effects.Germack (2017) [43] indicated that patient expectations were driving unnecessary prescriptions.Other studies emphasized the insufficient evidence regarding the efficacy of using antibiotics in dental care (Gottlieb 2017, Hoskin 2016, Yingling 2002) [44,57].These findings underscore the need for rational antibiotic use in dentistry. Spain Segura-Egea, J.J. (2022; 2017) [7,46] emphasized the effects of antibiotic prophylaxis on patients with compromised immunity and specific conditions like infective endocarditis or prosthetic joint replacements.The author also highlighted the overprescription of antibiotics in endodontic infections and the need for improved prescription habits and education.Domínguez-Domínguez, L. (2021) [17] revealed that 44% of dentists prescribed antibiotics for symptomatic irreversible pulpitis, with up to 27% not following current guidelines, indicating a need for improved antibiotic prescription habits among Spanish general dentists.Alonso-Ezpeleta, O. (2018) [38] found that dentists with postgraduate training in endodontics exhibited better adherence to international guidelines for antibiotic use.Martín-Jiménez, M. (2018) [39] assessed dental students' knowledge of antibiotic indications in endodontics.Segura-Egea, J.J. (2018) [46] published a position statement on the use of antibiotics in endodontics.Segura-Egea, J.J. (2010) [7] noted that while many members of the SECIB selected appropriate antibiotics, some still prescribed them inappropriately in the management of endodontic infections. Saudi Arabia The findings from multiple cross-sectional studies conducted in Saudi Arabia suggest that there is a concern regarding the appropriate prescription of antibiotics by endodontists and general dental practitioners (GDPs).While there is general adherence to global guidelines, instances of inappropriate antibiotic prescriptions were noted, particularly in cases of irreversible pulpitis, necrotic pulps without systemic involvement, and sinus tract infections.This indicates the need to improve knowledge and awareness among dental practitioners regarding the judicious use of antibiotics to combat antibiotic abuse and antimicrobial resistance, constituting a pressing issue in dental treatment practice in Saudi Arabia. Italy In the study by Di Giuseppe (2021) [21], a widespread practice of providing inappropriate antimicrobial prescriptions for prisoners was identified, indicating a need for diagnosis-specific monitoring and the implementation of prison-focused antimicrobial stewardship policies.Licata (2021) [22] emphasized the necessity of developing practical antibiotic prescription guidelines with clear indications and an easy-to-follow regimen.Sal-vadori's (2019) [35] findings highlighted the imperative to enhance the knowledge of Italian students regarding antibiotics and their appropriate use in endodontics.D'Ambrosio's (2022) [10] study demonstrated a consistent trend in Italy, like other countries, showcasing a high prevalence of antibiotic misuse and overuse among Italian dentists, who employed a variety of antibiotic management strategies. India In the study by Karobari, M.I. (2021) [23], awareness among dentists about antimicrobial prescription guidelines was found to be incomplete, indicating a requirement for further training and education to enhance evidence-based decision making to achieve improved practices and outcomes.Wasan H's investigation in 2017 revealed a pattern of frequent irrational prescription of antimicrobials for odontogenic conditions, emphasizing the immediate and sustained need for guidelines and educational intervention programs in dentistry.This approach is crucial for enhancing the quality of antimicrobial prescribing practices within the dental field.Garg AK's 2014 [52] study highlighted the issue of overprescription among oral healthcare providers in India, signifying a significant contribution to the global problem of antimicrobial resistance.The findings underscored the urgent necessity of raising awareness, both among the public and professionals, about the risks associated with antibiotic use. The literature highlights significant regional variations in antibiotic prescription practices.These variations are influenced by factors such as local guidelines, dental education levels, and regional healthcare policies.For instance, dentists with postgraduate training in endodontics showed better adherence to international guidelines, suggesting that advanced education positively impacts prescription practices. Dental Practitioner Experience The articles analyzed encompassed 44 articles.The responses available for analysis related to the number of prescriptions, ranging from 67 (minimum) to 45,240 (Maximum).The response rates varied, with an average of 61%, indicating diverse participation levels among professionals.Dentists had a mean antibiotic prescription rate of 23.76%, while specialists, others, and undergraduates showed rates of 22.42%, 41.78%, and 17.53%, respectively.Supplementary Table S3 provides a snapshot of antibiotic prescription patterns, highlighting variations in practices among different categories of dental professionals. Role of Education and Awareness The findings point to a critical need for enhanced education and awareness among dental professionals.There is a clear gap in understanding the appropriate use of antibiotics in dentistry, particularly in the treatment of irreversible pulpitis.This gap extends to the understanding of molecular advancements in pulpitis research, suggesting the need for alternative therapeutic approaches that could reduce reliance on antibiotics. Individual Study Findings The study by Abraham, S.B. (2020) [27] delves into the practices of antibiotic prescription in the context of endodontic infections in the UAE, showcasing a survey involving 174 respondents with a response rate of 70%.This research underscores the importance of responsible antibiotic use, as indiscriminate prescription can lead to the emergence of antibiotic-resistant microbes.It reveals the preferences of dental practitioners, with amoxicillin and erythromycin being popular choices, and indicates discrepancies between general dental practitioners and specialists.Additionally, the article identifies instances where antibiotics were prescribed incorrectly, notably in articles of irreversible pulpitis, necrotic pulps lacking systemic implications, and sinus tracts [27]. Meanwhile, Agnihotry A's studies from 2014, and 2019 address the use of systemic antibiotics for treating irreversible pulpitis and the associated concerns about antibiotic resistance.The 2019 study, rated as possessing low overall evidence quality, investigated pain relief outcomes between antibiotic and placebo groups [31], while the 2019 study underscored the inappropriate prescription of antibiotics for irreversible pulpitis, highlighting the knowledge gaps among dentists in this regard [33].These findings emphasize the need for responsible antibiotic prescription and more research to clarify the role of antibiotics in endodontic emergencies, especially irreversible pulpitis [85]. In Agwan MA's (2022) study, it was observed that 2% of the participants reported that they would prescribe antibiotics for irreversible pulpitis, a condition characterized by inflammation of the dental pulp.However, this practice deviates from clinical guidelines, as antibiotics are generally not recommended for pulpitis articles, given that the condition is primarily related to inflammation rather than bacterial infection.Instead, the primary approach for managing pulpitis should involve proper endodontic treatment, such as root canal therapy, which addresses the root cause of the issue [8,37]. Al Masan AA's (2018) study results indicate a significant focus on antibiotic prescription for conditions such as systemic complications (78%), acute apical abscesses (72%), and symptomatic apical periodontitis (28%).This study further highlighted variations in prescription practices between the Group G1 and Group G2 participants, with differences noted in various clinical scenarios.It is noteworthy that final-year undergraduate students seemed to be generally aware of the antibiotic resistance crisis, albeit with some gaps in their knowledge of antibiotic use guidelines for endodontic conditions.In contrast, general dentists displayed less awareness of antibiotic guidelines and sometimes deviated from them in their responses to clinical scenarios [37]. Furthermore, the short communication regarding the antibiotic prescription practices of dentists in Saudi Arabia [54] written by Sameer, E.A.H. (2013) found that a significant percentage of dentists prescribed antibiotics for endodontic conditions that typically do not require antimicrobial treatment.The rates of antibiotic prescription varied for different conditions, with some articles, like necrotic pulp with acute apical periodontitis and swelling, aligning with guidelines, while in others, there were deviations from recommended practices.This finding underscores the importance of educational initiatives for promoting rational antibiotic use in dental practice and combatting antibiotic resistance [18,38,47,86]. In a comprehensive analysis of the antibiotic prescription patterns of dental students, Arıcan, B. (2021) [15] collected data from 17 public and 3 private dental schools, accounting for 1113 final-year dental students.These students exhibited varying prescription behaviors across different clinical scenarios.Notably, 89.9% of the students prescribed antibiotics for acute apical abscess (AAA) articles with diffuse swelling, whereas 47.2% did so for AAA with localized swelling.Regional and university-type differences were evident in these patterns, with certain articles displaying significant variations.The students also exhibited diversity in their choice of antibiotic usage duration, with 41.7% opting for a 5-7-day period and 36.2%preferring to complete the entire course.Amoxicillin, co-amoxiclav, and clindamycin were favored antibiotics for patients without allergies, and prophylactic antibiotic use varied depending on the clinical condition.Awareness of antibiotic usage for post-endodontic scenarios showed some discrepancies, with students being less inclined to prescribe antibiotics for situations like irreversible pulpitis.These findings underscore the need for consistent guidelines and education in antibiotic prescription. Baudet (2020) [29] conducted a survey involving 775 dentists wherein 455 complete questionnaires were included in the analysis.The dentists predominantly worked as general dental practitioners (81.5%) in self-employed roles (77.0%) within urban areas (53.8%).They reported conducting an average of 47 scheduled consultations, 10 emergency consultations, and eight antibiotic prescriptions per week.While around 75.3% claimed to possess knowledge of national recommendations, only 32.2% specifically mentioned the French guidelines stipulated by the National Agency for medicines (ANSM).The primary reasons for prescribing antibiotics were abscesses, cervicofacial cellulitis, and pericoronitis.Amoxicillin was the most commonly prescribed antibiotic, often in the form of 1 g administered b.i.d. for 6 or 7 days.This study provides insights into antibiotic prescription patterns and awareness among practicing dentists [59]. In a study by Bolfoni, M.R. (2018) [42], 13,853 questionnaires were distributed, with 615 being completed, resulting in a response rate of 4.44%.The respondents had a diverse demographic profile.In articles of pulpitis, only 1.1% of the respondents prescribed antibiotics, while in situations involving irreversible pulpitis with acute apical periodontitis, 6.2% prescribed antibiotics.This study provides insights into antibiotic prescription habits among dental professionals. Furthermore, guidelines such as those recommended by the ADA advise against the prescription of antibiotics in articles of acute pulpitis, emphasizing the importance of prudent antibiotic use in dental practice (Carlsen, D.B. 2021) [14].A study conducted by Daher, A. (2015) reaffirmed the significance of appropriate treatment, as antibiotic-based pulpotomies were associated with a lower survival rate compared to calcium hydroxide treatment [87].Finally, D'Ambrosio, F. (2022) [10] conducted an online survey among Italian dentists to gauge their attitudes toward antibiotic prescription and awareness of antimicrobial resistance [63].This study revealed that the primary reasons for antibiotic prescriptions included abscesses, extractions, and pulpitis.Despite their high awareness (98.9%) of antimicrobial resistance, only a minority (7.4%) consulted guidelines for antibiotic prescriptions.These findings underscore the importance of enhancing awareness of and adherence to antibiotic prescription guidelines among dental professionals to combat antimicrobial resistance effectively. The study by Dahake, P.T. (2023) examined the prevalence of isolated bacterial species in the context of root canals.In this research, 50 teeth were examined, all of which contained both aerobic and anaerobic microorganisms [88].This study revealed the presence of various bacterial species, including aerobic, microaerophilic, facultatively anaerobic, and obligate anaerobic bacteria, as well as specific species such as Candida albicans (C.albicans), Bacillus subtilis (B.subtilis), Pseudomonas aeruginosa (P.aeruginosa), Staphylococcus aureus (S. aureus), Streptococcus mutans, Streptococcus mitis, and others.The antibiotic resistance profiles of these bacteria were assessed, highlighting varying sensitivities and resistances to antibiotics like clindamycin, metronidazole, and doxycycline.This comprehensive study provides insights into the diverse bacterial compositions within root canals and their antibiotic resistance profiles. The research conducted by Daher, A. (2015) [87] involved the treatment of primary molars in children and included a sample of 35 participants aged 3.6 to 9.4 years.These children had a total of 53 primary molars treated, with some undergoing CTZ pulpotomy and others receiving calcium hydroxide pulpectomy.The study analyzed various aspects of treatment outcomes, follow-up periods, and success rates.Notably, 62.2% of the primary molars treated with CTZ pulpotomy were rated as unsuccessful in the first year after the intervention.Tooth extraction was required for treatment failure articles, and radiographic and clinical aspects contributed to the categorization of articles as failures.The overall mean survival time for all treated molars was 15.2 months.The study also examined treatment outcomes based on treatment group and previous pulp diagnosis, revealing lower survival rates for articles with a necrotic pulp initial diagnosis and those treated with the mixed antibiotic paste used in CTZ pulpotomy.This research provides valuable insights into the outcomes of different dental treatments among pediatric patients [87]. The study by Yu, J. (2020) [50,89] conducted in Guangzhou aimed to assess the rational use of drugs, particularly analgesics and antibiotics, by dentists and their communication with patients regarding these medications.This research found that dentists in Guangzhou frequently prescribed amoxicillin, with percentages varying depending on dental conditions.For instance, amoxicillin was prescribed for 25% of articles involving acute pulpitis and for 80.1% of articles of acute apical abscesses.Furthermore, metronidazole was the second most recommended antibiotic, especially for articles of diffuse swelling after treatment of acute apical abscesses, with 89.6% of practitioners choosing this antibiotic.This study provides insights into prescription trends in Guangzhou, shedding light on the patterns of antibiotic usage in dental management. The study by Wasan, H. (2017) [50] investigated the impact of dental qualifications and practice settings on antimicrobial prescription practices among dental practitioners in Delhi and the National Capital Region (NCR) of India.Notably, it revealed that antimicrobial prescription for acute pulpitis was significantly higher among those pursuing postgraduate degrees (62.2%) compared to qualified specialists and dental graduates.This suggests variations in prescription practices based on qualifications, highlighting the importance of understanding the factors influencing antibiotic prescriptions among dental practitioners. Vessal G's (2011) [90] study in Shiraz, the Islamic Republic of Iran, assessed the knowledge and practices of dental practitioners regarding the therapeutic use of antibiotics for treating patients with dentoalveolar infections.The study revealed that 25% of the surveyed dental practitioners believed it was appropriate to use antibiotics to treat patients with acute pulpitis.This finding was in line with studies conducted in Yemen and Kuwait, which also reported similar percentages of dentists prescribing antibiotics for acute pulpitis.However, this percentage was lower (13%) in a study conducted in England, indicating variations in antibiotic prescription practices among different regions. In Vasudavan S's (2019) [30] study, in which 3434 surveys were distributed, with a response rate of 20%, the research focused on understanding antibiotic prescription patterns among different groups of dental practitioners in different experience brackets.For irreversible pulpitis, antibiotics were reported to have been prescribed in 45% of responses, with variations among different groups.Dentists with less than 10 years of experience (39%) prescribed antibiotics significantly less than those with 10 or more years of experience, highlighting the influence of experience on antibiotic prescription practices. Skucaite N's 2010 study aimed to characterize the antibiotic prescription patterns during root canal procedures as reported by Lithuanian general dental practitioners.Questionnaires were distributed to all 2850 registered Lithuanian dental practitioners, and responses from 1431 licensed general dental practitioners were analyzed.Approximately 2% of the practitioners prescribed antibiotics for symptomatic pulpitis [56].Dana R's (2019) study evaluated the knowledge and practices of Ontario physicians with respect to managing non-traumatic dental conditions, particularly antibiotic usage.With a 20.2% response rate from 1012 physicians, the study found that 57.4% of physicians prescribed antibiotics for irreversible pulpitis articles, with amoxicillin being the most prescribed antibiotic.This research provided insights into antibiotic prescription practices followed by physicians in Ontario when addressing dental conditions [36]. Several studies have examined the antibiotic prescription patterns for dental conditions, particularly pulpitis.Darkwah TO's 2021 study, conducted at the Ghana Police Hospital, analyzed 184 patient prescriptions (corresponding to 286 antibiotics) but did not specify the exact percentage of antibiotics prescribed for irreversible pulpitis or acute pulpitis [24].Darwish MA's 2021 study focused on dental students attending the University of Gezira, revealing that 30% of antibiotics prescribed for root canal treatments (RCTs) at Wad Madani dental teaching hospital did not align with the recent ADA guidelines [16].They also reported a lack of knowledge about antibiotic prescription guidelines among Sudanese dentists and dental students.Deniz-Sungur D's 2020 study, involving 1007 Turkish dentists, demonstrated that up to 10% of the participants prescribed antibiotics for symptomatic irreversible pulpitis [91].Di Giuseppe G's 2021 study conducted in Italian prisons found that 85.7% of prisoners diagnosed with symptomatic irreversible pulpitis with or without symptomatic apical periodontitis were prescribed antibiotics [21].Dias NM's 2022 research conducted in Colombia reported that 43.7% of dentists prescribed antibiotics for irreversible pulpitis with symptomatic apical periodontitis and that 57.2% prescribed them for symptomatic acute apical periodontitis [11].Finally, Dibaji F's 2021 cross-sectional study involving 400 general dentists in Iran indicated that antibiotic prescription ranged from 48.5% for articles of painful irreversible pulpitis to 97.3% for articles of pulp necrosis with acute apical periodontitis and preoperative symptoms [3].These studies provide valuable insights into antibiotic prescription practices for pulpitis in various regions and among different groups of dental practitioners.Drobac M's 2021 study conducted in Serbia involved 628 dentists with a 25.16% response rate, where 1.3% of the respondents indicated antibiotic reliance for symptomatic irreversible pulpitis [19].In D'Ambrosio F's 2022 study conducted in Italy, antibiotic prescription for pulpitis corresponded to a level of 14.1% [10].Fadare JO's 2017 study conducted in Nigeria analyzed 607 prescriptions, revealing that 8.5% of the treated patients received antibiotics for acute pulpitis [49].Fedorowicz Z's 2013 Cochrane review investigates the effectiveness and safety of oral antibiotics in treating severe toothaches caused by irreversible pulpitis, a condition resulting from nerve damage inside a tooth.The 'standard of care' involves the immediate removal of the affected pulp, but in some regions, antibiotics are still prescribed.This review, based on evidence available as of February 2019, includes one study with 40 participants who were administered either penicillin or a placebo in addition to painkillers.The findings indicate that antibiotics do not significantly reduce toothache caused by irreversible pulpitis, and there was no difference in painkiller use between groups.The study's limited size and low certainty of evidence emphasize the need for more high-quality research on antibiotic use for treating this condition [31]. Garg AK's 2014 study conducted in India found that 73.4% of dental practitioners preferred amoxicillin, with the majority prescribing antibiotics for irreversible pulpitis and acute apical periodontitis [52].Gemmell A's 2020 survey involving general dental practitioners showed that 25% frequently prescribed antibiotics for irreversible pulpitis [26]. Yingling NM's 2000 survey of American Association of Endodontists members reported that 16.76% prescribed antibiotics for irreversible pulpitis, while only 3.47% and 13.29% prescribed antibiotics for specific articles of irreversible pulpitis [57].Germack M's 2017 survey of endodontists indicated that antibiotics were prescribed for articles of irreversible pulpitis with mild symptoms (1.75%) and moderate symptoms (6.41%) [43].The findings from these studies highlight the variability of antibiotic prescription practices for pulpitis among dentists in different regions. Gottlieb, M. 2017 aimed to review the best available evidence on the utility of antibiotics for treating dental pain without evidence of an overt infection.There is insufficient evidence with which to support the use or disuse of empiric antibiotics to prevent pain or reduce infection rates.Further data are required to provide definitive recommendations.However, the use of empiric antibiotics is not without risks, and this should be considered considering the current evidence.Additionally, it is important to provide pain control and conduct a close follow-up with a dentist for a pulpectomy [44]. Karobari, M.I. 2021 conducted a survey among dentists around three different regions of the world: 26.7% of dentists were found to prescribe antibiotics for pulpitis [23].Khaloufi O 2022 aimed to evaluate the prescription attitudes of dental practitioners in Northern Morocco when treating pulpal and periapical pathologies.A total of 121 (55%) practitioners (63 females and 58 males) responded.The average age was 37 ± 0.4 years, with a minimum of 24 years and a maximum of 62 years.The distribution according to age group showed that more than 75% of practitioners were < 45 years old, 51 practitioners were between 25 and 35 years old, and 41 practitioners were between 36 and 45 years old [9].Marra F 2016 confirmed the existence of over-prescription due to the slow implementation of guidelines [75].Martín-Jiménez M's 2018 Spanish study found that for articles of irreversible pulpitis, up to 63% of students would prescribe antibiotics [39].Maslamani M's 2018 study found that of the 227 participants surveyed, 190 (83.7%) did not prescribe antibiotics for patients complaining of severe pain.Of the participants, 199 (87.7%) never prescribed antibiotics for reversible pulpitis with a normal periapical area [41]. Discrepancy between Guidelines and Clinical Practice The successful translation of clinical guidelines into practice requires a multifaceted approach that addresses not only the dissemination of guidelines but also the education and engagement of healthcare professionals, the fostering of a culture that embraces evidence-based practices, and effective communication with patients to manage expectations and build trust in the decision-making process. Following clinical guidelines, especially when it comes to not prescribing antibiotics for irreversible pulpitis, can be challenging for healthcare providers.One major problem is that not all doctors follow the guidelines the same way.Some may not want to change how they usually treat patients, especially if a change contrasts with their typical practices. Another issue is that it can be difficult for dentists to keep up with the latest information.New guidelines might not always make their way into regular practice because not everyone is aware of or educated about the changes in how they should be treating patients. Patient expectations also play a role.Sometimes, patients believe they need antibiotics, even if the guidelines suggest otherwise.Doctors might feel pressured to prescribe antibiotics just to make their patients happy.This situation is exacerbated when there is insufficient communication between doctors and patients about why antibiotics might not be necessary. Money and legal concerns can also affect decisions.Doctors might worry about getting in trouble or upsetting patients if they do not follow what is seen as normal, even if the guidelines stipulate that they should act differently based on the evidence.So, even if the guidelines recommend not using antibiotics to treat irreversible pulpitis, these various factors can make it challenging for doctors to follow such recommendations in real-life situations. Generally, the literature underscores a significant discrepancy between clinical guidelines and actual practices in the management of irreversible pulpitis.While guidelines generally advise against the routine use of antibiotics to treat this condition, the data indicate a prevalent trend of over-prescription across various regions.This disparity raises concerns about the effectiveness of and rationale behind current treatment approaches, especially considering the risk of antibiotic resistance. Autophagy The interesting concept of autophagy has been researched with respect to irreversible pulpitis, and it is worthwhile to shed light on this concept.Autophagy actively maintains cellular homeostasis by contributing to cellular metabolism, innate immunity, and cell survival.There is a significant relationship between autophagy and inflammation in infections, cancer, metabolic disorders, and liver diseases.It has been suggested that autophagy correlates with pulpitis.Most researchers have pointed out a close connection between autophagy and oral diseases such as periodontitis and pulpitis.Ye Yung (2023) screened nine hub lncRNAs as candidate regulators based on ceRNA networks, thereby offering a new reference for the further exploration of the association between autophagy and irreversible pulpitis [92].A similar line of research was reported by Qi, S. (2019) regarding the expression of proteins in pulpitis [93]. The levels of NKA, SP, IL-8, and MMP-8 vary depending on the clinical situation.For example, when the pulp tissue of symptomatic-irreversible-pulpitis-affected teeth and GCF specimens were compared to healthy tooth pulp tissue and GCF specimens, it wa sobserved that the levels of NKA, SP, IL-8, and MMP-8 increased dramatically.NKA, SP, IL-8, and MMP-8 levels were found to be considerably lower in GCF samples from teeth with symptomatic irreversible pulpitis 1 week after the inflamed pulp was removed.Finally, SP, IL-8, and MMP-8 levels were shown to be higher in pulp tissue samples from patients with symptomatic irreversible pulpitis who scored higher on pain scales than those who scored lower on pain scales [95]. Gene Expression and Biomarkers in Pulpitis A list of normalized differentially expressed (DE) genes was created in Liu, L's (2021) study in order to analyze the molecular pathways of pulpitis and find possible biomarkers for diagnosis [96].Antibiotics have a great influence on both the host's and micro-organisms' genetic characteristics (for example, regarding interactions between antibiotic drugs and resistance genetic mutations [97]).Biomarkers aid in the qualitative detection of antibiotic resistance genes [98]. Conclusions Based on the referenced studies and surveys, it was observed that healthcare providers often prescribe antibiotics based on uncertain diagnoses and the expectations of patients regarding these drugs.While antibiotics can reduce the risk of infection, their chronic use has led to a rise in resistant bacterial strains.Therefore, it is crucial for dental clinicians to be educated and trained in using antibiotics as a supportive measure rather than as a replacement for pain relievers.Clear clinical and prescription guidelines, along with accurate diagnostic techniques, are necessary to ensure the effective use of antibiotics without jeopardizing patient health.Individual health institutions and organizations involved in healthcare delivery should establish their own consensus guidelines for antibiotic prescription.This approach will enable the practice of 'precision dentistry' in antibiotic prescription. Recommendations The management of irreversible pulpitis is currently facing a critical challenge, as actual clinical practices often deviate from established guidelines.This issue mainly arises from inadequate adherence to these guidelines, excessive dependence on antibiotics, and educational gaps among dental professionals.The data highlight the urgent need for a unified approach to realign clinical practices with contemporary research and guidelines, focusing on the following areas: 1. Educational Efforts-Improving the training of dental professionals in understanding the development of pulpitis and the judicious use of antibiotics. 2. Supporting Guideline Compliance-Supporting adherence to clinical guidelines through ongoing professional development and regulatory initiatives. 3. Utilizing Molecular Research-Incorporating recent molecular research findings into clinical practice to provide more specific and effective treatments.4. Managing Patient Expectations-Instructing patients on the nature of dental conditions and proper medication usage to decrease antibiotic prescriptions driven by patient demand. Table 1 . Characteristics of the included studies. Table 2 . [62]s about prescribing antibiotics[62].Multiple antibiotics are superior to a single antibiotic.6: Bactericidal agents are always superior to bacteriostatic agents.7: Antibiotic dosages, dosing intervals, and the duration of therapy are established for most infections. 8: Bacterial infections require a "complete course" of antibiotic therapy. [58]cal and dental histories; • Rule out the presence of fever, malaise, fatigue, dizziness, or other disability; • Measure the patient's pulse and temperature (a normal temperature is 36.3-37•C);•Define the nature and extent of the swelling; • Identify the cause of the infection.During this process, determine whether the patient should be treated in a dental or hospital setting.This can be performed by checking for the following[58]:	2024-01-24T16:03:15.393Z	2024-01-01T00:00:00.000	{ "year": 2024, "sha1": "4004c8b03720ad179d8feaf5cc0eee81a28a1eb1", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/1422-0067/25/2/1357/pdf?version=1705936517", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "5395a9962e2b2953a18da38c93bdd9382c4d7671", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
53026932	pes2o/s2orc	v3-fos-license	Position as Well as Velocity Dependence of Spasticity—Four-Dimensional Characterizations of Catch Angle We investigated the muscle alterations related to spasticity in stroke quantitatively using a portable manual spasticity evaluator. Methods: Quantitative neuro-mechanical evaluations under controlled passive elbow stretches in stroke survivors and healthy controls were performed in a research laboratory of a rehabilitation hospital. Twelve stroke survivors and nine healthy controls participated in the study. Spasticity and catch angle were evaluated at 90°/s and 270°/s with the velocities controlled through real-time audiovisual feedback. The elbow range of motion (ROM), stiffness, and energy loss were determined at a slow velocity of 30°/s. Four-dimensional measures including joint position, torque, velocity and torque change rate were analyzed jointly to determine the catch angle. Results: The catch angle was dependent on the stretch velocity and occurred significantly later with increasing velocity (p < 0.001), indicating position dependence of spasticity. The higher resistance felt by the examiner at the higher velocity was also due to more extreme joint position (joint angle) since the spastic joint was moved significantly further to a stiffer elbow position with the higher velocity. Stroke survivors showed smaller ROM (p < 0.001), higher stiffness (p < 0.001), and larger energy loss (p = 0.005). Compared to the controls, stroke survivors showed increased reflex excitability with higher reflex-mediated torque (p < 0.001) and at higher velocities (p = 0.02). Conclusion: Velocity dependence of spasticity is partially due to joint angle position dependence with the joint moved further (to a stiffer position where higher resistance was felt) at a higher velocity. The “4-dimensional characterization” including the joint angle, velocity, torque, and torque change rate provides a systematic tool to characterize catch angle and spasticity quantitatively. INTRODUCTION Spasticity commonly occurs to patients with neurological disorders, such as stroke, spinal cord injuries, cerebral palsy, and multiple sclerosis (1)(2)(3). Spasticity is commonly defined as "a motor disorder characterized by a velocity-dependent increase in tonic stretch reflexes (muscle tone) with exaggerated tendon jerks, resulting from hyperexcitability of the stretch reflex, as one component of the upper motor neuron syndrome" (4). Various measures have been used to assess muscle alterations associated with spasticity. In the clinical setting, spastic muscle is usually evaluated by grading the resistance to a passive stretch felt by a clinician using the Ashworth scale, modified Ashworth scale (MAS), and the Tardieu scale (5,6). The felt resistance could be caused by a combination of neural and peripheral origins (i.e., biomechanical factors such as soft tissues or muscle properties). Although clinical measures are convenient to use, they can be subjective, less sensitive and qualitative rather than quantitative to varying degrees. Previous studies raised questions about reliability of the MAS assessment of spasticity (7)(8)(9)(10). On the other hand, the Tardieu scale has been suggested as an alternative to the MAS (6). Tardieu scale is conducted using various stretch velocities rather than using only one velocity in MAS while determining the angle where resistance felt (i.e., catch angle). It is argued that the MAS does not differentiate between spasticity and contracture, while the Tardieu scale is not confounded by the presence of contracture (11). However, with either scale, determinations of the catch angle (12,13) and range of motion (ROM) are influenced by stretch velocities and stretch force and subject to errors in reading the joint angle during assessments. A quantitative assessment with controlled passive stretches is needed to improve the reliability of the clinical measures. Well-controlled quantitative measures, based on motorized mechanical perturbations and electrophysiological approaches, are mostly used in laboratory settings (5,(14)(15)(16), but size and ease-of-use issues limit their applications in clinical settings (17)(18)(19). Several portable devices have also been developed and spasticity evaluations were performed by deriving viscous neuro-mechanical properties of the limb from passive movement kinematics and joint reaction torques (19)(20)(21)(22). However, those measurements did not translate easily to the common clinical assessments of ROM and catch angle. Reflex threshold measured in joint angle during passive movement has been used effectively to evaluate spasticity by investigating the onset of muscle activation to applied disturbance (23)(24)(25). In spite of the current development of spasticity quantification, in clinical setting thus far, clinicians evaluate spasticity based on how much resistance they feel as well as where they feel the reactive resistance while manipulating the joint quickly. The relation with regard to velocity dependence between stretch-induced muscle activation onset and the resistance (catch) felt by the clinicians in the stroke survivors has not been investigated thoroughly. Furthermore, it is not clear whether catch angle is also joint angle position dependent. In other words, whether joint angle might play a role in the increasing resistance felt by clinicians at a higher velocity and being judged as velocity-dependent spasticity is uncertain. A comprehensive but simple way considering stretch velocities, reflex-mediated muscle torque and joint angle is needed to assist clinicians understand the muscle alteration due to neurological disorders and interventions. Therefore, the purpose of this study was to introduce an innovative and quantitative way to depict the spasticity according to the concept of Tardieu scale and further examined the contribution of joint angle position dependence to the catch felt by the examiner. Subjects Twelve chronic stroke survivors (53.0 ± 8.5 years old, ten males and two females) who had a stroke more than 1 year (9.3 ± 5.6 years) and nine healthy controls (51.4 ± 24.9 years old, nine males) were included in this study. The stroke survivors with elbow flexors spasticity were recruited in the study. The subjects who had shoulder or elbow contractures were excluded from the participation. Table 1 depicts the characteristics of the stroke survivors. The healthy controls had no history of neurophysiologic or musculoskeletal disorders. The study was approved by the Institutional Review Board of Northwestern University. All subjects gave written informed consent before the experiment. Instrumentation The manual spasticity evaluator (MSE) used in this study was set up as a portable device to assess spastic muscles. A torque sensor (Transducer Techniques, CA, USA) and a hollow-shaft potentiometer (Vert-X51, Contelec AG, Switzerland) comprising MSE, were used to measure joint torques and joint positions respectively (26,27). Adjustable braces and supports were used to position the forearm and upper arm properly with respect to the MSE (Figure 1). Two mechanical stops were used to restrict the moving range of device that prevents over-stretching of the spastic joint. Biceps and triceps muscles activations were monitored using surface EMG electrodes (Bagnoli-8, Delsys Inc., Boston, USA). The torque, position and EMG signals were sampled at 1,000 Hz with a 16-bit resolution. A custom data acquisition program was used to provide real-time audio and visual feedback to help an examiner control the peak stretch velocity and the peak stretch torque (terminal torque) (26). When the examiner passively moved the subject's forearm, the target velocity and torque profile with boundary lines (e.g., ±10% of target velocity or target torque), as well as the instantaneous joint velocity and torque were displayed on the monitor. At fast velocities (90 • /sec and 270 • /sec), the data acquisition program provided audio feedback for controlling peak velocities during passive stretching. At the slow velocity of 30 • /sec, audio feedback was used to indicate that the designated torque limit had been reached and to stop the passive stretching. By doing so, the applied stretch force could be consistent while quantifying the spastic muscle each time. A test-retest reliability investigation of the MSE was performed on the healthy controls by the same rater twice with 1 day apart. Excellent reproducibility was found in measures derived from fast stretching (ICC= 0.88) and from slow stretch (ICC= 0.89 for passive ROM, 0.84 for stiffness and 0.75 for energy loss). Excellent intra-rater reliability was also shown while using MSE in the pediatric population (28,29). Experimental Procedures In a quiet room, a therapist assessed the spastic elbows of the stroke survivors using MAS (30). The therapist followed the procedure described previously (31) with the exception of the body position. In the current study, the subjects sat upright comfortably instead of lying down. The therapist stabilized the upper arm by holding it proximal to the elbow and moved the forearm in a quick passive motion (∼1 sec) throughout the available elbow range of motion from the end of flexion to maximal extension. During the experiment, the subject sat next to the MSE with the elbow flexion axis aligned with the rotation axis of the MSE. The shoulder was positioned at 75 • of abduction and the forearm and upper arm were secured to the supporting braces. Surface EMG electrodes were placed on the biceps brachii and triceps brachii with the reference electrode placed on the lateral epicondyle. In the clinical practice, the modified Tardieu R1 is the angle of the catch thought to be due to induced stretch reflex at an as fast as possible velocity. The passive ROM (R2) is graded under a slow velocity, which would not trigger the stretch reflex (12). In the current study, we chose 30 • /sec as the slow velocity to measure R2 and chose two fast velocities (90 and 270 • /sec) to detect the catch angle (R1) using MSE. The slow velocity of 30 • /sec was chosen because it did not induce stretch reflex during manual tests that may confound the R2 measurement. Two high velocities (90 and 270 • /sec) were chosen because 90 • (right angle) and its multiples are relatively easier for the rater to perceive during manual tests. Initially, the torque and position offset were recorded with the subject's elbow in the neutral position, defined as the position where subjects felt the most comfortable, not being stretched or restrained (79.6 • ± 10.9 • elbow flexion for stroke survivors, and 75.0 • ± 6.5 • elbow flexion for healthy controls with full extension defined as 0 • elbow flexion). To determine the passive ROM and stiffness of the elbow, we then moved the elbow at 30 • /sec until reaching a pre-defined torque (3 Nm) or a mechanical stop. Three trials of passive stretch separated by 1 min were performed at each of 90 • /s and 270 • /sec to evaluate the velocity-dependence of spasticity and catch angle. With practice, the examiner was able to control the peak velocity of passive stretch to match the target velocity as shown on the display (±10% of target velocity). One stretch cycle was defined from full flexion to full extension then back to full flexion. Since spasticity may be altered by repeated stretching (32), the elbow was not stretched more than three cycles in each trial. We also instructed the subjects relax during the tests. If voluntary muscle contractions to assist the stretch were detected by the examiner and shown as intermittent or continuous EMG activities of triceps brachii muscles, and/or EMG activities of the biceps brachii preceding the stretch initiation, the trial was discarded. The examiner would then let the subject rest before stretching the joint again. The MAS scores, the biomechanical measures and MSEmeasured R1 and R2 were taken by the same examiner. Data Analysis and Statistics Biomechanical Measurement: Torque and position signals were filtered with a low-pass cutoff frequency of 50 Hz. The derivative of torque with respect to time, dτ (t)/dt was calculated from the acquired torque signals. All the biomechanical measures captured at 30 • /sec were determined within the torque limits of 3 Nm, including the passive ROM [also described as the R2 angle (12)], elastic stiffness (K), energy loss, and elbow flexor torque at several joint angles. Stiffness, K, was defined as the slope of the torqueangle relationship of ascending arm at 70 • of elbow flexion, a common range among the subjects. The energy loss was the area between the ascending and descending limbs of the torque-angle curve during rotation (5). Since different subjects had different ROMs and limb inertias, the energy loss was normalized by the transverse inertia and the individual's range of motion. The transverse inertia was estimated from the length of the forearm, the perimeter of the elbow, and maximum forearm and wrist circumference (33,34). Joint angle position dependence of the resistance torque was evaluated at three selected elbow flexion angles (45 • , 60 • , and 75 • ) in the two groups and normalized by the body weight and height (35). The angles were near the end of stretch and around the common range (70 • ). EMG Signal Processing: The EMG signals were filtered with a passband of 10-450 Hz. To determine the onset of muscle activations (reflex-mediated responses) for verifying the catch angle, EMG signals were presented in a linear envelope (LE) form: with the filtered EMG data full-wave rectified and then low-pass filtered at 10 Hz. The onset of the reflex EMG was determined when the amplitude of the EMG LE was larger than the mean plus three standard deviations (SD) of the background EMG (36). The background EMG was measured during the quiescent period before the passive stretch. The elbow flexion angle corresponding to the reflex EMG onset was thus determined. Catch Angle Determination and Characterization of spastic muscles: Catch angle is where a sudden occurrence of increased muscle activations in response to a quick passive stretch, which leads to an abrupt stop or increased resistance (torque) before the joint rotation reaches the end of ROM (37). This behavior can be captured using MSE as shown in Figure 2: the elbow flexor torque increased with elbow flexors being stretched (flexion angle decreased in Figures 2A,B). When the passive stretch triggered a stretch reflex (EMG firing shown in Figure 2C), the elbow flexors contracted strongly causing the abruptly increased torque rate (dτ (t)/dt) as indicated by the second positive peak in Figure 2D where the catch angle was determined. Figure 2E shows that the examiner responded to the abruptly increased resistance with a decreased velocity to avoid over-stretching the joint (value of velocity changed toward zero). We then developed a systematic way to determine R1. Since dτ (t)/dt was affected considerably by the inertia during the initial acceleration (the first peak of dτ (t)/dt), the local minimum of velocity was selected as the first landmark (the dashed vertical line in Figure 2E), which occurred shortly after the catch. Next, the joint angle corresponding to the peak dτ (t)/dt in the 300-ms window preceding this landmark was determined as the catch, R1 (26). The ratio R1/R2 was derived through dividing the angular displacement between R1 and flexion angle by the overall ROM, to represent the portion, which is free from the catch. A four-dimensional display, including the variables of joint angle, velocity, torque (τ ), and dτ (t)/dt, was developed to illustrate the events involved in the catch that provides a more comprehensive quantification of spastic muscles group around the tested joint ( Figure 2F) at various stretch velocities. The width of the shaded area in Figure 2F represents velocity. Note the increased width as the high velocity was maintained. The torque increased as the elbow was moved into extension indicated by the dashed arrow. When a catch occurred, dτ (t)/dt increased abruptly and reached a local maximum (the relatively hot color of the dτ (t)/dt line). The abruptly increased resistance and the examiner's reaction to the catch resulted in a FIGURE 2 \| Representative kinematic, kinetic and EMG signals during the passive stretch. As the elbow was moved from 100 • flexion to extension (A), the elbow flexion torque τ (B) generated by the examiner on the stretched flexor muscles increased accordingly. Sequentially, the stretch induced a reflex response in the biceps (vertical line in C). The operator felt the sudden increase in resistance (vertical line in D) during the "catch," and responded by decreasing the stretch velocity (vertical line in E). A four-dimensional display (F) was developed to illustrate the aforementioned events. quick velocity reduction to a local minimum (choke, the narrow shaded area). Statistics: Since the data was not normally distributed, nonparametric statistics (Friedman test) were used for comparisons of catch angles at different stretch velocities with a significance level of p < 0.05. To investigate differences in the biomechanical measures (ROM, stiffness, torque at three joint positions, energy loss) and the velocity-dependent properties of muscle compared between the control and CVA groups, a Mann-Whitney Utest with a significance level of p < 0.05 was conducted. Spearman correlation was used for correlating the MAS with all biomechanical measures as well as the catch angle. Correlation was significant at p < 0.05. The intra-class correlation coefficient was chosen as the test statistic to evaluate the test-retest reliability. The two-way mixed model of intra-class correlation coefficient was used. An intra-class correlation coefficient ≥ 0.75 indicated significant reproducibility (38). SPSS software (SPSS Inc., Chicago, Illinois) was used to perform all statistical analysis. Figure 3C shows the representative curve in a healthy control, the dτ (t)/dt remained at a constant lower value (below 10 Nm/s) throughout the available range of motion. In addition, for healthy controls the joint resistance was much lower when compared to stroke survivors (p < 0.05). Dependence of R1 on Stretch Velocity and Velocity-Dependent Properties of Spastic Muscles During the passive elbow extension, the catch angle in stoke survivors was significantly smaller (elbow more extended, p < 0.001) with increasing stretch velocity ( Figure 4A). This indicates that the catch angle occurred later at faster stretch velocities. Furthermore, the R1/R2 was significantly higher at higher stretch velocities ( Figure 4B, p < 0.001). As expected, catch was not observed in any healthy controls. In the representative case shown in Figures 3A,B, catch occurred at 78.2 • and 58.8 • elbow flexion at the stretch velocities of 90 • /s and 270 • /s, respectively. As a feature of spasticity, the peak resistance torque during a stretch increased with the increasing stretch velocity in the stroke survivors (p < 0.005, Figure 5). Healthy controls also showed increased peak resistance at the velocity of 270 • /s when compared to the velocity of 90 • /s (p = 0.005). The slope of the relationship between the peak torque and the stretch velocity was significantly higher in stroke (9.34 nu × 10 −5 ± 4 × 10 −5 Nkg −1 deg −1 s) than that in healthy controls (4.99 × 10 −5 ± 2.95 × 10 −5 Nkg −1 deg −1 s; p = 0.02). Biomechanical Measures of the Spastic Muscles ROM measured at a controlled torque of 3 Nm was significantly reduced in the stroke survivors as compared to that of the healthy controls (74.2 • ± 21.5 • vs. 107.6 • ± 8.7 • , p < 0.001; Figure 6A). During extension with a 3 Nm torque limit, the stroke survivors stopped earlier at larger flexion angles (30.0 • ± 17.6 • ) compared to healthy controls (10.2 • ± 10.8 • ; p < 0.01). Figure 7 shows the examples of restricted ROMs for the severely spastic (MAS=3), mildly spastic (MAS=1) and healthy controls that were 36 • -93 • , 3 • -104 • , and −1 • -106 • elbow flexion, respectively. Stiffness measured at a prescribed elbow flexion angle of 70 • was significantly larger in stroke survivors when compared to healthy controls (Figure 6C; 0.058 ± 0.028 Nm/deg vs. 0.017 ± 0.008 Nm/deg, p < 0.001). The stiffness for the severely spastic, mildly spastic and healthy controls was 0.162 Nm/deg, 0.042 FIGURE 4 \| Dependence of catch angle on the passive movement velocity. The catch angle (R1) and the ratio of the angular displacement between catch angle and initial flexion angle over ROM (R1/R2) with a controlled torque limit are shown in (A,B), respectively. Each symbol represents a stroke survivor. Friedman test was used for multiple comparisons of catch angles at different stretch velocities with a significance level of p < 0.05. DISCUSSION This study demonstrates 4-D plot, a comprehensive, and systematic way, to investigate the group of spastic muscles around the elbow. Spasticity-related biomechanical characteristics, including joint ROM, joint torque, stiffness and energy loss, at various controlled velocities can also be acquired at the same single setting. During a controlled slow stretch, the MSE assesses biomechanical properties of the joint including the ROM, stiffness and energy loss using a controlled slow stretch and determine the catch angle at controlled fast stretch velocities. Convenient spasticity quantification has been a challenge. In order to evaluate stretch reflex responses accurately, the way to elicit spasticity should be standardized. Many factors including the pre-activation of muscles, position of the joints involved, and applied stretch torque and velocity, as well as the experience of clinicians may result in different outcomes and interpretations. Clinical measures, such as the MAS or Tardieu scales, have been used to identify the catch angle during passive stretch. However, the angles determined using these scales may not be accurate; they generally occur later than a biomechanicallydetected catch and suffer from poor inter-rater reliability (39). As shown in Figures 2, 3, the examiner reacted to the abruptly increased joint resistance by slowing or stopping the passive stretch. However, the catch occurred up to 300 ms prior to this slowing or stopping point. Therefore, peak dτ (t)/dt, instead of the stopping point, should be used as the indicator of the catch angle. In the current study, the instantaneous velocity change along with dτ (t)/dt were used to determine the catch angle reliably and to minimize potential human error. As one can see in Figures 2, 3, the human's reaction characterized as local minimum of speed (choke) was further away from the catch determined by dτ (t)/dt. The discrepancy between human's reaction and the true catch implies the potential human errors in the subjective clinical measures of "catch angle." As seen in the examples in Figures 2, 3, the differences ranged from 3 to 8 • . In a clinical setting, the catch angle reading is usually from eyeballing of a goniometer moving with the joint, which may introduce even a larger error in determining the catch angle. It should be noted that another peak of torque change rate, which occurred earlier during the passive stretch, was when an examiner overcame the resistance from the limb inertia and was not related to catch angle. Velocity-dependent increase in muscle tone is a key attribute to spasticity as shown in the current study as well that the stretch velocity has obvious effects on the normalized peak torque of catch (larger slope in Figure 5). However, the velocity dependence of spasticity might be partially due to joint angle position dependence. The delayed catch angle associated with fast velocities in our study showed the joint angle position dependence of the increased resistance. At a faster stretch velocity, the joint was quickly stretched further into an angle position where higher resistance existed. Assuming reflexmediated torque developed 60 ms after the stretch reflex was triggered, the joint would have been moved 10.8 • further in 60 ms at 270 • /s as compared to the stretch at 90 • /s ((270 • /s-90 • /s) × 0.06 s = 10.8 • ). The extra 10.8 • moved the joint to a stiffer position, which might make the examiner feel higher resistance at FIGURE 5 \| Velocity-dependent property of the muscle in subjects post-stroke (solid circles) and healthy controls (solid triangles). The dotted lines demonstrate the dependence on the velocity. Subjects post-stroke had a stronger velocity dependence compared to healthy controls (p = 0.02) and had higher torque at all velocities than healthy controls (p < 0.001). Asterisks () indicates significant differences between two velocities. FIGURE 6 \| Comparisons of passive properties of elbow flexors between the healthy controls (n = 9) and stroke survivors (n = 12). Patients post stroke showed reduced ROM (A), higher energy loss (B), increased stiffness (C), and increased resistance torque at several angles (D), represented as mean ± SE (standard error of mean). Asterisks () indicate significant difference (p < 0.05) between the two groups (D). Mann-Whitney U test with significance level of P < 0.05 was used to determine the differences. a faster velocity. Similar results were reported that greater catch angles were associated with higher applied angular velocities (28,40). Velocity-dependence of the catch angle further confirms that a standardized method to evaluate spastic muscles is essential since the interpretation of catch angle can be confounded by the stretch velocities. In the current study, passive properties were measured under real-time feedback control by moving the elbow slowly without eliciting a reflex response. The lack of a reflex response was corroborated by silence in the EMG signals of the stretched muscle. Significant changes in the passive properties of the spastic elbow of stroke survivors were observed when compared to healthy controls, including increased stiffness and flexors resistance, decreased ROM, and increased energy loss (Figure 6). Similar changes were also found in the spastic ankle of stroke survivors with hemiparesis (5). In general, the changes in stiffness and ROM were consistent with what have been reported previously (14,41,42). Since the supporting braces fixed to the MSE might hinder the ROM near the end of flexion, the value of elbow ROM shown in current study was smaller than the observations in previous studies (43)(44)(45). In addition, Figure 7 also shows that the reduced ROM was not only in one end, the stroke survivors may lose the range toward flexion or extension that can be relevant to daily functions. The correlation between passive stiffness and MAS demonstrates that the MAS is more closely related to the passive stiffness of the joint than to joint spasticity, even though it has been commonly used for assessing spasticity in both clinical and laboratory settings. MAS only includes as single stretch velocity and is scored by the amplitude of joint resistance that potentially make MAS reflect passive stiffness over spasticity. Because the felt joint resistance could be from either spastic responses and/or passive stiffness (46,47) and without various stretch velocities those could not be distinguished. The consequence of this ambiguous assessment may mislabel patients who have increased passive stiffness alone as having spasticity and being treated with inappropriate interventions. Damiano et al. indicated that evaluating patients at different velocities may help to distinguish passive stiffness from spasticity (46), which we adopted in our developed method for spasticity quantification. Administering the Tardieu scale using the MSE could provide proper spasticity characterization under various controlled velocities. The intra-rater reliability of clinical assessments can be improved using the MSE that contains accurate sensors and LIMITATIONS OF THE STUDY The brace of the system we used might prevent the all range of motion toward the end of flexion due to the contact of muscle bulks and brace. When there was no compromise using this system to assess the elbow flexors spasticity, one should interpret the passive flexion ROM with cautions. The sample size of the current study is relatively small. A larger size of sample should be considered in a future study. ETHICS STATEMENT This study was carried out in accordance with the recommendations of Northwestern University Institutional Review Board with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Northwestern University Institutional Review Board. AUTHOR CONTRIBUTIONS Y-NW, L-QZ, H-SP, and YR designed and carried out the experiment. Y-NW and L-QZ performed the data analysis. Y-NW, H-SP, YR, and L-QZ wrote the manuscript with support from J-JC and ER. FUNDING This work was supported in part by grants from the NSF (award no. IIP-0750515) and NIH (award no. R01-HD044295).	2018-10-26T13:03:52.163Z	2018-10-26T00:00:00.000	{ "year": 2018, "sha1": "8a114f62284daba426a9b430bbf5e89363a25e94", "oa_license": "CCBY", "oa_url": "https://www.frontiersin.org/articles/10.3389/fneur.2018.00863/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "8a114f62284daba426a9b430bbf5e89363a25e94", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
134486749	pes2o/s2orc	v3-fos-license	Study on Optimal Economic Evaluation of Industrial Drilling Platform for Shale Oil and Gas Development The development of shale oil and gas in China is at a critical stage, and the technology of factory drilling platforms, as the key technology of shale oil and gas development in North America, is worthy of in-depth study on its function, optimization mode and economic evaluation in China’s shale oil and gas development industry. The number of wells and the operation mode of factory drilling platforms will directly affect the economics of factory drilling platform technology, but there is a lack of technical and economic evaluation models of such platforms. Therefore, by analyzing the application of factory drilling platforms and the international advanced design method, a technical and economic evaluation model is established, and data from the Sichuan Shale gas field are calculated and analyzed. The research shows that the proposed economic evaluation and analysis method, and the calculation of the optimal number and operation mode of factory drilling platforms, can provide guidance for the overall deployment and design of this technology, and some relevant suggestions are proposed in the light of the problems related to the use of these platforms by Chinese oil and gas companies. Introduction From a global perspective, the successful mining of tight oil has brought about major changes in the world's oil and gas exploration and development, but is also gradually affecting the world's energy supply and demand pattern. According to the latest results of a global evaluation of tight oil and gas resources released jointly by the U.S. Energy Agency EIA and the American company Advanced Resources International (ARI), global tight oil technology can produce 47.3 billion tons of oil, accounting for 10% of total oil resources, and in the period 2018-2035, tight oil production is expected to more than double from the current 247 million tons to 514 million tons, mainly due to the high reserves of tight oil and to technological progress. China also has a wealth of tight oil resources, but due to technical constraints has so far only carried out experimental development. It is very important to choose the appropriate development technology and mode as the key factors to deal with the challenge of tight oil development. Based on systematic analysis of industrial drilling platforms, which are the key technology in the successful exploitation of tight oil abroad, this paper carries out an in-depth study to evaluate the economic benefits, future development prospects and problems faced with the technology, and provides a powerful reference for the development and production of tight oil in China. Technical requirements for shale oil and gas development China's tight oil resources amount to about 7 billion tons, of which the amount of technologically recoverable resources is about 2~2.5 billion tons, accounting for 9.3% of the world's tight oil resources, and mainly distributed in Songliao, Ordos, Sichuan and other basins, which have great potential for exploitation. With the increasing shortage of oil and gas resources and the intensifying gap between supply and demand, many countries have accelerated the development of tight oil. Among them, large-scale mining of tight oil in North America successfully reversed the 24-year decline in oil production in the United States, and American oil companies attached great importance to improving their rig efficiency and oil and gas well production during the previous period of low oil prices. Although the number of rigs used at that time decreased significantly, the oil and gas produced by each rig increased steadily. The was firstly because when a rig is moved to the core of the shale oil and gas, the output of that single well is improved, and secondly that when the operator gives priority to the elimination of old drilling rigs, the number left in operation decreases, so the oil and gas produced by the single drilling rig increases. The third reason is the use of factory drilling platform technology, which is key to improving the drilling efficiency and shortening the drilling cycle, such that the annual drilling output of a single drilling rig is increased. In China, the shale oil and gas industry is in the initial stage of large-scale development. With the increase of the reservoir depth, the degree of geological evolution is deepened, the structure is more complex, and the exploration technology requirements are higher, while there are inefficiencies in development, tension over land use, and high costs, etc., so there is need to optimize the drilling technology. Thus, factory drilling platforms are advantageous in solving these shale oil and gas development problems. Definition of factory drilling platform technology The basis of industrial drilling platform technology is a well layout based on a large-scale cluster of horizontal wells (a well pad). Since the application of "well factory" technology in shale oil and gas development in the United States, a new drilling platform has been generated, which is called the factory drilling platform. The factory drilling platform was first proposed by Schlumberger at the 2011 International Association of Drilling Contractors (IADC) Advanced Drilling Rig Technical Conference. This platform is a drilling process "factory", applied to the oil and gas field development process, which enables the use of repeatable experience and the mastery of downhole risks, with a specially manufactured drilling rig or specialized drilling technology using mechanized labor, instead of manual construction of the field production platform technology. The factory drilling platform technology synthesizes a remote operation drilling control center, field technology and real-time data control, and the technically optimized design of the factory drilling platform optimizes the number of wells operated, the selection of the drilling rig, the factory construction process and other parameters under the condition that the surface and underground well distance are known. The objective is to keep the cost of a shale oil and gas development project to a minimum, from a fast mobile drilling rig to a single well field of multiple wells for batch drilling completion and offline operations, in a pipeline-type way, in order to achieve side drilling, side fracturing, side production, and improve the efficiency of oil extraction. Application of factory drilling platform technology In the United States and Canada in the major shale oil and gas and dense hydrocarbon producing areas, the use of the factory drilling model to allow the rapid growth of wells has now exceeded 70%, and in some individual production areas has reached more than 90%, with a large number of retrofitted rigs and complete sets of custom-built drilling rigs put into use. Chevron not only uses factory rig technology to develop local shale oil and gas in the United States, but also applies it in other regions, drilling more than 300 wells a year in Thailand, where the company is using factory drilling platform technology to achieve the maximum efficiency with minimal investment, and all its platforms are 3 developed using the same process, practice and design. These factory rigs make Chevron the largest producer of crude oil and natural gas in Thailand. At present, North America has not only undergone a transformation of the existing drilling rigs, and the installation of rapid mobile devices formed by factory drilling rig platforms, but also has a complete set of customized new factory drilling platforms in operation. The successful development of a variety of advanced drilling rigs suitable for factory drilling platforms has provided equipment support, giving full play to the technical advantages of factory drilling, and has effectively promoted the popularization and application of factory drilling platform technology in North America. International optimized design method of factory drilling platform At present, the optimized design of advanced factory drilling platforms by international petroleum companies mainly includes the following: Development of multiple production layers for a single factory drilling platform. A single drilling platform is used to a) achieve joint development of multi-production layers, b) make full use of the well platform, c) reduce the spacing between the working wells and wells in the platform, d) increase the number of wells in a single operating platform, e) reduce the footprint of the drilling platform, f) reduce operating costs through sharing of land, drilling equipment, mud tanks and water treatment systems, and g) improve efficiency through factory operations. This collectively improves the overall benefit of the block. This approach is widely used in shale oil and gas development in the United States at Eagle Ford and the Permian Basin and at Montney, Canada. Laredo Oil Company in the Permian Basin, using a single well field multi-production layer development, operates 20 well factory platforms drilling 60 horizontal wells, with the development of 4 layers of shale oil and gas, and achieving 6%~8% lower operating costs. Flexible factory drilling platform. Due to the large variation of a reservoir within dozens of meters, the traditional factory rig may result in less contact area between some wells and the reservoir, or the quality of the reservoir in contact may be poor, so that the production capacity is not as expected. Chevron has developed a flexible factory drilling platform, using data collection from the first few wells to adjust the well structure and to position later wells more effectively in real time, so as to improve the economic benefits of well plant development. From a comparison of 12 well group platforms in the Permian Basin, it is found that although the net present value of stand-up wells is similar, the total net present value of a flexible factory rig is more than twice that of the traditional factory drilling platform owing to the use of spare well positions. Factory drilling platform batch drilling mode. This refers to the drilling operation of the surface, straight well section and horizontal well section of multiple wells in batches according to a certain order, and greatly improves the efficiency of drilling operations through the use of water operations. The batch drilling operation mode has the following features: First, one or a number of drilling rigs can be used to achieve the same well section in the same well group configuration of the same drilling rig and bottom drilling tool combination, saving a lot of time changing drilling tools; The second involves the use of a number of drilling rigs suitable for the simultaneous operation of 2~3 wells; it is common to use a single mobile drilling rig for groups of 4~6 wells; Third, the multi-mouth well open, cementing, in turn, second open, cementing, so that drilling, cementing, and logging equipment continues to run, reducing the non-production time, and improving operational efficiency; Fourth is the reuse of the drilling fluid, reducing the alternation of drilling fluid. ConocoPhillips, Statoil, Halliburton and Schlumberger have used this technology during drilling in the Eagle Ford shale oil and gas areas. Therefore, the construction of a factory drilling platform should include the following considerations: First, the large-scale deployment of cluster drilling platforms; Second, a custom special drilling rig, fracturing equipment, especially a mobile drilling rig, high-performance drilling pump and circulation system, and high-power density fracturing vehicle; Third, a drilling rig automation upgrade, top drive, automatic drilling, automated wellhead operation equipment, and a pipe automatic discharge system; Fourth is mature, practical, and integrated drilling technology standardization; Fifth is a large-scale complete automatic fracturing fluid set of technology; Sixth is clean production technology, a functional platform area design, clear sewage diversion technology, a non-landing circulatory system, electric oil power technology, noise suppression technology, debris treatment technology, drilling fluid recovery technology, fracturing fluid recovery technology, and waste treatment technology. Evaluation model The ultimate goal of the research and application of factory drilling platform technology is to reduce the cost of the project, shorten the engineering cycle, optimize the design of the platform by optimizing the surface and underground well distance through optimizing the number of wells, selecting the factory drilling rig, constructing the factory drilling platform and other parameters, thereby minimizing the cost of shale gas development projects. Therefore, the purpose of establishing a technical and economic evaluation model of the factory drilling platform and the contents of the economic evaluation model are as follows: To calculate and evaluate the average cost increase and decrease of a single well with different well spacings (300, 600 and 900 m), different factory drilling platform operation modes, different well numbers per platform (2, 4, 6,..., 20), and thus to select the optimal number of wells and the optimal operation mode for the platform. In the formula: Q is the increase or decrease of the engineering cost under the mode of the factory drilling platform (all costs in million yuan); ∆Q ZQ is the pre-drilling costs increase or decrease; ∆Q DR is the drilling costs increase or decrease; ∆Q FR is the fracturing costs increase or decrease; ∆Q RQ is the equipment upgrade and transformation costs, ∆Q ZD is the land requisition costs increase or decrease; ∆Q L is the well site road construction costs increase or decrease; ∆Q G is the fracturing water supply pipeline construction costs increase or decrease; ∆Q R is the drilling daily fee increase or decrease, ∆Q M is the drilling fluid cost increase or decrease; ∆Q C is the casing cost increase or decrease; ∆Q D is the orientation cost increase or decrease; ∆Q S is the fracturing construction cost increase or decrease; ∆Q DF is the fracturing dynamic demobilization increase or decrease; ∆Q ZS is the drilling plug demobilization increase or decrease. The calculation formulas for each sub-item in formula (1) are as follows. 1) The formula for the increase and decrease of pre-drilling costs is: The increase and decrease of the daily drilling fee is closely related to the shortening of the well construction period, The calculation formula is: 3) Considering the recycling of drilling fluid, the calculation formula of the average cost increase or decrease of single well drilling fluid on an industrial drilling platform is: ∆q i ) (4) In the formula: C iDM is the unit price of the drilling fluid for the i time (million yuan/m3).. ∆q i is the amount of drilling fluid used for the i time and repeated use (m3). 4) The calculation formula of the casing cost increase is: 5) The increase in directional costs is proportional to the increased directional operating time and is calculated as: ∆T) (6) In the formula: C iD is the target construction day fee for the first (million yuan/d); ∆T i is the first increase of the targeted construction time (d). 6) For the factory drilling platform equipment transformation and upgrade costs, the calculation formula is: In the formula: Q RQ is the total factory drilling platform equipment upgrade and transformation cost (million yuan/d). The calculation formula for the increase and decrease of the fracturing construction cost of the factory drilling platform is: Case study In order to verify the rationality of the technical and economic evaluation model of the factory drilling platform, the author uses data from the Fuling shale gas field. The basic data used in the example include the following: the underground well distance is 600 m, the horizontal section length is 1500 m, the well body structure, borehole track, drilling fluid system, drilling process parameters, as well as the fracturing segment quantity, liquid volume, sand volume, construction parameters and other process parameters are selected to refer to the technical scheme of mature standards in the work room. 1) Reference well selection The focal X well completed in 2016 (without the use of factory drilling platform technology) was used as the reference for the economic analysis. The well drilling and fracturing are based on the standard design and technology of the work room. The well drilling cycle is 75 days, divided into 18 sections of a single well fracturing project, of which the cost is 70 million yuan, including pre-drilling and land acquisition costs of 5 million yuan, a daily drilling rig fee of 10 million yuan (according to the duration of the 85 day measurement), a drilling fluid cost of 4 million yuan, and a directional well service fee of 1.2 million yuan. The casing cost is 4 million yuan, the drilling rig mobile equipment renovation cost is 3.5 million yuan, the fracturing construction cost is 10 million yuan (including fracturing vehicle fees, distribution costs, etc.), and the fracturing test pneumatic demobilization cost is 1 million yuan. 2) Economic evaluation of operation mode of factory drilling platform According to the drilling day quota, the directional daily fee, casing cost and the construction flow of the industrial drilling platform in Sichuan shale gas field, the increase or decrease of the engineering cost in the factory drilling platform mode is calculated using formula (1), and the results are shown in Table 1. The calculation results show that the average cost of a single well drilling and fracturing project with a four-well type factory drilling platform is 65 million yuan, while the total project cost of the reference well is 78 million yuan. This is a reduction of 13 million yuan. The model calculation value is 11.8989 million yuan, and the relative error is 8.47%. The calculated results are basically in agreement with the actual data in the field, which shows that the evaluation model established by the author is reasonable and reliable. As shown in Table 1 drilling platform, when the platform has fewer than 4 wells, the reduction of the total cost of the project increases greatly with increase of the number of platform wells. When the number of platform wells exceeds 6, the increase of the total cost reduction of the project slows down; when the number of platform wells exceeds 8, the reduction of the total cost of the project decreases with increase of the number of platform wells. Therefore, under the current well network conditions and process parameters, the optimal number of platforms for the operation mode of the factory drilling platform in Fuling shale gas field is 4~8. For economic evaluation of the operation modes of different industrial drilling platforms, there are two platform operating modes for dual drilling rigs in the shale gas field pipeline in Sichuan: i) Type 30 small drilling rig + type 50 large drilling rig factory drilling platform operation mode, and ii) Type 30 small drilling rig + type 70 large drilling rig operation mode. Using formula (1), the savings against the total cost of the project under the operating mode with four kinds of factory drilling platforms are calculated, and the results are shown in Figure 1. Figure 1, when the number of wells per platform is less than 6, the working modes of the platform with a single drilling rig and of the platform with a double drilling rig are similar, and the saving on the project cost is more significant than that of the whole towing factory drilling platform operation mode. When the platform has more than 10 well mouths, the operation mode with the double pipeline drilling rig with "type 30 drilling rig + type 70 drilling rig" has outstanding advantages in terms of project cost savings. Therefore, it is suggested to explore and test the operation mode of the double pipeline drilling rig of "type 30 drilling rig + type 70 drilling rig" in the deep shale gas well in the two phases of the Sichuan shale gas field. C. Conclusion With the aim of calculating the cost reduction of the average single well project, the technical and economic evaluation model of the shale gas factory drilling platform is established, and analysis and verification of the calculation are carried out using data from the Sichuan shale gas field. The results are in agreement with the field data, which verifies the rationality and reliability of the model. The calculation results show that the factory drilling platform working mode significantly reduces the cost of the pre-drilling engineering, drilling fluid, and fracturing construction and so on, but increases the cost of casing, directional construction and retrofitting of the drilling rig and fracturing equipment. According to this calculation, the optimal number of wells for factory drilling platforms in the Sichuan shale gas field is 4~8, and the optimal operation mode is the platform with a double pipeline drilling rig with "type 30 drilling rig + type 70 drilling rig". 3) The economic evaluation model established does not consider the economic benefit arising from the different production cycles, but can determine the optimal well layout of the factory drilling platform according to the requirements of production operations, the commissioning cycle and so on in practical application on site. Based on lessons from foreign experience and technology, research on the technology of factory drilling platforms has been carried out in China, mainly through the transformation of conventional land drilling rigs. This initially meets the needs of site construction, and has included pilot applications in individual blocks such as Sichuan, achieving better stage results. However, compared with foreign countries, there is still a clear gap in the technology, which does not fully meet the economic and safety requirements of the factory operation. V. Main problems and suggested improvements to domestic factory drilling platform technology A. Main problem At present, although the domestic industry has a more mature horizontal well drilling technology, preliminary results from the Sulige, Changning-Weiyuan factory drilling platform show it has partly achieved a shortening of the drilling cycle. However, in terms of efficient movement, scale of use, economy and safety, these factory rigs are far from meeting the needs of shale gas and tight oil development. 1) This form of factory drilling platform is still relatively single, and the factory operation flow needs to be further perfected. At present, the use of a single-row single drilling rig or a double-row double drilling rig, a single rig model equipped, has not really achieved factory operations, the relevant equipment transformation has not taken place, and the connection and supporting technical specifications do not fully meet the economic and security requirements for factory operation. 2) In the domestic industry, the number and application of complete sets of customized factory platform rigs is limited. Factory working rigs transformed from conventional land drilling rigs are widely used, but have some disadvantages, such as their large structure, the large number of modules and big footprint. 3) Modified factory rig platforms have a low equipment allocation rate and weak technical level. Drilling speed-up tools are single, mechanical drilling speeds need to be improved, and rotation guidance and other core equipment technology is still lacking. At the same time, the number of fast-moving electric drive rigs suitable for factory drilling is seriously limited, the performance of PDC bits and oil-based drilling fluid needs to be improved, while personalized drill bits and drilling fluids and other personalized technology development capabilities need to be strengthened, as does rotary-oriented drilling technology. B. Related suggestions The application of factory drilling platform technology to the development of unconventional oil and gas resources represents a change of production mode in the industry, which is no longer restricted to the traditional construction mode of production. Shale oil and gas development in China's oil and gas industry needs improved construction efficiency, shorter drilling cycles and reduced single well costs. At the same time, the use of advanced factory drilling platform technology can improve single-well production, while optimizing the cost per ton of oil is also an effective way to improve the efficiency. Aiming to overcome the problems of terrain limitation, development cycles and costs holding back shale oil and gas development in China's oil and gas industry, it is suggested to speed up the construction of factory drilling platform technology, enhance the development benefits, learn from successful experiences at home and abroad, carry out testing and mining, and realize platforms for well deployment and factory drilling and fracturing operations. 1) There is a need to strengthen the technology of shale oil and gas development, and popularize the use of factory drilling platforms. Aiming at the development of technology, equipment, management, operational integration and serialization of shale oil and gas, it is beneficial to initiate the large-scale reproduction and popularization of the technology and operations experience of factory drilling platforms, realize their large-scale application, and improve the overall strength and level of working teams. A corresponding technical system and specification should be established, forming an intensive, integrated and efficient factory drilling platform management mode. 2) Targeted design of factory drilling platform equipment, with the step-by-point transformation of factory operations, is needed. The development of factory drilling platform facilities suitable for the complex terrain of China's factory drilling platforms should be accelerated, taking account of the poor road transport conditions, small platform area and low number of wells in the platform. The development and use of a set of custom factory operating rigs with small structure sizes and high modularity should also be accelerated. Systematic modification and upgrading of existing drilling equipment will make the drilling equipment of movable value. It is necessary to gradually realize the operation mode of drilling cluster well groups, establishing safe operation of factory drilling platforms along with the relevant standards of environmental protection supporting equipment. Research and development in related technical equipment and well plant technology is needed, to improve the shale oil and gas development level and device level step by step. 3) Standardized management of factory drilling platforms should be realized, together with building a self-owned drilling rig brand. To shorten the delivery time of custom factory drilling platform equipment requires material procurement, unified specifications, improved capital turnover, a reduction in the costs of the procurement of different types of equipment, and moves toward simpler, unified transport and management. Drilling rigs suitable for factory drilling platforms will be developed into the company's own factory drilling rig brand, to realize the personalization and diversification of drilling rig design, to meet the sustainable development needs of enterprises, while further enhancing the company's influence in the design of drilling rigs in petroleum equipment enterprises. 4) Taking account of the influence of the domestic ground conditions and environment, the industrial operation mode of volume transformation is being explored. Multiple water source wells, software tanks and fracturing fluid buffer tanks are designed in the middle of the platform, and the water supply is concentrated to realize continuous mixing of non-storage. By controlling valve regulation, the multiple units on the ground can realize seamless connection between well operations and fracturing construction in the same platform, and improve the construction efficiency. Through platform digging and draining tank, to achieve the pressure after the centralized return of discharge, unified treatment, module capacity matching, the process of close connection, simple and flexible operation, high operating speed, smooth information transmission, the goal is to achieve batch, pipeline type factory operations.	2019-04-27T13:13:32.024Z	2019-03-01T00:00:00.000	{ "year": 2019, "sha1": "fc766d5741438970b9307ec0352cc28af6215552", "oa_license": null, "oa_url": "https://doi.org/10.1088/1742-6596/1176/3/032032", "oa_status": "GOLD", "pdf_src": "IOP", "pdf_hash": "22034852e248c02c4bca6d08e4632985727f23ae", "s2fieldsofstudy": [ "Economics", "Engineering", "Environmental Science" ], "extfieldsofstudy": [ "Physics", "Environmental Science" ] }
218460234	pes2o/s2orc	v3-fos-license	Recent decline in the use of invasive neurocritical care monitoring for traumatic brain injury: A case report Background: In this article, we discuss the dramatic decline in the utilization of invasive cranial monitoring of patients with traumatic brain injury (TBI). Case Description: A 52-year-old male presented with a severe TBI following a motor vehicle accident. The initial computed tomography scan showed a subdural hematoma, and the patient underwent a craniotomy. However, preoperatively, intraoperatively, and postoperatively, the critical care team never utilized invasive cranial monitoring. Therefore, when the patient expired several weeks later due to multiorgan failure, his death was in part attributed to the neurocritical care specialists’ failure to employ invasive cranial monitoring techniques. Conclusion: Evidence-based and defensive medicine, cost containment, and a lack of leadership have contributed to neurocritical care specialists’ increased failure to utilize invasive hemodynamic and neurological monitoring for TBI. INTRODUCTION Traumatic brain injury (TBI) causes significant morbidity and mortality in the United States; 1.7 million TBIs account for 275,000 hospitalizations and 52,000 deaths annually. Over the past few decades, patients with TBI were connected to various invasive hemodynamic and cerebral monitors including central venous pressure, pulmonary capillary wedge pressure, pulmonary artery catheter, electroencephalography, transcranial Doppler (TCD), ideal hemoglobin, brain tissue oxygen monitoring, and sensory evoked potentials. [3,9] A recent metaanalysis study by Stein et al. confirmed that invasive treatments of TBI patients resulted in 12% decrease in mortality and 6% increase in favorable outcomes. [6] However, despite these "benefits, " many centers are failing to utilize continuous invasive hemodynamic and cerebral monitoring critical care settings to maximize recovery from TBI. Guidelines are now primarily comprised of review articles and evidence-based practices with a lack of published evidence with respect to the Brain Trauma Foundation (BTF) guidelines. [2] CASE DESCRIPTION A 52-year-old Caucasian male sustained a TBI due to a motor vehicle accident. He had a Glasgow Coma Scale (GCS) of 13; his mean arterial pressure of 60 mmHg was maintained by a norepinephrine drip. When he developed a dilated left pupil with decerebrate posturing (GCS 5), he required intubation. e computed tomography (CT) showed a large left epidural hematoma (EDH) with a 2 cm midline shift warranting a craniotomy [ Figures 1 and 2]. During the surgery, no intracranial pressure (ICP) monitor was placed. Postoperatively, in the neurocritical invasive care unit, standard ASA monitoring included oxygen saturation (pulse oximeter), end-tidal CO2 monitoring, standard electrocardiographic, arterial line monitoring of blood pressure (BP), and serial arterial blood gas assessments were performed. e postoperative CT scans showed continued resolution of the EDH and improvement of the edema and midline shift (GCS of [6][7]. However, at the end of the 2 nd week, the patient expired due to complications of TBI (e.g., multiorgan failure). e main question is whether the use of an ICP monitor would have changed this patient's course and outcome. DISCUSSION e BTF most recently cited three parameters for management guidelines; CPP, ICP, and jugular bulb monitoring of arteriovenous oxygen content difference [ Table 1]. [2] ICP monitoring e BTF recommends that "ICP should be monitored in all salvageable patients with a severe TBI and an abnormal CT scan. " Furthermore, ICP monitoring is indicated in patients with severe TBI with a normal CT scan if two or more of the following features are noted at admission: age over 40 years, unilateral or bilateral motor posturing, or systolic BP <90 mmHg. A meta-analysis study by Stein et al. showed 12% decrease in mortality and 6% increase in favorable outcomes in patients with ICP monitors. [6] Jugular bulb venous saturation monitoring e jugular venous oxygen saturation (SjvO2) is an indicator of both cerebral oxygenation and cerebral metabolism. Intracranial pressure monitoring Level IIB Management of severe TBI in patients using information from ICP monitoring is recommended to reduce in hospital and 2-week postinjury mortality Recommendation from the prior (third) edition is not supported by evidence meeting current standards [1] Cerebral perfusion pressure monitoring Level IIB Management of severe TBI patients using guideline-based recommendations for CPP monitoring is recommended to decrease 2-week mortality [1] Advanced cerebral monitoring Level III Jugular bulb monitoring of arteriovenous oxygen content difference may be considered to reduce mortality and improve outcomes at 3 and 6 months postinjury [1] ere have been improvements in outcome by utilizing this method along with ICP/CPP monitoring in comparison with traditional methods alone. [5,7] Brain tissue oxygen tension Brain tissue oxygenation (Pti02) is a modality used to measure bedside focal brain oxygenation using a microcatheter inserted in the frontal white matter. Studies have demonstrated favorable outcomes in patients with combined ICP/CPP and Pti02-guided therapy. [3] Microdialysis monitoring of extracellular glutamate Several studies have implied a key role of glutamate, an excitatory amino acid, in the pathophysiology of a TBI. [3] One Class III study demonstrated that TBI patients whose glutamate levels normalize within 120 h of monitoring had lower mortality and better outcomes measured by the GCS at 6-month postinjury. [1] Cerebral autoregulation monitoring with TCD TCD measures systolic, mean, and diastolic cerebral blood flow (CBF) velocities and calculates the pulpability index from basal intracranial arteries and can be useful in patients with severe TBI to detect low CBF. Analysis of invasive neuromonitoring techniques e vicious cycle ensues in which the brain is left unmonitored due to evidence-based practices using outcome studies, despite articles demonstrating lack of randomization and hindsight bias (sicker patients were more likely to receive the invasive monitoring). [8] ere is no ideal single monitor that improves outcomes; rather, a combination of monitoring and interpretation/integration of data will help optimize the patient care. Shift away from invasive monitoring Several ideas may be postulated regarding the possible causes of shifting away from invasive monitoring and the lack of global protocols: (1) defensive medicine; (2) costeffectiveness; (3) lack of leadership; and (4) limited human resources. Why should a diagnostic test be limited to only addressing mortality reduction, and not toward the accurate detection of the diagnoses, and institution of optimal treatment? Certainly, as in this case, less monitoring does not lead to better outcomes. CONCLUSION For treating TBI, multidisciplinary efforts using multimodal approaches should be pursued. [4] Invasive hemodynamic combined with intracranial ICP monitoring together would likely optimize the approach to TBI.	2020-04-30T09:07:55.684Z	2020-04-25T00:00:00.000	{ "year": 2020, "sha1": "80409ec66fe6573e3b28b1a92172317f80c5ae71", "oa_license": "CCBYNCSA", "oa_url": "https://surgicalneurologyint.com/wp-content/uploads/2020/04/9980/SNI-11-85.pdf", "oa_status": "HYBRID", "pdf_src": "PubMedCentral", "pdf_hash": "0f49d782b1b2bd1cd574f3e85d68387a7310e9ed", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
25867409	pes2o/s2orc	v3-fos-license	Phorbol 12-myristate 13-acetate inhibits death receptor-mediated apoptosis in Jurkat cells by disrupting recruitment of Fas-associated polypeptide with death domain. Regulation of death receptor-mediated apoptosis is incompletely understood. Previous studies have demonstrated that phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, inhibits Fas (CD95)-mediated apoptosis in Jurkat (type II) cells but not SKW6.4 (type I) cells. In this study, we demonstrated that PMA also protects Jurkat cells from apoptosis induced by tumor necrosis factor-alpha and the tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL). Interestingly, PMA failed to protect Jurkat cells from apoptosis induced by other agents, including etoposide, camptothecin, and gamma-irradiation. Analysis of the initial events induced by agonistic anti-Fas antibodies revealed that PMA inhibited Fas binding to Fas-associated polypeptide with death domain (FADD) in Jurkat cells but not in SKW6.4 cells. Although the protein kinase inhibitor bisindoylmaleimide VIII increased apoptosis induced by agonistic anti-Fas antibody, tumor necrosis factor-alpha, and TRAIL, these effects were not observed with the protein kinase C inhibitor H7 and were not associated with increased FADD recruitment to Fas. These results indicate that PMA inhibits death signaling induced by a number of discrete receptors and suggest that the effects are mediated at the level of receptor-mediated adaptor molecule recruitment. Fas (also called CD95/APO-1) is a 45-kDa type I transmembrane protein that is expressed by a variety of cells (1). It belongs to a subgroup of the TNF-␣ 1 receptor/nerve growth factor receptor superfamily that also contains type I TNF-␣ receptor, death receptor 4 (TRAIL receptor 1), and death receptor 5 (TRAIL receptor 2) (2, 3). Each of these polypeptides contains a conserved cytoplasmic domain of ϳ80 amino acids called a death domain that is essential for cell death signaling (2,3). Fas participates in regulation of the immune system and in the response to certain anticancer drugs. Fas-mediated signaling is required for negative selection of T cells, termination of immune responses after antigen stimulation, and elimination of virally infected cells and tumor cells by cytotoxic lymphocytes (3)(4)(5)(6). In addition, a number of chemotherapeutic agents induce FasL synthesis (7)(8)(9)(10). Experiments utilizing blocking anti-Fas antibodies (11,12) or mice with mutant Fas receptor (12) have demonstrated an essential role for FasL-Fas interactions in 5-fluorouracil-induced cytotoxicity. The role of Fas in the action of other chemotherapeutic agents is less clear (13,14) but is currently under investigation in a number of laboratories. The Fas signaling pathway has been extensively characterized. Ligation of Fas by FasL or agonistic anti-Fas antibody results in sequential recruitment of FADD and procaspase-8 to the death domain of Fas to form the DISC (5,15). Juxtaposition of procaspase-8 molecules in the DISC is thought to initiate the autocatalytic processing and release of active caspase-8 into the cytoplasm (16). In so-called type I cells, caspase-8 activation at the DISC is sufficient to lead to direct proteolytic activation of caspase-3 (17,18), which then cleaves a number of different substrates to generate the apoptotic phenotype (19). In contrast, in type II cells, caspase-8 generation is not sufficient to activate caspase-3 (17). Instead, caspase-8 cleaves the cytosolic protein Bid to yield a fragment that facilitates translocation of Bax and Bak to the outer mitochondrial membrane (20 -22), where these proapoptotic Bcl-2 family members induce cytochrome c release (23,24) and subsequent triggering of apoptotic changes through the activation of caspase-9 (17,25). The events occurring after ligation of other death receptors appear to be similar. TRAIL is a TNF-␣ homologue that has been implicated in killing by immature natural killer cells (26) as well as interferon-stimulated monocytes and dendritic cells (27,28). Apoptosis induced by this cytokine involves ligation of death receptor 4 and/or death receptor 5 followed by recruitment of FADD and caspase-8 into a DISC (29 -31). While binding of TNF-␣ to type I TNF-␣ receptor first leads to recruitment of the type I TNF-␣ receptor-associated death domain protein TRADD (32), TRADD then serves as a platform to recruit other polypeptides, including FADD, into the complex (33,34). A number of previous studies have reported that PMA suppresses Fas-mediated apoptosis (35)(36)(37)(38)(39)(40)(41)(42)(43). Because PMA activates members of the PKC family, a group of 12 related lipiddependent serine/threonine kinases that play fundamental roles in signal transduction pathways that regulate growth and differentiation (44,45), it has been assumed that one or more PKC isoenzymes play a role in modulating the Fas pathway. How PKC activation alters Fas-mediated apoptosis, however, has remained unclear. Various studies have implicated PKC-induced activation of phosphatidylinositol-3 kinase (46) and the Raf 3 MEK1 3 ERK pathways (35,36,47,48) as well as p90 rsk -induced phosphorylation of BAD (49) in the inhibition of Fas-mediated signaling. On the other hand, PKC␣-mediated phosphorylation of Bcl-2 on Ser 70 reportedly increases the antiapoptotic function of Bcl-2 (50, 51), providing a potential explanation for the ability of PMA to block Fas-mediated apoptosis only in type II cells (42). In the present study, we examined the effect of PMA on apoptotic events in prototypic type I and type II cells. These experiments demonstrated that PMA inhibits induction of apoptosis by FasL, TNF-␣, and TRAIL, but not ionizing radiation or topoisomerase poisons, in type II cells. Additional experiments revealed that PMA inhibits the recruitment of FADD to the DISC in type II but not type I cells. These results provide new insight into the manner in which PKC regulates death receptor signaling in type II cells. EXPERIMENTAL PROCEDURES Materials-Reagents were purchased from the following suppliers: PMA, camptothecin, and Hoechst 33258 from Sigma; etoposide from Biomol (Plymouth Meeting, PA); enhanced chemiluminescence reagents, Sepharose-coupled glutathione, protein A, and protein G from Amersham Biosciences, Inc.; CH.11 IgM monoclonal anti-Fas from Kamiya (Seattle, WA); TNF-␣ and TRAIL from R & D Systems (Minneapolis, MN); apo-1-1 IgG 1 monoclonal anti-Fas, BisVIII, H7, calphostin C, and chelerythrin from Alexis (San Diego, CA); monoclonal antibodies to procaspase-8 and procaspase-3 from Pharmingen (La Jolla, CA); murine monoclonal E10 that recognizes ERK phosphorylated on Thr 202 and Tyr 204 as well as rabbit antiserum that recognizes modified and unmodified ERK from Cell Signaling Technology (Beverly, MA); rabbit polyclonal anti-mouse IgM from Dako; and horseradish peroxidaseconjugated anti-mouse IgG, IgG 1 , and IgG 2a from Southern Biotechnology (Birmingham, AL). Murine monoclonal antibodies that recognize poly(ADP-ribose) polymerase and heat shock protein 90 were gifts from Dr. G. Poirier (Laval University, Ste-Foy, Quebec, Canada) and David Toft (Mayo Clinic, Rochester, MN), respectively. The rabbit antiserum that recognizes a neoepitope at the C terminus of the caspase-3 large subunit was previously characterized (52). Induction and Assessment of Apoptosis-The human T lymphoblastoid cell line Jurkat and B lymphoma cell line SKW6.4 from the Amer-ican Type Culture Collection (Manassas, VA) were maintained at concentrations of Ͻ1 ϫ 10 6 cells/ml in RPMI 1640 medium containing 5% heat-inactivated fetal bovine serum, 100 units/ml penicillin G, 100 g/ml streptomycin, and 2 mM glutamine. To start the experiments, cells were treated with 50 nM PMA (or 0.1% dimethyl sulfoxide) for 3-5 min prior to the addition of a variety of death stimuli. After incubation as indicated in the figures, cells were sedimented at 200 ϫ g for 5 min and washed with ice-cold PBS. Two methods were then used to assess apoptosis. For flow cytometric analysis, cells were fixed in 50% ethanol for 30 min, washed with PBS, treated for Ն30 min at 20°C with 10 g/ml RNase A and 20 g/ml propidium iodide, and subjected to flow cytometry on a Becton-Dickinson FACScan using an excitation wavelength of 488 nm and an emission wavelength of 617 nm. Sample acquisition and analysis were performed with Becton-Dickinson CellQuest software. Cells containing Ͻ2 N DNA were considered apoptotic and expressed as a percentage of the total cells (53). Alternatively, cells were fixed in 3:1 (v/v) methanol/acetic acid, stained with 1 g/ml Hoechst 33258, and examined by fluorescence microscopy. Cells containing nuclei that were condensed or fragmented into multiple discrete fragments as illustrated in Fig. 2 were scored as apoptotic. At least 400 cells/sample were counted. Analysis of the DISC by Immunoprecipitation-Aliquots containing 5 ϫ 10 7 cells were treated with 500 ng/ml CH.11 anti-Fas antibody for 90 min in the absence or presence of 50 nM PMA. All further steps were performed at 4°C. Cells were washed once with PBS and lysed by incubation for 20 min in 1 ml of DISC buffer. Insoluble debris was removed by centrifugation for 15 min at 14,000 ϫ g. As a control, 1 ml of cell lysates from 5 ϫ 10 7 untreated cells were supplemented with 1 g of CH.11 antibody. After protein concentration of the extracts was determined, cell lysates containing 5 mg of protein in 1 ml DISC buffer were incubated with 10 g/ml rabbit anti-mouse IgM for 2 h. Aliquots of protein A-Sepharose (30 l) and protein G-Sepharose (10 l) were added for an additional 2 h. Immune complexes were then pelleted by centrifugation for 3 min at 14,000 ϫ g, washed five times with DISC buffer, and released from the beads by boiling for 5 min in SDS sample buffer. Samples were subjected to SDS-PAGE, transferred to nitrocellulose, and sequentially probed with reagents that recognize FADD, procaspase-8, and Fas. Blots were scanned using Adobe Photoshop and quantitated using NIH Image 1.61 as previously described (55). Procaspase-8/FADD Binding-The pGex4T-2 construct containing GST fused to full-length FADD was kindly provided by Dr. M. Peter (University of Chicago). Escherichia coli strain DH5␣ cells transformed with this construct were grown overnight and induced with 1 mM isopropyl-D-thiogalactoside for 3 h. GST-FADD was bound to glutathione-Sepharose beads according to the manufacturer's protocol and eluted by incubation with 10 mM glutathione in 50 mM Tris-HCl (pH 9.0). The eluates were dialyzed against PBS overnight. After Jurkat cells were treated with diluent or 50 nM PMA for 90 min, cell lysates were prepared by freezing and thawing cells in PBS containing the same protease and phosphatase inhibitors described for DISC buffer. Aliquots containing 5 mg of protein were incubated for 3 h at 4°C with purified GST-FADD. Glutathione-Sepharose was added for an additional 2 h. FADD-bound proteins were then pelleted by centrifugation for 3 min at 14,000 ϫ g, washed five times with PBS containing protease and phosphatase inhibitors, and released from the beads by boiling for 5 min in SDS sample buffer. Recovery of procaspase-8 was assessed by immunoblotting. RESULTS PMA Inhibits Fas-mediated Apoptosis in Jurkat Cells but Not SKW6.4 Cells-During Fas-mediated apoptosis, the events downstream of caspase-8 activation at the DISC are different in type I and type II cells (17,42). To confirm previous reports that PMA differentially inhibited Fas-mediated apoptosis in the two cell types, we treated prototypic type II (Jurkat) and type I (SKW6.4) cells with the agonistic anti-Fas antibody CH.11 in the absence or presence of PMA. Treatment of Jurkat cells with CH.11 resulted in the induction of apoptosis as indicated by the presence of increasing numbers of subdiploid cells after ethanol fixation followed by propidium iodide staining (Fig. 1A), by proteolytic cleavage of procaspase-8 and -3 (Fig. 1B, upper panels, lanes 6 -8), by cleavage of the caspase substrate poly(ADP-ribose) polymerase (Fig. 1B, lower panel, lanes 6 -8), and by the presence of apoptotic morphological changes after fixation and staining with Hoechst 33258 (Fig. 2A). When 50 nM PMA was added 3-5 min prior to CH.11, development of all of these signs of apoptosis was inhibited (Figs. 1, A and B, and 2A). Treatment of SKW6.4 cells with CH.11 also resulted in apoptosis ( Fig. 1, C and D). Compared with Jurkat cells, however, SKW6.4 cells showed little inhibition of apoptosis when PMA was added prior to CH.11 (Fig. 1C). Moreover, PMA only slightly inhibited the cleavage of procaspase-8, procaspase-3, and poly(ADP-ribose) polymerase in these cells (Fig. 1D). These results are consistent with a previous report that PMA fails to inhibit Fas-mediated apoptosis in SKW6.4 cells (41). PMA Inhibits Death Receptor-mediated Apoptosis but Not Mitochondria-initiated Apoptosis in Jurkat Cells-The observation that PMA inhibits Fas-mediated apoptosis in Jurkat cells, which require mitochondrial amplification of the DISC- initiated proteolytic signal (17), but not in SKW6.4 cells, in which apoptosis occurs independent of mitochondrial amplification (17), raised the possibility that PMA might be acting by inhibiting the mitochondrial pathway, possibly through an effect of PKC␣ on Bcl-2 phosphorylation (50,51). To assess this possibility, we treated Jurkat cells with etoposide, a chemotherapeutic agent that induces apoptosis through a pathway that involves mitochondrial release of cytochrome c (56) and caspase-9 activation (52, 57) but is independent of Fas (58) or FADD (59) function. Interestingly, PMA did not protect Jurkat cells from etoposide-induced apoptosis (Fig. 2B). Instead, nuclear fragmentation ( Fig. 2A) and poly(ADP-ribose) polymerase cleavage (Fig. 2C) were readily detectable in cells treated with etoposide and PMA. Based on current understanding of etoposide-induced apoptosis (52, 56 -59), these results argue against the possibility that PMA is exerting its effects on Fas-mediated apoptosis by inhibiting the mitochondrial pathway. When effects of other stimuli were evaluated, PMA also inhibited proteolytic cleavage of procaspase-8 and induction of apoptosis by FasL, TNF␣ and TRAIL in Jurkat cells (Fig. 2, B and C). In contrast, PMA did not protect from induction of apoptosis by camptothecin or ␥-irradiation. These results not only provide additional support for the view that topoisomerase poisons and ionizing radiation kill cells through a pathway that is distinct from the known death receptor pathways (13,14) but also indicate that PMA acts specifically on death receptor pathways rather than inhibiting the mitochondrial pathway. Failure of ERK Pathway Activation to Account for the Protective Effects of PMA-Previous studies have demonstrated that PMA treatment results in activation of the MEK1 3 ERK pathway in lymphoid cells (48,60,61). To evaluate the possible role of this signal transduction pathway in the PMA-induced inhibition of CH.11-induced apoptosis, Jurkat cells were treated with PMA in the absence or presence of the MEK1 inhibitor PD98059 (62,63). Treatment with PD98059 by itself had no demonstrable effect on these cells (Fig. 3). If PMA were acting through the MEK1 3 ERK pathway, PD98059 would be expected to abolish the effects of PMA. Contrary to this prediction, PD98059 had minimal effect on the CH.11/PMA combination (Fig. 3A) despite its ability to inhibit ERK phosphorylation in these cells (Fig. 3B). PMA Disrupts Fas Binding to FADD in Jurkat Cells but Not SKW6.4 Cells-Because the effect of PMA could not be explained by its effect on either the mitochondrial pathway of caspase activation or the mitogen-activated protein kinase pathway, we next evaluated the possibility that PMA might be altering death receptor signaling. To address this possibility, DISC components were analyzed in CH.11-treated SKW6.4 and Jurkat cells. When SKW6.4 cells were treated with CH.11, immunoprecipitation of the antibody-bound Fas followed by immunoblotting with anti-FADD antibody revealed that the DISC formed in Ͻ5 min (Fig. 4A). Treatment with PMA had little effect on the recruitment of FADD to Fas in these cells (Fig. 5A). Examination of Jurkat cells revealed a markedly different picture. Consistent with previous reports that DISC assembly in type II cells is difficult to detect (17), preliminary experiments indicated that the Fas-FADD interaction was almost undetectable 5 min after the addition of CH.11 to Jurkat cells (Fig. 4A, lane 7). Instead, FADD could be more readily precipitated with Fas 30 -90 min after the addition of CH.11 (Fig. 4A, lanes 8 -10). Control experiments indicated that the delayed and diminished DISC formation in Jurkat cells occurred despite the presence of levels of Fas, FADD, and procaspase-8 that were similar to SKW6.4 cells (Fig. 4B). This ability to detect DISC formation, although it was de-layed, provided the opportunity to assess the effect of PMA on Fas signaling events in these type II cells. In subsequent experiments, Fas immunoprecipitates prepared from cells treated for 90 min with diluent, CH.11, or CH.11 plus PMA were probed for the presence of FADD as well as procaspase-8. When Fas was immunoprecipitated by the addition of CH.11 to cell lysates prepared from untreated cells, FADD and procaspase-8 were undetectable in the immunoprecipitates (Fig. 5B, lane 1). When cells were treated with CH.11 for 90 min, FADD and procaspase-8 were both detected in the immunoprecipitates (Fig. 5B, lane 2). The addition of PMA prior to CH.11 resulted in a Ͼ90% decrease in the amount of Fas-associated FADD and procaspase-8 (Fig. 5B, lane 3). To rule out the possibility that this decreased recovery reflected PMA-induced decreases in FADD or procaspase-8 expression, levels of these polypeptides were analyzed in cell lysates obtained from the same experiment. As indicated in the lower panels of Fig. 5B, PMA treatment did not affect total cellular expression of either FADD or procaspase-8. The decreased recovery of procaspase-8 in the DISC after PMA treatment (Fig. 5B, lane 3) might reflect decreased binding of procaspase-8 to FADD in addition to the decreased Fas-FADD interaction. To examine this possibility, we assessed the ability procaspase-8 in extracts from control or PMA-treated cells to bind to GST-FADD in vitro. Results of this analysis (Fig. 5C) indicated that PMA had no effect on the binding of procaspase-8 to FADD. BisVIII Enhances Death Receptor-mediated Apoptosis without Enhancing DISC Formation-Based on the ability of PMA to inhibit death receptor-mediated apoptosis, we evaluated the possibility that inhibition of protein kinase C might enhance response to the same cytotoxic ligands. In a previous study, Zhou et al. (64) demonstrated that treatment of Jurkat cells with certain bisindoylmeleimides potentiated Fas-mediated apoptosis, whereas other protein kinase C inhibitors did not. Results shown in Fig. 6, A and B, demonstrate that BisVIII, the most potent of the bisindoylmeleimides, enhanced apoptosis after treatment of Jurkat cells with CH.11, TNF-␣, or TRAIL. In contrast, the protein kinase C inhibitor H7 failed to enhance apoptosis after any of these treatments. In further experiments (data not shown) the protein kinase C inhibitors calphostin C and chelerythrin induced apoptosis in Ͼ85% of Jurkat cells without the addition of death ligand, making it difficult to assess the effects of these particular protein kinase C inhibitors. To determine whether the effect of BisVIII might be due to enhanced DISC formation, cells were pretreated with BisVIII or H7 for 5 min before treatment with CH.11. Examination of whole cell lysates (Fig. 6C, lower panels) demonstrated that BisVIII altered the phosphorylation status of FADD, as indicated by a decrease in the slower migrating band and increase in the faster migrating band (65). This was reflected in the composition of FADD that was recruited to the DISC (Fig. 6C, upper panels). Nonetheless, the total amount of FADD in the DISC was not altered by treatment with BisVIII (Fig. 6, C and D). H7 (Fig. 6, C and D) and chelerythrin (not shown), had no effect on either phosphorylation or recruitment of FADD. These observations suggest that the protein kinase C inhibitors examined do not affect DISC formation. DISCUSSION A number of previous studies have demonstrated that PMA inhibits Fas-mediated apoptosis in type II cells. The mechanism of this effect, however, has remained incompletely understood. Moreover, it has been unclear whether this effect would be observed when other death receptors are ligated. In the present study, we have demonstrated that PMA treatment of type II cells also inhibits the effects of TNF-␣ and TRAIL but not etoposide, camptothecin, or ionizing radiation. In addition, we have demonstrated that PMA inhibits recruitment of FADD and procaspase-8 to the Fas-induced DISC. These observations have potentially important implications for current understanding of the regulation of death receptor signaling. The pathway leading from Fas ligation to caspase activation has been increasingly well characterized over the past 5 years (5,6,66,67). Initial events in this process involve binding of FADD to the death domain of Fas, followed by binding of procaspase-8 to the death effector domain of FADD. Once caspase-8 is activated, it can either directly activate procaspase-3 or cleave the cytosolic protein Bid to form a fragment that stimulates caspase activation through the mitochondrial pathway (22,25). After cells were treated with 500 ng/ml CH.11 for the indicated length of time, lysates containing 5 mg of protein were incubated with rabbit anti-mouse IgM followed by protein A-and protein G-Sepharose to immunoprecipitate Fas. Immunoprecipitates were subjected to SDS-PAGE and probed with anti-FADD antibody. B, whole cell lysates from SKW 6.4 or Jurkat cells were probed for FAS (immunoprecipitation followed by immunoblotting), FADD, procaspase-8, or heat shock protein 90 (immunoblotting). Equal amounts of protein cell lysate from the two cell lines were analyzed in each assay. These events can be regulated in a number of different ways. First, it has been suggested that the Fas receptor is preoligomerized and that mutations might affect this oligomerization state (68), although this preoligomerization has not been a universal finding (69). Second, it has been reported that Fas is a phosphoprotein whose signaling is regulated by an associated phosphatase (70). Third, the binding of FADD to procaspase-8 is inhibited by an endogenous polypeptide (Flice-like inhibitory protein) that bears death effector domains but no caspase active site (reviewed in Ref. 19). Fourth, it has been reported that caspase activation downstream of caspase-8 can be inhibited by inhibitor of apoptosis proteins (71). Finally, in type II cells, Fas-mediated apoptosis can be modulated by alterations that affect the mitochondrial pathway. For example, overexpression of Bcl-2 inhibits Fas-mediated apoptosis in type II cells (72)(73)(74)(75)(76). Previous studies have suggested that PMA might be exerting its effects by inducing phosphorylation and inactivation of the proapoptotic Bcl-2 family member BAD (35,49), possibly through activation of phosphatidylinositol 3-kinase (46). It has also been reported that PMA induces Bcl-2 phosphorylation (51) and inhibits Bid cleavage (35,42). Whether these effects explain PMA-induced inhibition of Fas-mediated signaling has remained unclear. In the present study, we utilized several approaches to address this issue. First, we examined the effect of PMA on other treatments. PMA inhibited the initiation of apoptosis by FasL, TRAIL, and TNF-␣ (Fig. 2, B and C). Although an effect of PKC activation on TRAIL-induced signaling was reported after completion of the present studies (77), to our knowledge this is the first demonstration that induction of apoptosis by FasL or TNF-␣ can be inhibited by PMA. In contrast, we observed that PMA failed to inhibit apoptosis induced by etoposide or ionizing radiation. These two stimuli initiate apoptotic signaling through a process that is independent of caspase-8 (52,78) and instead appears to involve caspase-9 as an initiating protease (59,79). Overexpression of Bcl-2 inhibits apoptosis induced by these treatments (80,81). The observation that PMA fails to protect cells from these treatments (Fig. 2, A and B) argues against the possibility that the effect of PMA on Fas-mediated apoptosis reflects enhanced Bcl-2 function. These same observations also argue against a critical role for BAD phosphorylation, another method of regulating the mitochondrial pathway, in PMA-induced protection. Second, we examined the effect of PMA on MEK-mediated events. Previous studies have suggested that activation of the MEK 3 ERK pathway protects cells from certain proapoptotic stimuli (82). Our results confirmed that PMA activated this pathway (Fig. 3B). Nonetheless, the MEK inhibitor PD98059 FIG. 6. BisVIII treatment enhances death receptor-mediated apoptosis but not DISC formation. A, effect of BisVIII and H7 on CH.11-and TNF-␣induced apoptosis. After a 5-min pretreatment with diluent, 10 M BisVIII, or 10 M H7, Jurkat cells were treated for 6 h with 12.5 ng/ml CH.11 or 10 ng/ml TNF-␣ plus 0.5 g/ml cycloheximide. At the end of the incubation, cells were fixed as illustrated in Fig. 2A and analyzed for apoptotic nuclear changes. B, effect of BisVIII and H7 on TRAIL-induced apoptosis. After a 5-min pretreatment with diluent, 10 M BisVIII, or 10 M H7, Jurkat cells were treated for 3 h with 6.25 ng/ml TRAIL and analyzed as in A. C, effect of BisVIII and H7 on DISC formation in Jurkat cells. Top, Jurkat cells (5 ϫ 10 7 ) were treated with diluent (lanes 1 and 4), 10 M BisVIII (lanes 2 and 5) or 10 M H7 (lanes 3 and 6). Beginning 5 min later, half of the samples were treated with 500 ng/ml CH.11 for 30 min (lanes 4 -6). After cells were lysed, CH.11 was added to samples in lanes 1-3. Fas receptors with associated DISC components were immunoprecipitated as described in Fig. 4A and sequentially probed with anti-FADD and anti-Fas. Bottom panels, whole cell lysates from the same cells were probed with anti-FADD or, as a loading control, heat shock protein 90 antibody. D, the total strength of the anti-FADD signal (top and bottom bands) in immunoprecipitate lanes 4 -6 of C was quantitated as described under "Experimental Procedures" and expressed in arbitrary units. diminished PMA-induced ERK activation without altering the effect of PMA on Fas-mediated apoptosis (Fig. 3A), suggesting that signaling through ERK is unlikely to account for the inhibitory effect of PMA on CH.11-induced apoptosis. Finally, we examined the effect of PMA on DISC assembly. The observation that PMA inhibited recruitment of both FADD and procaspase-8 to the DISC in Jurkat cells (Fig. 5B) provides an explanation for the PMA-induced inhibition of procaspase-8 cleavage (Fig. 2B), the previously reported PMA-associated inhibition of Bid cleavage in these cells (35,42), and the PMAinduced inhibition of Fas-mediated apoptosis in these cells (Figs. 1 and 2). Further analysis indicated that the potential ability of procaspase-8 to interact with FADD in Jurkat cells was unaltered (Fig. 5C), suggesting that the primary effect of PMA was to inhibit the Fas-FADD interaction in these type II cells. When similar experiments were performed in SKW6.4 cells, a prototypic type I cell line (17), a different picture emerged. We observed that DISC formation was much more rapid and extensive in SKW6.4 cells (Fig. 4A), raising the possibility that the distinction between type I and type II cells might reflect differences in the rate of trafficking of FADD to Fas. Moreover, we observed that PMA had no effect on FADD recruitment in SKW 6.4 cells (Fig. 5A), explaining why PMA has little effect on Fas-induced apoptosis in this cell line (Fig. 1C). Based upon a previous observation that BisVIII, which can inhibit certain protein kinase C isoforms, enhances Fas-mediated apoptosis (64), we evaluated the possibility that protein kinase C inhibitors might enhance DISC formation in Jurkat cells. The present analysis demonstrated that BisVIII enhanced the induction of apoptosis by TNF-␣ and TRAIL as well as CH.11 (Fig. 6, A and B). In contrast, H7 had no effect on induction of apoptosis by these death receptor ligands. These observations confirm the results of Zhou et al. (64) and extend them to additional death receptor ligands. In further experiments, we examined the effect of BisVIII and H7 on DISC formation. Neither of these agents altered the amount of FADD recruited to the DISC in the presence of agonistic Fas antibody (Fig. 6, C and D). Interestingly, BisVIII appeared to alter the phosphorylation state of FADD (Fig. 6C). Given the fact that FADD phosphorylation appears to be mediated by a cell cycleregulated kinase (65), this observation suggests that BisVIII might be altering the activity of kinases other than protein kinase C. While we cannot rule out the possibility that other protein kinase C inhibitors, particularly those selective for the isoform responsible for the protective effects of PMA, might alter DISC formation, these observations rule out the possibility that protein kinase C inhibitors in general are capable of altering proximal events during Fas-mediated signaling. Collectively, our observations have several potentially important implications. First, they confirm a recent report that protein kinase C can modulate TRAIL-induced apoptosis. Further experiments in our laboratory demonstrated that PMA also inhibits TRAIL-induced apoptosis in two other type II cell lines, HL-60 human leukemia cells and HCT116 colon cancer cells, 2 providing support for the view that events described in the present report are not unique to Jurkat cells. Second, the results described above indicate that protein kinase C activation simultaneously modulates sensitivity to multiple related cytokines, including FasL and TNF-␣, providing additional insight into the mechanisms responsible for the well established ability of PMA to inhibit killing by cytotoxic T cells and natural killer cells (83,84). Third, they provide an independent line of evidence supporting the view that certain chemotherapeutic agents induce apoptosis by a pathway that is distinct from that utilized by death receptor ligands (13,14,85). Finally, these results shift the focus of studies designed to investigate the effects of PMA on death receptor signaling. Although previous studies have emphasized effects of PMA on components further downstream in apoptotic signaling pathways, our results suggest that it might be important to determine how protein kinase C activation alters the ability of DISC components to interact with each other.	2018-04-03T02:29:19.431Z	2002-02-01T00:00:00.000	{ "year": 2002, "sha1": "1f1cae610b0c79b8fd6a1ace6224a8adba6c1b73", "oa_license": "CCBY", "oa_url": "http://www.jbc.org/content/277/5/3776.full.pdf", "oa_status": "HYBRID", "pdf_src": "Adhoc", "pdf_hash": "9b525499b4c4d10308f493f60655dc8499112f1e", "s2fieldsofstudy": [ "Biology", "Chemistry" ], "extfieldsofstudy": [ "Chemistry", "Medicine" ] }
8886262	pes2o/s2orc	v3-fos-license	Integrating fMRI and SNP data for biomarker identification for schizophrenia with a sparse representation based variable selection method Background In recent years, both single-nucleotide polymorphism (SNP) array and functional magnetic resonance imaging (fMRI) have been widely used for the study of schizophrenia (SCZ). In addition, a few studies have been reported integrating both SNPs data and fMRI data for comprehensive analysis. Methods In this study, a novel sparse representation based variable selection (SRVS) method has been proposed and tested on a simulation data set to demonstrate its multi-resolution properties. Then the SRVS method was applied to an integrative analysis of two different SCZ data sets, a Single-nucleotide polymorphism (SNP) data set and a functional resonance imaging (fMRI) data set, including 92 cases and 116 controls. Biomarkers for the disease were identified and validated with a multivariate classification approach followed by a leave one out (LOO) cross-validation. Then we compared the results with that of a previously reported sparse representation based feature selection method. Results Results showed that biomarkers from our proposed SRVS method gave significantly higher classification accuracy in discriminating SCZ patients from healthy controls than that of the previous reported sparse representation method. Furthermore, using biomarkers from both data sets led to better classification accuracy than using single type of biomarkers, which suggests the advantage of integrative analysis of different types of data. Conclusions The proposed SRVS algorithm is effective in identifying significant biomarkers for complicated disease as SCZ. Integrating different types of data (e.g. SNP and fMRI data) may identify complementary biomarkers benefitting the diagnosis accuracy of the disease. Background Schizophrenia (SCZ) is one of the most disabling and emotionally devastating illnesses. The global median lifetime morbid risk for schizophrenia is 7.2/1,000 persons [1]. Genetic factors play an important role in the development of schizophrenia. To date, over 1000 genes have been reported to associate with SCZ (http://www.szgene. org/default.asp) and many SNPs have been identified as biomarkers for the disease [2][3][4]. For example, Kordi-Tamandani et al. showed that that promoter methylation of the CTLA4 gene can increase the risk of SCZ disease [2]. Shayevitz et al. confirmed the gene NOTCH4 as a candidate gene for schizophrenia with genome-wide association studies (GWAS) [3]. Chen et al. stated that three SNPs spanning the MYO5B gene are significantly associated with SCZ: rs4939921, rs1557355 and rs4939924 [4]. Besides genomic data, fMRI is another widely used data modality in SCZ studies [5] [6]. To date, many methods have been proposed to integrate multi-types of data in SCZ disease study [7][8][9][10][11]. For example, Chen et al. proposed parallel independent component analysis (paraICA) to identify genomic risk components associated with brain function abnormalities and detected significant biomarkers from both fMRI data and SNP data that are strongly correlated [7]. Parallel ICA is an effective method for the joint analysis of multiple modalities including interconnections between them [8]. Utilizing this method, Meda et al. detected three fMRI components significantly correlated with two distinct gene components in SCZ study [11]. In this study, a novel sparse representation based variable selection (SRVS) method was proposed and applied to an integrative analysis of two types of data: fMRI and SNP, aiming to obtain comprehensive analysis. Sparse representation including compressive sensing has been widely used in signal/image processing and computational mathematics [12][13][14][15][16][17][18]. Candes et al. showed that stable signal can be approximately recovered from incomplete and inaccurate measurements [14]. Wright et al. proposed a sparse representation based clustering (SRC) for face recognition, demonstrating high classification accuracy [15]. In our recent works [16][17][18], we developed novel classification and feature selection algorithms based on sparse representation theory. We applied those methods to gene expression data analysis [16], to chromosome image classification [18], and to joint analysis of different data modalities (e.g. SNP data and gene expression data) [17], and achieved improved classification accuracies as well as better feature selections. In applications of sparse representation, The availability of a limited number of samples is an important issue (e.g., feature selection and signal recovery) [19][20] [21]. According to compressive sensing theory (e.g., the restricted isometry property (RIP) condition [23] [24] for signal recovery), the number of available samples should not be less than the number of signals to be selected/recovered. However, the number of features/variables in genomic data (e.g. SNP data) or medical imaging data (e.g. fMRI data) are usually significantly big than the number of samples. In those cases, the traditional methods for compressive sampling cannot effectively analyse the data. In a recent work, Li et al. [21] developed a voxel selection algorithm for fMRI data analysis. The method was based on sparse representation and is designed to get a sparse solution when sufficient samples exist. However, it may not handle the small sample problem described above. In this study, a novel sparse representation based variable selection (SRVS) algorithm was proposed to select relevant biomarkers from big data sets having small sample sizes. The analysis was obtained by using a window based approach, whose size determines the resolution of the variable selection. We first tested the SRVS algorithm on a simulated data set (size of 100 × 1e 6 , with 50 cases and 50 controls), demonstrating the multi-resolution characteristic of the method. Then the algorithm was applied to an integrative analysis of two real data sets: a SNP data set (size of 208 × 759075) and a fMRI data set (size of 208 × 153594). Using the proposed SRVS algorithm, biomarkers for SCZ were identified and validated. fMRI and SNP data collection A total of 208 subjects, after signing informed consent, were recruited in the study, including 96 SCZ cases (age: 34 ± 11, 74 males) and 112 healthy controls (age: 32 ± 11, 68 males). Both SNP and fMRI data were collected from each of those 208 subjects. The healthy controls have no history of psychiatric disorders and were free of any medical. SCZ cases met the DSM-IV diagnostic criteria for schizophrenia. After pre-processing, 153594 fMRI voxels and 759075 SNP loci were obtained for the following biomarker selections. Please refer to [22] for detailed description of data collection and pre-processing. Generalized sparse model To combine different data sets for integrative analysis, we consider the following model: where y ∈ R n×1 is the phenotype vector of the subjects; matrix X 1 ∈ R n×p 1 and X 2 ∈ R n×p 2 represent data sets of different modalities having normalized column vectors (e.g., \|\| * \|\| 2 = 1); X = [α 1 X 1 , α 2 X 2 ] ∈ R n×p ; α 1 + α 2 = 1, and α 1 , α 2 > 0 are the weight factors for X 1 and X 2 respectively. The measurement error ε ∈ R n×1 . We aim to reconstruct the unknown sparse vector δ = based on y and X , where δ 1 ∈ R p 1 ×1 , δ 2 ∈ R p 2 ×1 , and It can be proven that when p > 35n, the matrix X ∈ R n×p has the difficulty to satisfy the restricted isometry property (RIP) condition [24] for signal recovery. In this work, p = 759075 + 153594 = 912669 and n = 208. Thus p 35n = 7280. To overcome this problem, we propose the SRVS algorithm described as follows. SRVS algorithm To best approximate y with the model given by Eq. (1), we consider the following L p minimization problem: where \|\| * \|\| 2 represents L p norm; p ∈ [0, 1]. The SRVS Algorithm given below is used to solve the L p minimization problem and select the phenotype relevant column vectors out of X. It can be proven that, by using the SRVS algorithm, one can identify the significant variables with high probabilities. In addition, the SRVS algorithm can be shown convergent for any given k and ε, generating an effective solution for the sparse model specified by Eq. (2). In the following section, we discuss the sparsity control issue to determine the number of variables to be selected. Sparsity control using k In Step 2 of the SRVS Algorithm, we exploit Fisher-Yates Shuffling algorithm [31] with a window of length k to select X l ∈ R n×k from X ∈ R n×p . The length k determines the resolution of the SRVS algorithm. When k = p, the number of variables selected will be generally equal to the sample number n [23]. The smaller the k , the more the variables selected, and those variables generally include the variables selected with bigger k, as shown in Figure 1. This multi-resolution property enables us to select different number of variables at different significance levels. Further sparsity control using ε The parameter ε given in Eq. (2) can be used for further sparsity control. The magnitudes of entries of δ reflect the significance of the corresponding columns of X [21]. Thus, a threshold can be selected for δ using cross-validation [32]. Another way to determine a threshold is using the error term ε (as shown in Figure 2), which reflects the residual of \|\|y − Xδ\|\| [20]. When ε = 0, noises may be involved in the columns selected [20]. In this study, we set ε = τ \|\|y\|\| 2 . From Figure 2, we show that if the first 400 variables with amplitudes larger than 0.002 are selected (i.e. points (400, 0.002) on 'Regression coefficients' curve), it corresponds to the point (400, 0.4) on the 'Error term coefficient' curve; it indicates that with these 400 variables, the error term ε = 0.4\|\|y\|\| 2 . Validation To validate the variable selected using our proposed SRVS algorithm, we compared our selected SNPs and fMRI voxels with that of previous studies. In addition, we used the selected SNPs and fMRI voxels to identify SCZ patients from healthy controls with the sparse representation based classifier (SRC) [15] [18]. Then a leave one out (LOO) cross-validation approach was carried out to evaluate the identification accuracy. We compared the classification results with that of Li et al.'s method [21]. Furthermore, we compared the results of using variables selected from one type of data and that of both types of data. We also studied the influences of selecting different number of variables. Result We applied our SRVS method with the sparse model given by Eq. (1) to an integrative analysis of SNP and fMRI data sets. The results were compared with that of Li et al.'s method under different weighting factors. We also discussed the sparsity control issues using k and ε. Variable selection with different weight factors Sparse model given by Eq. (1) with different weight factors were solved by our proposed SRVS method and by Li et al.'s method, respectively, as shown in Figure 3. It can be seen that at the two ends (α 1 = 0.3 or 0.6), the variables were selected form one type of data. In each of the 16 trials given by Figure 3, we selected the top 200 biomarkers by our proposed SRVS method and by Li et al.'s method [21]. As shown in Figure 3, the weight factor has similar effects on the variable selection of the two methods. It was interesting to see that even though the number of SNPs was much larger than that of fMRI voxels (759075 vs. 153594), similar number of variables were selected from both data sets when weight factor α 1 for SNP data set was around 0.5 (0.46 for SRVS method with L 0 norms, and 0.47 for Li et al.'s method). This suggests that the two data sets may contain similar information for the SCZ case/control study. Comparison with Li et al.'s method We selected 200 variables (SNPs and fMRI voxels) in each trial by our proposed SRVS method and by Li's et al.'s method respectively, as shown in Figure 3. However, further study showed that the variables selected by the two methods were significantly different (overlap <10%) (see Figure 4). Thus it was necessary to validate and compare those different groups of variables selected. We first compared the selected SNPs and the corresponding genes with the publicly reported SCZ genes for both methods. Then we compared the brain regions identified using those two methods. In addition, we compared the classification accuracies using the variables selected by our proposed Table 1. It should be noted that even though both methods can identify gene 'OPCML', they recognized the gene through different SNPs (SRVS is by 'rs3026883' and Li et al.'s method is by 'rs1745939'). To further compare the two methods at different sparsity level, we studied more top variables in each of the 16 trials. To reach this purpose, we set ε = 0.3y 2 and k = 0.05 for SRVS method. For Li's method [21], the number of subjects selected in each run was one tenth of total number of subjects; and we set the threshold θ = 0.01(please refer to [21] for the meaning of θ). As a consequence, 500 to 800 variables (SNPs and fMRI voxels) were selected in each trial. In this case, our proposed method selected 20 reported genes. For Li et al.'s method, 14 reported genes were located, and 11 of the top 45 genes were identified by both methods [22]. However, the genes identified by the two methods have <10% overlaps. For the top 50 genes selected by the two methods, there was only one When comparing the fMRI voxels selected (follow the approach shown in Figure 3), we showed that the SRVS method were capable of selecting fMRI voxels that were clustered in specific regions, as shown in Figure 5 (a). Those voxels located within a same region will have high correlations with each other. Therefore the results indicate the capability of our proposed SRVS method in selecting significant biomarkers that are highly correlated. Further study showed that the brains regions selected by our proposed SRVS method were mostly reported being associated with SCZ [33][34][35], including temporal lobe, lateral frontal lobe, occipital lobe, and motor cortex (see Table 2). However, Li et al.'s method tended to select voxels that were scattered over different brain regions (see Figure 5 (b)). Besides, the brain regions selected by those two methods were largely different from each other. Thus we used multivariate classification approach to evaluate the effectiveness of the variables selected by two methods. Multivariate classification In this study, a LOO cross validation was carried out to evaluate the classification accuracy. In each run of the LOO validation, one sample was used for testing while the rest ones were used for variable selection. Results were presented in Figure 6. We showed that our proposed SRVS algorithm provided significantly higher classification ratios (CRs) (p − value < 1 e−11 ) for both the 200-selected-variable case and the 800-selected-variable case. However, using different number of top selected variables showed no significant differences for neither of the two methods (p-value > 0.1). From Figure 6 (a) we showed that the highest classification accuracy was achieved at the weight factor α 1 = 0.5, where around equal sized SNPs and fMRI voxels were selected by the SRVS method. At the two ends (α 1 = 0.3 or 0.6), the classification accuracies were relatively lower. This suggests that using biomarkers from both types of data may lead to better identification accuracy. Discussion In this study, we introduced a novel sparse representation based variable selection (SRVS) method, and applied it to an integrative analysis of SNP data and fMRI data. In the case of medical imaging data (e.g. fMRI data) or genomic data (e.g. SNP data), the number of samples tend to be much less than the number of variables (e.g. fMRI voxles; SNP loci). As a consequence, many of those variables are correlated and cannot be identified by traditional sparse signal recovery methods. The proposed SRVS method can identify significant variables with high probability, regardless of the coherence conditions required for exact signal recovery in compressive sensing. For example, significant fMRI voxels functionally correlated (within neighbour brain regions) were identified simultaneously by using our proposed SRVS algorithms (see Figure 5 (a)). This manifests the capability of out proposed SRVS method in handling big data set with small sample sizes. In addition, the proposed SRVS method can be generalized to integrate multiple data modalities for joint analysis and achieve comprehensive diagnosis. As can be seen from Figure 6 (a), the highest classification accuracy was achieved using approximately equal sized variables from both data sets, suggesting that using biomarkers from both types of data may lead to higher diagnosis accuracy. Another advantage of the SRVS method is its multiple detection resolutions. By choosing different values of widow length k one can select different number of variables at different significance level. Furthermore, the error term ε can be used for further sparsity control of the solution δ, selecting the most important variables. This multi-resolution characteristic of SRVS provides a flexible variable selection approach for big data sets. When compared to the previous SCZ studies, our method effectively identified more reported SCZ genes than Li et al.'s method. Furthermore, most of the brain regions identified using our proposed SRVS method are previously reported as SCZ associated brain regions. When using the selected variable to identify SCZ patients Anterior Cingulate R 7 The main brain regions selected using SRVS method from controls, our method generated significantly higher classification ratio than Li et al.'s method ( Figure 5 (b), p − value < 1 e−11 ). Those results demonstrated the effectiveness of our method. Conclusions Our proposed SRVS is effective in variable selection for complex disease as SCZ. The biomarkers selected generate better identification accuracy than that of Li et al.'s method. When combining information from fMRI data and SNP data for integrative analysis, higher identification accuracy can be achieved, demonstrating the advantage of the combined analysis.	2016-05-12T22:15:10.714Z	2013-11-11T00:00:00.000	{ "year": 2013, "sha1": "03243ec38d431a9d4eb2febb14a5b648ccd6751b", "oa_license": "CCBY", "oa_url": "https://bmcmedgenomics.biomedcentral.com/track/pdf/10.1186/1755-8794-6-S3-S2", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "42c39de6c844d05568f86fe537da1b52b9c8b545", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
69243288	pes2o/s2orc	v3-fos-license	SOME EPIDEMIOLOGICAL INDICATORS CHARACTERIZING THE SPREAD OF HEPATITIS A IN THE REGION OF STARA ZAGORA Introduction: Viral hepatitis A is a widespread infectious disease with a faecal-oral mechanism of transmission of the infection. Together with other food-borne and water-borne infectious diseases, it is a global health problem. Aim: To analyze some epidemiological indicators characterizing the prevalence of hepatitis A in the region of Stara Zagora for the period 2014-2016. Material and methods: Annual reports of the Regional Health Inspection Stara Zagora for registered cases of infectious diseases (statistical documentation Form 3 85 approved by the Ministry of Health), acute infectious diseases analyzes in Bulgaria of National Center of Infectious and Parasitic Diseases and official statistical information of National Center of Public Health and Analyses. Results: Viral hepatitis registered for the period is laboratory confirmed. The spread of the infected by gender shows a higher proportion of men 54.81% (336 units), compared with the infected women (45.19%, 227 cases). The highest incidence of Acute Viral hepatitis A is registered in the most risky and susceptible to intestinal infections age groups early childhood and school age. The incidence of Viral hepatitis type A in the region of Stara Zagora in 2016 is above the average for the country, unlike the previous two years. Three outbreaks of hepatitis A (2 in 2015 and 1 in 2016) were reported. Conclusion: Prophylaxis and prevention are crucial to reducing morbidity, as the disease affects human health and life and also leads to economic losses. This requires increased public awareness of viral hepatitis A, enhancing the population's health culture, personal hygiene, and strict control over nutrition and water supply in settlements. INTRODUCTION Viral hepatitis type A is a widespread acute infectious disease with a faecal-oral transmission of the infection and it is characterized by liver parenchyma damage and jaundice.Together with other infectious diseases transmitted mainly with food and water, it is a global health problem (1).The source of the infection is the sick person.The virus is emitted by the feces, urine and saliva of the sick person.It is spread sporadically and epidemically (2,3,4)._______________________ OBJECTIVE AND TASKS The purpose of this study is to analyze some epidemiological indicators characterizing the prevalence of viral hepatitis A in the region of Stara Zagora for the period 2014-2016. MATERIALS AND METHODS Annual reports of the Regional Health Inspectorate -Stara Zagora for the registered cases of infectious diseases (statistical documentation -Form 3 -85 approved by the Ministry of Health), analyzes of acute infectious diseases in Bulgaria of the National Center for Infectious and Parasitic Diseases and official statistical information of the National Center for Public Health and Analyzes are used (5)(6)(7)(8). RESULTS AND DISCUSSION For 2014-2016, 613 cases of acute viral hepatitis A were registered in the territory of the region of Stara Zagora.The relative share of cases of this type of hepatitis in the structure of the infectious pathology in the region of Stara Zagora is respectively: 3.41% -in 2014, 16.40% -in 2015; 5.78% -in 2016.(Figure 1).Compared with other hepatitis, its spread is the greatest (Figure 2).The gender distribution of acute viral hepatitis A among patients shows a higher relative share of sick men -54.26% (336 cases), compared with female patients (45.19% -277 cases) (Figure 3).During the observed period, all age groups in the region of Stara Zagora were affected , with highest incidence of acute viral hepatitis A in the age group 5 to 9 years, followed by the age group 0 to 4 years old and the group of 10-14 year olds.In the nursing age, one case was registered for 2015.The lower incidence of acute viral hepatitis A after 40 years of age can be explained the level of immunity tension as well as the high rate of asymptomatic infection in individuals over the age of 50 years (Figure 4).The significantly higher value of the indicator compared to the one in the country in 2015. (1.95% relative share of registered cases of acute viral hepatitis A in the country for 2015 and 16.40% for the region оf Stara Zagora) is due to the two epidemic outbreaks that occurred during the year.In the same year there was one died patient in the age group of 50-54 years (7).Epidemiological studies show that the outbreaks occur and spread mainly among the Roma population living in poor living conditions with poor sanitary and hygienic status, as well as in organized children and student collectives. Acute viral hepatitis A takes an important place in the structure of infectious pathology both in Bulgaria and abroad.It is one of the most common diseases for the United States for the period 1987-1997, as 28,000 cases per year are registered in the country.This raises the need from prophylaxis and prevention of the disease, involving the use of effective vaccines available in the United States since 1995.They enable public health authorities to significantly reduce the spread of the disease and eliminate it (13,14,15). Immunoprophylaxis, good tolerance and effectiveness of vaccines, their ability to protect the body from infection over a long period of time determine their application in Bulgaria.The application of this specific prophylaxis, the implementation of quarantine and anti-epidemic measures in the case of registered hepatitis A are part of the prevention of the disease. CONCLUSIONS In the region of Stara Zagora the highest incidence of acute viral hepatitis A was registered among the risk age groups susceptible to intestinal infections -early childhood and school age.In the organized children and school teams the contact-way is leading to the transmission of the infection.Epidemic outbreaks of acute viral hepatitis A are formed in environment with poor sanitation and hygiene habits and health culture. Prophylaxis and prevention are extremely important in reducing acute viral hepatitis A morbidity.This requires increasing the public awareness, enhancing the population's health culture for strict personal hygiene, public sanitary and hygienic standards, and strict Figure 1 . Figure 1.Relative share of acute viral hepatitis A in the structure of the common morbidity for the period 2014-2016 Figure 2 . Figure 2. Acute viral hepatitis A in the virus hepatitis group Figure 3 . Figure 3. Distribution of people with acute viral hepatitis A for the period 2014-2016 by gender Figure 4 . Figure 4.The spred of people with acute viral hepatitis A for the period 2014-2016 by age Figure 5 . Figure 5. Viral hepatitis A morbidity for the period 2014-2016 and water supply in settlements.	2019-02-19T14:06:44.934Z	2018-01-01T00:00:00.000	{ "year": 2018, "sha1": "cd0c5e36949ccbc5bf07009a470f8466c4b22c4b", "oa_license": "CCBYNC", "oa_url": "https://doi.org/10.15547/tjs.2018.s.01.002", "oa_status": "GOLD", "pdf_src": "ScienceParseMerged", "pdf_hash": "cd0c5e36949ccbc5bf07009a470f8466c4b22c4b", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Computer Science" ] }
199668688	pes2o/s2orc	v3-fos-license	Equivalence of Gibbons-Werner method to geodesics method in the study of gravitational lensing The Gibbons-Werner method where the Gauss-Bonnet theorem is applied to study the gravitational deflection angle has received much attention recently. In this paper, we study the equivalence of the Gibbons-Werner method to the standard geodesics method, and it is shown that the geodesics method can be derived with the Gibbons-Werner method, for asymptotically flat case. In the geodesics method, the gravitational deflection angle of particle depends entirely on the geodesic curvature of the particle ray in the Euclidean space. The gravitational deflection of light in Kerr-Newman spacetime is calculated by different technologies under the Gibbons-Werner framework, as an intuitive example to show the equivalence. I. INTRODUCTION Gravitational lensing plays an important role in gravitational theory. In theoretical physics, it is used to test the fundamental theory of gravity, where a famous example is that Eddingtonet al. [1,2] verified Einstein's general relativity by means of the deflection experiment of light in the solar gravitational field 100 years ago. In astrophysics and cosmology, it is used to measure the mass of galaxies and clusters [3][4][5], and to detect dark matter and dark energy [6][7][8][9][10]. In mathematics, it is related to singularity theory, topology and Finsler geometry [11][12][13][14][15]. Recently, Gibbons and Werner [11] introduced an elegant geometrical method of deriving the bending angle of light in a static and spherically symmetric spacetime. They used the famous Gauss-Bonnet (GB) theorem to a surface defined by the corresponding optical metric. Later, Werner [14] extended this method to the rotating and stationary spacetimes. In stationary spacetimes, the optical geometry is defined by the Randers-Finsler metric. Thus, Werner applied Nazım's method to construct an osculating Riemannian manifold where one can easily use the GB theorem. The work by Gibbons and Werner promotes the study of light deflection. On one hand, Jusufi et al. [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] studied the gravitational lensing not only in asymptotically flat spacetime but also in nonasymptotically flat spacetime such as a spacetime with cosmic string. Similar works can also be found in Refs. [32][33][34][35][36][37][38][39]. On the other hand, Ishihara et al. [40][41][42][43][44] studied the finite-distance corrections for gravitational deflection of light both for the weak and the strong deflection limit, where the source and observer are no longer assumed to be infinitely far apart from a lens. For a review on finite-distance corrections, we refer the reader to Ref. [45]. It is well known that there are many massive particles in our Universe, such as massive neutrinos. The study of gravitational deflection of massive particles allows one to understand the properties of the sources and these particles. In fact, the study of the massive particles lensing using traditional methods can be found in Refs. [46][47][48][49][50][51][52][53][54]. Moreover, two * taozhou@swjtu.edu.cn other routes have been established by applying the GB theorem to study the gravitational deflection of massive particles. The first route is related to the Jacobi metric of curved spacetime. To be precise, one can calculate the deflection angle of massive particles via applying the GB theorem to the surface defined by the Jacobi metric [55,56] for static spacetime and by the Jacobi-Maupertuis Randers-Finsler metric [57] for stationary spacetime. The second route is related to the optical media method. For static and spherically symmetric spacetime, Crisnejo and Gallo [58] used the GB theorem to study the gravitational deflections of light in a plasma medium and the deflection angle of massive particles. The finite-distance corrections of light with a plasma medium and the gravitational deflection of charged massive particles were studied quite recently [59,60]. For rotating and stationary spacetimes, Jusufi [61] used the GB theorem to study the deflection angles of massive particles by the Kerr black hole and the Teo wormhole, respectively, based on the corresponding isotropic type metrics, the refractive index of the corresponding optical media. Furthermore, the method in Ref. [61] was extended to distinguish naked singularities and Kerr-like wormholes [62], and to study the gravitational deflection of charged particles in Kerr-Newman spacetime [63]. In this paper, the method with the GB theorem to study the deflection angle shall be called the Gibbons-Werner method. It is worth investigating whether the Gibbons-Werner method [11] is equivalent to the standard geodesics method [64]. In fact, this topic has been discussed by some researchers. The first-order equivalence has been shown in Refs. [18,19,30,62], and the second-order equivalence has been shown in Refs. [56,58]. From a conceptual point of view, however, the two methods seem to be completely different. The Gibbons-Werner method shows that the deflection of particles (photon and massive particles) is determined by a quantity outside of itself relative to the lens [14,61], and thus the gravitational deflection angle can be regarded as a global topological effect, whereas the geodesics method is usually associated within a region from particles ray to lens. In the present paper, we will demonstrate the equivalence between the Gibbons-Werner method and the geodesics method for asymptotically flat spacetime, in terms of results and concepts. More specifically, the weak gravitational deflection of light in Kerr-Newman spacetime will be taken as a simple example. arXiv:1908.05592v2 [gr-qc] 11 Feb 2020 This paper is organized as follows. In Sec. II, we review the GB theorem and use the theorem to the lens geometry. Then, we show that the equivalence of the Gibbons-Werner method to geodesics method. In Sec. III, we give the Kerr-Newman spacetime as an example to show the equivalence. Finally, we summarize our results in Sec. IV. Throughout this paper, we use the natural units where G = c = 1 and the metric signature (−, +, +, +). II. THE EQUIVALENCE BETWEEN THE GIBBONS-WERNER METHOD AND GEODESICS METHOD A. The Gauss-Bonnet theorem Let D be a compact oriented two-dimensional Riemannian manifold with the Euler characteristic χ(D) and Gaussian curvature K, and its boundary ∂D is a piecewise smooth curve with geodesic curvature k g . Then, the GB theorem states that [11,65]: where dS is the area element of the surface, dσ is the line element along ∂D, and θ i is the exterior angle defined for the ith vertex in the positive sense. B. Application the Gauss-Bonnet theorem to the lens geometry Assume M be a two-dimensional smooth manifold with coordinates (x, y) and a Riemannian metricĝ ij . Now one can apply the GB theorem to the lens geometry in a region D ⊂ (M,ĝ ij ). For convenience, D is required to be asymptotically Euclidean and thus both the particle source S and the observer O are in the asymptotically Euclidean region. Let ∂D = γ g C i (i = 1, 2, 3) with the particle ray γ g and three curves C i . γ g is described by the impact parameter b, and the curves C i are defined by with the constant R > 0. Since the lens L is excluded in the domain D, χ(D) = 1. Additionally, as R → ∞, boundary curve intersections S, A, B and O are in the asymptotically Euclidean region, and thus one can have k g (C i ) = 0, θ S + θ A + θ B = 3π/2, and θ O = π/2 + α with the deflection angle α. Then the GB theorem becomes (2) Thus, the gravitational deflection angle can be written as Particle ray γg is a spatial curve and Ci are three curves defined by C1 : x = −R, C2 : y = −R and C3 : x = R with the constant R > 0. As R → ∞, the points of intersection S, A, B, and O are in the asymptotically Euclidean region, where S and O denote the particle source and the observer, respectively. L is the lens, b is the impact parameter and α is the deflection angle. as shown in Fig. 1. C. The equivalence between the Gibbons-Werner method and geodesics method In the discussion above, the Riemannian space (M,ĝ ij ) is somewhat arbitrary, which is asymptotically Euclidean and the condition of using the GB theorem is required. In the following, three cases will be discussed to show the equivalence between the Gibbons-Werner method and the geodesics method. In this case, the particle ray γ g is a spatial geodesic in (M,ĝ ij ), and Eq. (3) becomes Indeed, this is the original consideration of Gibbons and Werner [11,14] and for convenience we shall call it the narrow Gibbons-Werner method. In fact, many studies fall into this category. For light deflection, one has (M,ĝ ij ) = (M, g opt ij ), where g opt ij is the corresponding optical metric of curved spacetime. For massive particles, (M,ĝ ij ) = (M, j ij ), where j ij is the corresponding Jacobi metric of curved spacetime. In stationary spacetime, the optical metric (or Jacobi metric) is a Randers-Finsler metric. However, in these cases one can use the osculating Riemannian metric by Werner's method [14] or use Jusufi's method to avoid the Finsler metric [61]. 2. Case 2: K = 0, and kg(γg) = 0 Now, the particle ray is not geodesic in a curved space, and Eq. (3) can be written as where In Refs. [42][43][44], Ono et al. considered the so-called generalized optical metric space as the lens background, and used Eq. (5) to study the deflection angle of light in stationary spacetimes. 3. Case 3: K = 0, and kg(γg) = 0 In this case, we assume that M is Euclidean space, and Eq. (3) arrives at To our best knowledge, Eq. (6) has not been considered yet, and next it will be proved that this result is the same with the expression in the geodesics method. The line element of a three-dimensional Euclidean space is and a unit vector normal to the x − y plane is n n n = (0, 0, 1). The particle ray γ g can be denoted by y = y(x), and one can define its unit tangent vector as where denotes derivative with respect to x. Therefore, and one can obtain the geodesic curvature of γ g in the x − y plane as follows [65]: Then, one can calculate the deflection angle by which is nothing but the formula of calculating deflection angle with geodesics method in Refs. [50][51][52]. In short, the geodesics method just corresponds to special cases for the Gibbons-Werner method, where the GB theorem is used to Euclidean space. In other words, the geodesics method categorizes the deflection angle into the influence of geodesic curvature of particles moving in Euclidean space. Therefore, the geodesics method also has geometric meaning from the perspective of curvature. III. AN EXAMPLE: THE DEFLECTION OF LIGHT IN KERR-NEWMAN SPACETIME For the second-order post-Minkowskian approximation, the components of the metric of the Kerr-Newman spacetime in the harmonic coordinates (t, x, y, z) can be written as [66,67] where m and q are the mass and electric charge of the Kerr-Newman black hole, respectively. x x x = (x, y, z), r = x 2 + y 2 + z 2 and ζ i is the ith component of the gravitational vector potential ζ ζ ζ ≡ 2ma r 3 (y, −x, 0), where a is the angular momentum per unit mass. δ ij is the Kronecker symbol and the expanding parameter ε represents the black hole parameters m, a or q. The above metric is expanded as the power series of the parameters m, a and q, and O(ε 3 ) is the series with order greater than 2, such as m 3 , a 3 , q 3 , m 2 a, ma 2 , .... For stationary spacetime, its optical geometry defined by the Randers-Finsler metric takes the form [14,68] whereα ij is a Riemannian metric and β i is a one-form satisfyingα ij β i β j < 1. Consider a null curve in the Kerr-Newman spacetime, ds 2 = 0, and one can find a Randers-Finsler metric,α where In this subsection, we will apply Werner's method [14] to calculate the gravitational deflection angle of light. The light ray is geodesic in Randers-Finsler space, and therefore, Eq. (4) can be considered. To simplify, one can study the null geodesic in the equatorial plane. Chose z = 0 as the equatorial plane, and one can find the Kerr-Newman-Randers black hole optical metric as follows whereα ij and β i are the same as those in Eq. (14) except that i and j only run in {1, 2} here. The Randers-Finsler metric is characterized by the Hessian [14,68] where x ∈ M , and v v v ∈ T x M with T x M the tangent space at a given point. In order to obtain a Remannian metricḡ ḡ g, one can choose a smooth nonzero vector field V V V over M that contains the tangent vectors along the geodesic γ F such that In this construction, we can obtain a crucial result that the geodesic γ F of (M, F ) is also a geodesic γḡ of (M,ḡ), i.e., γ F = γḡ [14]. Following Werner [14], the osculating Riemannian manifold (M,ḡ ij ) can be used to calculate the gravitational defection angle of light. Near the undeflected light rays y = −b [50,51], one can choose the vector field as Using Eqs. (16), (17), and (18), finally the osculating Riemannian metric can be obtained as follows: with the determinant up to second order detḡ = 1 + 8m r + 6amy (22) and the Gaussian curvaturē In harmonic coordinates, Eq. (4) can be written as Here y 1 (x) denotes the light ray up to first order (see the Appendix A) Substituting Eqs. (22), (23) and (25) into Eq. (24), one can get the second-order deflection angle of light as follows: which is consistent with the results in Ref. [51]. B. The generalized optical metric method: K = 0, and kg(γg) = 0 In this section we consider the Riemannian space (3) M defined byα ij . The line element of (3) M is given by The light ray is the spatial curve in (3) M and following Fermat's principle, the motion equation of light ray is [42] de i dλ where e i ≡ dx i dλ , (3) Γ i jk denotes the Christoffel symbol associated withα ij , and \| denotes the covariant derivative witĥ α ij . The existence of β i illustrates that the orbit of light is not the geodesic in (3) M . Naturally, the contribution of geodesic curvature k g should be considered and we will use Eq. (5) to calculate the deflection angle. We focus on the motion of the light in the equatorial plane (z = 0). Then the geodesic curvature of curve γ g is given by [42] where ijk is the Levi-Civita tensor and N N N is a unit normal vector for the equatorial plane. Then, choose the unit normal vector as N p = − 1 √α zz δ z p , and one can obtain where zxy = − zyx = 1/ √ detα has been used and the comma denotes the partial derivative. With Eqs. (14) and (30), one can have where the first-order light ray in Eq. (25) has been used. According to Eq. (5), the deflection angle of the light can be divided into two parts. First, the Gauss curvature ofα ij is and one can calculate the part associated with Gauss curvature Second, from Eqs. (25) and (31), the part associated with geodesic curvature is Finally, the total deflection angle can be obtained as follows: which is consistent with the result in Eq. (26). C. The geodesics method: K = 0, kg(γg) = 0 From second-order light ray in Eq. (A3), the following relation can be obtained The deflection angle can be obtained by Eq. (6) Certainly, this expression is the same as the result obtained by Werner's method in Eq. (26) and by the generalized optical metric method in Eq. (35). IV. CONCLUSION In this work, we investigate the equivalence of the Gibbons-Werner method to the geodesics method in the study of gravitational lensing. It is shown that the geodesics method can be derived with the Gibbons-Werner method for asymptotically flat spacetime. In the Gibbons-Werner procedure, one can choose the Euclidean space as the lens background and the deflection effect is completely determined by the geodesic curvature of the particle's trajectory. Thus, one can choose arbitrary asymptotically Euclidean space as the lens background and the deflection angle can be written as α = α Gauss +α geod . The difference between these different background spaces is that the contribution on α Gauss and α geod is different. However, the total deflection angle is always constant. In practice, it is more convenient to use the geodesics method or the narrow Gibbons-Werner method. We can illustrate these two methods using the following formula The left side of the equation represents the geodesic method (α Gauss = 0, α = α geod ), while the right side represents the narrow Gibbons-Werner method (α geod = 0, α = α Gauss ). As an example to show the equivalence, we calculate the second-order gravitational deflection angle of light in Kerr-Newman spacetime, for three options with the Gibbons-Werner method, in the harmonic coordinates. More, the harmonic coordinates bring a lot of simplicity and overcome the cumbersome iterative in Ref. [56]. where p is the affine parameter in Kerr-Newman spacetime. With the boundary conditionsẏ\| p→∞ =ẏ\| x→∞ = 0 [51], one can get Finally, with the first-order parameter transformation dp = dx [51] and integrating y, one can get the second-order light ray as follows: where we have considered the boundary conditions y\| p→∞ = y\| x→∞ = −b [51].	2019-08-15T15:42:34.000Z	2019-08-15T00:00:00.000	{ "year": 2019, "sha1": "979e0ca50af689e29216ec9a551a60bd506fb7e3", "oa_license": null, "oa_url": "http://arxiv.org/pdf/1908.05592", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "bfb3d66ccbec72eab55e9ff58da3018f29cdbff9", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
226282084	pes2o/s2orc	v3-fos-license	A Poisson multi-Bernoulli mixture filter for coexisting point and extended targets This paper proposes a Poisson multi-Bernoulli mixture (PMBM) filter for coexisting point and extended targets, i.e., for scenarios where there may be simultaneous point and extended targets. The PMBM filter provides a recursion to compute the multi-target filtering posterior based on probabilistic information on data associations, and single-target predictions and updates. In this paper, we first derive the PMBM filter update for a generalised measurement model, which can include measurements originated from point and extended targets. Second, we propose a single-target space that accommodates both point and extended targets and derive the filtering recursion that propagates Gaussian densities for point targets and gamma Gaussian inverse Wishart densities for extended targets. As a computationally efficient approximation of the PMBM filter, we also develop a Poisson multi-Bernoulli (PMB) filter for coexisting point and extended targets. The resulting filters are analysed via numerical simulations. I. INTRODUCTION Multiple target filtering refers to the sequential estimation of the states of the current targets, which may appear, move and disappear, given past and current noisy sensor measurements. This is a key component in many applications such as selfdriving vehicles [1] and maritime navigation [2]. Multi-target filtering can be solved in a Bayesian framework by computing the posterior density on the current set of targets, given probabilistic models for target births, dynamics and deaths, and also models for the measurements, obtained from one or multiple sensors [3], [4]. The target birth model contains probabilistic information on where targets may appear in the surveillance area, and it enables the resulting filters to contain information on potential targets that may remain occluded [5,Fig. 6], which is of paramount importance in some applications such as self-driving vehicles. If the target extent is small compared to the sensor resolution, it is common to use point-target modelling. In this model, a target state typically contains kinematic information, such as position and velocity, and one target can generate at most one measurement at each time step [6]. Conversely, if the target extent is large compared to the sensor resolution, a better choice is to use extended target modelling [7]. Here the target state usually contains both kinematic information and information on its extent, e.g., represented by an ellipse [8]. In addition, each extended target may generate more than one measurement at each time step, represented via a Poisson point process (PPP) in the standard model [7], [9], [10]. For both point and extended targets with Poisson birth model and the standard measurement models, the posterior density is a Poisson multi-Bernoulli mixture (PMBM), which can be calculated by the corresponding PMBM filtering recursions 1 [5], [11], [12]. The PMBM has a compact representation of global hypotheses, representing undetected targets via the intensity of a PPP and making use of probabilistic target existence in each global hypothesis. The PMBM recursion can also handle a multi-Bernoulli birth model by setting the PPP intensity to zero and adding new Bernoulli components in the prediction step, resulting in the MBM filter [12], [13]. The MBM filter can also be extended to consider multi-Bernoullis with deterministic target existence, which we refer to as the MBM 01 filter, at the expense of increasing the number of global hypotheses [12,Sec. IV]. Both MBM and MBM 01 filters can consider target states with labels, and the (labelled) MBM 01 filtering recursion is analogous to the δ-generalised labelled multi-Bernoulli (δ-GLMB) filter [14], [15]. There are applications in which it is important to have more general models than the standard point and extended target models [3]. Specifically, there may be some targets that are small compared to the sensor resolution, while other targets are large, which implies that there are coexisting point/extended targets in the field of view. For example, in a self-driving vehicle application, pedestrians may be modelled as point targets while other vehicles as extended targets. The distinction between point and extended targets may also depend on the distance, as sensor resolution is usually higher at short distances. Therefore, it is of interest to develop multi-target filters that can handle coexisting point and extended targets. The extended target measurement models in [15], [16] are general enough to model measurements from coexisting point and extended targets, but no single-target state, dynamic model and filter implementations are presented for this case. In this paper, we fill this gap and propose a PMBM filter for coexisting point and extended targets. In order to do so, we first develop a PMBM filtering recursion for a generalised measurement model, in which each target generates an independent set of measurements with an arbitrary distribution, and clutter is a PPP. With a suitable choice of the target-generated measurement distribution, this generalised model recovers the standard point and extended target measurement models. As a result, the PMBM filter with the generalised measurement model can be used to address multi-target filtering problems with point and extended targets [5], [11], [12], and more general problems. For example, the generalised measurement model can also be used for diffuse multipath [17], extended targets composed of point-scatterers [7], and point targets with stationary landmarks, modelled as extended targets. The resulting PMBM recursion has a track-oriented form that enables efficient implementation [18]. Based on the developed PMBM filtering recursion, the second contribution is to derive a PMBM filter for coexisting point and extended targets. In this setting, each Bernoulli contains probabilistic information on target existence and type, either point or extended target. The implementation is provided for a linear Gaussian model for point targets [19] and a Gamma Gaussian Inverse Wishart (GGIW) model for extended targets [5], [7], [20]. Finally, we explain how a PMBM density in this context can be projected onto a Poisson multi-Bernoulli (PMB) density [11]. Performing this projection after each update provides us with a PMB filter, which is a fast approximation to the PMBM filter. Simulation results are provided to analyse the performance of the filters. The rest of the paper is organised as follows. Section II introduces the problem formulation and an overview of the solution. The update for the PMBM filter with generalised measurement model is derived in Section III. Section IV explains the PMBM filter for coexisting point and extended targets, and the PMB projection. Simulation results and conclusions are given in Sections V and VI, respectively. SOLUTION This paper deals with multiple target tracking with both point and extended target models. This section presents an overview of a PMBM filter with a generalised measurement model that will be used to model coexisting point and extended targets in Section IV. We introduce the models in Section II-A and the PMBM filter overview in Section II-B. A. Models A single target state x ∈ X , where X is a locally compact, Hausdorff and second-countable (LCHS) space [3], contains the information of interest about the target, for example, its position, velocity and extent. The set of targets at time k is denoted by X k ∈ F (X ), where F (X ) represents the set of finite subsets of X . A main novelty in this paper is the development of a PMBM filter with a generalised measurement model. Here, the set X k of targets at time step k, is observed through a set Z k ∈ F (R nz ) of noisy measurements, which consist of the union of target-generated measurements and clutter, with the model: • Each target x ∈ X k generates an independent set Z of measurements with density f (Z\|x). • Clutter is a PPP with intensity λ C (·). It should be noted that the standard point and extended measurement models [5], [11] can be recovered by suitable choices of f (Z\|x). We also consider the standard dynamic model for targets. Given the set X k of targets at time step k, each target x ∈ X k survives with probability p S (x) and moves to a new state with a transition density g (· \|x ), or dies with probability 1−p S (x). At time step k, targets are born independently following a Poisson point process (PPP) with intensity λ B k (·). B. PMBM posterior In this paper, we show that, for the above-mentioned measurement and dynamic models, the density f k\|k (·) of X k given the sequence of measurements (Z 1 , ..., Z k ), where k ∈ {k − 1, k}, is a PMBM density. This section provides an overview of the PMBM posterior and its data association hypotheses. The PMBM is of the form [11], [12] f where λ k\|k (·) is the intensity of the PPP f p k\|k (·), representing undetected targets, and f mbm k\|k (·) is a multi-Bernoulli mixture representing potential targets that have been detected at some point up to time step k . Symbol denotes the disjoint union and the summation in (1) is taken over all mutually disjoint (and possibly empty) sets Y and W whose union is X k , i.e., X k is fixed, and Y and W free. In the PMBM posterior, there are n k\|k Bernoulli components and for each Bernoulli there are h i k\|k possible local hypotheses. By selecting a local hypothesis a i ∈ 1, ..., h i k\|k for each Bernoulli, we obtain a global hypothesis a = a 1 , ..., a n k\|k ∈ A k\|k , where A k\|k is the set of global hypotheses. Each global hypothesis represents a multi-Bernoulli distribution. The i-th Bernoulli component with local hypothesis a i has a density where r i,a i k\|k is the probability of existence and f i,a i k\|k (x) the single target density. The weight of global hypothesis a is w a k\|k and meets where w i,a i k\|k is the weight of the i-th Bernoulli with local hypothesis a i , and a∈A k\|k w a k\|k = 1. The set of feasible global hypotheses is defined as in the extended target case [5], [21]. We denote the measurement set at time step k as Z k = z 1 k , ..., z m k k . We refer to measurement z j k using the pair (k, j) and the set of all such measurement pairs up to (and including) time step k is denoted by M k . Then, a single target hypothesis a i for the i-th Bernoulli component has a set of measurement pairs denoted as M i,a i k ⊆ M k . The set A k\|k of all global hypotheses meets A k\|k = a 1 , ..., a n k\|k : a i ∈ 1, ..., h i k\|k ∀i, That is, all measurements must be assigned to a local hypothesis, and there cannot be more than one local hypothesis with the same measurement. More than one measurement can be associated to the same local hypothesis at the same time step. Each global hypothesis therefore corresponds to a unique partition of M k [5, Sec. V], and the number of global hypothesis is the Bell number of \|M k \|. At each time step, each non-empty subset of Z k generates a new Bernoulli component, corresponding to a potential target detected for the first time or clutter. This implies that, at each time step, 2 m k − 1 new Bernoulli components are generated. It should be noted that the prediction step of a PMBM density is closed-form for the standard dynamic models [11], [12], and is not affected by the choice of measurement model. Therefore, the next section focuses on the update and we omit the details for prediction, which can be found in [11], [12]. MODEL This section provides the PMBM filter update step with the measurement model in Section II. We denote a Kronecker delta as δ i [·], with δ i [u] = 1 if u = i and δ i [u] = 0, otherwise. Also, given two real-valued functions a (·) and b (·) on the target space, we denote their inner product as A. Update The update of the predicted PMBM f k\|k−1 (·) after observing Z k is given in the following theorem. Theorem 1. Assume the predicted density f k\|k−1 (·) is a PMBM of the form (1). Then, the updated density f k\|k (·) with set Z k = z 1 k , ..., z m k k is a PMBM with the following parameters. The number of Bernoulli components is n k\|k = n k\|k−1 + 2 m k . The intensity of the PPP is For Bernoullis continuing from previous time steps i ∈ 1, ..., n k\|k−1 , a new local hypothesis is included for each previous local hypothesis and either a misdetection or an update with a non-empty subset of Z k . The updated number of local hypotheses is h i k\|k = 2 m k h i k\|k−1 . For missed detection hypotheses, i ∈ 1, ..., n k\|k−1 , a i ∈ 1, ..., h i k\|k−1 , we obtain Let Z 1 k , ..., Z 2 m k −1 k be the nonempty subsets of Z k . For a Bernoulli i ∈ 1, ..., n k\|k−1 with a single target hypothesis a i ∈ 1, ..., h i k\|k−1 in the predicted density, the new local hypothesis generated by a set Z j k has a i = a i + h i k\|k−1 j, r i,a i k\|k = 1, and For the new Bernoulli initiated by subset Z j k , whose index is i = n k\|k−1 + j, we have two single target hypotheses (h i k\|k = 2), one corresponding to a non-existent Bernoulli and the other Theorem 1 is proved in Appendix A. We can see that the updated PPP intensity in (7) corresponds to the predicted intensity multiplied by the probability of not receiving any measurements. This is expected as the PPP contains information on the undetected targets. Misdetection hypotheses lower the probability of existence of the Bernoullis via (9) and (11). If f (∅\|x) does not depend on x, the single-target densities of misdetection hypotheses remain unchanged, see (12). For the update of a previous Bernoulli component with subset Z j k , the updated Bernoulli has a probability of existence equal to one. Each non-empty subset Z j k ⊆ Z k creates a new Bernoulli component. If \|Z j k \| > 1, the existence probability r i,2 k\|k of the new Bernoulli component is one, which implies that, conditioned on the corresponding hypothesis, this Bernoulli represents an existing target. If \|Z j k \| = 1, the existence probability r i,2 k\|k of the new Bernoulli component depends on the clutter intensity λ C (·), as this Bernoulli may correspond to a target or to clutter. The higher λ C (·), the lower the probability of existence of this potential target. B. Relation to standard point/extended target models In the standard point target measurement model, a target x is detected with probability p D (x) and, if detected, it generates one measurement with density l(·\|x). This model is obtained by setting If we use the above definitions of local and global hypotheses and Theorem 1 for point targets, many of the global hypotheses contain local hypotheses where more than one measurement is associated to the same Bernoulli at the same time step. Since this is impossible according to (23), all these hypotheses would obtain weight zero. A more convenient way to handle point targets is to exclude these hypotheses from the set A k\|k that we consider, see [11]. In the standard extended target model, a target x is detected with probability p D (x) and, if detected, it generates a PPP measurement with intensity γ (x) l(·\|x), where l(·\|x) is a single-measurement density and γ (x) is the expected number of measurements. We can recover this model by setting In this case, Theorem 1 becomes the standard extended-target PMBM update in track-oriented form [5], [21]. C. Discussion We have shown that the update of a PMBM prior with the generalised measurement model in Section II is also PMBM. The proposed measurement model contains the standard point target and extended target measurement models as particular cases, and can be used for other types of measurement modelling. For example, another important special case is that each target could generate a union of independent Bernoulli measurements, which can model extended targets that consist of reflection points [22], [23]. It can also model extended targets with binomially distributed target-generated measurements [24]. The considered measurement model also allows us to model coexisting point and extended targets, for example, modelling radar returns from vehicles (extended targets) and pedestrians (point targets), as will be explained in Section IV. It can also model scenarios in which far-away targets produce point-target measurements and targets that are sufficiently close produce extended-target measurements, for example, by setting a distance threshold, which may depend on the target extent, to switch between both types of model. The proposed PMBM update requires PPP clutter, which can be relaxed in Bernoulli filters [25]. We would also like to remark that we have presented the results for PPP birth density, as we think this is generally the most suitable birth process, due to the lower number of generated hypotheses [12], [13]. Nevertheless, the presented results also hold for the following cases. For multi-Bernoulli birth, the above equations are valid, by setting the Poisson intensity equal to zero, and adding the Bernoulli components for new born targets in the prediction step [12], [13]. In this case, the posterior is a multi-Bernoulli mixture (MBM), which can also be represented as MBM 01 [12, Sec. IV]. For multi-Bernoulli birth, one can also uniquely label each Bernoulli component, for which the labelled MBM 01 recursion would correspond to the δ-GLMB filter recursion [15]. IV. PMBM FILTER FOR COEXISTING POINT AND EXTENDED TARGETS This section presents the PMBM filter, and a track-oriented PMB filter, for coexisting point and extended targets. The single target space for point targets is R nx , which represents the kinematic state (e.g. position and velocity). We model extended targets with the GGIW model [16], whose space is where R + represents the positive real numbers and S d + the positive definite matrices of size d, which is the dimension of the extent. The single target space for coexisting point/extended targets is then X = R nx X e , where stands for union of sets that are mutually disjoint, i.e., X = R nx ∪ X e and R nx ∩ X e = ∅ [3]. Other works with this type of hybrid space are for example [3], [26]- [28]. If x ∈ X e , then x = (γ, ξ, X), where γ represents the expected number of measurements per target, ξ is the kinematic state and X is the extent state that describes the target's size and shape. It should be noted that, though not necessary, it is also possible to include a class variable in the target space to distinguish between point and extended targets, as in interacting multiple models [29], see Appendix B. This appendix also explains the corresponding single-target integral. We use a measurement model that corresponds to the standard point and extended target measurement models depending on the type of target we observe. That is, for x ∈ R nx , f (Z\|x) is given by (23) with a probability p D (x) = p D 1 of detection, l(z\|x) = N (z; H 1 x, R) where H 1 is the measurement matrix, R is the noise covariance matrix, and N (·; x, P ) is a Gaussian density with mean x and covariance P . For x ∈ X e , f (Z\|x) is given by (24) The rest of this section is organised as follows. Section IV-A presents the considered single-target densities. The update and the prediction are provided in Sections IV-B and IV-C. The PMB approximation is addressed in Section IV-D. Target state estimation is explained in Section IV-E. Practical aspects are discussed in Section IV-F. A. Single-target densities We develop a PMBM implementation in which we propagate a Gaussian for single target densities and a (factorised) GGIW density for extended target densities [7], [20], [30]. In a factorised GGIW density, the distributions for γ, ξ and X are independent, which has computational and practical benefits The Gaussian density for x ∈ X with mean x i,a i ,1 for x ∈ R nx and zero for x ∈ X e . Note that N p (·) is zero evaluated at x ∈ X e , as N p (·) represents point targets. The Gamma density with parameters α > 0 and β > 0 is denoted as G (·; α, β). The inverse Wishart density on matrices in S d + with v > 2d degrees of freedom and parameter matrix . Then, the GGIW density for x ∈ X with parameters is for x ∈ X e and zero for x ∈ R nx . The single-target density of the i-th Bernoulli and local hypothesis a i is where c i,a i k\|k and 1 − c i,a i k\|k are the probabilities that the target is a point-target and extended target, respectively. The PPP intensity is a mixture where n p k\|k is the number of components with point-targets, with weight w p,q k\|k , mean x p,q,1 k\|k and covariance P p,q,1 k\|k , and n e k\|k is the number of components with extended targets, with weight w e,q k\|k and parameters ζ e,q k\|k . It should be noted that n p k\|k q=1 w p,q k\|k and n e k\|k q=1 w e,q k\|k represent the expected number of undetected point and extended targets, respectively. B. Update We represent the update of a GGIW density with parameters ζ i,a i k\|k−1 with a given measurement set Z j k as a function ζ e,q k\|k , e,q k\|k = u e ζ i,a i k\|k−1 , Z j k where ζ e,q k\|k is the updated GGIW and e,q k\|k the marginal likelihood, see Appendix C. The Kalman filter update of a Gaussian density with mean x i,a i ,1 k\|k−1 and covariance P i,a i ,1 k\|k−1 and measurement z is represented as are the updated mean and covariance, and i,a i ,1 k\|k is the marginal likelihood, see [19] for details. We apply Theorem 1 to obtain the specific parameters of the updated PMBM provided in the following lemma. Lemma 2. The updated PMBM with a prior PMBM described by (1), (28) and (29), with measurement set Z k = z 1 k , ..., z m k k has the structure in Theorem 1 with the following parameters. The number of PPP components is n p k\|k = n p k\|k−1 and n e k\|k = 2n e k\|k−1 . For point targets, For extended targets and q ≤ n e k\|k−1 , we have ζ e,q k\|k = ζ e,q k\|k−1 , w e,q k\|k = 1 − p D 2 w e,q k\|k−1 . For q > n e k\|k−1 ,q = q − n e k\|k−1 , ζ e,q k\|k , e,q k\|k = u e ζ e,q k\|k−1 , ∅ w e,q k\|k = p D 2 e,q k\|k w e,q k\|k . For missed detection hypotheses of previous Bernoullis, The detection hypotheses of a previous Bernoulli with a subset Z j k , with Z j k = m j k , has r i,a i k\|k = 1, and For the new Bernoulli initiated by subset Z j k , the single target density corresponding to an existing Bernoulli is ζ i,2,q k\|k , i,2,q 2,k\|k = u e ζ e,q k\|k−1 , Z j k (48) r i,2 k\|k = 1 and c i,2 k\|k = 0. For m j k = 1, Z j k = {z}, we have x i,2,q k\|k , P i,2,q k\|k , i,2,q 1,k\|k = u p x p,q,1 k\|k−1 , P p,q,1 k\|k−1 , z Lemma 2 is obtained by using Theorem 1 and the GGIW and Gaussian updates [5], [19]. We can see that the number of components in the PPP corresponding to extended targets doubles in the update. This is due to the fact that the likelihood for misdetection for extended targets, see (24), has two terms 1 − p D 2 and p D 2 e −γ . The first term corresponds to a misdetection obtained through the detection process modelled by p D 2 , whereas the second term corresponds to a misdetection obtained when the detection PPP generates zero measurements [5], [16], [32]. These terms create two updated PPP components for each prior PPP component. For the same reason, in the update of previous Bernoullis with a misdetection, the extended target updated density is a mixture of two GGIW, see (35). As only the Gamma distribution differs in the two updated GGIWs, we apply merging for Gamma densities [33] to obtain an updated single-target density of the form (28). For the detection of previous Bernoullis, the hypothesis represents with probability r i,a i k\|k = 1 that there is target. If m j k > 1, the target is an extended target with probability one (c i,a i k\|k = 0). If m j k = 1, the target may be a point or an extended target. For the new Bernoulli components, if m j k > 1, the local hypotheses represent an existing extended target with probability one. For m j k = 1, the new Bernoulli may represent clutter, a single target or an extended target. All possible clutter events are accounted for in the hypotheses with m j k = 1 and so do not need to be duplicated in events with m j k > 1. We can also see that the single target density (47) for new Bernoulli components is a mixture for both point and extended targets. To obtain an updated density as in (28), we perform merging of the Gaussian mixtures and merging of the GGIW mixtures [33], [34]. It should be noted that, if the probability of detection is nonconstant, it can be approximated as a constant at the predicted means for point and extended targets for each hypothesis [5, Tab. IV]. Then, we can perform the corresponding updates in Lemma 2. C. Prediction We consider that the probability of survival is a constant p S (·) = p S and linear/Gaussian dynamics for point targets. That is, for x ∈ R nx , we have where F is the transition matrix and Q is the process noise covariance matrix. For GGIW targets, there are several dynamic models [7], [8]. In the simulations, we use the one in [5]. We also assume that a point target cannot become an extended target and vice versa. The target birth intensity is of the form We apply the PMBM prediction step [11], [12] to obtain a PMBM with the following parameters. Given a singletarget filtering density f i,a i k−1\|k−1 (·) of the form (28), then the predicted density is of the same form with where p p (·) and p e (·) denote the Kalman filter [19] and the extended target GGIW prediction [5, Tab. III], respectively. The predicted PPP is and ζ e,q k\|k−1 = p e ζ e,q k−1\|k−1 . While this prediction step assumes that there is no dynamic change between point and extended targets (e.g., the targets are pedestrians and vehicles), in some applications, a point target may become an extended target if it gets sufficiently close to the sensor. In this setting, one should design the corresponding transition density to capture this. D. PMB approximation It is also useful to consider a PMB approximation to the PMBM (1) to develop a faster algorithm. If we perform this approximation after each update, we obtain the corresponding PMB filter [11], [35]. Given an updated PMBM (1) with k = k, the track-oriented PMB approximation is where f i k\|k (·) is a Bernoulli density with probability r i of existence and single target density p i (·) such that The PMB approximation (59)-(60) minimises the Kullback-Leibler divergence (KLD) on a single target space augmented with an auxiliary variable, which represents if the target remains undetected or corresponds to the i-th Bernoulli component [36]. We can see that (62) is a mixture over all local hypotheses and that the PPP part of the PMBM (1) is not affected by the PMB approximation. In the implementation for coexisting point-extended targets, we are interested in single target densities of the form (28). By using moment matching (KLD minimisation) for the mixture in p i (·), we obtain the single-target density where m G (·) is a function that obtains the mean and covariance of a Gaussian mixture with weights [37]. The function m GG (·) obtains the GGIW parameters that minimise the KLD from a mixture with weights β i,a 1 GG , ..., β i,a h i GG (normalised to sum to one) and parameters ζ i,a 1 k\|k , ..., ζ i,a h i k\|k [33], [34]. E. Target state estimation Given a PMBM posterior, we can apply several estimators to estimate the current set of targets, see details in [12, Sec. VI]. We proceed to explain how Estimator 1 in [12, Sec. VI], which is the one we use in the simulations, is adapted to deal with the single-target space X = R nx X e . We first obtain the global hypothesis with highest weight and select its Bernoulli components whose probability of existence is above a threshold (0.5 in the simulations). For each of these Bernoulli components, which have densities of the form (28), we estimate a target state, which may be a point or an extended target. If the probability of being a point target is c i,a i k\|k > 0.5, then we estimate a point target located at the mean x i,a i ,1 k\|k . Otherwise, we estimate an extended target with kinematic and extent states located at the mean [8] (71) F. Practical aspects As in other multiple target filters with data associations, the number of global and local hypotheses increases unboundedly in time. Therefore, in practice, it is necessary to perform approximations, with the objective of only propagating hypotheses with relevant weights. In fact, due to the structure of the hypotheses of Theorem 1, the way to handle the data association problem with coexisting point and extended targets is quite similar to the extended target case [5], [7]. In our implementation, the PMBM posterior is represented by a list of Bernoullis i ∈ 1, ..., n k\|k , where each of them contains their local hypotheses with their parameters, a global hypothesis table, which contains indices to local hypotheses of each Bernoulli, and a vector with the global hypotheses weights. To deal with the data association problem at each update, we first perform gating to obtain two sets of measurements: 1) measurements that are in the gate of at least one previous Bernoulli, and 2) measurements that are only in the gate of the PPP components. Measurements that do not fall into these categories are discarded. For the set of measurements in group 1), we first generate possible partitions of this set using the DBSCAN algorithm with distance thresholds between Γ d,min and Γ d,max , with a step size of ε d [38], [39]. The minimum number of points to form a region, which is a parameter of the DBSCAN algorithm, is set to 1 to capture point-target measurements. Among the possible partitions generated by the multiple runs of DBSCAN algorithms, there may be repeated ones, so we keep the unique ones and we obtain the unique subsets of measurements in these partitions. These subsets are then used to generate the updated local hypotheses for previous Bernoullis, see (8)- (16). A new Bernoulli component is also created for each unique subset of measurements that is in the gate of a GGIW PPP component. For each previous global hypothesis and partition, obtained by DBSCAN, we run Murty's algorithm [40] to find the global hypotheses with highest weights. For the set of measurements in group 2), which may correspond to newly detected targets, we run the DBSCAN to obtain possible partitions. Each of these partitions in theory gives rise to different global hypotheses corresponding to new born targets. We simplify this procedure by finding the partition with highest weight and only generating the Bernoulli components that are generated by the sets in this partition [5]. These new Bernoulli components are added to all the global hypotheses, whose weights remain unchanged. We would like to point out that, while DBSCAN is a fast method for clustering, it is agnostic to target shape. Therefore, in difficult scenarios, it may be suitable to consider further partitions using additional methods that account for target shape, for example, prediction partition and expectation maximisation partition [32], [41]. We also perform pruning of global hypotheses with low weights, and pruning of Bernoulli components with low existence probabilities [12], [13], [42]. A pseudocode of the resulting PMBM update is provided in Algorithm 1. The PMB filter performs the same PMBM update and it is then followed by the PMB approximation, see Section IV-D. It is also possible to approximate the PMB marginal data association probabilities directly using belief propagation [43]- [45]. V. SIMULATIONS In this section, we assess the PMBM and PMB filters for coexisting point and extended targets via numerical simulations 2 . In this section, we refer to these filters as pointextended PMBM and PMB (PE-PMBM and PE-PMB) filters. The filters are implemented with the following parameters: maximum number of hypotheses 20, threshold for pruning the PPP weights 10 −5 , threshold for pruning Bernoulli components 10 −3 and threshold for pruning global hypotheses 10 −3 . The DBSCAN algorithm [38] is run with distance thresholds between Γ d,min = 0.1 and Γ d,max = 12, with a step size of ε d = 0.1. We have also implemented a point-extended MBM (PE-MBM) filter, see Section III-C. Extended target filters can in principle deal with point-target detections, as they do not place zero probability to this event. Therefore, we compare the proposed filters with extended target PMBM and PMB filters, which we refer to as E-PMBM and E-PMB filters [5], [39]. We proceed to discuss the models and the simulations results. All the units in this section are given in the international system. A. Models We consider a point target state [p x ,ṗ x , p y ,ṗ y ] T , which contains position and velocity in a two-dimensional plane. Point targets move with a nearly-constant velocity model with Figure 1, which has been obtained by sampling from the dynamic process. The E-PMBM and E-PMB filters are recovered by setting the birth intensity for point targets to zero in the PE-PMBM and PE-PMB filters. B. Results We first show the ground truth and the estimate of the set of targets at time step 52 in an illustrative run with the PE-PMBM filter in Figure 2. We can see that the each extended target generates several measurements and are detected. The ellipses of the estimated targets are reasonably accurate. The point target generates a single measurement at this time step, and it is also detected. Its estimate is close to its true state. In this scenario, the class probability quickly reaches either zero or one for the considered targets, classifying all targets correctly. We evaluate filter performance via Monte Carlo simulation with 100 runs. We compute the error between the true set of targets at each time and its estimate using the generalised optimal subpattern assignment (GOSPA) metric with parameters α = 2, p = 2, c = 10, and its decomposition into localisation errors and costs for missed and false targets [46]. The base metric for target states is the Gaussian Wasserstein distance, which measures error for position and extent [47]. In the base metric, we consider a point target as an extended target with extent zero. The root mean square GOSPA (RMS-GOSPA) error against time and its decomposition are shown in Figure 3. We can see that PE-PMBM and PE-PMB filter perform quite similarly and outperform E-PMBM and E-PMB. PE-MBM performs quite similarly to PE-PMBM and PE-PMB but does not detect one of the targets at time step 1, as the birth model sets the maximum number of new born targets to one. For PE-PMBM and PE-PMB, missed target errors are higher when new targets are born. False target errors are higher when targets die and when targets get in close proximity. Localisation errors are higher at the beginning of the simulation, and when targets get in close proximity, as the data association problem is more complicated. ET-PMBM and ET-PMB also behave quite similarly and have more difficulty in detecting the point targets, so they show a higher missed target error at some time steps. In addition, the localisation error is also higher at some time steps, as point targets are estimated with a certain extent, which increases the error compared to the ground truth. To provide more complete simulation results, we show the RMS-GOSPA errors, along with the GOSPA error decomposition, considering all time steps for different values of the probability of detection and clutter rate in Table I. Due to space constraints, we do not show E-PMB, which behaves quite similarly to E-PMBM. In this table, "Tot.", "Loc.", "Fal." and "Mis." refer to total GOSPA, localisation, false target and missed target costs, respectively. The filters with coexisting point extended targets consistently provide more accurate results, especially due to a lower number of missed targets. The PE-PMBM and PE-PMB filters provide quite similar results though the PE-PMB filter is slightly better. While the PE-PMBM filter provides the closed-form solution to the filtering recursion, we apply approximations and an suboptimal estimator, so the PE-PMB filter may work better in some scenarios. Decreasing the probability of detection or increasing the clutter rate, the GOSPA error for all filters increases, mainly due to a rise in missed target cost. VI. CONCLUSIONS We have derived the update of a PMBM filter with a measurement model that can consider point and extended targets, and we have shown that the updated posterior is also a PMBM. We have also proposed an implementation of the resulting PMBM recursion to consider coexisting point and extended targets. In order to do so, we first set the suitable single-target space and single target densities, which are based on Gaussian densities for single targets, and GGIW densities for extended targets. Finally, based on the previous results, we have explained how to obtain a computationally-lighter PMB filter for coexisting point and extended targets. We think there are many lines of future work. In many applications, there are coexisting point and extended targets, and one can perform research into tailored measurement and target models for each application. Another line of future work is to extend the above results to consider PMBMs on sets of trajectories, with coexisting point and extended targets, to provide full trajectory information [21], [27], [28]. In this appendix, we prove Theorem 1, which provides the update step, making use of probability generating functionals (PGFLs). A PGFL is an alternative representation of a multiobject density, in the same way as Fourier and z-transforms are for signals defined in the time domain. For a multi-object density f (·), its PGFL G f [·] is given by the set integral [3] where h (·) is a unitless function of state space, and h X = x∈X h(x) , h ∅ = 1. The test function for PGFLs related to densities defined for targets and measurements are denoted as h (·) and g (·), respectively. Given the PGFL G f [·], we can recover its multi-object density f (·) by the set derivative [3] f A. PGFLs of targets and measurements The density (1) in PGFL form is represented as [11] where Given the multi-target state X, measurements from each target are independent, and there is also independent PPP clutter. Therefore, the PGFL G Z [g\|X] of the measurements given X is the product of PGFL where G[g\|x] is the PGFL of f (Z\|x). B. Joint PGFL of targets and measurements The joint PGFL of measurements and targets is [3], [11] ∝ exp λ C , g + λ k\|k−1 , hG[g\|·] We denote the first line of (83) as which represents the joint PGFL of measurements (including false alarms) and targets in the PPP, up to a proportionality constant. We also denote which represents the joint PGFL of measurements (not including false alarms) and the i-th potential target. Then, using (5), we can write (83) as Applying the product rule [11,Eq. (31)], we obtain The sum in (89) where we have applied (73 is the PGFL of a weighted Bernoulli distribution, i.e., a distribution of the form [11, Lem. 2] This lemma therefore proves how to update a previous Bernoulli with a misdetection or a detection hypothesis in Theorem 1. 2) PPP update: We now turn to calculating the update of the PPP in (89) via the set derivative of F 0 [g, h], see (84). The proof of Lemma 4 is in Section A-D. Following (89) , we evaluate the first factor of (101), F 0 [g, h], at g = 0 to obtain associations to previous Bernoulli components, as in done in Theorem 1. D. Set derivative of F 0 [g, h] We prove Lemma 4 by induction. The set derivative of F 0 [g, h], see (84), with respect to a set with \|W \| = 1 is straightforward, as there is a single partitioning of a one element set. For induction, we assume that the lemma holds up to a given size \|W \|, and we show that it holds for W = W ∪ {z}: (115) Each step in the previous derivation results from the product rule [3]. From (102), we have ∂ ∂{z} d V [g, h] = d V ∪{z} [g, h]. In addition, each partitioning ofW consists of either a partitioning of W with an additional single element subset {z}; or a partitioning of W , adding element z to one of the existing subsets [3, App. D.2]. Since the top line in (116) handles the former case and the bottom line handles the latter case, we find that (116) is equivalent to F 0 [g, h] P ∠W V ∈P d V [g, h], which proves Lemma 4. B. Relation to spaces in interacting multiple models We explicitly relate the space of coexisting point extended targets, X = R nx X e , in Section IV to spaces used in interacting multiple models [29], which usually include a class variable to distinguish between different models. Given x ∈ R nx X e , we know if x represents a point target or an extended target as R nx and X e are disjoint. Therefore, it is not necessary to extend the single target space with a class variable to distinguish both types of targets. Nevertheless, it is possible to add a class variable c such that the single target state becomes y = (c, x), where c = 0 for point targets and c = 1 to extended targets. In this case, the single target space is ({0} × R nx ) ({1} × X e ) and the PMBM filtering recursion remains unchanged. APPENDIX C For completeness, in this appendix, we provide the (approximate) single extended target update for factorised GGIW priors [5], [30]. The resulting expressions are provided in Table II. The update for the parameters of the Gamma distribution is exact due to the Poisson-Gamma conjugacy.	2020-11-10T02:00:59.043Z	2020-11-09T00:00:00.000	{ "year": 2020, "sha1": "42a31141d1aacddc104c78c4810e9875476b23df", "oa_license": null, "oa_url": "http://arxiv.org/pdf/2011.04464", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "42a31141d1aacddc104c78c4810e9875476b23df", "s2fieldsofstudy": [ "Computer Science", "Mathematics" ], "extfieldsofstudy": [ "Computer Science", "Mathematics" ] }
235285248	pes2o/s2orc	v3-fos-license	A numerical study of tidal run up and inundation impact using logical tool-less than equal Kendal Regency as Special Economic Zone and Industrial Park is stimulating the rapid coastal urban growth. This condition initiates the high risk of disaster in the northern Central Java. The research aims to model the tidal run-up and spatial inundation distribution using numerical calculation of logical tool-less than equal and to identify the land uses that are affected by the tidal run-up and inundation in the Kendal coastal area. This research conducted by the digital elevation model in 7 m spatial resolution and the highest high-water level (HHWL) data in the one-year analysis. Furthermore, the data processing was run using Less than equal tools. The result showed that the HHWL condition in 2018 reached up to 0.35 m. Considering the HHWL data, the eastern coastal area of the Kendal Regency is massively being affected by the tidal run-up and inundation. The furthest distance of the tidal run-up and inundation reached up to 3.7 km. There are six land uses affected, i.e., built-up area (0.04%), garden (2.01%), dry land (4.89%), grassland (5.09%), a fish pond (41.95%), and paddy field (4.24%). Introduction Tidal floods, a condition where sea tides inundate a land area for a long time, can negatively affect life in coastal areas [1,2,3]. Seawater that pushes inland toward the drainage system in the settlement is likely to slow down the movement of wastewater, decreasing environmental quality. Soil degradation due to contamination by highly saline water continuously shrinks the agricultural land in coastal areas. Nearby communities also have to bear substantial economic losses as they have to allocate expenses for building embankments or raising the floor to prevent seawater flowing into the house [4,5,6]. Kendal Regency on the northern coast of Java Island is prone to tidal floods [7]. Its nearly level beach morphology is composed of alluvial fan deposits from several major rivers (i.e., Bodri, Kalikutho, Kendal, and Blorong), and it creates an environment where seawater can quickly enter the mainland during high tides. Damages to mangrove ecosystems due to abrasion and human intervention in coastal areas have erased the existence of natural barriers to tidal floods [8,9]. Coastal reclamation for expansion of the residential regions, special economic zones, and industrial parks also potentially change the direction of tides and broaden the inundated area landward. Modeling the spatial distribution of tide propagation and inundation is an effort to reduce disaster risk in the future. Knowledge of inundated locations provides a basis for identifying at-risk objects. The next step includes calculating the amount of loss, which can be estimated by mapping the affected areas. Tide propagation and inundation in various regions on the north coast of Java Island have been modeled [10,11], but for Kendal Regency, it has not been exposed in detail. The rapid development of the geographic information system (GIS) technology offers many significant benefits, mainly for solving environmental and disaster-related problems [12,13,14]. Sources of elevation data with high resolution and records of highest tides are now available and sufficient for inputs to the modeling of tide propagation and seawater inundation with minimal errors. Many types of software are highly facilitative of running the mathematical process in inundation modeling. This study was designed to examine the areas affected by tide propagation and seawater inundation based on the outcome of the Logical Math tools with the Less Than Equal feature in ArcGIS. This method has faster and more straightforward steps to predict areas inundated by tidal floods. This method is also become a new method to be conducted to predict the inundation in Kendal coastal area. Methods Tidal run-up modeling was prepared with tidal data and Digital Elevation Model (DEM). The daily tidal information was obtained from the Ministry of Maritime Affairs and Fisheries (KKP). It consists of hourly records of sea level in one day for one month. The one-year tidal data analysis pinpointed the highest sea level, which was later used as the basis for tidal run-up modeling. The DEM data retrieved from the Geospatial Information Agency (BIG) has a 7x7m 2 resolution. It is created from various satellite images, such as IFSAR, TERRASAR, and ALOS PALSAR. There are several methods capable of estimating the propagation of tidal wave energy, including algorithms. In this case, a simple algorithm was employed to see and measure the propagation of high (+h) and low (-h) tides. The algorithm was determined using the Logical Math Tools-Less Than Equal in ArcGIS (Equation 1). ≤ ±ℎ ………………………………………………………………… (1) The algorithm formulated in ArcMap was processed to produce a raster-based visual model of seawater propagation. This model was then analyzed and transformed into a thematic map of the highest tidal inundation in the coastal area of the Kendal Regency ( Fig.1) automatically using ArcGIS software. The 2016 land use map acquired from BIG was overlaid with the tidal run-up map to identify the affected areas and their at-risk elements quantitatively. Study Area Kendal is a regency in Central Java Province that has been designed as a specific economic zone and industrial park according to the national development program. As stated in the local regulation [15], Kendal is a priority area for industrial activities, and the government has prepared 2,600 ha of land for this designation [16]. This program also takes into account the geographical position of Kendal, which is in the middle of the northern part of Java Island. Another consideration is its high accessibility through the North Coast Road, the most crowded land transportation lane in Indonesia [17]. Results and Discussion Based on a simple algorithm in the Logical Math Tool-Less Than Equal, the modeling showed that tidal propagation and inundation harmed six land uses, most of which were in the north of the North Coast Road. The land uses were built-up area or building, plantation, dry agricultural land, grassland, pond, and paddy field, as shown in Table 1. Referring to the area and the percentage of affected areas, ponds were the most widely affected land use. Tidal propagation and inundation deteriorate the land quality of the fish ponds and threaten their sustainability [18]. The other adverse impacts are as follows: (1) weakened pond embankments due to increased inundation, (2) decreased pond water quality due to the high supply of saline water from tidal propagation, (3) disrupted waste disposal from the ponds that eventually threatens their quality. Figure 2 compares the total area of land uses with the total area of land uses by tidal propagation and inundation on the coast of Kendal Regency, i.e., north of the North Coast Road on Java Island. Table 1 and Figure 2 show that the built-up area, plantation, dry agricultural land, grassland, and paddy field are affected. Here, the built-up areas are not limited to settlements and public facilities but also buildings in general. The impact of tidal propagation and inundation on structures includes less optimal drainage system due to inundation-which decelerates the movement of wastewater, and subsequently, decreases environmental quality. Another impact is the environmental degradation of the built-up area-which damages properties, furniture, and other appliances [4,6] and corrodes iron-based articles [19,20,21]. As for the cultivation area, the impact includes wilting and any disruptions to the growth stage that lead to plant death. Almost all terrestrial plants are intolerant of the high salinity in seawater [18]. The spatial distribution of tidal propagation and inundation in the Kendal Regency has reached a dangerous level. The model revealed that tides propagated 3.7 km inland and even put the Kendal Industrial Park, a local and national asset, at risk. This threat is shown in Figure 3. As a result, the management board of this estate needs to pile up a mass of land periodically to minimize the impact of tidal propagation and inundation. The local government can use the tidal propagation and inundation modeled in this study as consideration for regional management. With a specific reference to tidal propagation and inundation events, the planned management can reduce the adversity that these disasters may cause. Conclusion The tidal propagation and inundation in Kendal Regency affected six land uses. In the north of the North Coast Road, the most significant percentage of the damaged areas (41.95%) was fish ponds and then followed by grassland (5.09%), dry agricultural land (4.89%), paddy field (4.24%), plantation (2.01%) and built-up area (0.04%). Records showed that the impact of these disasters was as far as 3.7 km inland. As such, a further study of regional management that takes tidal propagation and inundation into account becomes necessary, notably because these disasters do not cause a minor impact. Acknowledgement This research was developed from a thesis by the first author entitled "The Process and Impact of Coastal Dynamics in Central Java and the Special Region of Yogyakarta" as grant of Pendidikan Magister menuju Doktor untuk Siswa Unggul or Magister to Doctoral Education for Excellent Student from Directorate General of Resources for Science Technology and Higher Education of the Republic of Indonesia in contract number 2016/UN1/DITLIT/DIT-LIT/LT/2018. This research was also partially funded by the Faculty Grants of Faculty of Social Science, Universitas Negeri Semarang. The authors would like to express their gratitude to all colleagues and lecturers that had contributed to develop this research.	2021-06-02T23:44:02.771Z	2021-01-01T00:00:00.000	{ "year": 2021, "sha1": "5a0aca86d290d2974fb15bea0d06b0a228aa164e", "oa_license": null, "oa_url": "https://doi.org/10.1088/1755-1315/683/1/012062", "oa_status": "GOLD", "pdf_src": "IOP", "pdf_hash": "5a0aca86d290d2974fb15bea0d06b0a228aa164e", "s2fieldsofstudy": [ "Environmental Science", "Engineering" ], "extfieldsofstudy": [ "Physics" ] }
222842289	pes2o/s2orc	v3-fos-license	A task-sharing intervention for prepartum common mental disorders: Feasibility, acceptability and responses in a South African sample Background Peripartum common mental disorders (CMD) are highly prevalent in low- and middle-income countries (LMIC) such as South Africa. With limited public mental health resources, task sharing approaches to treatment are showing promise. However, little is known about the feasibility and acceptability of, as well as responses associated with problem-solving therapy (PST) for the treatment of prepartum CMD symptoms in South African public health settings. Aim To investigate participants’ preliminary responses to a task sharing PST intervention, and to evaluate the feasibility and acceptability of the intervention. Setting A Midwife and Obstetrics Unit attached to a Community Health Centre in a Western Cape district. Methods Using mixed methods, 38 participants’ responses to a PST intervention, and their perceptions of its feasibility and acceptability, were explored. Primary outcomes included psychological distress (Self Reporting Questionnaire; SRQ-20) and depression symptoms (Edinborough Postnatal Depression Scale; EPDS). Semi-structured interviews were conducted three after the last session. Six stakeholders were also interviewed. Results Significant reductions were seen on EPDS (Cohen’s d = 0.61; Hedges g = 0.60) and SRQ-20 (Cohen’s d = 0.68; Hedges g = 0.67) scores. The intervention’s acceptability lay in the opportunity for confidential disclosure of problems; and in relieving staff of the burden of managing of patients’ distress. Barriers included lack of transport and work commitments. Conclusion Results support task sharing PST to Registered Counsellors to treat antenatal CMDs in perinatal primary health care settings. Research is needed on how such programmes might be integrated into public health settings, incorporating other non-specialists. Introduction Perinatal common mental disorders (CMDs), such as depression and anxiety, are highly prevalent in low-and middle-income countries (LMICs) 1 and are associated with a range of adverse outcomes for mothers and infants. 2,3 Yet, in LMICs, up to 90% of people who could benefit from mental health treatment do not receive care. 4 In South Africa, three out of four people with CMDs do not receive treatment. 5 As a means to address this treatment gap, task-sharing mental health interventions to non-specialist health workers (NSHW) has garnered increasing attention. 6 By extension, there is a growing body of evidence showing that task-sharing interventions to treat perinatal CMDs are feasible to deliver, acceptable and also effective. 7 Several systematic reviews have investigated the effectiveness of task-sharing mental health interventions in LMICs. 8,9 One systematic review of 13 studies found that task-sharing interventions improved maternal mental health, which had a positive impact on infant development and health. 7 Correspondingly, improving mothers' ability to respond to their infants' needs also improved maternal mood. 7 Similarly, another review pooled data from 10 trials of psychosocial interventions delivered by NSHW in community settings and antenatal Background: Peripartum common mental disorders (CMD) are highly prevalent in low-and middle-income countries (LMIC) such as South Africa. With limited public mental health resources, task sharing approaches to treatment are showing promise. However, little is known about the feasibility and acceptability of, as well as responses associated with problem-solving therapy (PST) for the treatment of prepartum CMD symptoms in South African public health settings. Aim: To investigate participants' preliminary responses to a task sharing PST intervention, and to evaluate the feasibility and acceptability of the intervention. units aimed at reducing perinatal CMDs. 10 They found that, compared to usual care, interventions led to an overall reduction in CMD symptoms when using continuous data for symptomology. 11 As one of the World Health Organization's (WHO) Mental health Gap Action Programme (mhGAP)-recommended treatments, 12 problem-solving therapy (PST) has found significant support as an easily adaptable, user-friendly and task-sharable psychotherapy. 13,14,15,16 Evidence suggests that it is an effective treatment for several CMDs, including mood, 17 anxiety, 18 psychological distress 19 and substance use disorders 20 in a broad range of sociocultural settings. 21 In LMICs, the evidence for PST is growing. In Zimbabwe, Chibanda et al. 13 found that three to six sessions of PST delivered by lay workers significantly reduced CMD symptoms in a sample of 320 adults. Another study showed that levels of psychological distress were significantly lowered using a PST intervention in a South African sample of 103 participants. 19 Also in South Africa, a trial of blended motivation interviewing and PST amongst 335 patients attending emergency care reported significant reductions in substance use and depression 3 months post-enrolment. 22 Notably, Chibanda et al. 23 found that, at 6 weeks post-intervention, depression scores of a group of women receiving a PST intervention were significantly lower than those who received antidepressant medication. Given the comparatively recent recognition of the burden associated with maternal mental illness in LMICs, 1 there are extensive gaps in our knowledge. Firstly, limited research has been conducted on the efficacy of task-sharing evidencebased interventions that are integrated into antenatal primary healthcare. Whilst there is evidence to support the use of PST to treat depression, 14 the evidence for its application to peripartum CMDs is limited internationally and absent in South Africa. Secondly, little is known about the feasibility and acceptability of mental health interventions that are integrated into antenatal healthcare services, for both participants and stakeholders. As such, this article aims to describe (1) women's preliminary responses to the PST intervention, (2) to explore women's perceptions of the intervention's feasibility and acceptability, and (3) to explore healthcare providers' perceptions of barriers to and facilitators of integrating a PST intervention into midwife and obstetrics unit (MOU) services. Setting Data were collected at a MOU that serves a large district in the Western Cape province of South Africa, with a primarily low-income population of more than 300 000 people. 24 Midwife and obstetrics units fall under the governance of the Western Cape's Department of Health and provide a range of perinatal services at primary care level, including antenatal check-ups, deliveries by midwives and postnatal care for mothers and infants. They are usually attached to a primary healthcare in areas that were classified as 'black African' or 'coloured' i under the apartheid regime, serving previously disadvantaged communities. Eighty-four per cent of the South African population are dependent on government-funded health services. 25 Design and procedures We employed a mixed-methods design comprising two phases. In phase 1, quantitative data were collected to measure participants' preliminary responses to the intervention, whilst qualitative data were collected to explore women's perceptions of its feasibility and acceptability. This phase addressed the study's first two aims. In phase 2, qualitative methods were again used to collect data concerning the MOU personnel's (stakeholders) perceptions of the barriers to and facilitators of intervention delivery. This phase addressed the study's third aim. Given the small sample sizes in both phases, qualitative methods for examining feasibility and acceptability were deemed most appropriate. Phase 1: Participant responses to and perceptions of feasibility and acceptability Participants: Over a period of 1 year, a purposive sampling was used to recruit 38 pregnant women to participate in the study (see Figure 1). To be eligible to participate in the study, women had to be pregnant, at least 18 years old, must have registered for care at the facility and must have scored 15 or more on the Edenborough Postnatal Depression Scale (EPDS) during the standard intake interview conducted by the intake nurse. All eligible women were given a referral to the registered counsellor. Those who accepted the referral were asked to provide written informed consent to participate in the study. Procedure: Following the recruitment all participants completed a baseline assessment. Immediately following the assessment, they received the first of three PST sessions. The second and third sessions were scheduled about a week apart from each other. Patients deemed at risk for suicide, or with signs and symptoms of other serious pathology, were referred for specialist care. Three months after their last PST session, participants were asked to return to the MOU where the baseline questionnaire was re-administered by two research assistants. Short qualitative interviews explored the acceptability and perceived benefits of the intervention and asked for suggestions regarding the content and procedural improvements. All interviews were audio-recorded and transcribed. At the baseline and follow-up assessments, the participants were given a grocery store voucher as a token of thanks for their time. i. The authors recognise the deeply and historically problematic nature of this; however, the capacity to monitor developments in health and socio-economic disparities, which originated from such a classification system, is made possible by the continued use of these markers in South Africa Intervention The PST intervention used in this study was adapted from Sorsdahl et al. 20 Focus groups were conducted with 12 women who met the inclusion criteria to gather feedback on the intervention and how it should be adapted for pregnant women. Adaptations were primarily target population-related and included the addition of information about the experience of pregnancy. An outline of the session content and procedures is presented in Table 1. All sessions incorporated worksheets intended for use during the session, as well as homework assignments. Intake nurses were trained to use the EPDS, incorporate it into standard assessment procedures and make referrals to the registered counsellor. In addition to her formal Bachelor of Psychology degree training, the registered counsellor also received a 3-day training course in maternal mental health and 18 h of training in the PST model and manual. She also received at least 1 h of clinical supervision per week from the first author, a registered clinical psychologist. Procedural matters, case management and fidelity to the therapeutic protocol were addressed in supervision. A random review of recorded sessions did not reveal any protocol drift. Measures The primary outcome of this study was psychological distress. The secondary outcomes included perinatal depression, functional impairment, substance use involvement, perceived stress and perceived social support. Psychological distress: The Self-Reporting Questionnaire (SRQ-20) 26 is a 20-item screening tool designed to screen for symptoms associated with a range of CMDs. A cut-off value of ≥ 8 was used to determine caseness, producing the binary categories of 'high' (≥ 8) and 'low' (≤ 7). 27 The SRQ-20 has satisfactory sensitivity and specificity. 28 Symptoms associated with perinatal CMDs: The EPDS 29 is a 10-item scale that screens for symptoms of perinatal CMDs in the last 7 days. It is one of the most validated tools in LMICs. 30 A cut-off value of ≥ 15 was used to determine caseness, yielding binary categories of 'high' (≥ 15) and 'low' (≤ 14). 31 Functional impairment: The Sheehan Disability Scale (SDS) 32 was used to assess functional impairment in three inter-related domains: work/school, social and family life. On a scale of 0 (being 'not at all') to 10 ( being 'extremely'), participants were asked to rate the degree to which symptoms had disrupted their lives in these domains. Substance use involvement: The Alcohol, Smoking and Substance Involvement Screening Test (ASSIST) 33 was used to investigate self-reported substance use. Substance involvement scores are generated for each substance used in the 3 months prior to the interview. Scores of ≤ 3 (10 for alcohol) indicate a low risk for substance-related health problems, whilst scores between 4 and 26 (11-26 for alcohol) reflect a moderate risk and that ≥ 27 reflect a severe risk. 33 Perceived stress: The Perceived Stress Scale (PSS) 34 asks participants to respond on a scale of 0 ('never') to 4 ('very often') to a series of 10 questions examining the levels of perceived stress. Perceived support: The Multidimensional Scale of Perceived Social Support (MSPSS) 35 is a 12-item scale that asks participants about their current perceptions and experiences of being supported or assisted by family members, significant others and friends. The participant would be reminded of the PST model and a list of adaptive coping strategies would be discussed. A problem from the 'not important' category would then be selected and the ways in which coping strategies might be applied to these would be discussed. In addition, a problem from the third group would be selected and together the registered counsellor and the participant would develop a step-by-step plan to solve the problem. Again, the participant would be reminded of the model and then more coping strategies would be discussed. Thereafter, a problem from the 'important but unsolvable' category would be selected from the list made in the first session, and ways of coping with this problem would then be discussed. Again, a problem from the third group would also be selected and together registered counsellor and the participant would develop a step-by-step plan to solve the problem. Phase 2: Stakeholder perceptions of the intervention's feasibility and acceptability Participants: A purposive sampling was used to recruit participants, as all stakeholders who were identified as being the most directly involved in or impacted upon by the project were invited and agreed to participate. They comprised three staff members who were most involved in the screening of participants at their first antenatal visits, the primary liaison person and acting head of the MOU, the Community Health Centre's social worker and the registered counsellor who delivered the intervention (see Table 2). Procedure: Face-to-face interviews were arranged with stakeholders, who provided informed consent to participate under conditions of anonymity and voluntary participation. As the registered counsellor was under the clinical supervision of the first author at the time, she was interviewed by a clinical psychologist independent of the study. This was done to avoid any conflict of interest, as well as to minimise interviewer and response bias. A semi-structured interview schedule was used to guide the interviews with stakeholders. It included questions regarding stakeholders' perceptions of the intervention's utility to the service, as well as the impact of intervention on their day-to-day duties. All interviews were audiorecorded and transcribed. Once transcribed, the audio recordings were destroyed and all identifying information was removed from the transcribed material, which was kept on a password-protected computer. Data analysis for phases 1 and 2 Quantitative data were analysed using SPSS version 23.0. Participants' socio-demographic data were analysed with descriptive statistics. We used both paired t-test and the Wilcoxon's signed-rank test to assess the initial effect of the intervention on the primary and secondary outcome variables. The last observation carried forward method was used to impute missing data. We reported effect sizes using Cohen's d and Hedges' g for small samples. Qualitative data were analysed in NVivo 11 using the framework method. 35 This approach to thematic analysis involves a series of stages that include familiarisation with the material, coding the transcripts, developing an analytical framework, applying the analytical framework by indexing, charting data into the framework matrix and interpreting the data. Ethical consideration Ethical approval to conduct the study was obtained from the Faculty of Health Sciences Research Ethics Committee (HREC) at the University of Cape Town. Permission to collect data at the midwife and obstetrics unit was also obtained from the Western Cape Department of Health as well as the facility management. Written informed consent was obtained from all the participants. In order to protect the identities of the stakeholders, identification codes were omitted from quotes included in this article. Women's preliminary responses to the problemsolving therapy intervention Of the 38 women who participated in the study (see Table 3 for a description of socio-demographic characteristics of the participants), 15 Figure 1). Sixteen participants were lost to follow-up because of withdrawal from the study, relocation to another area, a change in contact number or scheduling conflicts. Of those who participated in the follow-up interview, 10 (45.5%) completed all three sessions. Addressing the study's first aim, which sought to investigate women's preliminary responses to the intervention, several significant gains were seen on both primary and secondary outcome measures, as reflected in Table 4 Feasibility and acceptability of the intervention ii Data from interviews with women who participated in the intervention (participants) and staff members involved in the delivery of the project (stakeholders) highlighted several emergent themes, addressing the study's second and third aims regarding the intervention's feasibility and acceptability. Perceptions of the intervention's acceptability and usefulness Most of the participants felt that they derived some benefit from the intervention, with nearly all participants reporting that they would recommend such a programme to friends. The opportunity to hear another perspective, to talk about ii.The term 'participants' is used to denote women who received the PST intervention, whilst the term 'stakeholders' represents staff who were involved in the delivery of the intervention. past experiences or to have time for themselves was critical. The opportunity to confide in a non-judgmental person, who was not previously known, was deemed particularly helpful: 'I found out I was pregnant. I didn't want the baby, all of that and -it was so painful for me but -really after talking to her itjust to speak to someone that's not family, man, someone you don't know, really helped, it's almost like it's just a burden off your shoulder. Since the first session we had, I could see a light again and I could actually feel that this is my baby …' (Participant #26, aged 28) From these descriptions, PST-specific factors of the intervention may be less important to some participants than having the registered counsellor's impartial and empathic ear. Other participants valued the problem-solving approach itself. Some participants expressed appreciation for its pragmatism, whilst others referred directly to the problem-solving aspects of the intervention as useful, such as the development of better coping mechanisms: Reports on improved self-efficacy that in turn led to more positive feelings about themselves, such as 'being stronger' and feeling like 'a better person', were described. For some participants, these benefits were linked to improved relationships, whilst others found that they were more able to seek out social experiences than before: A few participants spoke about using what they had learnt from the intervention to help others, going so far as to make copies of the booklet for friends. One participant even arranged to meet with her friends at the time that her weekly appointment with the registered counsellor had been due, to teach them the PST techniques. On the other hand, two participants reported that they did not find the intervention helpful at all (one of them still attended all three sessions). Whilst both seemed to suggest that their objections were related to the registered counsellor's style, it is possible that the PST model was a poor fit for them, as evident from the following comment: 'there wasn't really space for me to talk, we were just reading out of the booklet'. Despite one participant's appreciation for the ways in which the model helped her better manage distressing thoughts, she also made an important observation about the limits of counselling interventions for someone who lives in poverty. In this instance, this participant highlighted the tension between the need to think about how to find money and the anxiety and stress that these thoughts generated for her: From the stakeholders' perspective, most seemed to feel that the programme lightened their own workloads, as it gave them a resource to refer distressed patients to, instead of having to manage the patients themselves. Several stakeholders described how interactions with distressed patients could be burdensome and stressful for the staff, in that containing the patient took time and energy from their own limited resources, as highlighted in the following extract: 'And I'm just asking [the patient] "so why are you smoking such a lot!" … Noooo, but then I end up having to hear about her being abused as a child and her husband is hitting her. And one question led to all of that … Sometimes you just don't ask … Sometimes you just say, "I just want to get through the day, I'm not going to ask".' (Stakeholder #1) All the stakeholders talked about the ways in which having a counsellor at the MOU relieved some of the demands that distressed patients represented. This seemed to be the most significant and meaningful contribution that the programme made to staff: Perceived barriers and challenges Participants who had missed appointments with the registered counsellor or had prematurely terminated their participation provided several reasons for doing so. Although a few participants highlighted how stigma associated with attending counselling sessions at the MOU might be a barrier, such as 'people will think something is wrong with you, structural barriers, mainly related to financial constraints and lack of transport, were the most commonly cited. Childcare and work commitments also presented obstacles to attending sessions: 'Okay all her available times, then I was busy -it was either work or I had to be by my child's school.' (Participant #24, aged 23) The stakeholders highlighted several challenges associated with the programme, many of which appeared to be related to an overburdened system. As referrals increased, the registered counsellor had less time to immediately see all patients referred to her and in some instances she would need to arrange an appointment on another day instead. When asked about what she thought was problematic about the programme, one stakeholder had this to say: 'The amount of patients that was referred … Because I don't think [the Registered Counsellor] could keep up with all the patients. And then she used to say she can't see somebody now, she must get a date or whatever, then the staff just stopped referring.' (Stakeholder #1) The role that the programme played in relieving the staff members seemed to be echoed in this way and perhaps highlighted a sense of inadequacy or anxiety about having the capacity to manage a distressed patient. One stakeholder alluded to this: Every stakeholder made reference to the overburdened and understaffed state of the system and the consequent demands placed on staff members. Interestingly, whilst the value of the programme for the staff appeared to be in the relief it offered from the demands of distressed patients, feeling overburdened may have, in and of itself, represented a barrier to the acceptability of the programme. In this vein, two stakeholders expressed frustration at their colleagues for their unwillingness to participate in projects in general: 'I think people work in little squares and they're just concerned about what happens in their little square.' For one of the stakeholders, the reticence to participate was observed in colleagues, whilst frustration was understandable and explained by staff being overburdened and overworked: 'I know, it also has to do with the lack of staff and the amount of clients that need to be seen. So I also understand that perhaps people feel overwhelmed and so [speaking as if an affected staff member] "I don't really want to become too interested in something else because even if I want to do that I must still see to the fifty that's waiting for me".' (Stakeholder #2) http://www.phcfm.org Open Access The registered counsellor also reported that the intake nurses felt that the screening and referral process was burdensome: 'Those that were involved -let's just say the intake nurses found it to be a "las" (burden) -it's extra work for them, [as if quoting the staff] "I've got to refer, I've now got to give you updates on my numbers, now you want to give them a sticker" -you know if we didn't have stickers they had to fill in the forms for me by hand, so needed dates of birth, surnames, contact details.' (Stakeholder #3) The lack of space at the MOU was another obstacle to the acceptability of the programme, as stated by one stakeholder: 'The only thing here is the space thing you understand?' However, one stakeholder felt that the lack of space was sometimes used as a reason to prevent new programmes from being adopted, as new programmes often represent additional work. In this way, physical space may well have represented staff members' capacity -in terms of time and energy -to accommodate the additional duties that programmes often bring with them: 'It's difficult because they agree to a lot of things and then when it needs to happen, there's no space available, people don't want to share their space.' (Stakeholder #2) Recommendations for improvement of the intervention Given that a majority of participants stated that the intervention was acceptable the way that it was, few recommendations for improvements were made. Of those who provided recommendations, many stated that group sessions would be beneficial in providing support and that the intervention should be made available at other locations. This recommendation was to address the practical difficulties in accessing the clinic, or to protect participants from the stigma associated with receiving mental health services: 'Ja [yes] house-visiting and stuff like that, that will help, for me because I am staying very far from the clinic. Maybe have it at other clinics.' (Participant #15, aged 38) Several participants stated that the number of sessions was inadequate and that more or longer sessions would improve the intervention: 'Longer sessions because you just deal with this and now you get to, not a breaking point but you know, you get to a point where you think, where you feel there's still a lot for you to resolve but the sessions is too short.' (Participant #20, aged 31) Despite some ambivalence, all stakeholders stated that having a counsellor at the MOU was essential. Stakeholders stated that they needed someone who would attend specifically to patients' mental health, worrying that in the meantime, as the programme has terminated, staff might not detect problems: ' Two stakeholders stated that stigma associated with mental illness needed to be addressed in order to improve the programme. Both felt that being seen by other patients to use the service made the women feel self-conscious and therefore less inclined to take up counselling: 'Maybe they don't want to be seen -I think -when people don't want to be seen -maybe they thought "this one knows me and they know I'm coming for this and I'm coming for that" -see them in a certain time or maybe give them appointments to come.' (Stakeholder #4) Despite the concern about space, one solution offered by several stakeholders to ensure that all patients are seen on the day was to have more counsellors available so that a walk-in service could be made possible. However, one stakeholder stated that a walk-in service would mean that counselling is treated as a 'crisis service'. She opined that this would send the wrong message to women about taking care of their mental health: 'I don't know how effective that is also um -because it also creates the wrong perception with the client in terms of intervention and what can be done -and again in my opinion I think it would be better to say to people you know, mental health is a thing that you should pay attention to continuously and not only when you are in crisis or when there's a problem.' (Stakeholder #2) Discussion This is the first study in South Africa to investigate the feasibility, acceptability and preliminary responses to an adapted PST intervention for psychologically distressed pregnant women. Quantitative data provide initial support for the potential benefits of the intervention for reducing symptoms of psychological distress and improving functioning. In line with findings from other studies, 13,19,23 this study's results showed reduced symptoms of CMDs and psychological distress. Improved functioning was seen on the work, social and family/home dimensions of the SDS. Qualitative data supported the feasibility and acceptability of the intervention, with participants reporting reductions in distress and improvements in social functioning, whilst stakeholders were generally positive, reporting some relief from having to manage patients' psychological distress. Retention rates of almost 40% for the full intervention, whilst not high, appear to be in line with prepartum mental health interventions conducted in other settings. 8,36 One systematic review found that low-income women are more likely to discontinue therapeutic treatments prematurely, mainly for financial reasons. 37 Indeed, a lack http://www.phcfm.org Open Access of transport or money and work commitments were reported as barriers by the participants of this study, as has been found in other studies. 37,38,39 The implications for policy then are that simply increasing the number of available services or human resources for mental health is not adequate. Addressing these barriers might include developing after-hours services, providing transport coupons or delivering interventions at patients' homes, when appropriate and possible. As many participants could not be reached for follow-up interviews, it is difficult to know the full range of barriers to care that they experienced. Given that these participants are known to have been distressed, it is also plausible that unresolved symptoms served as barriers to care. Furthermore, some participants suggested that stigma associated with seeking mental healthcare may have represented a barrier to some women. This is a widely recognised barrier and has been noted in several studies. 40,41,42 Programmes aimed specifically at reducing stigma and increasing awareness amongst peripartum women may be important contributions to maximise the success of future interventions. For participants, the intervention's acceptability seemed to lie primarily in the opportunity to talk confidentially to a non-judgemental and empathic person about their problems. Whilst the PST model seems to have had an influence on many participants' thinking, this appears to have been a secondary benefit for some. This is consistent with evidence from studies to show that task-sharing PST interventions are generally acceptable and feasible to intervention participants in primary care. 22 However, these findings seem to point to the primary importance of a trusting relationship with an empathic counsellor. Given that task-sharing studies often prioritise intervention models over counsellor skills and qualities, this may have significant implications for future research as well as for practice. For stakeholders, the programme was generally perceived as expanding and improving the quality of services provided by the facility. Having a professional resource to refer to seemed to relieve them of the pressures of managing distressed patients during the course of routine care. Mental health problems appeared to add to the burden of care experienced by MOU staff who reported not having the time, capacity or skills to manage psychologically distressed patients. In this respect, the intervention was widely deemed to be acceptable by stakeholders. To our knowledge, other studies have not found this. However, the overburdened state of primary healthcare systems might in and of itself represent a barrier to the successful integration of programmes that rely on staff members' participation. Similar South African studies have shown that stakeholders experience the inclusion of new interventions into usual care as generating additional burden, and that staff buy-in is central to the success of programmes. 20,43 These findings have important implications for practice and policy. Developing interventions that staff members experience is helpful to their work and not burdensome is likely to be essential to the sustainability of mental health programmes that are integrated into primary care. There are some limitations of this study. The main limitations are the small sample size and the absence of a control group, restricting our ability to comment on the effect of the intervention. Furthermore, it is possible that participants might have experienced spontaneous remission of symptoms and the study's positive outcomes simply reflect that. However, these findings suggest that a scaledup randomised controlled trial of a task-sharing PST intervention to reduce psychological distress amongst pregnant women might have positive outcomes. In addition, both women who participated in the intervention and stakeholders involved in its delivery generally found value in the programme. Conclusion Despite the study's limitations, in combination with the qualitative data, the outcome data from this study support the feasibility and acceptability of this task shifting brief intervention as well as its potential to effect positive outcomes in the treatment of prepartum psychological distress and CMD symptoms. Perhaps most significantly, the results of this study suggest that integrating mental healthcare interventions into primary care services may improve the mental health of services users, in addition to reducing the burden that patient's psychological distress may represent for healthcare providers. Data availability statement Data for the study will be made available upon written request, with the permission of all authors. Disclaimer The views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of any affiliated agency of the authors.	2020-10-17T13:06:21.995Z	2020-10-01T00:00:00.000	{ "year": 2020, "sha1": "20078844d94efe70161323f96d442484fec913bb", "oa_license": "CCBY", "oa_url": "https://espace.curtin.edu.au/bitstream/20.500.11937/85686/2/85508.pdf", "oa_status": "GREEN", "pdf_src": "PubMedCentral", "pdf_hash": "03afecc902f3a13d4486fb0ff3421b5d98db3375", "s2fieldsofstudy": [ "Medicine", "Psychology" ], "extfieldsofstudy": [ "Medicine" ] }
44580836	pes2o/s2orc	v3-fos-license	Profiling Protein Kinases and Other ATP Binding Proteins in Arabidopsis Using Acyl-ATP Probes* Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)1 probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding proteins. Our search for labeled peptides upon in-gel digest led to the discovery that the biotin moiety of the labeled peptides is oxidized. The in-gel analysis displayed kinase domains of two receptor-like kinases (RLKs) at a lower than expected molecular weight, indicating that these RLKs lost the extracellular domain, possibly as a result of receptor shedding. Analysis of modified peptides using a gel-free platform identified 242 different labeling sites for AcATP in the Arabidopsis proteome. Examination of each individual labeling site revealed a preference of labeling in ATP binding pockets for a broad diversity of ATP binding proteins. Of these, 24 labeled peptides were from a diverse range of protein kinases, including RLKs, mitogen-activated protein kinases, and calcium-dependent kinases. A significant portion of the labeling sites could not be assigned to known nucleotide binding sites. However, the fact that labeling could be competed with ATP indicates that these labeling sites might represent previously uncharacterized nucleotide binding sites. A plot of spectral counts against expression levels illustrates the high specificity of AcATP probes for protein kinases and known ATP binding proteins. This work introduces profiling of ATP binding activities of a large diversity of proteins in plant proteomes. The data have been deposited in ProteomeXchange with the identifier PXD000188. ATP binding and hydrolysis are the driving processes in all living organisms. Hundreds of cellular proteins are able to bind and hydrolyze ATP to unfold proteins, transport molecules over membranes, or phosphorylate small molecules or proteins. Proteins with very different structures are able to bind ATP. A large and important class of ATP binding proteins is that of the kinases, which transfer the gamma phosphate from ATP to substrates. Kinases, and particularly protein kinases, play pivotal roles in signaling and protein regulation. The genome of the model plant Arabidopsis thaliana encodes for over 1099 protein kinases and hundreds of other ATP binding proteins (1,2). Protein kinases are involved in nearly all signaling cascades and regulate processes ranging from cell cycle to flowering and from immunity to germination. Many protein kinases in plants are receptor-like kinases (RLKs), often carrying extracellular leucine-rich repeats (LRRs). The RLK class contains at least 610 members (3), including famous examples such as receptors involved in development (e.g. BRI1, ER, CLV1) and immunity (e.g. FLS2, EFR). Other important classes are mitogen-activated protein (MAP) kinases (MPKs) (20 different members), MPK kinase kinase kinases (MAP3Ks) (60 different members (4)), and calcium-dependent protein kinases (CPKs) (34 different members (5)). Because of their diverse and important roles, protein kinases have been intensively studied in plant science. The current approach is to study protein kinases individually-a daunting task, considering the remaining hundreds of uncharacterized protein kinases. New approaches are necessary in order to study protein kinases and other ATP binding proteins globally rather than individually. ATP binding activities of protein kinases and other proteins can be detected globally by acyl-ATP (AcATP) probes (6, 7) (Fig. 1A). AcATP binds to the ATP pocket of ATP binding proteins and places the acyl group in close proximity to conserved lysine residues in the ATP binding pocket. The acyl phosphonate moiety serves as an electrophilic warhead that can be nucleophilically attacked by the amino group of the lysine, resulting in a covalent attachment of the acyl reporter of the AcATP probe on the lysine and a concomitant release of ATP. The reporter tag is usually a biotin to capture and identify the labeled proteins. Labeled proteins can be displayed on protein blots using streptavidin-HRP. However, because AcATP labels many ATP binding proteins and protein kinases are of relatively low abundance, mass spectrometry is more often used to identify and quantify labeling with AcATP probes. The analysis is preferably done using Xsite, a procedure that involves trypsination of the entire labeled proteome, followed by analysis of the biotinylated peptides rather than the biotinylated proteins (8). This "KiNativ " approach provides enough depth and resolving power to monitor ϳ160 protein kinases in a crude mammalian proteome (7). Of the 518 human protein kinases (9), 394 (76%) have been detected via AcATP labeling (6). KiNativ has mostly been used to validate targets of human drugs that target protein kinases using competitive labeling experiments. This approach has been used to identify selective inhibitors of, for example, Parkinson's disease protein kinase LRRK2 (10), the BMK1 and JNK MAP kinases (11,12), and the mTOR kinase (13). Importantly, the correlation of the biological activity of protein-kinase-inhibiting drugs with inhibitor affinity detected using KiNativ is better than that achieved when affinities are determined by assays using heterologously expressed protein kinases (7). This improved correlation illustrates that assays in the native environment provide a more realistic measure of protein kinase function. In addition to characterizing inhibitors selectively, AcATP probes can also display differential ATP binding activities of protein kinases. For example, labeling with AcATP probes during infection with dengue virus displayed a 2-to 8-fold activation of a DNA-dependent protein kinase (14) Similarly, AcATP labeling revealed an unexpected Raf kinase activation in extracts upon protein kinase inhibitor treatment (7). In conclusion, profiling with AcATP probes is a powerful approach for monitoring protein kinases and offers unprecedented opportunities to identify selective protein kinase inhibitors and discover protein kinases with differential ATP binding activities. In this work, we introduce AcATP profiling of plant proteomes. In addition to the analysis of labeled peptides, we characterized labeling using gel-based approaches and discovered that biotin is often oxidized in this procedure. We also performed an in-depth analysis of labeling sites in proteins other than protein kinases, which had not been done before. We discuss labeling outside known nucleotide binding pock-ets and investigate the correlation of labeling sites with protein abundance. We describe 63 labeling sites of known nucleotide binding pockets, of which 24 represent a remarkable diversity of protein kinases, including several LRR-RLKs. This work launches a new approach to study ATP binding proteins in plant science. EXPERIMENTAL PROCEDURES Plant Growth Conditions-Arabidopsis thaliana ecotype Col-0 was grown in a climate cabinet in a 10-h light regime at 22°C and 60% relative humidity. Probes-Biotin-AcATP was synthesized following the procedures described by Patricelli et al. (6). Briefly, N-(ϩ)-biotinyl-6-aminohexanoic acid (30 mg, 0.085 mmol) was suspended under an argon atmosphere in anhydrous dioxane/N,N-dimethylformamide/dimethyl sulfoxide (1:1:1, 3 ml) and cooled to 0°C. To this ice-cold solution were then added triethylamine (47 l, 0.34 mmol) and iso-butyl chloroformate (33 l, 0.255 mmol), resulting in the appearance of a precipitate. The resulting suspension was stirred at 0°C for 20 min, allowed to warm up to room temperature, and then stirred for an additional 1.5 h. ATP triethylammonium salt (69 mg, 0.085 mmol) dissolved in anhydrous dimethyl sulfoxide (1 ml) was added to the reaction mixture. After 18 h, the reaction was stopped by the addition of water (4 ml), and the mixture was extracted with ethyl acetate (3 ϫ 4 ml). The aqueous layer was frozen and subsequently lyophilized. The resulting solid was suspended in water (1 ml), transferred to a reverse-phase C 18 flash-column (LiChroprep ® RP-18, 40 -63 m, Merck) pre-equilibrated with 5% acetonitrile in water and eluted with 30% acetonitrile in water. Product-containing fractions were pooled, frozen, lyophilized, and subsequently stored at Ϫ20°C (yield: 4.3 mg, 5.1 mol, 6%). The identity of the product was confirmed via liquid chromatography electrospray ionization mass spectrometry analysis on a Thermo Scientific LCQ FleetTM electrospray ionization spectrometer equipped with an Eclipse XDB-C18 5-m column from Agilent, Santa Clara, CA and using a linear gradient of solvent B (5 mM NH 4 OAc in acetonitrile) in solvent A (5 mM NH 4 Labeling with BHAcATP-Arabidopsis proteome was extracted from 4-week-old Arabidopsis rosette in 50 mM Tris pH 7.5. The lysate was cleared via centrifugation and subjected to gel filtration using DG10 columns (Bio-Rad). Labeling was performed in 50 mM Tris pH 7.5, 10 mM MgCl 2 , unless specified otherwise. The lysate was incubated with 5 or 10 M BHAcATP or dimethyl sulfoxide for the no-probe control at room temperature for 15 min. For inhibition experiments, the lysate was preincubated with the different nucleotide-based inhibitors at a final concentration of 10 mM for 30 min before labeling with BHAcATP for 15 min. The labeling reaction was stopped by the addition of gel loading buffer containing SDS and DTT. Proteins were separated on 12% acrylamide gel and transferred onto PVDF membranes. The protein blot was probed with streptavidin-HRP (Ultrasensitive, Sigma) and detected with enhanced chemiluminescence (SuperSignal West Femto Chemiluminescent Substrate, Thermo Scientific). Affinity Purification and Identification of Labeled Proteins-After labeling of Arabidopsis leaf extract (1 ml 5 mg/ml protein) for 1 h, the lysate was desalted using a DG10 column (Bio-Rad). The eluate was incubated with 100 l neutravidin beads (Thermo Scientific) in the presence of PBS and 0.2% SDS for 1 h. The beads were washed twice with 0.2% SDS, twice with 6 M urea, and twice with water and eluted with 50% formic acid. Proteins were separated on protein gels and stained with SYPRO Ruby (Invitrogen). Bands of interest were excised from both samples and subjected to in-gel digestion with trypsin. In-gel Digest and MS-LC-MS/MS analysis was performed on an LTQ Velos (Thermo) coupled to an EasyNanoLC (Proxeon/Thermo) using a "Top 20 " data-directed acquisition. LC was performed on a C18 column (10 cm ϫ 75 m) with a gradient of 5%-40% acetronitrile in 0.1% formic acid for 30 min, followed by a wash with 95% acetonitrile. The data-directed acquisition included active exclusion for mass (Ϯ40 ppm) and time (60 s). Spectra were searched using Mascot 2.3 using a fixed modification of cysteine (57.02 Da for carbamidomethyl) and variable modifications of methionine (15.99 Da for oxidation) and lysine (339. 16 and 355.16 Da for BHAc and OBHAc labeling, respectively). Mass tolerance was set at 0.3 Da for the precursor ions and 0.4 Da for fragment ions. Up to two missed cleavages for trypsin were permitted, as the labeling would prevent cleavage at the labeled lysine. The MS2 spectra were searched against TAIR10 pep 2012 (November 2012), containing 35,386 proteins of Arabidopsis thaliana, supplemented with a small database with 1095 artifact proteins and a reversed decoy database of the same proteins (total: 72,962 entries). Peptides were retained with Mascot scores of Ͼ41, which is above the Mascot significance level. Hits to the decoy database within this selection were given a 0.3% false discovery rate for all peptides and a 0.7% false discovery rate for non-redundant peptides. Proteins were selected having a minimum of two different peptides, as shown in supplemental Table S1. This selection for two peptides per protein removed all the false positive hits to the decoy database. Proteins were ranked on their score and manually selected considering the occurrence of the protein in the rest of the gel and its expected molecular weight and overall score and spectral count (supplemental Table S2). Only robustly identified proteins that were highly enriched in the gel slice relative to other gel slices are reported. Labeling with Desthiobiotin-acyl-ATP-Analysis of desthiobiotinacyl-ATP (DBAcATP)-labeled Arabidopsis leaf extracts was performed as described elsewhere (6,7). Briefly, lysates from Arabidopsis leaves were generated via bead-based agitation in kinase buffer (20 mM HEPES pH 7.8, 150 mM NaCl, 0.1% Triton X-100). The resulting lysates were gel filtered into 20 mM HEPES pH 7.8, 150 mM NaCl, 1% v/v phosphatase inhibitor mixture II (Calbiochem) to remove endogenous competing nucleotides prior to labeling with 20 M DBAcATP probe for 15 min in the presence of 20 mM MnCl 2 . The labeled extracts were denatured and reduced (6 M urea, 10 mM DTT, 65°C for 15 min) and then alkylated with iodoacetamide (40 mM, 30 min at 37°C). Following another gel filtration step (into 10 mM ammonium bicarbonate, 2 M urea) to remove unreacted reagents and lower the urea concentration, the samples were digested with trypsin. DBAcATP-labeled peptides were enriched on streptavidin resin (Thermo) and eluted using 0.1% TFA in 50% acetonitrile. MS analysis was performed on a Thermo-LTQ linear ion trap instrument in a data-dependent mode as described for the initial characterization (7) using a mass range from 500 -1800 m/z. The MS2 spectra were searched using SEQUEST against the TAIR9 pep 20090619 database (June 2009) containing 32,769 proteins of Arabidopsis thaliana. MS2 searches included fixed iodoacetamide modification of cysteine (57 Da for alkylation) and variable modifications of methionine (16 Da for oxidation) and lysine (196 Da for DBAcATP labeling). Up to three missed cleavages with trypsin were allowed, and non-tryptic or half-tryptic peptides were excluded. Mass tolerance was set at 3 Da for the precursor ions and 1 Da for fragment ions. This high mass tolerance for precursor ions had to be used because most of the precursor ions are detected with multiple charges, and the mass tolerance in SEQUEST is not charge state specific. A probability score was calculated for each labeled peptide as described previously (15). This score is based on Xcorr, delta-Cn, and peptide mass/length and compared with the distribution of false positive scores generated by searching with incorrect masses of the modification (ϩ10 and Ϫ10 Da from the correct value). The resulting MS2 spectra were assembled in one Excel file, and all spectra with Ͻ95% probability were removed. Peptides that were detected in only one of the four MS runs were discarded. The resulting list of 242 peptides included peptides that are unique (u) in the Arabidopsis proteome or that match multiple genes (ambiguous (a)). Some peptides match multiple gene models of a single gene (isoforms (i)). These isoforms are indicated in a supplemental table but were treated as identifiers for specific genes. Supplemental Table S3 contains the lists, with 10,465 spectral counts with individual scores with probabilities of Ͼ75%. Supplemental Table S4 summarizes the spectral counts of the 242 labeled peptides with Ͼ95% probability. Analysis of Labeling Sites-In order to map the labeling sites onto protein structures, we searched the Protein Data Bank database for sequences that were homologous to the labeled proteins. Sequences for which a co-crystal with a nucleotide was available were aligned with the identified proteins, and the orthologous position of the labeled lysine was indicated in the alignment. This residue was selected in the protein structure using PyMol, and the distance to the nearest phosphate of the bound nucleotide was measured. Comparison with Protein Levels in the AtProteome Database-For every labeled protein in supplemental Table S4, we retrieved the number of spectral counts from leaves from the AtProteome database (48). For ambigious peptides, only the protein with the highest number of spectral counts was selected. In Silico Analysis of Labeling Sites-To predict the labeled peptides for all the protein kinases in Arabidopsis, we retrieved protein sequences of all protein kinases from the Arabidopsis thaliana kinase database. Sequences for each subfamily in the AthKD database were retrieved and aligned using MultiAlign. The labeled peptide that was experimentally identified was highlighted in the alignment, the orthologous Lys residues were indicated, and Lys and Arg residues were highlighted in the alignment. Tryptic peptides containing the labeling site were selected from the alignments. The orthologous labeled Lys and the Arg/Lys-Pro sites were not considered as trypsin cleavage sites, and miscleavages were ignored. The selected peptides were ranked on sequence, and redundancy for each peptide sequence was counted (n proteins carrying the same labeled peptide). Ile/Leu residues were not discriminated in this analysis, as they have the same mass. Protein kinases with a unique labeled lysine (n ϭ 1) were counted. The other protein kinases contain ambiguous peptides (n Ͼ 1). This analysis was done for peptides carrying Lys1, Lys2, Lys3, and Lys3. RESULTS In this study, BHAcATP was used to label Arabidopsis leaf extracts. BHAcATP is composed of ATP, an acyl phosphate linker, and a biotin tag (Fig. 1A). Incubation of leaf proteomes with BHAcATP followed by the detection of protein blots with streptavidin-HRP revealed numerous biotinylated proteins when compared with the no-probe control (Fig. 1B). Signals at 55, 45, and 43 kDa were hallmark signals ( Fig. 1B; white, black, and gray arrowheads, respectively) later identified as ATPB/RBCL, PGK1, and a mixture of ATP binding proteins, respectively (see below). The endogenously biotinylated proteins 3-methylcrotonyl-CoA carboxylase (MCCA) and biotin carboxyl carrier protein (BCCP) appeared as background signals at 80 kDa and 34 kDa, respectively, in both the labeled samples and the no-probe control. However, both these proteins are also ATP binding proteins, and the MCCA signal is mixed with methionine synthase, an ATP binding protein (see below). Labeling Is Dependent on pH and Divalent Metal Ions-Different labeling conditions were tested in order to further characterize BHAcATP labeling. Labeling with BHAcATP depends on pH. Weak or no labeling occurs at acidic pH (pH 3-5), and strong labeling occurs at neutral to basic pH (pH 7-10; Fig. 2A). We chose pH 7.5 as the standard labeling condition to mimic the conditions of the plant cytoplasm. The addition of MgCl 2 or MnCl 2 greatly increased labeling, whereas adding the chelating agents EDTA and EGTA decreased labeling (Fig. 2B). Interestingly, additional 60-and 70-kDa signals appeared when MnCl 2 was added, and two 65-kDa signals appeared when MgCl 2 was added (Fig. 2B, solid circles). The MnCl 2 -induced signals were suppressed more effectively by the addition of EGTA than of EDTA, whereas the MgCl 2 -induced signals were suppressed equally by both EDTA and EGTA. These results are in agreement with the fact that EGTA has a higher affinity for Mn 2ϩ than EDTA. The increased labeling upon the addition of divalent metal cations and the corresponding decrease upon treatment with chelating agents are consistent with the role of divalent ions in ATP binding. Labeling Is Suppressed with ATP-like Nucleotides-We next tested whether labeling would be suppressed by nucle-otides. Pre-incubation with ATP or ADP strongly suppressed BHAcATP labeling, whereas AMP did not suppress labeling (Fig. 2C), suggesting that phosphates at the ␤ and ␥ positions are essential for the suppression of BHAcATP labeling. In addition to ATP, CTP and, to a lesser extent, GTP and TTP also suppress BHAcATP labeling (Fig. 2C). Notably, NADP, but not NAD, also suppresses BHAcATP labeling (Fig. 2C), indicating the importance of a phosphate moiety at the 2Ј position in the nucleotide to suppress labeling (supplemental Fig. S1). Interestingly, nucleotide triphosphates (NTPs) were more potent inhibitors than their deoxynucleotide counterparts (dNTPs; Fig. 2D), suggesting that the 2Ј hydroxyl group on the sugar moiety of the nucleotide also plays a role in determining the ability to compete for labeling (supplemental Fig. S1). In conclusion, these data indicate that labeling is specific because it can be competed with ATP analogues. BHAcATP Labels ATP Binding Proteins-To identify the proteins labeled by BHAcATP, the labeled proteins were affinity purified using streptavidin beads, separated on protein gel, and stained (Fig. 3A). Bands were excised, treated with trypsin, and analyzed via LC-MS/MS. Peptides with high Mascot scores (Ͼ41) and proteins with at least two unique peptides were retained. A total of 112 proteins were identified in the BHAcATP-labeled sample, but only 13 of these proteins were also identified in the no-probe control (supplemental Tables S1 and S2). The no-probe control contained proteins FIG. 1. Structure and mechanism of labeling with BHAcATP. A, BHAcATP contains ATP, an acyl phosphate reactive group, and a biotin tag. When BHAcATP binds to the ATP binding pocket of a protein, the amino group of the nearby lysine reacts with the carbonyl carbon, which results in the covalent binding of the biotin tag to the protein while ATP is released. B, typical BHAcATP labeling profile of Arabidopsis leaf proteome. Arabidopsis leaf extracts were labeled with BHAcATP and the biotinylated proteins were detected on protein blots using streptavidin-HRP. Coomassie Brilliant Blue staining indicates equal loading. Asterisks indicate endogenously biotinylated proteins MCCA and BCCP. White, black, and gray arrowheads indicate bands containing ATBPϩRBCL, PGK1, and a mix of ATP binding proteins, respectively. Abbreviations: MCCA, 3-methylcrotonyl-CoA carboxylase; BCCP, biotin carboxyl carrier protein; ATPB, chloroplastic ATPase; RBCL, ribulosebisphosphate carboxylase; PGK1, phosphoglycerate kinase-1. such as the endogenously biotinylated protein MCCA and abundant proteins such as RBCL. We made a selection of the 112 proteins by selecting proteins with the highest spectral counts, supplemented with detected protein kinases and proteins for which a labeling site was identified (see below). The resulting selection consisted of 38 proteins that could be assigned to the 13 different bands (supplemental Table S2 and Fig. 3B). Some of these proteins also were assigned to the bands in Fig. 3A. These data indicate that the abundant 55-kDa signal (band 5) was caused by labeling of a subunit of the chloroplastic ATPase (ATPB) and the large subunit of ribulose-1,5-bisphophate carboxylase oxygenase (RBCL). This signal also contained peptides from the beta subunit of the mitochondrial ATP synthase, glycinamide ribonucleotide synthetase, and CPK9. The 45-kDa signal (band 7) was caused predominantly by phosphoglycerate kinase (PGK1), though this signal also contained spectra from other proteins. The 40-kDa signal (band 8) contained a mixture of chloroplastic sedoheptulose-1,7-bisphosphatase; chloroplast RNA binding protein; fructose-biphosphate aldolase-2; subunit A of glyceraldehyde 3-phosphate dehydrogenase; and several other proteins, including two protein kinases (LRR1 and PK), inositol-1,3,4-trisphosphate-5,6-kinase, and an uncharacterized carbohydrate kinase. Weaker signals in the upper 70-to 90-kDa region of the gel contained methionine synthase (band 1), MCCA (band 1), formyltetrahydrofolate synthetase (band 2), transketolase (band 2), heat shock proteins 70 (HSP70, band 2) and 60 (HSP60, band 3), thioglucoside glucohydrolase 2 (band 3), F-box protein GRH1 (band 3), Ser/Thr/Tyr kinase STY8 (band FIG. 2. BHAcATP labeling depends on conditions. A, BHAcATP labeling is dependent on pH. Arabidopsis leaf extracts were labeled with BHAcATP at various pH values, and biotinylated proteins were detected from protein blots using streptavidin-HRP. B, labeling is affected by MnCl 2 and MgCl 2 . Arabidopsis leaf extracts were labeled with 5 M BHAcATP in the presence (ϩ) or absence (-) of 10 mM MnCl 2 or MgCl 2 and/or 20 mM EDTA or EGTA. Biotinylated proteins were detected on protein blots using streptavidin-HRP. MnCl 2 -and MgCl 2 -induced signals (solid circles) are suppressed by EDTA and/or EGTA. C, BHAcATP labeling is outcompeted with a variety of nucleotide compounds such as GTP, CTP, TTP, ATP, ADP, and NADPH, but not with AMP and NAD. D, the nucleotide triphosphates (NTPs) GTP, CTP, TTP, and ATP were more efficient in competing labeling than their deoxynucleotide forms (dNTPs). For C and D, Arabidopsis leaf extracts were preincubated with nucleotide inhibitors for 30 min before labeling with 5 M BHAcATP. Asterisks and arrows are as described in Fig. 1. 3), and subunit B of glutamyl-tRNA aminotransferase (band 4). Weak signals at 50 kDa (band 6) contained a subunit of a biotin carboxylase (CAC2), a MAP3K protein kinase (VIK1), a monodehydroascorbate reductase (MDAR6), and CPK11. Weak signals in the 35-to 40-kDa region contained peroxisomal NAD-malate dehydrogenase-2 (band 9), an LRR-RLK (band 9), and phosphopantothenate-cysteine ligase (COAB, band 10). The signal at 33 kDa (band 11) contained predominantly avidin, but also present were subunit O 2 of photosystem II and non-intrinsic ABC protein-7. Finally, the weak signals at the bottom of the gel (27-30 kDa) contained chloroplastic glutamine synthetase (GS2, band 11), chloro-plast RNA-binding protein (band 12), carbonic anhydrase (band 12), glutathione transferase (band 12), and a UMP/CMP kinase (PYR6, band 12). In conclusion, the vast majority of the identified proteins in the AcATP-labeled samples are ATP binding proteins (Fig. 3B). These ATP binding activities include a variety of protein kinases (STY8, CPK9, VIK1, CPK11, RLK-LRR, and LRR1), ATP-based transporters (ATPB, ATP synthase, and non-intrinsic ABC protein-7), metabolite kinases (carbohydrate kinse, PGK1, inositol-1,3,4-trisphosphate-5,6-kinase, and PYR6), and metabolic enzymes and chaperones using ATP (glycinamide ribonucleotide synthetase, MCCA, formyltetra- Tables S1 and S2 for more details. B, spectral counts of proteins in each of the 13 bands. Summarized are band numbers (column 1), accession codes (column 2), protein names (column 3), spectral counts in no-probe control (column 4), and the AcATP-labeled sample (column 5). The spectral counts also include BHAc-and OBHAc-modified peptides, indicated separately in columns 6 and 7. Expected ATP binding based on literature data is indicated in column 8 (ATP binding activity reported (ϩ) or not reported (NR)). The theoretical MW for each protein is given in column 9. Deviations from the actual MW are printed in bold. Protein kinases are printed in red. This table is a selection of the most abundant spectral counts per band, supplemented with detected protein kinases and proteins for which labeled peptides were detected. Proteins that were detected in more than one band are shown only in the band with the highest spectral counts. For a complete list of identified proteins, see supplemental Tables S1 and S2. C, AcATP labeling site in HSP60. The orthologous labeling site of AcATP in HSP60 of Arabidopsis is indicated in the crystal structure of the highly homologous GroEL of E. coli (1sx3, 16) containing ATP. The labeled lysine was located from the aligned protein sequences and highlighted in the structure using PyMol. The lysine is 7.03 Å from the gamma phosphate of ATP. hydrofolate synthetase, HSP70, HSP60, CAC2, MDAR6, peroxisomal NAD-malate dehydrogenase-2, and GS2). Putative Receptor Shedding of RLKs-We next examined whether the experimental molecular weight (MW) of the proteins identified from the protein gel (Fig. 3B) corresponded to the theoretical MW calculated from the protein sequence. We noticed a good correlation for nearly all the detected proteins, with the striking exception of two LRR-RLKs that had a calculated MW of 68 kDa but migrated in the region of 40 kDa (Fig. 3B). These were two highly homologous LRR-RLKs carrying six extracellular LRRs, a transmembrane domain, and a cytoplasmic kinase domain (Fig. 4). The extracellular domain also contains multiple putative N-glycosylation sites, which causes LRR-RLK proteins to migrate with an apparent MW that is typically 20 to 30 kDa larger than their calculated MW. Peptide coverage (Fig. 4 and supplemental Table S1) suggests that these labeled proteins consist of the full protein kinase domain but lack the extracellular domain. The detection of half-tryptic peptides of the C-termini of both these LRR-RLK proteins indicates that the C-terminus is intact and that a significant truncation must have occurred from the N-terminus. Interestingly, the calculated MW of the cytoplasmic domain is 37 and 38 kDa for At3g02880 and At5g16590, respectively, which coincides with the observed MW of these proteins. Taken together, these data indicate that these LRR-RLKs exist in leaves as kinase domains lacking the extracellular domain. Detection of an Oxidized Biotin Moiety-The searches mentioned above included a variable modification of lysine residues with the probe and allowed two trypsin miscleavages. Searches with the theoretical BHAc modification on lysine (339.16 Da) did not lead to hits with Mascot scores above the threshold (Fig. 3B). However, during more liberal searches we noticed that BHAc modifications appeared, but only in pep-tides that included an oxidation associated with the presence of a methionine in the peptide sequence (data not shown). This observation sparked the idea that the modification of the probe is associated with an additional oxygen. Indeed, when we searched the data with an oxygen added to the BHAc modification (OBHAc, 355.16 Da), we were able to identify modified peptides for nine different proteins with relatively high Mascot scores (41-71) (Fig. 3B). One of those labeled peptides is from HSP60 (At3g23990), a well-described ATP binding protein. To determine the location of the labeling site in the protein relative to ATP, we searched the protein database for proteins homologous to HSP60 and identified GroEL, an HSP60 protein from Escherichia coli, for which an ATP-bound crystal structure is available (1sx3 (16)). HSP60 and GroEL proteins share 56% amino acid identity, including the region where the labeled lysine is located. The amino group of the labeled lysine is located 7.0 Å from the gamma phosphate of the bound ATP in GroEL (Fig. 3C). Thus, labeling in HSP60 occurred at the expected labeling site. We next studied the fragmentation spectrum of the labeled peptide of HSP60 further to identify the location of the oxygen within the labeled peptide. The labeled peptide in HSP60 has the sequence VTKDGVTVAK and was detected with a Mascot score of 41.4 with a parental ion of 686.93 m/z (z ϭ 2) (Fig. 5). Nearly all b-and y-ions were detected in the fragmentation spectrum, locating the labeled lysine including the oxygen at the third position in the peptide: VTK * DGVTVAK. The spectrum also contained three unexplained peaks in the low-mass range: 243. 15 439. 35 Da, corresponds to the labeled version of the commonly observed immonium ion variant for lysine. High-energy collision-induced dissociation fragmentation of lysine residues typically results in an unsaturated cyclic 84 Da fragment, known as a piperidine or tetrahydropyridine ion (17). As the lysine has lost a proton during the labeling reaction, the observed 83 Da distance is explained, indicating that such fragmentation and subsequent cyclization of lysine do not cause a loss of the biotin. Taken together, these data demonstrate that the additional oxygen is located at the biotin, presumably on the sulfur. The 243.2, 356.5, and 439.3 Da ions were also found in spectra of the other labeled peptides, indicating that oxygen is consistently present on the biotin moiety. Xsite Identifies 242 Labeling Sites-The observation that most of the 112 BHAcATP-labeled proteins are ATP binding reflects the potency of the probe to target ATP binding proteins in a crude proteome. However, only a few of these 112 proteins are protein kinases. Furthermore, some of the BHAcATP-labeled proteins are not annotated as ATP binding proteins, and therefore the labeling sites on these proteins are uncertain. To address both issues, we analyzed the labeled proteome more deeply by analyzing the labeled peptides. For this we used a variant of the AcATP probe, DBAcATP (Fig. 6A) (7). Desthiobiotin cannot be oxidized and has a lower affinity for streptavidin, resulting in a more efficient elution of peptides for identification. After labeling with DBAcATP, the proteomes were digested with trypsin, and the biotinylated peptides were purified using streptavidin beads and analyzed via LC-MS/MS (Fig. 6B). We recorded 10,465 peptide spectra, corresponding to 567 different peptides carrying a modified lysine residue (Fig. 6C). Peptides with probability scores greater than 95% (15) and those that were identified in two or more reactions were selected (Fig. 6C), resulting in 6992 spectra corresponding to 242 peptides (supplemental Table S4). DBAcATP Labels a Diversity of Protein Kinases-We first analyzed the data for peptides derived from protein kinases. Annotation through the PFAM database revealed that 24 of the 242 labeled peptides originated from at least 21 different protein kinases ( Fig. 7A; supplemental Table S4). This list contains seven RLKs, of which six carry extracellular LRRs. The detection of RLKs is remarkable, because we did not enrich for membranes. In addition, we identified protein kinase peptides from several MPKs, CPKs, PTI-like kinases, and other Ser/Thr protein kinases. We also detected AvrPphB susceptible 1 (PBS1) (18), VH1-interacting kinase (VIK) (19), AT6 protein kinase (20), and proline-rich extension-like receptor kinase-1 (PERK1) (21). Of the 24 protein kinase peptides, 19 were unambiguous and were derived from only one protein kinase (black accession numbers in Fig. 7A). Four proteinkinase-derived peptides were ambiguous, as the peptide sequence was identical in two or more protein kinases in the TAIR10 protein database (gray accession numbers in Fig. 7A). Two protein-kinase-derived peptides were identified for two protein kinases (top in Fig. 7A), indicating that DBAcATP labels these protein kinases at two positions. In the case of PERK1, two overlapping peptides containing the same labeled lysine were detected. We marked the identified protein kinases in the Arabidopsis thaliana kinase database, which classifies all the 1099 protein kinases of Arabidopsis into classes, groups, and families based on sequence similarities. We identified representatives of 11 different protein kinase families belonging to 10 different groups and the three major protein kinase classes (Fig. 7B). This indicates that AcATP probes are not family specific and can be used to profile ATP binding activities of the majority of Arabidopsis protein kinases. Alignment of the identified protein-kinase-derived peptides showed that these peptides fall into four groups containing four different labeled lysine residues, Lys1-4 (Fig. 7C). When mapped on the protein kinase domains, Lys1 is positioned in the beginning of the protein kinase domain, Lys2 in the middle, Lys3 in the second quarter, and Lys4 in the third quarter of the protein kinase domain (Fig. 7A). Lys1 and Lys2 are also part of the conserved protein kinase motifs II and VIb, respectively (22). These two lysines are also present in the PTO kinase, a tomato protein kinase involved in immunity, for which a structure is available (2qkw) (23). Both Lys1 and Lys2 reside in the ATP binding pocket of the PTO kinase, at distances of 7.43 and 4.32 Å from the gamma phosphate of the bound ATP ( Fig. 7D and supplemental Fig. S2A). The closer proximity of Lys2 seems reflected in the higher spectral count of Lys2-containing peptides relative to Lys1-containing peptides. Modeling of PTI1-1 indicates that Lys3 locates in an extended loop that might fold back on the ATP binding pocket (supplemental Fig. S2B), whereas modeling of VIK1 indicates that Lys4 is located in close proximity to the gamma phosphate of bound ATP (supplemental Fig. S2C). To determine how common these labeled lysine residues are within the protein kinase family of Arabidopsis, we counted these lysines in the alignments of each protein kinase family. We found that Lys1 and Lys2 were conserved in 70% and 47% of the 1099 protein kinases, respectively. Lys3 in PTI kinases and Lys4 in VIK1 were less conserved among protein kinases (3.5% and 5.0%, respectively). Thus, the frequencies of the theoretically labeled lysine in protein kinases correspond to the frequencies of the different labeling sites detected in protein kinases (supplemental Fig. S6). DBAcATP Also Labels Other ATP Binding Proteins in Their ATP Binding Pockets-When ranked according to peptide frequency, peptides from protein kinases are not among the top 20 most frequent peptides (Fig. 8A). The most frequent peptide from a protein kinase is that from LRR1 (At5g16590) on position 22 with 67 spectral counts ( Fig. 8A and supplemental Table S4). The most frequently detected labeled peptide is from PGK1, with 968 spectral counts. PGK1 is also labeled at two additional lysine residues with 196 and 97 spectral counts, respectively. The second most frequent labeled peptide is from chloroplast ATP synthase subunit beta (ATPB), with 256 spectral counts. Other proteins in the top 20 are acetyl coenzyme A (CAC2), subunit B of Glu-tRNA aminotransferase, an ATP synthase, ribose-phosphate pyrophosphokinase 4, the small subunit of ribulose bisphosphate carboxylase, cell division cycle 48 (CDC48), a GTP binding protein (At1g30580), and UMP/CMP kinase (PYR6) (Fig. 8A). Most, but not all, of these proteins were also detected abundantly using the in-gel approach (Fig. 3). Although these proteins are unrelated and catalyze very different reactions, ATP is a common substrate, consistent with the reactivity of the probe (supplemental Table S5). Protein structures of homologs of PGK1, ATPB, CAC2, and ribose-phosphate pyrophosphokinase 4 contain the labeled Lys in close proximity to the terminal phosphate of the bound nucleotide (supplemental Fig. S3). Note that CAC2 is a biotin binding protein, but the labeled Lys is close to the bound ADP (supplemental Fig. S3C). This demonstrates that the labeling of these proteins is FIG. 6. Identification of labeled peptides by Xsite. A, structure of desthiobiotin-acyl-ATP (DBAcATP). The desthiobiotin affinity tag is used because it cannot be oxidized and has a lower affinity to avidin, which facilitates efficient peptide elution. B, leaf extracts were labeled with DBAcATP and trypsin-digested, and the labeled peptides were purified and analyzed via LC-MS/MS. C, pipeline for analysis of Xsite reactions. A total of four Xsite reactions were analyzed. Peptides were selected for (i) having labeled lysines with probability scores greater than 75%, (ii) having Ͼ95% probability, and (iii) being found in at least two of the four Xsite reactions. To select peptides from PKs, PFAM analysis was performed on the list of labeled proteins, resulting in a list of 558 spectra corresponding to 24 different peptides from PKs. in accordance with the labeling mechanism of AcATP probes. However, not all proteins in this list are known to bind nucleotides. Most evident is the large subunit of Rubisco (RBCL), which is labeled at 13 different lysine residues that are scattered on the surface of the RBCL protein (supplemental Fig. S4). Potential of DBAcATP Labeling in Arabidopsis Proteome-We extended the analysis of labeling sites in homologous protein structures and found that 63 of the 242 labeled peptides resulted from labeling of a lysine residue in a known nucleotide binding pocket at Յ10 Å from the phosphate of the bound nucleotide (supplemental Fig. S5; blue in Fig. 8A). To estimate the potential of DBAcATP labeling in the Arabidopsis proteome, we retrieved for each of the 63 labeled peptides the corresponding PFAM domain. A total of 26 different PFAM domains corresponding to 13 different protein clans plus six uncategorized PFAM families were identified (supplemental Table S6). The Arabidopsis genome encodes for a total of 1683 proteins carrying these 26 different PFAM domains. The largest PFAM domains are those of protein kinases (PF00069 ϭ 813 members and PF07714 ϭ 388 members), followed by AAA ATPases (147 members). Because all 1683 of these proteins are probably binding ATP and representatives were found to be labeled by DBAcATP, we predict that the Arabidopsis proteome contains at least 1683 putative targets that could be labeled in the ATP binding pocket by DBAcATP. Preferential Labeling of ATP Binding Proteins-To investigate whether protein kinases and other ATP binding proteins are preferentially labeled relative to highly abundant proteins, we plotted the spectral counts of the labeled peptides against the spectral counts detected for these proteins in leaf proteomes of the AtProteome database (Fig. 8B). We chose this approach to ensure the inclusion of low-abundance protein kinases, which were detected by Baerenfaller et al. (48) with over 1300 MS runs. The leaf proteomes that they used are virtually the same as the ones we used for our studies. Interestingly, this graph splits the labeled peptides into three groups. Peptides from protein kinases cluster in group 1 at low protein levels and medium labeling. Peptides from other ATP binding proteins are in group 2 and show medium protein levels and medium to high labeling. Finally, the third major group contains proteins with high protein levels and low to medium labeling. Importantly, the vast majority of these proteins were not labeled in ATP binding pockets. This illustrates that DBAcATP shows a high selectivity for labeling ATP binding proteins, as only highly abundant proteins are labeled outside known ATP binding pockets. Interestingly, protein kinases were detected at a moderate labeling/protein ratio relative to labeling events in ATP binding pockets of non-protein kinases (0.5 Ϯ 0.57(n ϭ 15) versus 2.86 Ϯ 0.68(n ϭ 34)) (Fig. 8C). These data suggest that the protein kinases have a lower affinity for ATP or that that part of the protein kinase pool cannot be labeled. In order to investigate this in more detail, we extracted spectral counts for 494 protein kinases from leaf proteomes of the AtProteome database (48). Interestingly, of the top 20 protein kinases that are most frequently detected in leaf proteomes, only 8 were detected upon labeling (supplemental Fig. S7), and these data show no correlation between spectral counts in the AtProteome database and spectral count frequency upon AcATP labeling. Furthermore, inspection of the protein kinase sequences revealed that most of the undetected labeled peptides fell in a MW range that should have been detected in our assay. These data reinforce the idea that not all kinases can be labeled in a given extract, possibly because they do not bind ATP. However, such data should be interpreted with extreme caution, because different peptides have different ionization potentials, and the absence of a labeled peptide does not mean that the protein is not labeled. DISCUSSION We have reported the first in-depth analysis of targets of an AcATP probe in a plant proteome. By comparing a gel-based identification platform for labeled proteins with a gel-free platform for labeled peptides, we have demonstrated the advantages and limitations of these complementary approaches. The analysis of labeling sites using the protein database showed that AcATP probes target predominantly, but not exclusively, the ATP binding pockets of a broad range of protein kinases and other unrelated ATP binding proteins. The labeled proteins include a few well-characterized ATP binding proteins, as well as many ATP binding proteins that have not been studied so far. A Comparison of Two Analysis Platforms-The data generated by the gel-based and gel-free platforms are complemen- FIG. 7. Protein kinases labeled with DBAcATP. A, protein domains are indicated as boxes, with protein kinase domains containing bars representing identified labeled peptides containing Lys1 (blue), Lys2 (red), Lys3 (green), and Lys4 (black). Protein kinases that match the same peptides are indicated with gray accession numbers and summarized as At4g01370 ϩ 3 ϭ At4g01370, At3g45640, At2g43790, and/or At1g01560; At3g20410 ϩ 1 ϭ At3g20410 and/or At1g50700; At3g17410 ϩ 1 ϭ At3g17410 and/or At1g48210; and At1g73660 ϩ 2 ϭ At1g73660, At5g11850, and/or At1g18160. Common names of protein kinases are indicated, as well as corresponding spectral counts. B, classification of the Arabidopsis protein kinases according to the Arabidopsis thaliana Kinase Database (AthKD). The identified labeled protein kinases are indicated with red arrowheads. Accession numbers and common names are shown. Detected protein kinases represent 11 families that belong to 10 groups and 3 classes. C, alignment of identified labeled peptides containing four different labeled lysines. Lys1 and Lys2 belong to protein kinase motifs II and VIb, respectively. Color coding is the same as in A. Labeled lysine residues are printed in bold. D, position of the two conserved labeled lysine residues in the ATP binding pocket of LRR-RLK protein At3g02880, based on the crystal structure of the highly homologous protein kinase PTO (23). The distances from the labeled Lys to the gamma phosphate of the bound ATP molecule are shown. FIG. 8. Spectral count analysis of labeled peptides. A, labeled peptides ranked on spectral count frequencies. Labeling sites in the ATP binding pocket of protein kinases and other nucleotide binding proteins are indicated in red and blue, respectively. The top 20 peptides with the highest spectral counts are shown in the box, along with their accession numbers, their common names, the peptide sequences (labeled Lys, bold), and their spectral counts. Distances from the nucleotides to the labeled Lys are indicated in Å. NR, not reported nucleotide binding site. Ambiguous peptides are summarized as follows: At3g12780 ϩ 1 ϭ At3g12780 and/or At1g56190; At5g08670 ϩ 2 ϭ At5g08670, At5g08680, and/or At5g08690; At1g67090 ϩ 3 ϭ At1g67090, At5g38430, At5g38420, and/or At5g38410; At3g09840 ϩ 2 ϭ At3g09840, At3g53230, and/or At5g03340. B, correlations between ATB binding activity and protein abundance splits the labeled peptides into three groups. The frequency of spectral counts of the labeled peptides is plotted against the spectral counts of the corresponding proteins detected in leaf proteomes (extracted from the AtProteome database). For ambiguous labeled peptides, the protein with the highest spectral count was selected from the AtProteome database. Note that for some proteins (e.g. RBCL) multiple labeled peptides were detected. Group numbers 1-3 are explained in the main text. C, average spectral count ratio between labeling (by AcATP) and abundance (extracted from AtProteome database) for the three groups of labeled proteins. Error bars represent S.E. for n proteins (indicated below the bars). tary, and each platform has its advantages and disadvantages. Although many proteins were detected using both approaches, several were detected by only one approach. HSP60 and HSP70, for example, were detected only using the in-gel approach, whereas CDC48 and MPKs were detected only via the gel-free approach. Gel-based identification resulted in the detection of 112 proteins, of which only 9 were protein kinases. Although the vast majority of the detected proteins were absent in the no-probe control, the labeling site could be determined for only nine proteins, based on the detection of labeled peptides. This number would have been even lower if we had not realized that biotin is somehow oxidized during this procedure. By contrast, sequencing the labeled peptides using Xsite/KiNativ via the gel-free approach identified more protein kinases and also determined labeling sites in each of the labeled proteins. Although this gel-free approach seems more powerful, it cannot discriminate between proteins if the labeled peptide is identical. For example, the labeled peptides do not discriminate between CPK9 and CPK11 because the labeled peptide is identical, whereas both proteins were distinguished via the gel-based approach. The gel-free approach also does not provide the MW of the labeled protein, and this can be very important information. For example, we noticed that two labeled LRR-RLKs migrated not at the expected 67-68 kDa, but at 40 kDa, indicating that they had lost the extracellular LRR domain. At this stage we cannot exclude the possibility that this processing occurred upon protein extraction. It is interesting to point out that it was recently discovered that Xa21, an LRR-RLK from rice, is proteolytically cleaved during signaling to release a cytosolic protein kinase domain that migrates to the nucleus to phosphorylate transcription factors (24). A similar receptor-shedding mechanism might be at work for the two LRR-RLKs detected on our study. Detection of an Oxidized Biotin Moiety-Our analysis of biotinylated proteins via in-gel digest and LC-MS/MS revealed no peptides that were labeled with BHAc but several peptides that were labeled with OBHAc, the oxidized version of BHAc. To our knowledge, this type of modified biotin has not been reported before during proteomics, and this discovery might have important implications for the detection of other modifications containing biotin. We speculate that the oxygen is located on the sulfur of biotin, resulting in a biotin sulfoxide (25). This modification was also observed, for example, during the synthesis of oligonucleotides (26), and would not occur on desthiobiotin. At this stage we do not know when during the in-gel procedure the oxidation occurs. Searches on the occurrence of oxidized biotin on proteins labeled with other probes will tell how common this modification is. How to Detect More Protein Kinases?-We detected 24 labeled peptides from protein kinases, but there are 1099 protein kinases encoded by the Arabidopsis genome (1). There are several reasons why no more protein kinases were detected. First, most of the protein kinases are not expressed in tissues that we used for the labeling experiments. Second, peptides from some protein kinases are too small or too large to be detected with the chosen MS settings. However, in silico analysis indicates that the majority (95%) of the theoretically labeled peptides of Arabidopsis protein kinases are in the range of 5 to 30 amino acids. Third, some protein kinases cannot be discriminated based on the sequence of the labeled peptides. Nonetheless, in silico analysis showed that 64% of the Arabidopsis protein kinases contain a putative labeled lysine in a peptide sequence that is unique in the Arabidopsis proteome (supplemental Fig. S6). Fourth, MS is not sensitive enough to detect more labeled peptides from protein kinases in an unbiased mode. Indeed, inclusion lists coupled to retention times and searches for marker ions in the MS2 mode can tremendously increase the number of identified protein kinases. The added search modes have increased the number of robustly detected protein kinases to 160 protein kinases per cell line (7). Thus, we expect that we will be able to detect labeling of over 100 different protein kinases from a single proteome with the implementation of empirical searches. The wider range of detection will increase the power of protein kinase profiling in plants tremendously. Profiling Arabidopsis Protein Kinases-We have detected 24 labeled peptides that originate from protein kinases. Analysis of the labeling sites in these protein kinases showed that AcATP probes react with lysines that reside at four distinct positions. Lys1 and Lys2 reside in the ATP binding pocket in motifs II and VIb, respectively, and were previously described to be labeled by AcATP probes (6). Lys3 and Lys4 were detected in PTI1-like kinases and MAP3K VIK, respectively, and had not been characterized before. Although crystal structures are not available for representatives of these protein kinase subfamilies, modeling indicates that regions carrying Lys3 and Lys4 might indeed be able to fold back on the ATP binding pocket. The reactivity of Lys3 and Lys4 indicates that even though these lysines are poorly conserved, they might be in close proximity to ATP and might play important roles in PTI1-and VIK-like protein kinases. The absence of any other labeled peptide from the detected protein kinases illustrates the remarkable selectivity of the AcATP probe to target the ATP binding pocket of these proteins. The labeled protein kinases are remarkably diverse and contain representatives of all major classes of Arabidopsis protein kinases. The list includes RLKs, Pelle-like kinases, MAPKs, MAP3Ks, CPKs, PTI-like kinases, and PERK1. The diversity of the protein kinases illustrates the wide range of protein kinases that can be labeled with AcATP and is consistent with the fact that all protein kinases (by definition) bind ATP and most of them carry lysine residues to stabilize the phosphate. The data are also consistent with studies of AcATP probes on animal proteomes, where Ͼ75% of the human protein kinases have been detected (6). Thus, AcATP probes have the remarkable potential to label nearly all protein kinases not only of mammals, but also of other organisms. The detection of several RLKs with relatively high spectral counts is noteworthy because these proteins are thought to be low in abundance and we did not enrich for membrane proteins. Functions have been described for several of the detected protein kinases. MPK3, -4, -6, and -11 are involved in immune signaling and control gene expression though WRKY transcription factors (4,27,28). PBS1 is involved in the perception of bacterial pathogens secreting the AvrPphB effector (29,30). AvrPphB cleaves PBS1, and this activates resistance protein RPS5. Calcium-dependent kinase CPK3 is involved in stomatal closure, herbivore defense, and salt stress acclimation and is a positive regulator of sphingolipid-induced cell death (31)(32)(33). PTI1-2 interacts with oxidative stress-response protein kinase OXI1 and integrates phosphatidic acid signaling with reactive oxygen signaling (34). VIK is a MAP3K involved in the uptake of glucose into the vacuole (19), and AT6 is a MAP3K that acts as a negative regulator of salt tolerance (20). PERK1 is thought to be involved in sensing cell wall stress during wounding and pathogen infection (21,35). The detection of these characterized protein kinases via AcATP labeling is consistent with their presence and activities in the Arabidopsis rosette used for our studies. The other two-thirds of the detected protein kinases have been described only by transcript levels and phylogeny. Our data demonstrate that these uncharacterized protein kinases are present and able to bind ATP. Labeling of Other ATP Binding Proteins-Previous studies with AcATP probes have been mostly focused on the labeling of protein kinases. In this study we also examined the labeling of proteins other than protein kinases. An in-depth analysis of the labeled peptides revealed that AcATP probes label an astonishing diversity of protein families. Most of the labeled proteins are able to bind nucleotides, and analysis of structural data indicates that AcATP probes label these nucleotide binding proteins predominantly inside ATP binding pockets. The structural diversity of the nucleotide binding proteins is remarkable, as illustrated by the fact that they belong to 13 different protein clans in the PFAM database (36). Several different ATP binding proteins have been identified with high spectral count frequencies. PGK1 is the top hit among the labeled proteins. PGK degrades ATP during glycolysis (37). Subunit B of glutamyl-tRNA aminotransferase is an Asn/Gln-tRNA amidotransferase involved in the transamidation of misacylated Asp-tRNA or Glu-tRNA forming a correctly charged Asn-tRNA or Gln-tRNA, a reaction that requires ATP (38,39). CDC48 encodes for cell division cycle protein 48 and is important in cell division, expansion, and differentiation. Arabidopsis plants carrying mutations in CDC48-encoding genes show impaired seedling development and defective pollen and embryo development (40). CDC48 is an AAA-type ATPase and belongs to a large superfamily containing a highly conserved triple-A domain that binds and hydrolyzes ATP (41). CAC2 is the biotin carboxylase subunit of the heterodimeric, biotin-containing enzyme chloroplastic acetyl-coenzyme A carboxylase, which is involved in the synthesis of fatty acids in Arabidopsis (42) and uses ATP for the conjugation of a carboxylate-containing molecule to an amino-or thiolgroup-containing molecule (43). CAC2 contains a phosphatebinding loop and a Mg 2ϩ -binding site and has two alpha-beta subdomains that grasp the ATP molecule. This "ATP-grasp " domain is common to a superfamily that also contains Dalanine:D-alanine ligase, glutathione synthetase, and carbamoyl phosphate synthase. PYR6 is a uridine 5Ј-monophosphate (UMP)/cytidine 5Ј-monophosphate (CMP) kinase that converts UMP/CMPs to uridine and cytidine diphosphates, respectively. PYR6 orthologs in bacteria and yeast are required for cellular proliferation and RNA and protein synthesis (44,45). PYR6 has also been detected in the mature pollen of Arabidopsis, consistent with its role in cell proliferation and division (46). Taken together, these five structurally and functionally diverse examples illustrate the broad range and relevance of ATP binding enzymes that we can monitor using AcATP probes. Labeling Outside Known ATP Binding Pockets-Our analysis of labeling sites also showed that a large number of labeling sites are not located in known nucleotide binding pockets. It is possible that several of these labeling sites are nucleotide binding sites that have not yet been described. However, the correlation between spectral counts and transcript levels indicates that labeling outside known nucleotide binding pockets is typically detected for highly abundant proteins, such as Rubisco. An obvious explanation for the labeling of abundant proteins outside nucleotide binding sites would be that solvent-exposed lysine residues have a low basal reactivity toward AcATP and that this causes aspecific labeling of highly abundant proteins. Strangely enough, however, AcATP labeling of abundant proteins nevertheless requires divalent ions and can be competed with nucleotides that do not carry an acyl reactive group. These data support the exciting idea that ATP-competable AcATP labeling sites might identify novel nucleotide binding sites in proteins. Interestingly, a recent crystal structure of rice Rubisco with NADPH revealed that two of the labeled lysines are in close proximity to the phosphate in NADPH (supplemental Fig. S4D) (47). Thus, labeling outside known nucleotide binding sites is an interesting topic for further studies. Future Prospects-AcATP labeling is a powerful way to monitor ATP binding activities in native proteomes. Besides the majority of the 1099 protein kinases of Arabidopsis, AcATP can potentially label another 482 ATP binding proteins, representing another 11 different protein families. Although AcATP probes also label outside known nucleotide binding sites, the preferential labeling of nucleotide binding proteins in ATP binding pockets demonstrates the high selectivity of the AcATP probe. An interesting future prospect will be to study the changes of ATP binding activities of proteins upon an external stimulus. Our data suggest that labeling not only reflects the abundance of the ATP binding proteins, but also is affected by the ATP binding activity of the proteins. ATP binding can be dynamic and dependent on the cellular state. However, further studies on changes in ATP binding activities upon a stimulus must involve pair-wise comparisons of the same labeled peptide under different conditions, and this requires further development of quantitative methods such as stable isotope labeling of amino acids in cell culture and the use of isotope-labeled probes. We aim to use AcATP labeling in the near future to identify selective inhibitors of Arabidopsis protein kinases and to study the activation of protein kinases upon external stimuli.	2018-04-03T05:49:03.357Z	2013-05-29T00:00:00.000	{ "year": 2013, "sha1": "6731a066591f3cff68923d069610e247de2fad74", "oa_license": "CCBY", "oa_url": "https://doi.org/10.1074/mcp.m112.026278", "oa_status": "HYBRID", "pdf_src": "Highwire", "pdf_hash": "dfbdec796c75b03c3d35a0102a9960a69b5f2988", "s2fieldsofstudy": [ "Biology", "Chemistry" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
10681027	pes2o/s2orc	v3-fos-license	Effect of Word Complexity on L2 Vocabulary Learning Research has shown that a number of factors, such as maturational constraints, previous language background, and attention, can have an effect on L2 acquisition. One related issue that remains to be explored is what factors make an individual word more easily learned. In this study we propose that word complexity, on both the phonetic and semantic levels, affect L2 vocabulary learning. Two studies showed that words with simple grapheme-to-phoneme ratios were easier to learn than more phonetically complex words, and that words with two or fewer word senses were easier to learn that those with three or more. Introduction There is much computer-assisted language learning (CALL) literature that explores effective methods of teaching vocabulary. In recent studies conducted using the REAP system, which finds documents from the internet to teach vocabulary, we have shown that speech synthesis reinforces written text for learning in reading activities (Dela Rosa et al., 2010), and we have also shown that contextsensitive dictionary definitions afford better vocabulary learning for L2 language students (Dela Rosa and Eskenazi, 2011). One issue that remains to be explored in this context: determining what factors make an individual word easier to learn. We propose that word complexity, on both the phonetic and semantic levels, can affect how easily an L2 vocabulary word can be learned. In this paper we first discuss past work on factors that impact vocabulary acquisition in intelligent tutoring environments, and then explore work on defining the complexity of a word with respect to vocabulary learning. Next we describe two classroom studies we conducted with ESL college students to test the effect of word complexity on L2 vocabulary learning. Finally we examine our results and suggest future research directions. Background Many studies have been conducted to investigate the relationship between different variables and second language learning. For example, the age of the foreign language learner is often pointed to as a major factor in determining whether an individual will be successful in learning a new language (Marinova-Todd, 2000). In the domain of L2 vocabulary instruction, researchers have shown that factors such as maturational constraints, attention, previous language background, and order of acquisition, can all affect L2 vocabulary acquisition (Oxford and Scarcella, 1994). Additionally, another factor that affects L2 vocabulary learning is the number of exposures of a practice item that a student receives during learning activities. In a study on the effects of spacing and retention of vocabulary pairs, Pavlik and Anderson (2005) showed that each time an item is practiced, it receives an increment of strength, but these increments decay as a power function of time. Furthermore, it is generally accepted that reading is beneficial to vocabulary acquisition (Perfetti, 2010). One group of factors in foreign language vocabulary instruction that has often been overlooked is at the level of the individual word, such as word complexity. In sections 3.2 and 3.3, we describe two simple measures of phonetic and semantic word complexity that were examined during our classroom studies. There have been work on defining the complexity of a word, such as Jakielski's (1998) Index of Phonetic Complexity, but we do not know of work that measures the effect of word complexity on L2 vocabulary learning. Classroom Study Setup In order to determine the effect that word complexity, in both the phonetic and semantic levels, have on L2 language learners, we conducted two in-vivo studies with ESL students at the English Language Institute of the University of Pittsburgh. The first study focused on the effect of phonetic word complexity on vocabulary learning. The second study explored the effect of semantic word complexity, in the form of the number of senses a word has, on vocabulary learning. Both studies and the tutoring system that was used are described in the next sections. Overview of the Tutoring System The tutoring system, REAP, is a web-based language tutor developed at Carnegie Mellon that harvests documents from the internet for L2 vocabulary learning and reading comprehension (Heilman et al., 2006). It has been used as a testing platform for cognitive science studies. This system has the ability to provide reader-specific passages by consulting profiles that model a reader's reading level, topic interests, and vocabulary goals. The system's interface has several features that enhance a student's learning experience. One key feature is that it provides users with the ability to listen to the spoken version of any word that appears in a document, making use of the Cepstral Text-to-Speech system (2001) to synthesize words on the fly when clicked on. Additionally, students can look up the definition of any of the words they encounter while reading the documents using an embedded electronic dictionary. The system also automatically highlights focus words, i.e. the words targeted for vocabulary learning in a particular reading. Study 1: Phonetic Complexity In Study 1, we looked at the effect that phonetic complexity, one measure of a word's complexity, has on learning a word, and whether this complexity causes a word to be learned more easily when multimodal input is provided in the form of written text accompanied by spoken text generated through speech synthesis. To measure a word's phonetic complexity, we used the ratio of a word's graphemes to phonemes, where words with a ratio closer to 1 were simpler than those with a ratio much greater or less than 1. Note that for this study, simple letters have been used as the grapheme units. For example, the word cat has a simple one-toone mapping between its graphemes and phonemes (C A T vs. K AE T), while other words like borough and index have a more complex relationship (B O R O U G H vs. B ER OW, and I N D E X vs. IH N D EH K S), with grapheme-tophoneme ratios greater than 1 and less than 1 respectively. For this study, there were 21 intermediate-level ESL college students at the University of Pittsburgh's English Language Institute whose native languages included Arabic, Chinese and Spanish. Weekly group readings were given as class activities, centered on a total of 18 focus words, followed by practice closed cloze questions (multiple-choice fill-in-the-blank with 5 answer choices provided, and distractors coming from the Academic Word List or words that are similar but do not fit the blank properly) on the focus words that appeared in the particular reading. The focus words used in this study were taken evenly from the following word groups:  Words with grapheme-to-phoneme ratio equal to 1 [6 words]  Words with grapheme-to-phoneme ratio greater than 1 [6 words]  Words with grapheme-to-phoneme ratio less than 1 [6 words] A pre-test was administered at the beginning of the study, consisting of closed cloze questions about the focus words. A similar set of questions was presented to the students during the post-test, which occurred one week after the last reading activity. Between the pre-test and post-test, 6 reading activities were administered, one per week, each focused on a single document. This activity typically took students 20-30 minutes to complete. Study 2: Semantic Complexity In Study 2, we investigated the effect that multiple word-senses, another measure of word complexity, have on learning a word. There were 21 intermediate-level ESL college students at the University of Pittsburgh's English Language Institute, whose native languages included Arabic, Chinese, Korean, and Spanish. As in Study 1 there was a pre-test, a post-test, and a series of weekly documents to be read featuring the focus words. In total there were 26 focus words, all of which were taken from the Academic Word List and 7 weekly reading activities. With respect to word complexity, the focus words were divided into the following groups:  Results The results of both of our studies showed that the use of the tutoring system significantly helped students improve their performance on the vocabulary tests, as made evident by the average overall gains between the pre-test and post-test (p < 0.001). Note that the error bars shown in this section show the standard error. Also note that normalized gain, the measurement being used to describe improvement in both studies, is given the by the following: If the post-test score is greater than the pre-test score, then Normalized gain = (post-test scorepre-test score) / (maximum-possible-scorepre-test score) Otherwise, Normalized gain = (post-test scorepre-test score) / (pre-test score) In Study 1, the average normalized gain between the pre-test and post-test was 0.2563 (± 0.0466). Figure 1 illustrates the differences in vocabulary gain when the gains are separated by word condition type. The average gains per condition are 0.2222, 0.1270, and 0.1191 for the conditions of grapheme-to-phoneme ratio = 1, grapheme-to-phoneme ratio > 1, and grapheme-tophoneme ratio < 1 respectively. In Study 2, the average normalized gain between the pre-test and post-test was 0.5323 (± 0.0833). Figure 2 illustrates the impact of word sense complexity on vocabulary gains. With respect to word sense complexity, the average gains per condition are 0.2495, 0.4163, and 0.1699 for the 1-sense, 2-senses, and 3-or-more senses conditions respectively. Discussion The results of both studies tend to confirm our initial hypotheses and suggest that word complexity, in the forms of phonetic complexity and the number of word senses a word has, does make a significant difference in how easily an L2 vocabulary word is learned. In Study 1, we see that the 'simple' words (those with grapheme-to-phoneme ratios equal to 1), afford more learning than the more 'complex' words, as made evident by the difference in gains between the pre-test and post-test (p < 0.04) shown in Figure 1. This result suggests that the phonetic complexity of a word may play a role in learning that word in an intelligent tutoring environment. In Study 2, the words with many senses (3 or more) have significantly lower gains than words with 1 or 2 senses (p < 0.05). There was no significant difference in gains between words with 1 word sense and words with 2 word senses, as shown in Figure 2. This result seems to suggest that words with 2 or fewer word senses are generally easier for L2 students to learn than those with 3 or more word senses. This could be because a student has a harder time choosing the correct meaning of a word amongst many choices. Fewer choices seem to afford more learning than showing just the right one, which may indicate that by comparing two meanings with the meaning in the document, the student is actively constructing her knowledge of the word. Dela Rosa and Eskenazi (2011) found that giving students only the correct meaning of a polysemous word afforded less learning than giving them several meanings in a ranked order. Conclusion and Future Directions This paper demonstrates that word complexity can affect how easily an L2 vocabulary word can be learned. We proposed two dimensions of word complexity, one based on the complexity of a word's grapheme to phoneme ratio, and another based on the number of meanings a word has. Two in-vivo studies were conducted with ESL college students to test our hypothesis. Our results suggest that word complexity on both the phonetic and semantic level does have an effect on L2 vocabulary learning. A future research direction that this work suggests is the search for other measures of word complexity, such as a more complex measure of grapheme to phoneme ratio, for example taking into account the ambiguity of a particular grapheme, or more complex measures of semantic complexity, like one that may take the average number of synonyms a word sense has, to determine their effect on learning using an intelligent tutoring system. This information could help define different ways to teach different words, providing more scaffolding for harder words, for example. We would also like to investigate whether the average aggregate vocabulary learning trends of different native language groups correlates with different measures of word complexity, and thus might reveal a relation between the structure of L1 and difficulties in L2 vocabulary learning. Finally we would like to investigate whether providing examples of focus word usage prior to or following a reading activity is beneficial to vocabulary learning.	2014-07-01T00:00:00.000Z	2011-06-24T00:00:00.000	{ "year": 2011, "sha1": "80871b0a8df7f069fc171f1749f039a9b6babe37", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "ACL", "pdf_hash": "d4080401492905275e6faa12ef3adafaccd29203", "s2fieldsofstudy": [ "Linguistics" ], "extfieldsofstudy": [ "Computer Science" ] }
253743781	pes2o/s2orc	v3-fos-license	Changes in behaviour drive inter-annual variability in the at-sea distribution of northern gannets The at-sea distribution of seabirds primarily depends on the distance from their breeding colony, and the abundance, distribution and predictability of their prey, which are subject to strong spatial and temporal variation. Many seabirds have developed flexible foraging strategies to deal with this variation, such as increasing their foraging effort or switching to more predictable, less energy dense, prey, in poor conditions. These responses may vary both within and between individuals, and understanding this variability is vital to predict the population-level impacts of spatially explicit environmental disturbances, such as offshore windfarms. We conducted a multi-year tracking study in order to investigate the inter-annual variation in the foraging behaviour and location of a population of northern gannets breeding on Alderney in the English Channel. To do so, we investigated the link between individual-level behaviour and population-level behaviour. We found that a sample of gannets tracked in 2015 had longer trip durations, travelled further from the colony and had larger core foraging areas and home range areas than gannets tracked in previous years. This inter-annual variation may be associated with oceanographic conditions indexed by the North Atlantic Oscillation (NAO). Our findings suggest that this inter-annual variation was driven by individuals visiting larger areas in all of their trips rather than individuals diversifying to visit more, distinct areas. These findings suggest that, for gannets at least, if prey becomes less abundant or more widely distributed, more individuals may be required to forage further from the colony, thus increasing their likelihood of encountering pressures from spatially explicit anthropogenic disturbances. Introduction It is widely accepted that seabirds have developed flexible foraging strategies as a mechanism with which to respond to seasonal and/or annual variation in the abundance and distribution of prey (Weimerskirch et al. 2005). For example, in response to poor prey availability, seabirds may exploit more predictable prey types, lower in energetic value (Wanless et al. 2005), or they may increase foraging effort . They may do this by varying their time budget while at sea (Ronconi and Burger 2008), or by increasing the duration or range of foraging trips (Garthe et al. 2011;Monaghan et al. 1994;Uttley et al. 1994). However, this variability in foraging behaviour can have consequences for reproductive success (Becker et al. 2007). This is because seabirds are central place foragers during the breeding season, constrained to return to the colony regularly throughout the incubation and chick-rearing period. Thus, increased foraging trip duration may result in both parents undertaking simultaneous foraging trips, Abstract The at-sea distribution of seabirds primarily depends on the distance from their breeding colony, and the abundance, distribution and predictability of their prey, which are subject to strong spatial and temporal variation. Many seabirds have developed flexible foraging strategies to deal with this variation, such as increasing their foraging effort or switching to more predictable, less energy dense, prey, in poor conditions. These responses may vary both within and between individuals, and understanding this variability is vital to predict the population-level impacts of spatially explicit environmental disturbances, such as offshore windfarms. We conducted a multi-year tracking study in order to investigate the inter-annual variation in the foraging behaviour and location of a population of northern gannets breeding on Alderney in the English Channel. To do so, we investigated the link between individuallevel behaviour and population-level behaviour. We found that a sample of gannets tracked in 2015 had longer trip 1 3 156 Page 2 of 15 leaving eggs or chicks unattended at the nest and subject to attacks by predators or conspecifics (Lewis et al. 2004). Therefore, energy limitation, predation or competition can have implications on reproductive success. Ultimately, as long-lived animals, seabirds will prioritise their own survival over that of their offspring and abandon breeding attempts when prey availability is very low (Ponchon et al. 2014). Variation in oceanic conditions may influence the spatial or temporal availability of prey (Burke and Montevecchi 2009;Chavez et al. 2003). An example of this is the North Atlantic Oscillation, a climatic event where fluctuations in atmospheric pressure at sea level result in warmer, wetter and windier climates (Hurrell 1995), with warmer sea temperatures in years with a high NAO index (Sims et al. 2001). Years of high NAO have been associated with lower overwintering survival (Votier et al. 2005) and breeding performance of seabirds (Paiva et al. 2013a, b;Thompson and Ollason 2001). While research efforts have focussed on linking variation in oceanographic conditions to productivity at the population level, the role of intra-and interindividual variation in behaviour has received little attention (Wakefield et al. 2015). Indeed, in most cases variation amongst individuals in the population has been overlooked, under the classical assumption that individuals in a population behave in similar ways. Yet variation in foraging behaviour can occur both within and between individuals (e.g. Barlow and Croxall 2002;Kato et al. 2000;Woo et al. 2008). However, few studies have looked at how intra-and inter-individual variation differs between years, and what the consequences of this may be at a population level. Inter-annual variation in both the abundance and distribution of prey might lead to variation in inter-individual variability in the size, location and overlap of foraging areas (Fig. 1). Low inter-individual variation in trip duration or foraging area may occur because prey is concentrated in particular areas, attracting all individuals in a population. Alternatively, this may be because prey is sparsely distributed, and all individuals in the population have large searching areas, i.e. all individuals are going everywhere. Alternatively, high inter-individual variation suggests that prey is abundant in their distribution, either patchy or dispersed (Fig. 1). Additionally, intra-individual consistency in foraging locations of seabirds has been observed at various temporal scales across months and years in some individuals, yet others show high intra-individual variability (Ceia Fig. 1 Four hypothetical scenarios to describe the distribution of prey (blue dots) and the foraging location of seabirds (red circles); a low resource + high patchiness = small foraging area and high inter-individual overlap, b low resource + low patchiness = large foraging area and high inter-individual overlap, c high resource + high patchiness = small foraging area and small inter-individual overlap, d high resource and low patchiness = large foraging area and small inter-individual overlap Wakefield et al. 2015). These diverging strategies suggest that some individuals in a population may have greater specialisation with regards to diet and habitat use than others (Bearhop et al. 2006). This interindividual variation is essential to consider when tracking studies are used to identify important areas for conservation, because often only a small proportion of the population is tracked, and few studies take into account how well the sampled individuals represent the foraging locations of the entire population (Soanes et al. 2013a). If the foraging locations of the tracked individuals do not represent those of the entire population, then important at-sea locations may be overlooked, which may be crucial when tracking data are used to identify important areas for marine spatial planning (Soanes et al. 2013a). However, by using what we know about the size and location of the foraging areas of tracked birds, it is possible to incorporate this limitation and predict the size of foraging areas used by the entire population (Soanes et al. 2013a). Consistency in foraging locations as a result of individual dietary and habitat specialisation has been observed in northern gannets Morus bassanus (Patrick et al. 2014;Wakefield et al. 2015). This challenges their traditional classification as generalist predators that feed on a variety of pelagic fish and fisheries discards (Nelson 1978;Votier et al. 2010). Additionally, northern gannets, and congeneric populations, show inter-annual variation in foraging behaviour and reproductive success as a result of sea temperature, primary productivity and the type and abundance of prey (Angel et al. 2015;Garthe et al. 2011;Montevecchi 2007). However, most studies overlook the link between this individual consistency and inter-annual variation. This is important because while northern gannet populations are increasing at an average of 3 % per year across the UK and Ireland (Wanless et al. 2006), they have high conservation importance due to their restricted ranges, with 75 % of the world's population breeding in Europe (Gremillet et al. 2006). Consequently, there is concern that populations may be impacted by anthropogenic pressures such as over-fishing of prey stocks (Gremillet et al. 2015), changes in the bycatch policy (Votier et al. 2013) or the installation of windfarms (Furness et al. 2013). To understand how gannets are going to be affected by these pressures, a better understanding of inter-annual variation in foraging behaviour at both the individual and the population level is required. For example, if in years of low prey availability all individuals in the population visit larger areas, then all individuals may have an increased risk of interacting with these pressures. Conversely, if inter-annual variation in foraging behaviour is driven by individual birds visiting different areas, then spatial pressures may have differential effects on individuals in the population. Here we use 4 years of tracking data to investigate the inter-annual variation in the foraging behaviour and space use by a population of northern gannets breeding on Alderney, Channel Islands. Alderney's population may be particularly vulnerable due to its position near the southern limit of the species range (Brown et al. 1996), the overlap in home range with offshore developments (Soanes et al. 2013b) and the limitation in extending its range due to competition from conspecifics in nearby colonies (Wakefield et al. 2013). We investigate the link between individual-level and population-level behaviour and explore NAO as a potential driver of this variability. Specifically, we determined whether in years where the population has a larger foraging area, if this is driven by individual birds diversifying to visit more different, distinct patches of high prey availability (e.g. Fig. 1d), or by each bird increasing its own foraging area to overlap with the foraging area of the entire population, suggestive of low prey availability (e.g. Fig. 1b). Data collection Fieldwork was conducted at the breeding colony of northern gannets on Les Etacs, Alderney (49°42′N, 2°14′W), during the early chick-rearing period in early June of 2011 and 2013-2015. All procedures were licensed by the States of Alderney. Birds with chicks approximately 2-4 weeks old were captured at their nest using a noose pole. GPS data recorders, logging positions every 2 min (IgotU GT 120 (2011), IgotU GT-600 (2013GT-600 ( -2015, Mobile Action Technology) packaged in plastic heatshrink, were attached to the base of the tail using Tesa Extra Power tape. The devices weighed 22 g or 33 g, ~1 % of the body mass of an average gannet (3.3 kg, Wanless and Okill 1994). Loggers were removed 2-3 weeks later and birds not recaptured would have lost their devices within approximately 1 month (pers obs). Devices of 1 % body mass have previously been shown to have no effect on foraging duration, breeding success or body condition in northern gannets (Hamer et al. 2000). Breeding success was monitored at the colony in 2013-2015 as per the UK seabird programme monitoring methods handbook (Walsh et al. 1995). At the start of the chick-hatching period, five plots were designated, each containing 50 Apparently Occupied Sites (AOSs), and the number and age of the chicks were recorded every 7-10 days throughout the breeding season. The number of chicks which fledged in each plot were divided by 50 and averaged across the five plots in order to obtain a value of chicks fledged per pair for the colony. Due to the inaccessibility of the colony, these productivity counts were conducted via a telescope from the main island of Alderney; thus, only nests on the edge of the colony could be observed, probably resulting in a biased sample of newer, less successful breeders (Nelson 2002). Consequently, estimates of fledging success obtained in the present study may not be comparable to those obtained elsewhere. However, this potential bias should remain consistent between years, allowing for inter-annual comparisons on a relative basis. Data processing and analysis GPS positions were interpolated to every 10 s using the adehabitatLT package (Calenge 2006) in R (R Core Team 2013) to account for missing data associated with diving behaviour or occasional missed GPS locations. The colony was defined as Les Etacs rocks (49.705 N, 2.239 W) with a 30 m surrounding buffer, based on personal observations of gannet behaviour, and for each bird, each trip was defined as all points between leaving and returning to this area. Trip characteristics including: duration (hours); trip length (total distance, km); maximum distance from the colony (km); and directness (trip length/maximum distance from the colony) were calculated for each trip for each bird. Directness is a measure of deviation from a straight line, with a value of 2 representing direct movement between the colony and furthest point, and anything above this representing a less direct track. A frequency histogram of trip duration showed a clear bimodal distribution. One mode represented trips up to 40 min in duration, whereas the second mode represented trips lasting many hours. Foraging trips were, therefore, defined as any trip over 40 min in duration to discount birds loafing adjacent to the colony, or short periods of flight following disturbance at the colony. General linear mixed effects models were used in package nlme (Pinheiro et al. 2016) to identify inter-annual variation in trip characteristics. Year was the fixed effect and individuals were included as random effects to account for pseudo-replication. Post hoc Tukey tests were conducted in package multcomp (Hothorn et al. 2008) to identify between which years differences lay, and least squared means were calculated using package lsmeans (Lenth 2016) to calculate annual mean values of all trip characteristics. Warwick-Evans et al. (2015) showed that individual gannets increase time allocation in spatial locations where they forage more frequently, and that 5 × 5 km is the most appropriate scale at which to capture this behaviour. Thus, the R package Trip (Sumner 2011) was used to calculate the proportion of time spent (s) in each 5 × 5 km cell of a pre-defined grid around the colony for each bird for each year (Fig. 2). These proportions were then averaged across all birds for each year to define the most important foraging areas for the population. The cells used were ranked in order of time spent and the top 95 % were defined as the home range area (HRA) and the top 50 % the core foraging area (CFA). The CFA and HRA for each year were plotted in ArcGIS (ArcGIS version 10.2), and the size of these areas was calculated. Additionally, the cells in which individual birds spent the top 50 and 95 % of their time were calculated in order to measure inter-individual variation in CFA and HRA. Again, time spent in each grid cell can be used as a proxy for foraging behaviour, because individuals of this species spend more time in areas with increased foraging activity . To quantify the interactions between northern gannets breeding on Alderney, and windfarms proposed for development in the English Channel, the number of foraging trips which overlapped with proposed development sites (downloaded from 4cOffshore 2015) in each year was calculated using Arc-GIS (Fig. 2). In order to calculate how well the individuals that we tracked represented the HRA and CFA of the entire population in a specific year, we followed the methodology devised by Soanes et al. (2013a). For each year independently, the HRA and CFA were calculated initially for one individual and subsequently for an increasing number of individuals. The individuals included in each calculation were sampled at random from all of the tracked birds, until the total number of gannets tracked that year had been sampled. These data were then bootstrapped 10,000 times using R package boot (Canty and Ripley 2014) to determine the mean values of CFA and HRA. These data were then fitted to the Michaelis-Menten model as per Soanes et al. (2013a). This model uses information about the size of the CFA and HRA of the tracked birds to predict the size of these areas for an increasing sample size, and ultimately for the entire population. This allows us to extract the asymptotic value of the y axis (a) i.e. the size of the CFA/HRA predicted for the entire population, and the value at which half of the maximum response is attained (b) i.e. the number of individuals necessary to sample in order to represent half of the CFA/HRA for the entire population (Fig. 3). Thus, the value of b can be used to describe inter-individual variation in the location of CFAs and HRAs. Values of a and b were then used to extrapolate the CFA and HRA for the entire population of approximately 10,000 birds breeding on Alderney, for that specific year. We then calculated the proportion of the population-level CFA and HRA that was represented by our sample of gannets for each year independently. Additionally, this approach can inform us of the number of trips necessary to sample from an individual in order to represent half of its individual CFA or HRA (Soanes et al. 2013a). Thus, at the individual level, b can be used to describe intra-individual variation, or consistency, in the location of CFA and HRA. For example, if the entire CFA or HRA of an individual could be determined from just one trip, then the value of b would be low, intra-individual variation in terms of the location of CFA or HRA would be low, and consistency would be high. Thus, this approach was used to determine how well the trips we sampled from each individual represented the entire foraging area for that individual, and also how consistent each individual was between trips. In 2011, only four individuals recorded three or more trips; thus, the Michaelis-Menten equation could only be fitted for these four individuals, and conclusions about consistency within individuals in 2011 should be interpreted cautiously. In order to evaluate the overlap in space use between sampled individuals, the number of birds that used each 5 × 5 km grid cell in their CFA or HRA within a single year was calculated. Subsequently, in order to evaluate overlap in space use between years, the number of years that each 5 × 5 km grid cell was used was calculated. Additionally, for each pairwise combination of 2 years, and in both directions, the proportion of cells that were used in the populations mean HRA and CFA in year X that were also used in year Y was calculated in order to investigate the sample overlap in foraging locations between specific years. Given that the sample of the population we tracked did not represent the entire population, we calculated the population overlap using the equation. where O is the sample overlap (%) and S Y2 is the percentage of the total predicted HRA or CFA in our second year sample (See Appendix 1). This calculation assumes that for both CFA and HRA areas which are visited but not observed are as likely to have been visited as those which have been visited and observed, i.e. detection rate is equal in overlapping, and non-overlapping cells. Foraging habitats of northern gannets have previously been linked to chlorophyll a, sea surface temperature, bathymetry and copepod abundance (Hamer et al. 2000;Scott et al. 2013;Votier et al. 2010). Thus, further evidence to support these links is not addressed in this study. Additionally, this study deals with predicted population metrics, and thus, an index of oceanographic conditions at a larger scale is more relevant; therefore, the June NAO index, downloaded from www.cgd.ucar.edu/cas, was used as an index of annual oceanographic conditions. Warmer sea temperatures in years with a high NAO index influence the type and abundance of fish communities (O'Brien et al. 2000;Planque and Taylor 1998), which in turn influence the foraging behaviour of seabirds (Garthe et al. 2011). Results Northern gannets tracked on Alderney between 2011 and 2015 consistently foraged within the English Channel, though they were also recorded, on occasion, in the North Sea (Fig. 4). From 2011 to 2015, mean (±SE) trip duration Population overlap = O × 100/S Y2 Fig. 3 A hypothetical relationship between the number of individuals sampled and the size of the core foraging area for seabirds showing high and low inter-individual variation in core foraging area locations changed from 16.6 ± 2.1 to 27 ± 1.4 h, corresponding to a shift in mean length from 331 ± 34 to 476 ± 22 km, and mean maximum distance to the colony from 106 ± 9.9 to 135 ± 7 km, respectively. Northern gannets overlapped with windfarm sites less often in 2011 and 2014, than in 2013 and 2015 (Table 1). Inter-annual variation in foraging areas Both the CFA and the HRA of tracked gannets varied between years (Fig. 4). While commonly used areas around the North coast of France in the CFA and around Alderney in the HRA were observed in multiple years, sampled birds used relatively few areas consistently in all 4 years of study, especially in terms of CFA (Fig. 5). Scaling these samples up to population-level predictions also revealed differences between years in the extent of predicted CFA and HRA (Table 2). Predicted CFA was greater in 2015 than 2011, 2013 and 2014, respectively, with an increase in size of 30 % from smallest to largest. Similarly the predicted HRA was greater in 2015 than 2013, 2014 and 2011, respectively, with an increase in size of 60 % from smallest to largest (Table 2). A similar pattern was seen in terms of population and sample overlap in the number of grid cells used in different years. For CFA, 2015 encompassed a greater proportion of cells than the other 3 years (Table 3). More dramatically, HRA in 2015 was predicted to encompass all of the cells also predicted to be used by the birds in 2014 and 2011, and nearly all of those used in 2013 (Table 3). A value of >100 % was calculated for the population overlap as a Inter-annual variation in foraging trip characteristics We found strong evidence of inter-annual variation in trip duration, trip length, maximum distance from the colony, core foraging area and home range area from the tracked gannets (Fig. 6). In addition there was weak evidence of inter-annual variation in the directness of foraging trips (Fig. 6). Broadly speaking, trips in 2015 were longer in duration, distance travelled, maximum distance from the colony, directness, and birds had larger CFA and HRA than (Table 4). Additionally, there was inter-annual variation in the size of the predicted CFA for individual birds (Fig. 7a), and the higher mean and larger error bars in 2015 suggest that the CFA for individual birds was larger with higher inter-individual variation in size than in subsequent years. The predicted HRA for individual birds was not significantly different between the years; however, the large variation within years in these values suggests that the inter-individual variation in the size of HRA was also considerably higher in 2013 and 2015 (Fig. 7b). The number of trips necessary for a sample to represent half of both CFA and HRA for individual birds (b) predicted using the Michaelis-Menten equation did not vary significantly between the years, suggesting that between trips individual birds were similarly consistent in their habitat use between years. However, the within-year variation surrounding these values represents the inter-individual variation in consistency, i.e. some birds were very consistent in their foraging locations, whereas others were more variable. This variation was also lowest in 2011 and 2014 which suggests there was smaller inter-individual variation in the consistency of the location of HRA of individuals in those years (Fig. 7d). Discussion Seabirds are known to exhibit inter-annual variation in foraging behaviour at the population level, and intra-and inter-individual flexibility; however, few studies link the two. We show strong evidence of inter-annual variation in the size and location of core foraging areas and in foraging trip characteristics recorded from a sample of northern gannets breeding on Alderney, Channel Islands. Gannets tracked in 2015 undertook trips with a longer duration, length and maximum distance from the colony as well as larger CFA and HRA than those recorded in other years. This corresponded with a lower breeding success than previously recorded. This large foraging range in 2015 combined with the largest overlap of HRA and CFA between individuals suggests that all individuals travelled extensively in search of prey. Thus, inter-annual variation in the size of the foraging area for the entire population is driven by individual birds visiting larger areas in all of their trips, not by individual birds diversifying to visit more, different areas ( Fig. 1), which is indicative of low prey availability. Overlap of a 50 % core foraging area and b 95 % home range cells used by the tracked sample of northern gannets breeding on Alderney in 1 (grey), 2 (pale blue), 3 (mid blue) or all 4 (dark blue) years of study Inter-annual variation in foraging areas and trip characteristics Variation in physical oceanographic processes can alter the distributions of plankton and fish and, thus, prey availability to seabirds (Shealer et al. 2002) resulting in inter-annual variation in foraging locations for many species (Burke and Montevecchi 2009). Seabirds have developed a flexible foraging strategy as a mechanism with which to deal with this spatial and temporal variation in prey distribution Weimerskirch et al. 2005) and the inter-annual variation in foraging areas and trip characteristics of Alderney's northern gannets may be explained by this. Reduced prey availability can result in longer foraging trip duration, range and core foraging area in seabirds Suryan et al. 2000). Thus, the longer foraging trips and larger CFAs from gannets tracked in 2015 than those tracked in 2011 and 2014 may be due to lower prey availability as a result of oceanographic conditions (Burke and Montevecchi 2009). The June NAO index in 2013 and 2015 was higher than in 2011 and 2014 (Table 2), which is consistent with years of increased trip duration and range. This suggests that the NAO might be influencing the type, abundance and availability of prey and, thus, seabird foraging behaviour in the English Channel. Sea temperature may influence the structure of fish communities (Perry et al. 2005), and in warmer temperatures some prey may occur deeper, potentially becoming unavailable to seabirds (Montevecchi 2012). Links between NAO and the distribution of other seabirds such as Cory's Shearwater Calonectris borealis (Paiva et al. 2013a) and Macaronesian Shearwater Puffinus baroli have been shown. Additionally, northern gannets have been observed to travel further with a larger home range in years where larger pelagic fish were more abundant than small fish (Garthe et al. 2011), potentially explaining the larger CFA and HRA in 2015 when the NAO index was high. However, the NAO index was even higher in 2013, and although trip duration was longer and CFA and HRA were larger than in 2011 and 2014 when the NAO indexes were negative, they were not as extreme as in 2015; this suggests that other factors, such as increased fishing activity, or increased patchiness of prey, were also involved; however, we could not evaluate this further within the scope of our study. The combination of the increased foraging range and large overlap of HRA and CFA between individuals in 2015 implies that all individuals had large searching areas, i.e. all birds were going everywhere in search of prey (e.g. Fig. 1b), rather than to consistent individual-specific foraging areas (e.g. Fig. 1d). This suggests that prey was widespread and thinly dispersed, which is consistent with the less direct path between the colony and foraging areas observed in that year. Gannets showed the most direct path between the colony and the foraging areas in 2013, again suggesting that prey may have been in more predictable locations in that year (Pettex et al. 2010). Trip duration was higher and CFA and HRA smaller in 2013, than in 2011 and 2014 and this, combined with a more direct commuting path, suggests that gannets were foraging in more predictable locations, further from the colony in 2013 (e.g. Fig. 1a). However, the directness of foraging trips may also be related to other behaviours such as wind direction (Gremillet et al. 2004), or following fishing vessels (Votier et al. 2010), or conspecifics (Buckley 1997). The lower HRA combined with fairly direct trips and shorter trip durations in 2011 and 2014 suggest that birds were foraging at predictable locations with higher prey availability closer to the colony in these years. Breeding success was also lower in 2013 and 2015 than in 2014 and may be a result of the increased foraging trip duration in those years. If adults have had to travel further from the colony in order to forage, they may have failed to return with sufficient food for chick provisioning (Baird 1990), or at a sufficient rate in order to maximise reproductive success (Suryan et al. 2002). Additionally chicks left unattended at the colony are open to attacks by predators or conspecifics (Lewis et al. 2004). In general, there was little overlap in the locations of sampled CFA between years, with only 8 of the 5 km by 5 km cells being used in all 4 years. This suggests that the distribution of prey varied between the years. However, the 5 km × 5 km cells used for these analyses are small in comparison with the scale of some Area Restricted Search (ARS) behaviour observed in gannets (Hamer et al. 2009); thus, overlap in foraging location at these larger scales is omitted. However, a previous study of the foraging behaviour of Alderney's gannets found that this was the most efficient scale to capture their search behaviour . Furthermore, we know that our sample under-represents the population CFA and HRA and that sample overlap is thus lower than population overlap (Table 2). Thus, we can assume that more cells are actually visited in multiple years. Overlap in HRA between the years was much larger, as birds tended to commute along similar paths to reach foraging areas, particularly towards Northern France and south-west UK where foraging occurred in all 4 years. In fact, sampled birds in 2015 used all of the HRA cells used in 2011 and 2014, and most of those used in 2013. This is further evidence that it was necessary for these gannets to travel further in order to forage in 2015, and thus, prey items were more widely dispersed. Intra-and inter-individual variation Gannets tracked on Alderney in 2011 required fewer trips to be tracked in order to represent half of the CFA of individual birds than in subsequent years, i.e. these birds displayed lower intra-individual variation (higher consistency) in the location of the CFA of individual trips than those tracked in later years (Fig. 7c). However, these results were not significant, probably due to the low sample size of individuals with multiple trips recorded in this year. The values of b, in terms of CFA, were similar amongst the subsequent 3 years, and thus, inter-annual variation in this intra-individual variation cannot be confirmed. The low inter-annual variation in b in terms of HRA illustrates that intra-individual variation in the location of the HRA was similar between years. However, the variability in this value, described by the error bars, was considerably larger in 2013 and 2015, than 2011 and 2014, demonstrating higher inter-individual variation in their intra-individual variation in 2013 and 2015. Gannets tracked on Alderney in 2015 displayed lower inter-individual variation in the locations of CFAs and HRAs than in previous years, as described by the low b value (Table 2). Low levels of inter-individual variation in the location of CFAs suggest either that prey is concentrated in small areas, attracting all individuals (e.g. Fig. 1a), or that prey is sparsely distributed and all individuals in the population have large searching areas. The low inter-individual variation observed in 2015 combined with the larger CFA strongly suggests that, of these two alternatives, this inter-individual variation was driven by individual birds visiting larger areas (Fig. 1b). Combining this low interindividual variation with the large overlap in CFA between sampled individuals in that year, we can suggest that the inter-annual variation in the size of the CFA for the entire population is also driven by individual birds visiting larger areas, and not by individual birds visiting more, different areas (Fig. 1d). Consistency in foraging locations within and between individuals has been shown in northern gannets (Patrick et al. 2014;Wakefield et al. 2015) and other seabirds (Irons 1998;Weimerskirch 2007) and may be due to individual specialisation in diet (Bearhop et al. 2006;Patrick et al. 2014;Woo et al. 2008) or predictability of prey patches (Hamer et al. 2001;Weimerskirch 2007). However, this consistency is rarely considered at an inter-annual level, although Wakefield et al. (2015) demonstrated that gannets show intra-individual consistency in foraging areas across years, due to long term dietary specialisation, and site familiarity gained in early life. Our data suggest that in the more challenging foraging conditions of 2013 and 2015, more individuals in the population were generalist in terms of foraging locations, however this may be due to selecting different proportions of individuals with different foraging strategies, in terms of generalist or specialist, in different years. Limitations and implications Predictions from the Michaelis-Menten equation indicate that in no year did our sample of gannets fully represent either the HRA or CFA predicted for the entire population breeding on Alderney. This is likely to be the case in the majority of seabird tracking studies as devices can be costly, and logistics of getting to colonies may limit the frequency of fieldwork, which can result in only sampling a small proportion of the population. The relative importance of this limitation depends on the question being asked. If differences in the trip characteristics between groups, for example males and females (e.g. Cleasby et al. 2015), are being investigated, then it could be assumed that underrepresentation of the entire population in terms of trip characteristics would not be biased in either direction, and thus would not influence the conclusions. However, if the location of CFAs or HRAs is being explored, then this can have important consequences, particularly if tracking studies are being used to identify important areas for conservation or marine spatial planning. For example, the proportion of birds/trips entering windfarm sites in this study are also likely to be underestimated, which, in turn, may have implications when predicting the impacts from these devices. In this study, the number of birds necessary to track in order to represent the CFA for the entire population varied annually, as a result of differences, between years, in the inter-individual variation in the location of CFA. It would have been necessary to track many more birds in 2011 than in the subsequent years. However, only 2.4 trips per individual were recorded in 2011, considerably fewer than in subsequent years, and this supports the idea that gannets display intra-individual variation in foraging locations and highlights the importance of sampling multiple trips per individual (Soanes et al. 2013a). This inter-annual variation in the number of birds necessary to track to represent the CFA of the whole population was also observed in years where similar numbers of trips per individual were recorded (2013)(2014)(2015). This indicates that inter-individual variation in the location of CFA differs between years, and should be an important consideration in tracking studies. Gannets tracked in 2015 undertook foraging trips with a longer duration and length and a larger CFA and HRA than gannets tracked in previous years. These inter-annual differences in foraging behaviour are driven by differences in the intra-and inter-individual variation in foraging behaviour and location between the years, and may be associated with variation in oceanographic conditions, and a lower breeding success (Becker et al. 2007;Garthe et al. 2011). Years with sparsely distributed or low abundance of prey, may become more frequent as a result of exploitation by commercial fisheries or climate change (Perry et al. 2005). This may result in increased trip duration, potentially leading to lower reproductive success through both energy limitation and predation or competition (Lewis et al. 2004). Additionally, if core foraging areas and home range areas of individual birds increase, then more individuals are likely to encounter pressures from spatially variable anthropogenic disturbances, such as the development of windfarms. Indeed, gannets tracked in this study overlapped with windfarm sites less often in 2011 and 2014, than in 2013 and 2015, in terms of both birds and trips. Furthermore, intra-specific competition from the large North Sea gannetries may limit the foraging range of Alderney's gannets (Wakefield et al. 2013). Interestingly, Alderneys gannets show consistency in their westward boundaries, most likely because the gannets from Les Sept Iles forage in the western English Channel (Gremillet et al. 2006), thus limiting the potential range of Alderney's gannets. If North Sea gannets limit the northern boundaries, then Alderney's gannets may be forced to alter their time budgets or prey type in years of poor food availability. This may have negative impacts on reproductive success, as alternative prey items may have a lower energetic value, or altered time budgets may be more energetically costly. Acknowledgments The project was funded by a CASE PhD studentship from the Natural Environment Research Council and the Alderney Commission for Renewable Energy. Permission to carry out field work was granted by The States of Alderney. The Channel Island Bird Ringing Scheme gave permission to ring the gannets. We would also like to thank Tim Morley, Holly Marshall and Jenni Godber for help in the field. Funding This study was funded by a CASE PhD studentship from the Natural Environment Research Council and the Alderney Commission for Renewable Energy (Grant number NE/K500975/1). Compliance with ethical standards Conflict of interest All authors declare that they have no conflict of interest. Ethical approval All applicable international, national and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Appendix: Overlap equation The equation to calculate the overlap in cells used for the CFA/HRA between years, for the entire population is: Population overlap = O × 100/S Y2 , where O is the observed overlap (i.e. the % overlap in cells used for the CFA/HRA between years in our tracked birds) and S Y2 is the percentage of the total predicted CFA/HRA sampled in year 2. Y1 and Y2 represent year 1 and year 2 respectively. This equation was derived from a series of possible overlap scenarios: 1: If a cell was used in the CFA/HRA in year 1 and year 2 (Population overlap = Y1Y2) then the cell could be observed in year 1 and year 2 (Y1Y2), observed only in year 2 (-Y2), observed only in year 1 (Y1 -), or not observed at all (--). Alternatively, if a cell was used in the CFA/HRA in year 1 but not in year 2 (population overlap = Y1 -) then the cell could be observed only in year 1 (Y1 -), or not observed at all (--). PopulaƟon Overlap Observed overlap Probability C ount Y1 Y2 qp 1 p 2 n 1 -Y2 qp 2 (1-p 1 ) n 2 Y1Y2 Y1 -qP 1 (1-p 2 ) + (1-q) p 1 n 3 Y1 --q(1-p 1 ) (1-p 2 ) + (1-q) (1-p 1 ) n 4 Y1 -p 1 p 2 p 2 (1-p 1 ) P 1 (1-p 2 ) (1-p 1 ) (1-p 2 ) p 1 (1-p 1 ) q (1-q) Let q be the probability that a cell that was visited in Y1 was also visited in Y2 (i.e. the population overlap), let p 1 be the probability of observing a cell that was visited in year 1, and p 2 be the probability of observing a cell that was visited in year 2 (i.e. the proportion of the total predicted HRA or CFA that was sampled). Then the probability of each outcome can be calculated as follows: Thus, the proportion of cells observed in year 2 which overlap with cells observed in year 1 = n 1 /(n 1 + n 3 ) (i.e. the number of cells visited and observed divided by the total number of cells visited, whether or not they were observed), thus the percentage overlap (O) = 100 × n 1 /(n 1 + n 3 ). We know that n 1 = nqp 1 p 2 , where n is the total number of cells and that Thus so q = O/(100 × p 2 ). We know that S Y2 = p 2 × 100 Thus population overlap (%) = O × 100/S Y2 .	2022-11-22T15:40:54.393Z	2016-06-18T00:00:00.000	{ "year": 2016, "sha1": "6de4917da861fe15040d671da3eb1f240fbcbeb1", "oa_license": "CCBY", "oa_url": "https://link.springer.com/content/pdf/10.1007/s00227-016-2922-y.pdf", "oa_status": "HYBRID", "pdf_src": "SpringerNature", "pdf_hash": "6de4917da861fe15040d671da3eb1f240fbcbeb1", "s2fieldsofstudy": [ "Environmental Science", "Biology" ], "extfieldsofstudy": [] }
211161049	pes2o/s2orc	v3-fos-license	A Systematic Literature Review Paper on Online Medical Mobile Applications in Malaysia Abstract—The introduction of mobile devices to the worldwide market has marvelous possible to disturb the way Health care is providing. In this paper, we will overview the work done in the five years from 2014 to 2018 in the field of mHealth in Research perspective. For that purpose, we choose the Scopus database to review the past research published on mHealth in Malaysia. For that purpose, the quantitative review has been observed in bibliometric analysis and a Qualitative review is done through systematic review in the order through PRISMA (Preferred Reporting Items for Systematic Review and Meta-Analysis). After the selection of 58 papers, the process based on the different steps. In the first step, the corresponding to Microsoft excels in a descriptive analysis of the published literature on mobile Healthcare in the field of online Healthcare like the distribution of the year, distribution of subjects and distribution of the author. Quantitative studies are 15 in number collected from the past literature, the researcher used the quantitative method for measuring the results Quantitative research collects data that will be processed to understand the indicators, overall trends, and requirements of the market. The qualitative studies collected from past research were 27 in numbers, collected studies were processed on the excel sheet to find out the areas discussed in the past. Traditional Health Care of Malaysia is top of the list in the world, but the mHealth still needs to improve in the region. Past studies in the mHealth are discussing physical health and wearable devices in detail with connectivity to smartphones but serious diseases are cover in very some studies. Like diabetes and HIV apps and patient are not highlighted in the collected data. Patient record management and coordination with families are also part of some research studies and that is very important for recovery in some cases. Introduction The Malaysian health sector scored 95 percent out of 100 in the recent medical rankings, Malaysia takes the top spot in the Healthcare category of our Annual Global Retirement Index. The healthcare in the Southeast Asian gem is simply world-class with up-to-date and sophisticated infrastructure. With 13 JCI accredited hospitals in the country and almost every doctor fluent in English. In fact, most doctors were trained in the UK, U.S., or Australia so communicating is flawless. Malaysia is one of the top tourist countries with medical assistance and facilities, The health facilities include in one of the top ranks in the report of Joint Commission International and two top score hospitals are available in Kualalumpur and two others are Penang, both cities are hustling and bustling with the tourists from all over the globe. Experts are normally choosing the hospital which is easily available and having quality services according to the requirements of patients and tourists in case of emergency, whether it may be private or public hospital. It is commonly believed that private hospitals are more expensive than public hospitals, but private hospitals standards are much similar to Western Healthcare Centers and hospitals. Some people are convinced on Private hospitals are very much relative fees with the Public hospitals and very economical for international Tourists due to more value of their currencies as compare to Malaysia. In Malaysia, you don't need to make a long appointment and wait for specialist doctors and any referral from the Government for emergency treatment. Its very simple to take a receipt from the receptionist and wait for your number for Doctor/specialist of choice. Malaysia healthcare sector is much cheaper as compared to many other countries of the world with high-level facilities, normally its very much equal to the same amount at home and fractionally more at the hospital for the local people. In Malaysia the pharmacist, medical staff and nurses are more trained and skillful with well-informed information about the latest changes in the medical field. Malay people are very simple and well-mannered people and genuinely take interest in that to impress, they are very friendly people and have a smile on their faces most of the time. ("The Best Healthcare and Healthcare Systems in the World 2019," ) IL Malaysia Correspondent Keith Hockton, who lives in Penang says, "Recently, I decided on a whim to have a medical. I'd never had one done before and as I had a free morning, I decided just to pop into the Lam Wah Eee Hospital. I was already registered and found myself sitting outside a GP's office, not five minutes after arriving. Within an hour, I had been examined by a doctor, had an ECG and blood and urine tests done…and I was on my way home. "The total cost of the visit was just $44. The doctor who had examined me called me later that afternoon with the results. It's this level of service that makes medical in Malaysia not only an attractive option but also a non-scary one. It's all so easy." Most people from neighbor countries like Indonesia, Hong Kong, and Singapore have come to Malaysia for medical assistance due to low medical expenses and high rated health care services. Usually all doctors and specialist can speak English and most are professionally trained in the USA, UK, and Australia, that's why they are familiar with the Western standard of health care. Also, many of the hospitals in Kuala Lumpur and Penang are JCI accredited, meaning that they are considered to meet the gold standard in healthcare throughout the globe. More than 800,000 foreigners seek treatment in the hospitals in Penang and Kuala Lumpur every year. There are specialists in every hospital, but unlike in the U.S., you don't have to wait for months to get an appointment. Just turn up to the hospital, register, then take a number and wait your turn. If you are then referred to another doctor or need to get an X-ray or scan, that will also happen on the same day in the same place. Prescriptions in Malaysia cost a third of what you pay at home. But it's not just the cost that's attractive; it's the service. The pharmacists, like the rest of Malaysia's medical staff, are well trained and informed. Malaysians are friendly people, but it's the genuine interest that they take in you, no matter how small or large the issue, which impresses. It takes you back to a time when personal service meant something. That same service is alive and well here. There are doctor's clinics throughout the country, which are perfect places to get treatment for something minor like a cold, flu, or sinus infection. They usually charge $10 and because these are small clinics you won't have to wait if you would in a busy hospital. But for anything more serious, it's best to go to a specialist or general practitioner in one of the many top-notch hospitals in the country. A first-time doctor or specialist visit is usually between $15 to $65 with follow-up visits around $11 to $28. If you are admitted, the overnight stay will cost roughly $55 to $200 for a private room per night. Many of the hospitals offer health screening packages which include a physical, chest X-ray, ECG, blood work (43 different tests), abdomen ultrasound, and a vision test. More specific tests can be added on, but the basic package starts at less than $120. Dentistry in Penang is just as high quality. Just like the doctors, most are schooled in the West and speak English. The technology is the same, and in some cases more advanced than at home, depending on the office you go to. Cleanings start at $22 at a modern office with state-of-theart equipment, and it's only ("Healthcare in Costa Rica -International Living Countries," )$29 for a filling. Porcelain crowns start at $400, all just a fraction of the cost in the U.S. There is a two-tier healthcare system in Malaysia; government-run universal healthcare and a co-existing private healthcare system. Expats can choose whatever hospital they want and pay out of pocket if they don't have insurance. Most expats choose to go to the private hospitals (which tend to be more expensive) instead of the public ones and will still save money when they pay out of pocket for most minor visits. Private health insurance is available, and many expats take out policies for any major health issues. International insurance companies like AIG, BUPA, and Cigna offer various plans for expats-some include medical coverage while you travel as well. ("Healthcare in Costa Rica -International Living Countries," ) The introduction of mobile devices to the worldwide market has marvelous possible to disturb the way Health care is providing. By 2021, The smartphone devices will expectedly to reach 1.5 Of these mobile devices, Smartphone and applications will most popular technology in the near future (Nussbaum et al., 2019). Since the internet creation and availability in every corner of the world, its great use, most importantly in the developing countries of the earth has generated another way of living life (Nussbaum et al., 2019). One of the more powerful instrument is Mobile Health; Internet developed a high reputation in telemedicine and telehealth, now present in every modern health care organization the mHealth corner is growing gradually (Martínez-Pérez, de la Torre-Díez, & López-Coronado, 2013). Developing the field of mobile Health or Telehealth, eHealth has arrived as the new phenomena, technology, and commerce, with commerce and technology as tools in the service of health (Oh, Rizo, Enkin, & Jadad, 2005). In this paper, we will overview the work done in the last five years from 2014 to 2018 in the field of mHealth in Research perspective. For that purpose, we choose the Scopus database to review the past research published on mHealth in Malaysia. Methodology For the development of theory, a literature review is playing the role of facilitator, that is also helping to fulfill the possible research gaps, and highlight the areas where more or further research is needed within the available literature that are cover the topic (Y. Chen & Zheng, 2019). The main objective of the paper is to provide a systematic and realistic sketch of the mHealth in the last five years within Malaysia. For that purpose, the quantitative review has been observed in bibliometric analysis and a Qualitative review is done through systematic review in the order through PRISMA (Preferred Reporting Items for Systematic Review and Meta-Analysis) (Y. Chen & Zheng, 2019). There are four main steps for Literature review, Quality assessment, eligibility and inclusion criteria and the study included in qualitative and Quantitative relevant synthesis studies is employed ( Figure 1). The descriptive literature reviews minimize the bias in a systematic review to identification, selection, synthesis and a brief summary of different studies. The summarize the important results of the literature through the systematic review is not only done but also differentiations among the studies (Nussbaum et al., 2019). The study of a research topic evaluation, top trends and using frequent methodologies in bibliometrics use of Quantitative analysis to measure the importance of publications in the field-specific (J. Chen, Zhu, Liu, Chen, & Yang, 2018). Literature Research The possible comprehensive literature from the published research is collect from Database Scopus on the topic of mHealth in the years 2014 to 2018. The filtration of the language is used during the collection of data from the database, the papers are collected and selected only from Malaysia perspective of mHealth with filter option in the database. The all possible journals of the Scopus database are used for the collection of data, for the maximum collection of relevant data, the filter on the subject wise research is not utilized. The keywords are used for the data collection and search is "mobile AND healthcare", the total results were displayed by the database 21258 and when filter used for the years from 2014 to 2018, the result was found 10318. The document type for this review paper has only selected the Article and when the option used for the article only the surprising results were changed to 5902. The conference papers and review papers are not selected for the review. In the last when the country/region is selected for the review paper, only 58 studies were remains type for this review paper has only selected the Article and when the option used for the article only the surprising results were changed to 5902. The conference papers and review papers are not selected for the review. In the last when the country/region is selected for the review paper, only 58 studies were remains. Quality assessment The review paper is based on original articles, all the review papers, and conference papers were excluded during the process. The duplication of the papers was also checked very keenly to avoid the duplications of papers. Moreover, the abstract and conclusion were screened the narrow down the available records. Additionally, reference and citations were also checked. Eligibility and inclusion criteria The identified and available literature, papers were moved forward through a very comprehensive and highly accurate selection process. The articles in the languages are used and the English language is selected for the collected papers. The all possible journals in Scopus were selected for the data collection process. This paper reviews the mHealth literature and health-related applications are also included in the collection of data. Some of the studies also discussing the mobile wearable devices in the mHealth were also selected. During the selection articles with no open access and with open access are also selected. The number of qualitative studies is higher the quantitative in the study. The duplications of the papers are strictly monitored during the process. Studies included in qualitative synthesis After the selection of 58 papers, the process based on the different steps. In the first step, the corresponding to Microsoft excels to a descriptive analysis of the published literature on mobile Healthcare in the field of online Healthcare like the distribution of the year, distribution of subjects and distribution of the author. In the other step, the content analysis was done to identify and analyze the main research streams, reporting the absolute way on the different areas and also mentioning the future opportunities and challenges to research (Rodrigues & Mendes, 2018). Descriptive analysis The figure shows that there are many publications on the topic of mHealth many numbers of publications are published in the last five years. The mHealth is very much getting popularity as the digitalization of the economy growing day by day very rapidly. In this paper, the main concept is to find the overall work done on the mobile healthcare sector over the years. The Malaysian healthcare sector is the world best due to the resources and trained medical staff with highly scientific equipment at affordable prices ("The Best Healthcare and Healthcare Systems in the World 2019,"). The World Health Organization (WHO) Global Observatory for e-Health (GOe) defined m-Health or mobile health as medical and public health practice supported by mobile devices, such as mobile phones, patient monitoring devices, personal digital assistants (PDAs), and other wireless devices ( Year base The Subject base The subject base chart is explained that the number of publications related to different subjects. The highest articles are related to medicine subject. The medicine subject is at the top numbers with 23 % articles. The collected data is not limited to one subject that presents the diversity of the study. The medicine field researchers are working more efficiently on mHealth to open new opportunities for the patients. The second on the list of publications in the last five years is computer science subject. the subject is covering 21 % publication are that is very high and important figure due to the involvement of computer and internet in the mHealth. The Engineering base studies are 19% and the average is high as compare to the other subjects. The Biochemistry, Genetics and Molecular Biology are 5 large studies contained in the current study, with 6% publications. Agricultural and Biological Sciences, Materials Science, Pharmacology, Toxicology and Pharmaceutics and Social Sciences are having 4 % articles from each subject. The other areas are also having articles in the study. Cited by The graph of the most cited articles in the study shows that the Advances in Smartphone-Based Point-of-Care Diagnostics is the article, which is cited 78 times and the author of the article is Husain, W with the other co-authors. The main author is affiliated with the School of Computer Sciences, Universiti Sains Malaysia, Penang, Malaysia. The citation of the article is very high as compared to the other articles collected in the review paper. Organizational decision to adopt hospital information system: An empirical investigation in the case of Malaysian public hospitals is the second-highest cited article with 42 times. The author Ahmadi, H., Faculty of Computing, Unversiti Teknologi Malaysia. The third most cited paper is Smart environment as a service: Three-factor cloud-based user authentication for telecare medical information system, that is cited 39 times and the other of the study is Siddiqui, Z. Gamification Solutions to Enhance Software User Engagement-A Systematic Review is the fourth most cited study collected for the review paper from the Scopus database. There are some other high studies shows in the graph. The number of high cited studies are collected from the Scopus and reviewed in the study. Journal base The journal base graph of the study shows that international Journal of Interactive Mobile Technologies articles were 5 and journal of Telecommunication, Electronic and Computer Engineering were also having 5 articles. Both journals published articles are collected from the Scopus database. The mHealth is very much gaining the placements in the journals due to its importance in the research world. The International Journal of Human-Computer Interaction, Journal of Telecommunication, Electronic and Computer Engineering and PLoS ONE are the journals having the 4 papers each. The journals are very much placing research on the mHealth. Classification The published articles are processed in the Microsoft Excel sheet and open the process of classification for further review. The three main categories are decided to analyze and understand the work has been done. The past published literature is discussed in detail to find out the required information and future base agenda for the new researchers. Method base classification The detail and deep discussions are done in this section after the classified categories of the already work done. Quantitative method Quantitative studies are 15 in number collected from the past literature, the researcher used the quantitative method for measuring the results Quantitative research collects data that will be processed to understand the indicators, overall trends, and requirements of the market. An analytic create questions to find out the relationship between the factors involved in the survey (Song & Zhang, 2019). The researcher has permission in quantitative research to find the theories are created deliberately to find some solutions to the problem. For that, there is the importance of performance and reason for performance, secondly available information is initially due to the statistic that is easily calculate able and summarizes (Song & Zhang, 2019). The numeric data examination is done through mathematical standard procedures (Kim et al., 2002) and the last procedure is an arithmetic term used in quantitative research. Most of the collected Quantitative papers Health care of the patients and use statistical tools to find the results. Mobile Health can be accessed every android mobile user, now the mobile devices are very much easier to use with applications, to develop and improve the affordability, availability, and quality of the services provided by the devices in healthcare services (Marrie et al., 2019). Now the more powerful operating systems of the smartphones make users to install additional software and big data storage and fast processing capabilities. Due to the developed and more updated capabilities, smartphones are converted into handheld computers (Marrie et al., 2019). The clinical practice and research in the field accepted the great power of mHealth in the current time. By reshaping the communication and interaction between patient, doctors, and researchers to get more reliable data for quantitative research. The developments are significant and very relevance's to healthcare (Cvrkel, 2018). Some of the collected studies also discuss adult Mobile Health care, the adult is suspectable of mental incapacities and disabilities, Globally the using of mHealth in the older population is increasing quickly, as compared to any other age group, the reason behind that is life expectancy and a decrease in the birth rate (Alam, Hoque, Hu, & Barua, 2020a). The mHealth is a revolution in health care, m health can control the Heart Fails through self-management. Mobile phones are the ideal mediums to intervene in the Heart Fails. The mobiles phones are used as the large monitoring system and also smartphones can deliver the Heart Fail related educational messages for the patients (Cajita, Hodgson, Budhathoki, & Han, 2017). This does not improve the quality of management but also control the mortality and quality of life. Some of the studies also discuss the very important matter of maternal health or pregnant women cares in past research. The pregnancy challenge of life not for a single but two lives are on stake. We need to set out and understand the needs and demands of a pregnant woman. In order to design a much better health management application to measure the realities and develop a food plan for maternal during the days of pregnancy (Peyton, Poole, Reddy, Kraschnewski, & Chuang, 2014). Some studies talked about mHealth applications for health care. The advancement of Mobile applications in mobile health is improving individual health, quality of life, shaping a healthy lifestyle and remote living patients monitored. The rule-based system is one of the most relevant components such as applications to overcome deductive mechanism reasoning ("Healthcare in Costa Rica -International Living Countries," 2018). Five billion smart users are using the phones worldwide and that is 85% used for commercial use. Approximately 63 % of Malaysians are using the smartphones, almost every smartphone user is using the WhatsApp application that is one of the most compatible application. WhatsApp application is very popular Malaysia and world third leading smartphone internet using nation ( The Mobile Applications usage is significantly growing, the number of applications during the year 2010was 5820, that was on the health and wellness ("Research \| mobi health news," 2011), during the year 2013, the number of applications was raised up to 17000 medical apps globally (Liu, Lin, & Sadeh, 2014). The mobile applications are consistently gain the confidence and reliability from the patient prospectus. The number of users is still trusting very much to adopt the technology base application to enhance health care. By the year 2015, 500 million people are utilizing the services of the mobile apps and confidence is increasing day by day due to advancement in the android and IOS applications operating systems (Hageman, 2016). The mHeallth apps are more reliable and help in finding the instant solution in case of emergency. The researchers need to find mobile apps for the distance living community to find the access for medical assistance. The mobile apps are also helpful to save time of doctors and the patients. The applications are easily available in the play store and users can install. Qualitative method The qualitative sampling strategies for the qualitative reject by the many researchers due to not using the criteria of universal principle, but some researches appreciate the idea in qualitative research and called it a treasury idea for novel researchers (Curtis, Gesler, Smith, & Washburn, 2000). In the current years, most of the qualitatively oriented health researchers are gaining popularity due to adding the values for the health care sector. The qualitative evidence in the process is stressed by the WHO (World Health Organization) for usefulness to assess the needs, the experience of the stakeholders and expectations of the patient, that is very much complex for the healthrelated decision making (Curtis et al., 2000). The qualitative studies collected from past research were 27 in numbers, collected studies were processed on the excel sheet to find out the areas discussed in the past. Patient management is very much disor-ganized and high error in clinical communications due to incompleteness. Traditional face to face meetings, appointment timings issues, and telephonic communications was very much hectic and adverse for the patients. The opportunity for the advancement and development in Clinical management was needed (McElroy, Ladner, Saf, & 2013). The innovative and highly sophisticated advancement in the field of Clinical communications changes the perception and future of Patient management. The highspeed internet, smartphones connectivity with wifi and cheap internet packages for the android and iPhones make access to information easier and immediate responses on the situations (Ai et al., 2011). Clinical Communications are also revolutionized due to the technology advancements, now patient management system makes access to a large number of medical information, imaging reports are available instantly that saves time and give ultimate solution in minimum time (International Living, 2018). According to WHO, patient care with the help of the mobile devices and smartphones are the new horizon of mHealth for clinical communications, there are a number of tools like WhatsApp, WeChat, tango, Viber or line allowing health care professionals to create contact with the patients (Free et al., 2013). The applications are not only facilitating the message and call feature but also offering the video and audio messages. The practitioner can easily generate groups and allow multiple users to interact with each other (Beratarrechea et al., 2013). Mobile applications not only used for the social media level but some of the top business organizations apps also discussed in the literature, the apps are specifically designed for the purpose of medical assistance. The Zigbee (XBee) is also discussed in the study to control the high blood pressure, this is measuring the blood pressure without any usage of wire, the system is consist of the devices (Ling, Tan, Wong, & Lee, 2015). The developing countries are moving to adopt mHealth facilities very quickly due to less amount of infrastructure and high-volume population. mHealth not only achieve healthcare-related targeted but also integrated health system management such as sustainable health goals (Ling et al., 2015). The mHealth and health basic system is very much important for the government and policymakers to identify the future need and demand for health care of public like financial risks protection, quality essential of healthcare and affordable medicines for everyone (Istepanian, Jovanov, & T, 2008a). Self-observing devices are also nowadays coming up rapidly to monitor personal health schedules. The wearables are very smart and connected with smartphones to monitor and store the record. Wearable devices are maintaining records of fitness, calories burned and gain (Istepanian et al., 2008a). mHealth wearable devices are very much effective in disease removal to enhance the level of fitness. Development of mHealth devices open the options of personal doctor to maintain selfcare (Alam, Hoque, Hu, & Barua, 2020). Wearable devices or tools of mHealth are provided the real-time data and health measurements that is user engagement. The engagement is defined as the way people are using resources and smartphone applications (Istepanian et al., 2008a). Collected research discusses wearable devices to a real-time game-changer in individual health care. Qualitative studies are more talking about the mHealth applications for wearables and smartphone applications use for individual fitness and basic healthcare issues. There is still the development of mHealth applications are not at an appropriate level. The researchers are lacking to discuss maternal health, old age citizens health care and serious illness. The researcher needs to extend research find out batter way to create the growth. Segmentation and context The most important section of the study is to the discussion made upon segment and context discuss in past literature from Scopus database drive papers. Most of the literature is not able to explain or define mHealth in a convincing manner. The term mHealth is driven near in twenty century from the landscape of health technologies and the discipline to which they were used. The comprehensive definition or domain are not explained but the elements and tools are suggested. In this study, we will look upon the ontology of mHealth and construct of the term from the collected data (Istepanian, Jovanov, & T, 2008). The (Istepanian et al., 2008) defines the mHeallth " smartphone or mobile computing, medical sensor and communication technologies for healthcare". The article suggested that "endless mobility and wireless global healthcare connectivity". The recent most articles are defining as the process is moving towards the targeted personalized system with adaptable elements with compatibility with future updated 4G and 5G networks. This definition is very much concentrating on the hardware and network transitions with the passage of time connectivity with medical transitions. The World Health Organization (WHO) is still missing the standardized definition of mobile health or mHealth (WHO, 2011). The mobile devices, patient monitoring devices, personal digital assistants are promoting or supporting medical practices or applications, radio messages, mobile messages, and mobile phone support 3G and 4G networks. The WHO also including the Global Positioning System (GPS) and Bluetooth technology(WHO, 2011) (Speciale & Freytsis, 2013). Mobile health segment is frequently discussed in the context and focus section of the collected data from past articles. The usage of information technology (IT) was developing after the twenty century raised many questions about the health care of individuals and masses. The innovation needs to enhance the healthcare facilities effective tools of health was a great debate. The mHealth concept was to approach the rural areas for health care awareness and possibilities (WHO, 2016). A study discusses the integration of Malay Traditional Medicines with modern medicines in postnatal care. Traditional Malay medicines and western medicines are usually based in a different system and in isolation, but the concept of the researcher was to integrate services through an application MyPostnatalSys. The study concludes that healthcare will get the benefit from the standardization of exchanging the tools and information with an individual's health cares (Istepanian et al., 2008). Another area is discussed in the collected data is about Atrial Fibrillation which is an unfamiliar medical condition for the general patients, AF is very dangerous for the low literacy patients about the medical situation (Ayyaswami, Padmanabhan, Crihalmeanu, et al., 2019). Cardiovascular diseases also reported by some of the groups during the collected data (Ayyaswami, Padmanabhan, Patel, et al., 2019). The applications are needed to improve the quality and educate the patient about self-care for AF. Studies determine the accuracy and effectiveness of mobile apps in google play store and also in the apple app, the mobile health apps are growing more and more in users. The content and accessi-bility to users are measures in the studies and it is found that most apps are lacking scientific validation and need reading-grade beyond the high education (J. Chen et al., 2018). Weight management is also discussed by some studies, smartphones and wearable devices are very much effective in weight control and physical health care. (Dounavi & Tsoumani, 2019) believes that chronic disease is very effectively controlled through weight management. In the last few years, researchers are reviewing the literature about weight management and generating awareness about healthy foods and achieving physical health with the use of applications to control weight. Some studies in Malaysia talks about mental health too, mental health is a very hot topic in recent years due to anxiety and depression. Clinicians moved some portable applications and devices as a tool to aid and support in the rehabilitation of patients (Torous, Staples, Reports, & 2015). The patients are feeling not good and confidence to attend face to face therapy, results are very much effective to reduce the mental illness. Professional believes that when mobile applications used with medication to decrease the mental illness much more result-oriented (Alamri, 2015). In the literature, a study discusses the Coronary Heart Diseases, in middle-income countries and middle east countries heart disease is very dangerous and most killer cause of the death (Motlagh, O'Donnell, & Yusuf, 2009). In addition, Cardiovascular disease is one of the most killer cause in Malaysia also due to its dangerous history in patients. The researcher introduces the idea of IHeart mobile application that is a location-based cardiac emergency system to observe and monitor the victims of hypertension and arrhythmia. iHeart is designed to monitor patients, it must create a relationship between patient, Health care professionals and affected families for needs and feedback (Motlagh et al., 2009). The segment of the study overview most of the apps that are designed and identify in collected past data from the Scopus database. Mobile applications are very much effective for the patient health care in Malaysia, development of networks into 4G and 5G create more options for users to adapt the technology base apps. Some of the researches talk about the mHealth is needed to create value for patients. Mobile applications are in good quality, but some time google play store showing applications with low quality and demanding too much personal information, that causes security concerns for the users. The applications are needed to improve more and reliability of the users. Conclusion After processing 58 research papers from the Scopus database, cannot avoid the evidence and the majority of Articles are dominant in mHealth through long term management. However, mHealth apps are still needed to improve in many dimensions. No dout about it mHealth is a hot topic in the current scenario, WTO believes that mHealth apps have the ability to change traditional health care trends into mHealth centers. In Malaysia, mHealth apps are very common and the availability of internet with android smartphones are growing day by day. Traditional Health Care of Malay-sia is top of the list in the world, but the mHealth still needs to improve in the region. Past studies in the mHealth are discussing physical health and wearable devices in details with connectivity to smartphones but serious diseases are cover in very some studies. Like diabetes and HIV apps and patient are not highlighted in the collected data. Patient record management and coordination with families are also part of some research studies and that is very important for recovery in some cases. The security concerns are related to mHealth apps and some people are not trusting apps due to too much information asked by applications during installation. Researchers need to point out the agenda in future studies to overcome the concerns of users about security issues. Normally mHealth applications are very much user-friendly and people with a basic education can use very comfortably the features of apps. Some applications are still very much complicated, especially with old people and uneducated users of smartphones. The collected studies are explaining that apps need to be more and more user-friendly because its help to deliver batter mHealth among the citizens of Malaysia.	2020-01-23T09:07:47.487Z	2020-01-21T00:00:00.000	{ "year": 2020, "sha1": "3c3f49c29ad9cb402307f7c8af845ad3e7fa08c4", "oa_license": "CCBY", "oa_url": "https://online-journals.org/index.php/i-joe/article/download/12263/6379", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "dd8ada00e43383fadb6da6eb89e45b508d9e643a", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Computer Science" ] }
56237741	pes2o/s2orc	v3-fos-license	Patterns of change in cytochrome c oxidase redox status Investigators using mono channel near infrared spectroscopy (NIRS) have reported different patterns of change in cytochrome c oxidase (Cyt) in similar studies of tissue ischaemia. We investigated whether there were distinctive differences in NIRS signals obtained simultaneously from different sampling sites during the same imposed physiological intervention within the same subject. Methods: Subjects were 36, healthy, 10 kg, commercial swine undergoing cardiopulmonary bypass to initiate 3 to 7 periods of 7.5 minutes of circulatory arrest. Each arrest was initiated at one of 81 combinations of high, normal, or low levels of core temperature, haematocrit, pH, and serum glucose. Each combination was repeated twice, yielding 162 NIRS data sets. Results: Six distinct patterns of change of Cyt were found. Typically, brain Cyt quickly became reduced shortly after the start of arrest, muscle Cyt did not start becoming reduced until after 31⁄2 minutes of arrest, and spinal cord Cyt either did not change status or became gradually reduced throughout the period of arrest. The brain response may reflect strong oxygen dependence, while the muscle response may indicate a dependency buffered by myoglobin stores, and the spine response may indicate a low concentration of available Cyt that is too diffuse to be rapidly influenced by changes in oxygen availability. Conclusion: Multi-channel NIRS is needed for systemic evaluation of respiration at the cellular level in clinical settings. Distinctive Cyt patterns of change occur in different organs at the same time, in response to circulatory arrest. Introduction This study examines whether changes in electron transfer within Complex IV of the mitochondrial respiratory chain in response to total circulatory arrest differs in the spine, brain and muscle as measured by near infrared spectrophotometry. Noninvasive near infrared spectrophotometry (NIRS) has now been available for experimentation in animals and humans for several decades.To date, NIRS has been used primarily to measure patterns of change in the concentrations of oxygenated hemoglobin (HbO 2 ) and de-oxygenated hemoglobin (Hb) in the blood, and to measure the redox status of cytochrome a,a 3 (Cyt).Cytochrome a,a 3 , residing on the cristae of the intracellular mitochondria, is the terminal enzyme in the respiratory chain and, as a catalyst promoting the release of energy, its redox status reflects mitochondria oxygen supply and demand [7]. There have been a number of trials validating the NIRS cytochrome a,a 3 signal, but results have sometimes varied between investigators [1][2][3]5,9,11,13].For example, it was found that Cyt redox status corresponds directly with changes in HbO 2 in human muscle, but the opposite was found in horse muscle [4,10]. In this study, we used NIRS to investigate whether there were distinctive differences in the Cyt redox patterns of change obtained simultaneously from diverse sampling sites during the same imposed physiological intervention within the same subject. Method The subjects were 36 healthy 10 kg juvenile Yorkshire swine.The experimental protocol was approved by the University of British Columbia's Animal Care Committee.All animals were anesthetized, intubated, mechanically ventilated, and placed on mechanical cardiopulmonary bypass. All animals had the sensor array of a NIRO-300 (Hamamatsu Photonics KK, Hamamatsu City, Japan) spectrophotometer positioned on the intact skin of the skull at the intersection of the left-eye/left-ear lateral and longitudinal axes.It was oriented towards the NIRO-300's emitter which was placed similarly at the right-eye/right-ear axis intersection 55 mm from the sensor.The second channel of the NIRO-300 was placed with its sensor on the intact skin over the mid thoracic spine and its emitter on the intact skin over the spine 65 mm caudal.The emitter and detector optodes of a NIRO-500 (Hamamatsu Photonics KK, Hamamatsu City, Japan) spectrophotometer were placed on the intact skin along the midline of the right hind flank with a 40 mm inter-optode spacing.Once in place, the sensor/emitters on the brain, spine, and muscle were covered with light occluding masks.Both the NIRO-500 and NIRO-300 were set to collect data continuously at 1 Hz. During bypass, each animal had two or more 7½ minute periods of circulatory arrest.Before each period of arrest, the animal's body temperature, haematocrit, pH, and glucose were adjusted to match a randomly chosen combination of ranges.Body temperature was established at 22, 18, or 14 • C, haematocrit at 14, 20, or 26%, pH at 7.25, 7.40 or 7.55, and glucose was established at 4.7, 6.0 or 7.25 mmol/l.There were 81 combinations of physiologic parameters that were repeated twice among all 36 animals. Blood samples were taken to measure sodium, potassium, bicarbonate, glucose, pH, haematocrit, partial pressure of carbon dioxide, partial pressure of oxygen, base excess, and oxygen saturation.These were recorded by iSTAT (Abbott Laboratories, Abbott Park, Illinois USA) point-of-care analyzers within three minutes of withdrawal. Data were analyzed using Excel-XP (Microsoft Canada Co. Mississauga, Ontario), and SPSS 11.01 (SPSS Inc., Chicago IL) was used for the 4-way ANOVA testing. Results There were 11 (7%) spine and muscle graphs that were incomplete because of blood and fluid seepage contaminating the sensor(s) or because the skin adhesive failed and the sensor(s) became displaced.These partial data sets were excluded from the analysis. The HbO 2 and Hb patterns of change were the same for all trials and had the same pattern of change in the brain as in the spinal cord and muscle.During the period of arrest, HbO 2 concentration decreased while Hb concentration increased. The Cyt patterns of change in the brain, spine, and muscle could be categorized into 6 distinctive patterns as shown in Fig. 1.The distribution of the Cyt patterns of change obtained given by the percentage of classification category is shown in Fig. 2. There was no period of circulatory arrest in which the brain, spine, and muscle all had the same Classification Type of Cyt pattern of change.In approximately 35% of muscle data sets, 15% of cerebral data sets, and 75% of spine data sets, Cyt redox status either did not change or became gradually reduced.Most (44% of 162) muscle data sets did not show reduction until after the first 3½ minutes of arrest, while most (49% of 162) brain collections showed reduction soon after the start of arrest. In the majority (60%) of spine collections, Cyt changes (Type I) were markedly different from the majority (50%) in the brain (Type VI), and the majority (43%) in muscle (Type V), with a rate of change in the spine at least 100 times slower than either the brain or muscle. Discussion This study used 3 NIRS channels to simultaneously monitor brain, spine, and muscle tissue oxygenation status and found that the patterns of change in Cyt redox always differ between organs. Conventionally, it is assumed that all Cyt enzymes are structurally alike whether isolated from wheatgrass root, bovine heart, or bacteria; and by corollary, behave alike.While their spectra have been found to be alike in the neonatal pig, adult rat, and purified enzyme, it remains possible that significant spectral differences still exist [2,8].Investigators do not yet agree on the electron exchange rates, or the conformation of the molecule, or its electron transfer pathways and proton interactions [12,14].Thus a static model of Cyt is not yet complete, while a behavioral model is only in its early stages of understanding [6].The behavior of this enzyme in isolation may be entirely different from that of an extensive Cyt in vivo cohort.Given these many uncertainties in understanding Cyt, it is possible that while Cyt enzymes may be alike between species, their patterns of change may be unalike given different tissue environments. The four circulatory arrest parameters (temperature, haematocrit, pH, and glucose) were established and measured in circulating blood.However, interstitial pH and glucose were not measured and may have differed from the blood.The 4-way analysis of variance (ANOVA) results shown in Table 2 indicate that only temperature is related to the rate of change in Cyt regardless of sampling site.This more likely indicates that additional trials are needed in order to increase the power of detection within this test, rather than that any cross relationship exclusively does not exist. The finding that 6 Cyt patterns of change are common to each organ and that only 6 patterns were found in 162 trials, suggests a potential usefulness in developing artificial intelligence software for automated interpretation of NIRS results.A graphical index of typical patterns of change versus species, versus organ, and versus intervention, could be created to enhance the interpretation of clinical NIRS observations. Conclusion Multi-channel NIRS is needed for systemic in vivo evaluation of respiration at the cellular level.The respiratory chromophore, cytochrome c oxidase, has nine typical patterns of change in redox status none of which appears simultaneously in the brain, spinal cord, and/or muscle as a response to circulatory arrest. Fig. 1 . Fig. 1.Types of cytochrome a,a 3 patterns of change during 7½ minutes of circulatory arrest.The rates of change for the slopes shown are given in Table1. Fig. 2 . Fig. 2. Histogram of the occurrence of types of patterns of change given in Fig. 1 from 162 brain, 131 brain, and 151 muscle trials among 36 healthy anesthetized swine. Table 2 Summary of the 4-way ANOVA testing for the rates of change as a dependent variable with temperature, haematocrit, pH, and serum glucose as independent variables	2018-12-15T21:44:58.635Z	2004-01-01T00:00:00.000	{ "year": 2004, "sha1": "d2fd71bec93939026b0820bdf6efa7514e1b8bbf", "oa_license": "CCBY", "oa_url": "https://downloads.hindawi.com/journals/jspec/2004/605373.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "d2fd71bec93939026b0820bdf6efa7514e1b8bbf", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Chemistry" ] }
196549942	pes2o/s2orc	v3-fos-license	Effect of glabridin on collagen deposition in liver and amelioration of hepatocyte destruction in diabetes rats Abnormalities in insulin hormone levels leads to a hyperglycemic condition of diabetic mellitus. Hyperglycemia seriously induces organ and system destructions. The excessive accumulation of collagen fiber deposits occurs in inflammatory and reorganization processes of chronic liver diseases in type I insulin-dependent diabetes. Regarding the research objective, glabridin (GLB), an active compound of licorice, was used as a daily supplement (40 mg/kg) in order to decrease hepatocyte destruction and collagen deposition in liver tissue of diabetic animals induced by streptozotocin. A total of 40 were randomly allocated to five groups (each, n=10), control, control treated with GLB (GLB), diabetic rats (DM) injected with single dose of streptozotocin (60 mg/kg) to induce a diabetic condition, diabetic rats receiving GLB (DM+GLB; 40 mg/kg) and diabetic rats treated with glibenclamide (DM+GL; 4 mg/kg). Characteristic histopathological changes in liver cells and tissues of rats were determined by Masson's trichrome staining and transmission electron microscopy (TEM). Western blotting was used to detect the expression of the key markers, collagen type I and fibronectin proteins. The histological investigation of liver tissue of the DM group revealed that the collagen fiber deposition was increased in the periportal, pericentral and perisinusoidal spaces compared with controls. Hepatocytes appeared as small and fragmented cells in TEM examination. Collagenization of the perisinusoidal space was recently demonstrated to represent a new aspect of the microvascular abnormalities and liver fibrosis. Healthy hepatocytes with round nucleus were observed following supplementation of glabridin. In addition, collagen fiber deposition was reduced in the area adjacent to the perisinusoidal space. The expression of collagen type I and fibronectin decreased strongly following glabridin supplementation in DM+GLB rats compared with DM rats, indicating that the hepatic tissue reorganization regained its normal morphology. These findings suggest that it may be beneficial to examine the role of glabridin as a therapeutic agent in diabetes treatment in future research. Introduction Extracellular matrix (ECM) accumulation or changing of the condition of the extracellular matrix is a common pathological reaction to tissue abuse acting as hyperglycemia (1). ECM is dissimilar quantitatively and qualitatively within various tissue and organs. The changes of which lead to diabetes-induced organ and system failure, resulting in nephropathy, retinopathy, and diabetic cardiomyopathy (2). It comprises protein fibers such as collagens and elastins implanted in an amorphous compound of proteoglycan molecules. Chronic hyperglycemia can cause disturbances of morphology and biochemistry of ECM that are associated with a lost function in target organs and induces the increase of reactive oxidative species (ROS) production (3). Furthermore, the excessive accumulation of fat and oxidative stress can influence diabetic pathology and complications, including diabetic liver damage (4). Oxidative stress occurs when the production of ROS exceeds its clearance. It constitutes a characteristic feature of hepatic injuries (5). At this time, the hepatocytes are damaged and Kupffer cells are activated. The inflammatory cells and platelets release cytokines and growth factors that result in fibrogenesis (6,7). Fibrosis is the deposit of connective tissue by the liver in response to injury commonly found in most chronic inflammatory liver diseases. Liver fibrosis entails substantial alterations in both composition and amount of the deposited ECM (4). This process comprises an inflammatory response and limits ECM deposition. The inflammatory cytokines and growth factors induce hepatic stellate cell (HSC) activation and cellular production such as transforming growth factor (TGF)-β (8), collagen (type I, III, and IV) (9), and fibronectin (10,11). TGF-β creates an accumulation of pro-fibrotic factors at the site of injury, which is common in several human fibrotic states (12). Collagens type I and IV are associated with the healing process of diabetic liver tissue (6). Therefore, the appearance and detection of collagen deposition in liver tissues might be the key markers for diagnosis (13,14). Fibronectin participates in the progression of the fibrotic state and supports other matrix proteins correlating with cell cycle development. Furthermore, it also cooperates in cell proliferation and adhesion (15). The deposition of collagen is the initiating event triggering liver fibrosis (16,17). Concerning acute hepatic injury, HSCs (HSCs) differentiate from pericytes into myofibroblasts for reconstruction of the ECM. Conversely, in chronic hepatic injury, HSCs occupy the space of Disse to serve the progression of excessive ECM production and decrease ECM metabolism (11). Movement and aggregation of HSCs at the area of tissue reconstruction activate ECM deposition and alter its degeneration (9). If the hepatic injury continues, the liver regeneration eventually fails, and abundant ECM fills the place occupied by hepatocytes leading to collagenization of liver tissue (16). Licorice has been reported to protect against hepatic injury (18). It also prevents oncogenesis caused by abnormal hormones or toxic substances and ameliorates free radical-induced oxidative of kidney damage (19). Glabridin ishe main active component of licorice (Glycyrrhiza glabra). It is a polyphenolic flavonoid that displays estrogenic, antimicrobial, anti-fatigue and anti-proliferative activity activities in human breast cancer cells (20). In addition, it reduces inflammation and alters melanogenesis (21). Glabridin displays potent antioxidative and superoxide-scavenging activities in biological membranes (22). It can prevent mitochondrial lipid peroxidation and protect respiratory enzyme activities against oxidative stresses in the mitochondrial electron transport system (22). Accumulating lines of evidence demonstrated that licorice has anti-inflammatory, anticancer, antioxidant and antimicrobial effects (20,23). In particular, Jung et al (24) have previously evaluated the hepatoprotective effects of licorice extract and suggested that it could reduce liver injury by enhancing antioxidant and anti-inflammatory capacity in alcohol induced fatty liver disease. In addition, Wu et al (25), reported that the hypoglycemic effect of glabridin increased body weight, glucose tolerance and superoxide dismutase (SOD) activities in the liver, kidney and pancreas, whereas decreasing fasting blood sugar levels and malondialdehyde (MDA) content in the liver, kidney and pancreas in the STZ induced diabetic mice for 28 days. The morphological changes of liver cells and tissues that may result from the treatment and supplementation with glabridin remain to be elucidated. The present study aimed to evaluate the efficiency of glabridin on restoration and improvement of diabetic liver tissue on histological, ultrastructural changes and to determine the collagen type I and fibronectin protein expressions in streptozotocin (STZ)-induced diabetic rats. The results indicated that glabridin from licorice may affect the collagen deposition and the ECM accumulation and reverse the patterns of area-based liver tissue reorganization. Therefore, glabridin may have potential to repair the damaged diabetic liver. Materials and methods Induction and assessment of diabetes. The present study used 8-week-old male Wistar rats (weight, 200-250 g) provided by the Southern Laboratory Animal Facility, Prince of Songkla University (Hatyai, Thailand). A total of 40 rats were housed in a controlled animal laboratory environment and maintained under a humidity of (50±10%) in a 12-h light/dark cycle (25±2˚C), with ad libitum access to standard rat chow and water. The experimental protocol used was approved by the Animal Ethics Committee of the Prince of Songkla University. Experimental diabetic rats were induced by single dose intraperitoneal injection of STZ (60 mg/kg; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) dissolved in 0.1 mol/l citrate buffer. Control rats were injected with citrate buffer alone. Blood sugar level was measured and analyzed by one-touch glucometer (Accu-Check Active ® ; Roche Diagnostics GmbH, Mannheim, Germany). Rats with blood sugar level >250 mg/dl were used as the diabetic group. In order to monitor blood glucose levels, blood glucose was tested every week for 8 weeks. Control and diabetic rats were randomly divided into five groups (each, n=10; Fig. 1): Control rats receiving a balanced standard diet; glabridin control rats receiving the same diet supplemented with glabridin (GLB; purified >98% by high-performance liquid chromatography analysis; Shaanxi Langrun Biotechnology Co., Ltd., Xi'an, China) in 0.5 ml of 0.5% Tween 80 solution; diabetic rats receiving a balanced standard diet (DM); diabetic rats receiving a balanced standard diet supplemented with glabridin (DM+GLB) in 0.5 ml of 0.5% Tween 80 solution; and diabetic rats treated with glibenclamide (Sigma-Aldrich; Merck KGaA; DM+GL) 4 mg/kg in 0.5 ml of 0.5% Tween 80 solution in order to demonstrate the effectiveness of glabridin. All animals were clinically observed and weighed on a weekly basis. Following 8 weeks of glabridin supplementation, the animals were sacrificed and blood was drawn from the heart into sample tubes for screening of liver function. The liver function test was analyzed by the Southern Lab Center Saha Clinic (Songkhla, Thailand; using a Siemens ADVIA 1800 System Analyzer; Siemens Healthineers, Erlangen, Germany). Livers were removed, dissected, and immediately fixed in 10% formalin at room temperature for 24 h as a preparation for histological experiments. Masson's trichrome staining. Following fixing the hepatic tissue samples in 10% formalin, they were dehydrated in graded series of ethanol through 70, 80, 90, 95 and 100% with two changes for 1 h each. Three changes of xylene as clearing reagent for 30 min each were performed. Hepatic tissues were then embedded in paraffin, sliced into 5-µm sections, and stained with Masson's trichrome at room temperature for 2 h. All hepatic sections were examined and images were captured via light microscopy (magnification, x20 and x60; BX-50; Olympus Corporation, Tokyo, Japan). Thickenings of the perisinusoidal, periportal and pericentral spaces (26) were measured and analyzed by Olympus cellSens software version 1.12 (Olympus Corporation). Transmission electron microscopy (TEM). Liver tissue samples, 1 mm 3 in size, were taken from the livers. They were immediately fixed in 2.5% buffered glutaraldehyde for 2 h at room temperature. Subsequently, the specimens were immersed in 1% osmium tetroxide, dehydrated, infiltrated with propylene oxide, and embedded in pure Araldite 502 resin polymerized at room temperature for 24 h, 48˚C for 48 h and 60˚C for 48 h. Then, thin sections (0.5-1.0 µm) were stained with Toluidine blue for 2 min at room temperature, used as a guideline to identify the area of interest and further sections. Ultrathin sections ~60 nm were processed using an ultramicrotome. Ultrathin sections were then spread mostly on 200 or 300 mesh copper grids and stained with uranyl acetate and lead citrate solutions both at room temperature for 15 min. The section were examined and images were captured using TEM (TEM-JEM2010; JEOL, Ltd., Tokyo, Japan). Western blot analysis of collagen type I and fibronectin. Liver lysates were prepared on ice-cold RIPA buffer (Sigma-Aldrich; Merck KGaA) supplemented with 1x protease inhibitor cocktails (EMD Millipore, Billerica, MA, USA). Homogenates were centrifuged at 14,000 x g for 30 min at 4˚C to collect supernatants. The protein concentration of the supernatant was assessed using a BCA protein assay kit (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Protein samples (10 µg) of liver tissues from control, GLB, DM, DM+GLB and DM+GL rats were diluted 1:2 in 2X treatment buffer (0.125 M Tris-HCl, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol and 0.2% bromophenol blue) and boiled for 5 min. Protein samples were separated by 12% polyacrylamide gel electrophoresis and then transferred (100 V, 0.35 A and 300 W for 1.5 h) onto nitrocellulose membranes (GE Healthcare, Chicago, IL, USA). The membrane was blocked with 5% non-fat dry milk in 0.1% Tween phosphate buffer (PBS-T) for 60 min at 4˚C. Nitrocellulose blot was then probed with monoclonal antibodies for collagen type I (1:3,000; cat. no. ab34710), anti-fibronectin (1:3,000; cat. no. ab2413) and β-actin (1:5,000; cat. no. ab8227; all Abcam, Cambridge, MA, USA) for 24 h at 4˚C. After incubating the sample with the primary antibody, the blot was washed thrice in PBS-T, and then incubated for 2 h at room temperature with a goat anti-rabbit horseradish peroxidase-conjugated immunoglobulin G (1:10,000; cat. no. ab6721; Abcam) which was used as a source of secondary antibodies. Finally, the membranes were exposed to film for collagen type I and fibronectin detection by enhanced chemiluminescence (ECL) method. The ECL detection system and ECL film (GE Healthcare) were used to visualize the presence of proteins on the nitrocellulose blots. The intensity of western blot bands was quantified by densitometry, using Scion Image 4.0 software (Scion Corporation, Frederick, MD, USA). Statistical analysis. Data are expressed as the mean ± standard error of the mean. Statistical analysis was performed using one-way analysis of variance and Bonferroni's post hoc test. P<0.05 was considered to indicate a statistically significant difference. Table I. Following 1 week, rats receiving STZ injection exhibited an increase in blood glucose levels. They were significantly elevated in DM rats (P<0.001) as compared with control rats until termination of the study at 8 weeks. The blood sugar levels of DM+GLB and DM+GL rats were reduced when compared with DM rats. There was a significant decrease in blood sugar levels in DM+GLB rats (5 weeks, P<0.01; 6-8 weeks, P<0.001) and in DM+GL rats (P<0.001) at 5-8 weeks when compared with DM rats. There were no significant differences observed in the blood sugar levels between DM+GLB and DM+GL rats for 8 weeks. The liver function test results of blood samples at 8 weeks are presented in Table II. The glutamate oxaloacetate transaminase (SGOT), glutamate pyruvate transaminase (SGPT) and alkaline phosphatase (ALP) levels in DM, DM+GLB and DM+GL rats were increased when compared with control rats. SGPT and ALP were significantly increased in DM rats (P<0.05, and P<0.001 respectively) when compared with control rats. Following administration with glabridin and glybenclamide, the three parameters were markedly decreased in DM+GLB and DM+GL rats when compared with DM rats; however, no significance differences were identified among DM, DM+GLB and DM+GR rats. Effect of glabridin on blood sugar levels and liver function test. Blood sugar levels in all animals were presented in Histological observations of liver tissues with Masson's trichrome staining. Hepatocytes and blood vessels of the livers in all rats throughout the 8-week experiments were radially arranged in the liver lobule (Fig. 2). A cellular plate formed of hepatocytes was directed from the periphery of the lobule to its center, which was primarily one cell thick and situated between a system of capillary sinusoids, forming a meandering and porous like structure. The spaces between these hepatic plates contained irregular dilated vessels, which were capillaries and sinusoids. The sinusoids ran in the direction of the center, where they drained into the central vein ( Fig. 2C and F). The portal triad contained a portal venule (a branch of the portal vein), an arteriole (a branch of the hepatic artery) and bile ducts (portion of the hepatobiliary system; Fig. 2A, B, D and E). The loose connective tissue containing collagen fibers were also present and located around every blood vessel, keeping the vessels in place. Collagen was stained and indicated in blue or very light green. Hepatocyte nuclei could be observed as dark red structures within cells whereas the cytoplasm was stained red. The spaces between the formed elements of the tissue were filled with the matrix. Liver tissues of DM rats revealed by collagen fiber deposition were increased in the portal triads ( Fig. 2G and H) and in the areas around the boundary of hepatocytes along the sinusoidal spaces (Fig. 2I). The blood sinusoids around the central vein were dilated (Fig. 2I). Few sinusoids were opened into the central vein. Edema appeared in the hepatocytes. The hepatocyte nuclei displayed signs of pyknosis. Hepatocytes were large polyhedral cells with large round nuclei (Fig. 2L). In addition, a number of hepatocytes contained small dense nuclei with eosinophilic cytoplasm. Another finding was that lipids accumulated in the hepatocytes and formed non-membrane bound vacuoles with peripheral nuclei. The hepatocytes radiating from the central vein exhibited a regular pattern following glabridin supplementation. The portal triad contained a portal venule, an arteriole, a bile duct and lymphatic vessels ( Fig. 2I and K), which were similar in the normal portal triad of control rats. Blood sinusoids were located between plates of hepatocytes. Administration of glabridin in DM+GLB (Fig. 2L) and glibenclamide in DM+GL (Fig. 2O) rats lowered the increase of central vein and sinusoidal dilatation, lipid accumulation, pyknotic nuclei of hepatocytes and inflammation in the area of portal triad. Decreased collagen fiber deposition in the portal triad in DM+GLB and DM+GL rats ( Fig. 2I and K) were a signal of reduced fibrosis. Therefore, glabridin attenuated the severity of liver damage resulting from STZ-induced diabetes. The area of collagen deposition in the liver tissue, stained in blue, were quantified by thickness in three zones, area of portal triad (periportal area or periportal space; PT), area around the boundary of hepatocytes along the sinusoidal space (perisinusoidal area or perisinusoidal space; PS) and around the central vein (pericentral area or pericentral space; PC) of all animal groups. The thickness of these three different zones were measured ( Histological changes in liver cells and tissue with TEM technique. TEM was used to observe differences in hepatic cells and tissues (Figs. 3 and 4). Micrographs of normal livers in control (Fig. 3A) and GLB (Fig. B rats exhibited round whereas Golgi apparatus were dispersed in the cytoplasm. The hepatic sinusoids were lined with thin endothelial cells. The perisinusoidal space was located between hepatocytes and The nuclei of the HSCs were visible with heterochromatin. Its cytoplasm was abundant and indented by fat droplets (Fig. 4A and B). TEM examination revealed the pathological hepatocytes and liver tissues in DM rats. Hepatocytes were small and shrunken as fragmented cells. The irregular nucleus of hepatocytes exhibited damage with finger-like projections of the nuclear membrane. The degeneration and disorganization of cytoplasmic organelles was also presented (Fig. 3C). Many mitochondria of hepatocytes became swollen while huge lipid vacuoles were deposited in the cytoplasm of hepatocytes. Nuclear membranes were disrupted and nuclear chromatins were exposed. Endoplasmic reticulum cisternae and Golgi apparatus were dilated and destructed with irregular lamellar organization and large dilatations (Fig. 3D). Blood sinusoids around the boundary of hepatocytes were also dilated (Fig. 3D). Collagen fiber deposition was increased in the area adjacent to the perisinusoidal space in DM rats ( Fig. 4C and D). The variable size of collagen bundles was observed in the perisinusoidal space. After supplementation of glabridin for 8 weeks, the hepatocytes and HSCs had a similar appearance as that of the control group. Healthy hepatocytes with round nuclei indicated the regenerated hepatocytes of DM+GLB rats and DM+GL ( Fig. 3E and F, respectively). The cytoplasm contained light and dark stained swollen mitochondria and few vacuoles. TEM examination revealed the collagen production in the intracytoplasmic compartment of HSCs and these collagen fibers were radiated from cytoplasm toward the liver parenchyma. Collagen fiber deposition was decreased in the area adjacent to the perisinusoidal space in DM+GLB and DM+GL rats. Western blot analysis of collagen type I and fibronectin. The collagen type I and fibronectin protein bands were observed and visualized using monoclonal anti-collagen type I and anti-fibronectin antibodies. Western blot analysis established the specific collagen type I protein band at molecular weight 130 kDa and a fibronectin protein band of molecular weight 220 kDa on films via ECL in control, GLB, DM, DM+GLB and DM+GL samples from rat livers (Fig. 5). These specific proteins represented the expression of type I collagen and fibronectin. The bands from DM revealed more intense protein expressions than that those of the control, GLB, DM, DM+GLB, and DM+GL rats on ECL films. In addition, the positive control expression and characterization of β-actin protein were revealed at molecular weight 43 kDa. The β-actin protein was further used for protein analysis. The collagen type I and fibronectin expressions decreased following treatment with glabridin and glyburide when compared with DM animals. The amount of collagen type I and fibronectin proteins in DM rats were significantly increased compared with control (P<0.001). In DM+GLB rats, the quantity of collagen type I and fibronectin significantly increased when compared with control (P<0.001). However, the amount of collagen type I and fibronectin in DM+GLB were significantly decreased compared with DM rats (P<0.001; Fig. 5). Discussion The diabetic model was developed by the administration of STZ to male rats. It was revealed that STZ-injected rats produced significantly increased levels of blood glucose. The increase of blood glucose levels stimulated the sorbital pathway and protein kinase C (PKC), which may lead to the production of growth factor and cytokines. Direct high glucose concentration which induces mitogen-activated protein kinase (MAPK) and PCK activation. Supplementation of glabridin revealed that the blood sugar levels were decreased. The effectiveness of glabridin from licorice extract and against the diabetogenic effects of streptozocin has also been established in rats (26). For screening liver disease, liver function test are commonly used in clinical practice. In the present study, liver function tests were performed to observe the effects of STZ-induced diabetes on the liver at 8 weeks following STZ treatment. The levels of SGOT, SGPT and ALP were increased in the livers of DM rats when compared with control rats. Following glabridin supplementation, they were decreased when compared with DM rats. SAKP is a membrane bound enzyme glycoprotein enzyme (27). Expression of this enzyme is increased when the bile ducts become blocked. SGOT and SGPT are both enzymes that are mainly found in mitochondria of the liver (28). If liver damage is present, the enzymes are released into the bloodstream following liver cell death (29). Abnormally high concentrations of SGOT and SGPT in the blood may indicate damage to the liver (30). SGOT is normally present in liver, heart, skeletal muscle, kidneys, brain and red blood cells (31). SGOT is elevated with liver damage or heart attack. Elevated SGOT levels are not specific for liver damage, and SGOT has also been used as a cardiac marker (32). By contrast, SGPT is normally found largely in the liver. Therefore SGPT is a much more specific indicator of liver dysfunction (33). The results from the histological observation of liver sections of DM rats indicated that sinusoids around the central veins and central veins were dilated. Edema appeared in the hepatocytes. The hepatocyte nuclei were evident of pyknosis. In addition, some hepatocytes were containing small dense nuclei and disruption of nuclear membranes. Another finding was that lipids accumulated in the hepatocytes and formed non-membrane bound vacuoles with peripheral nuclei. According to the results, the presented pyknosis of hepatocyte nuclei and edematous hepatocytes were displayed by continuous hepatocytes injury and death. It was evident that there were lipid droplets accumulating in the cytoplasm of hepatocytes (34). The accumulation of cytoplasmatic lipid droplets in hepatocytes is a situation that evokes the transformation into fatty liver. It is possible that this situation arises from an increased incorporation of fatty acids into the liver, which could have been a consequence of the hypoinsulinemia and the decreased capacity to excrete of lipoprotein secretion resulting from an insufficient production of apolipoprotein B (35). Characteristics of a fatty liver include the intracellular accumulation of triglycerides resulting from an increased liponeogenesis and an increased triglyceride uptake (36). Concomitantly, the hepatic secretion of very low-density lipoproteins decreases (37). The mechanism of liver damage includes cellular necrosis and inflammation, both of which are consequences of the increased mitochondrial oxidative stress from the triglyceride metabolism and the generation of free radicals in the peroxisomes (37). Another factor that increases mitochondrial oxidative stress is the increased production of adipokines (cytokines produced by the adipocytes) including tumor necrosis factor-α (TNF-α) and leptin (38). These chemical mediators, which are produced during inflammation and cell necrosis, activate HSCs that respond by expressing collagen, connective tissue growth factor and aggregation of extracellular matrix components, thus leading to fibrosis (39,40). Furthermore, the hepatic glycogen content of the STZ-treated animals decreased substantially. It is plausible that the displacement of glycogen in the hepatocyte cytoplasm is the result of lipid droplet accumulation (41). In addition, there are also anatomical changes of the sinusoidal capillarization. Sinusoidal endothelial cells are reported to lose fenestrae, basement membrane accumulates and sinusoidal outlets become sparse (42). These pathological alterations disturb the normal function of the sinusoid (42). Besides, capillarization of the sinusoid may disturb the exchange of several bioactive substances between the hepatocytes across, perisinusoidal space, and sinusoidal blood (43). All of these processes are to contribute to the hepatic fibrosis. Furthermore, DM rats revealed that collagen fiber deposition was increased in the PT, PS and PC areas. Adipokines are known to activate HSCs and induce them to increase production of connective tissue growth factor, collagen, and to accumulate extracellular matrix components, in this way, favoring fibrosis (44). HSCs, also called Ito cells or perisinusoidal cells, are pericytes found in the space of Disse, the hepatic space surrounding the sinusoids (45). Alteration in the microenvironment within the perisinusoidal space caused by activated HSCs facilitates the development of liver fibrosis (45). The number of activated HSCs increases in areas of inflammation (46), emphasizing the importance of resident HSCs migration into the PS during the progression of liver fibrosis. Accordingly, the number of activated HSCs is substantially reduced during the regression of liver fibrosis. This occurs by the induction of apoptosis or cellular senescence, which returns cells to a quiescent state (47). The HSCs thus constitute the main cell type participating in liver fibrosis. HSCs become activated following hepatic damage. This process is characterized by proliferation, changes in contractility, and chemotaxis. The activated HSCs secretes collagen to the scar tissue, with cirrhosis as a potential outcome (39). HSCs are also stimulated by other cells, including hepatocyte, T-lymphocyte, and Kupffer cells. The systems of stimulation are groups of cytokine and inflammatory secretions that include TNF-α, TGF-β, insulin-like growth factor, interleukin-6, and interferon-γ (13,14,48). TGF-β constitutes an important mediator of liver fibrosis (49)(50)(51). TGF-β causes an increase of ECM protein production that results in myofibroblast differentiation of HSCs (52). Thus, TGF-β serves an important role in proliferation, differentiation, and morphogenesis. In hepatic fibrosis there is an increase of TGF-β expression, which can be understood as a homeostatic response to repair damage tissue (53). Fibronectin a non-collagenous glycoprotein serving several functions, is synthesized and secreted by hepatocytes, endothelial cells, macrophages and fibroblasts (53). Fibronectin expression is associated with normal processes such as differentiation, and to pathological processes such as cellular damage, hepatic fibrosis and repair (54). The most abundant collagen types found in a healthy liver are the fibril-forming types, i.e. collagen types I and III (55). During fibrogenesis, as the collagen becomes integrated into the ECM, there is an eight-fold increase in types I and III (55). In addition, the ratio of collagen type I/III is changed from 1:1 in healthy liver to 1:2 in the cirrhotic liver (55). Recent studies have demonstrated that the cause of the development of DM and its complications are lipid peroxidation that leads to the formation of ROS (56). An increase in ROS generation together with a decrease in the activity of antioxidant system causes an imbalance that leads to oxidative stress (57). The high level of blood sugar that occurs in diabetes results in oxidative stress and weakens the capacity of endogenous antioxidant substances due to the synthesis of several reducing sugars via both the glycolytic and polyol pathways (57). In the present study, the demonstration of the therapeutic effect of glabridin and the drug, glibenclamide, were similar. Glibenclamide is used most commonly as a standard drug in STZ-induced diabetes (58) and used as positive control compared with glabridin from licorice. It is an effective drug to facilitate insulin release from β cells (59). The mechanism of action of glibenclamide in hyperglycemic conditions is to lower blood glucose via stimulating insulin production from the existing β cells of pancreas in STZ diabetic rats (59). In addition to this direct action, it also exhibits pancreatic effects. This drug binds to the sulfonylurea receptor 1 in the pancreatic β cells (60). This inhibition causes cell membrane depolarization and opens the voltage dependent calcium channels (61). It is one of the leading treatments for diabetes that can increase intracellular calcium concentration in β cells and subsequently stimulates the release of insulin (62). Glabridin can be used as a complementary and alternative therapy due to its reduced cost compared with other pharmaceutical agents and easier access to diabetes treatment (25). The hypolipidemia effect and the marked decrease in artherogenic indexes by glabridin in STZ-treated rats reduced the incidence of artherosclerosis in diabetic patients (63). Additionally, the beneficial therapeutic effect has been reviewed for the active compound from licorice; glabridin significantly elevates SOD activities which lowers MDA content of kidney, pancreas and liver. Hence, it may be concluded that part of the antioxidative activities of glabridin in STZ-induced diabetic mice may result from its hypoglycemic effects (25). Therefore, glabridin may prevent the progression of diabetic liver fibrosis, which probably acts by enhancing anti-oxidative and anti-inflammatory capacity. In conclusion, the present findings suggest that glabridin has the potential to recover the damaged liver tissue of STZ-induced diabetic rat. The 40-mg/kg dose of glabridin from licorice results in a suitable outcome to ameliorate the pathological changes in diabetes rat hepatocytes and collagen accumulation in the extracellular matrix of the liver. Furthermore, the method of light microscopy, TEM and western blot analysis clearly elucidated the decrease of collagen deposition in the liver and was beneficial in establishing glabridin as a therapeutic target for diabetes treatment. The results of the present study suggest that further study is required to assess the mechanism and pharmacological actions of glabridin, and to assess if it may be beneficial as a therapeutic agent in the treatment of diabetes.	2019-07-15T00:39:37.722Z	2019-06-11T00:00:00.000	{ "year": 2019, "sha1": "8c752b12da5d1d46da20864198b2527e38acdf64", "oa_license": "CCBYNCND", "oa_url": "https://www.spandidos-publications.com/10.3892/etm.2019.7664/download", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "8c752b12da5d1d46da20864198b2527e38acdf64", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [ "Chemistry", "Medicine" ] }
52879271	pes2o/s2orc	v3-fos-license	Air embolism following bronchoscopy with fine needle aspiration: An unexpected complication Flexible fibreoptic bronchoscopy with fine needle aspiration is a common procedure, useful in the diagnosis and assessment of lung disease. There are known complications associated with such a procedure that are well documented in the literature. However, there are only four cases of air embolus following fine needle aspiration during bronchoscopy described in the literature. Due to the varying clinical manifestations of the complication, it remains underrecognized by the clinical community and was not described at all by the most recent British Thoracic society 2013 statement on bronchoscopy. The following two case reports describe incidences where air emboli ensued following bronchoscopy with fine needle aspiration. They examine four notable, and arguably avoidable, risk factors that can exacerbate an air embolus and offer guidance on both imaging and treatment for any physician faced with a corresponding clinical picture. Introduction Flexible fibreoptic bronchoscopy with Fine Needle Aspiration (FNA) of lymph nodes is a widely accepted and safe procedure which is useful in the diagnosis and assessment of lung disease. Minor complications include vasovagal spasm, bleeding and arrhythmias with an incidence of 6.5% [1]. Major complications are much less common but include respiratory arrest, pneumonia, pneumothorax and major bleeding occur at 0.5% [2]. Systemic air embolus is an extremely rare complication of computed topography (CT) guided transthoracic biopsy, reported to be around 0.02%-0.07% [3][4][5][6]. In a prospective study of complications following Flexible Fiberoptic Bronchoscopy in 908 patients there were no instances of neurological events [7]. To our knowledge only five cases of air embolus following fibreoptic bronchoscopy have been reported [8][9][10][11][12][13] (Table 1). Furthermore, in the recent British Thoracic society statement 2013 on bronchoscopy there was no mention of air embolus as a complication of fibreoptic bronchoscopy [14]. We are describing two cases of air embolus causing a cerebral vascular accident because we feel that this complication is under recognised and under reported and can potentially be fatal [10]. Case report 1 A 69 year old gentleman was referred to the rapid access unit for recurrent chest infections. It was subsequently noted on CT of the thorax that the patient had an enlarged right hilar lymph node measuring 2.5 × 1.6cm, with multiple mediastinal lymph nodes, as well as evidence of centrilobular and paraseptal emphysema (Fig. 1). He was subsequently referred for bronchoscopic evaluation using a standard (adult size) bronchoscope. FNA was performed using 1.1mm 19-gauge needle. During the procedure the patient became hypoxemic and unresponsive. Hemiparesis was evident on the left side. A CT brain and CT Intracranial Angiogram, at the time of the event, was reported as no evidence of acute ischaemia or intracranial occlusion. Although, in retrospect, it appears that there was effacement of the cerebral sulcus on the right side, in keeping with acute ischaemia. The only finding was of air passing through the base of the skull, along the lacerum segment of internal carotid artery (Fig. 2). AIt was noted that the chest tube was noted to be oscillating with respiration and was no longer bubbling, suggestive of a resolution of the air leak. There was no evidence of bleeding in the chest tube or in the tube site. Moreover, the patient was conversing and had a complete resolution of her shortness of breath and chest pain. Despite absence of identifiable clot in the middle cerebral artery on the cerebral angiogram the decision was made to thrombolysis the patient based on the severity of the hemiparesis and the identifiable onset of the neurological symptoms. Almost immediately after the thrombolysis was administered haemoptysis ensued. This was further complicated by the patient developing generalised tonic-clonic seizures. Rapid intubation secured the airway. A repeat CT chest was performed identifying new onset mediastinal haemorrhage (Fig. 3) and evidence of diffuse pneumomediastinum (Fig. 4) Haemoptysis resolved within 2 hours spontaneously and seizures ceased in 24 hours after Phenytoin loading. A further 24 hour period saw the patient extubated, regaining full consciousness, with no cognitive impairment and minimal residual left-sided hemiparesis. Repeat CT brain showed some subtle effacement of the gyral pattern in the right cervical hemisphere over the vertex but no low-density change in brain parenchyma. However, subsequent Magnetic Resonance Imaging (MRI) found right cerebral hemisphere watershed ischaemia with foci of acute infarction, consistent with the left-sided hemiparesis demonstrated. It was concluded in retrospect that this was an air embolus ( Fig. 5). Stroke rehabilitation was performed, and full recovery was made within two weeks. A repeat CT thorax showed no progression of his mediastinum lymphadenopathy, thus excluding any possibility of malignant lesion of the node. Unfortunately, there were no facilities to enable hyperbaric oxygen treatment at this hospital and by the time the patient was stable enough for transfer, their neurological status had improved considerably. Case report 2 69-year-old male with a history of productive cough and recent admission for pneumonia went on to have a CT thorax that reported as a 3 × 2.1 cm soft tissue mass at the right hilum with adjacent hilar lymphadenopathy and mild pulmonary fibrosis on CT thorax (Fig. 6). Due to a history of bowel cancer, with sigmoid colectomy two months prior, there was a concern for metastases to the lungs. FNA was performed using 1.1mm 19-gauge needle. The patient became hypoxemic on the table and became unresponsive. Reversal of Midazolam sedation was attempted with no marked response. Similarly, to the first case, there were no facilities to enable hyperbaric oxygen treatment at this hospital. However, the patient did regain consciousness, but suffered a left-sided hemiparesis. CT brain and CT angiogram intracranial showed marked evidence of pneumocephalus in the right middle cerebral artery (MCA) and within the extra-axial space on the right side, characteristic of air emboli (Fig. 7). Similarly to the first case, the patient made a full recovery with the assistance of a physiotherapist and occupational therapist within a week and due to a lack of adequate facilities, the patient was not treated with hyperbaric oxygen therapy but rapid neurological improvement was observed clinically. Discussion A small number of air emboli cases feature in the literature, but it is said that the number is underreported as asymptomatic patients will go undiagnosed [6,15]. We feel that our two cases are instructive in this respect as they highlight this potential, albeit rare, risk of bronchoscopy. In doing so, physicians will become increasingly aware of it but also aware that a full recovery can be made. There is a wide spectrum of possible presenting symptoms that a patient can manifest with, thus making diagnosis difficult [3,4]. In fact, in our first case we felt that the patient had suffered an acute ischaemic cerebrovascular accident, that was not a direct consequence of the bronchoscopic procedure. The patient was thrombolysed as per acute stroke guidelines as we did not recognise air embolus as aetiology until we reviewed the case retrospectively [16]. Therefore, it is vital that physicians are mindful of and can recognise the symptoms of a potential air embolus. The onset of neurologic symptoms (confusion, personality change, dizziness, visual disturbance, or paraesthesia); delayed recovery from anaesthesia; cardiovascular instability and the presence of air bubbles in retinal vessels on fundoscopy, are some quick and non-invasive indicators that should alert the doctor to the possible occurrence of an air embolus [5]. The second case on the other hand was immediately recognised as an air embolus and was consequently managed accordingly from the start. It is important to note that both patients, despite demonstrating very significant neurological deficits, recovered completely despite no targeted intervention directed against the air embolus. It is not yet clear how an air embolus is induced while performing FNA during bronchoscopy. The literature yields some hypotheses on causation related to the FNA procedure. First, air may enter the pulmonary system through the needle. If atmospheric pressure exceeds pulmonary venous pressure, air emboli can traverse down the pressure gradient and establish themselves in the pulmonary veins. This can occur if the patient were to inhale deeply during procedure. Second, if internal airway pressure distal to the scope were to rise, the risk of embolus is thought to increase. For example, actions that mimic valsalva manoeuvre, such as coughing and straining, during the procedure, can create a sudden pressure increase distal to the needle, thus inducing embolus [3][4][5][6][17][18][19][20]. A potential learning point for physicians is to consider increasing sedation in patients who show signs of airway resistance during the procedure, including coughing, straining or deep breathing, to reduce risk of embolus. It is of note that in both our cases the patients had severe episodes of coughing during the performance of the FNA procedure. In addition, patients with chronic obstructive pulmonary disease (COPD) or those on positive pressure ventilation should also be considered for increased sedation as distal pressure gradient may be exacerbated due to increased air trappings [17]. A further hypothesis suggests that a needle may penetrate simultaneously at an air-containing space, such as a nearby pulmonary alveolar space or bronchus, and a nearby pulmonary vein, which can create a communicating fistula [3][4][5][6][17][18][19][20]. Lastly, radiology literature suggests an association between the size of the needle used and increased incidence of air emboli [6]. Diagnosis of an air embolus is primarily clinically suspected, but can be difficult. However, second to clinical suspicion, brain and CT scan can provide a definitive diagnosis by showing air bubbles in the cerebral vessels, aorta, pulmonary veins and left atrium and ventricle [21]. However, a pivotal point is that an air embolus less than 1.3cm may not be detected by CT brain or CT intracranial angiogram and will only be seen on MRI. Therefore, to be aware of the need for an MRI, as opposed to a CT brain or intracranial angiogram, if you experience a patient with clinical symptoms that allude to a cerebral vascular accident is vital to obtain definitive exclusion criteria. Case 1 is a clear example of where the air embolus was initially overlooked due to the embolus size likely being < 1.3cm and thus not appearing on CT imaging. If patients are correctly diagnosed as having an air embolus as opposed to a cerebral vascular accident, correct treatment can be initiated and is shown to decrease mortality rate to 7% [5,6]. Hyperbaric oxygen therapy is considered the gold standard for treatment of systemic air embolism [22]. By breathing 100% oxygen increases ambient pressure above that of the atmosphere and consequently the size of gas bubbles decreases. This is because the increased ambient pressure compresses the bubble (air embolus), reducing its size. Secondly, it also induces systemic hyperoxia which has the following effects. 1) Oxygen replaces Nitrogen in the air bubble by diffusion. 2) A large quantity of oxygen can then dissolve into the plasma. 3) Increased oxygen is also diffused out into the tissue [18]. In addition, the patient can be placed in the left lateral decubitus position with a lowered head [5,6,15,20]. In summary, air embolism is an underrecognized and potentially disastrous complication of FNA during bronchoscopy. Summary conflict of interest statement The authors declare no conflicts of interest. • has drafted the submitted article or revised it critically for important intellectual content • has provided final approval of the version to be published; • has agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the • has provided final approval of the version to be published; Acknowledgements • has agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved	2018-10-11T13:15:26.283Z	2018-09-20T00:00:00.000	{ "year": 2018, "sha1": "47ae1961e218679b0ecb04e43b13f6d4b71f807d", "oa_license": "CCBYNCND", "oa_url": "https://doi.org/10.1016/j.rmcr.2018.09.012", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "f54a065df4970a09d11adfd8457fb7099654bc57", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
244710913	pes2o/s2orc	v3-fos-license	Burden of puerperal sepsis and its associated factors in Ethiopia: a systematic review and meta-analysis Background Puerperal sepsis is a genital tract infection that can occur from amniotic fluid rupture to six weeks after birth. Maternal complication associated with puerperal sepsis includes prolonged hospital stay, septicemia, disseminated intravascular coagulation, pelvic inflammatory disease, infertility, and death. Even though, puerperal sepsis is the fourth leading cause of maternal morbidity and mortality in Ethiopia the overall prevalence of puerperal sepsis and its associated factors are not studied at the national stage. As a result, this systematic review and meta-analysis bring out the pooled prevalence of puerperal sepsis and its associated factors in Ethiopia. Methods A variety of data sources such as Pub Med, Web of Science, Science Direct, Embase, Google Scholar, HINARI, and Ethiopian universities online repositories were searched to identify the primary studies which were used for this systematic review and meta-analysis. The article search was conducted from February10/2021-March 10/2021. The quality of the selected primary studies was assessed using the Newcastle - Ottawa quality assessment Scale (NOS). Data extraction was done with Microsoft Excel and then exported to STATA 11 version statistical software for analysis. The Cochran (Q-test) and I2 test statistics were used to assess the heterogeneity of the studies. Publication bias was evaluated by the eggers regression test. Subgroup analysis was performed with region and sample size category. Result In this review, a total of 2222 respondents were involved from seven studies. The pooled prevalence of puerperal sepsis was 14.811% (95%CI; 8.46: 21.16; I2 = 94.2, P ≤ 0.001). Cesarean section delivery (CSD) (OR = 3.26, 95%CI: 1.90, 5.61), membrane rupture≥24 h (OR = 4.04, 95%CI: 2.54, 6.42), being multiparous mother (OR = 3.99, 95%CI: 1.82, 8.78), vaginal examination≥5 times (OR = 3.15, 95%CI: 1.17, 8.52), and anemia (OR = 5.68, 95%CI: 4.38, 7.36) were factors significantly associated with puerperal sepsis. Conclusion The prevalence of puerperal sepsis was high in Ethiopia. CSD, membrane rupture≥24 h, being multiparous mother, vaginal examination≥5, and anemia were factors associated with puerperal sepsis. Appropriate standard infection prevention techniques during CSD shall be practiced to reduce the maternal burden of puerperal sepsis. The unnecessary vaginal examination should be discouraged during the intrapartum period. Besides this, routine Iron sulfate supplementation and counsel on iron reach foods during ante partum and postpartum shall be considered for all mothers. Supplementary Information The online version contains supplementary material available at 10.1186/s13690-021-00732-y. Background Postpartum endo-mayometrities is a genital tract infection that can strike at any point between the rupture of the membranes or labor and the 42nd postpartum day. Puerperal sepsis is diagnostic if the woman has at least two of the following clinical features: Pelvic pain, Fever (oral T o ≥ 38.5 C 0 ), abnormal vaginal discharge, foul odor vaginal discharge, and delay in the involution of the uterus within six weeks of giving birth [1]. Even if maternal mortality is somewhat reducing globally, the majority of maternal deaths occur at the time of giving birth. Puerperal sepsis is avoidable leading cause of maternal morbidity and mortality [2]. Globally; 75,000 maternal deaths occur in a year as a result of puerperal sepsis: among this; 11.6% in Asia, 9.7% in Africa, 7.7% in South America, and 7.7% in Caribbean countries [3,4]. After postpartum bleeding, unsafe abortion, and pregnancy-induced hypertension, puerperal sepsis is the fourth greatest cause of maternal mortality [5]. Maternal complication associated with puerperal sepsis includes prolonged hospital stay, septicemia, disseminated intravascular coagulation, pelvic inflammatory disease, infertility, and death [6]. In sub Saharan countries including Ethiopia; there is lack of data on puerperal sepsis. In poor resource setting after delivery mothers discharged to their home after short period of follow up and this might not afford enough time to exclude evidence of puerperal sepsis prior to discharge from the health facilities. Ethiopia has agreed to implement the sustainable development goals (SDGs) to decrease maternal mortality to < 70/100,000 live births through 2030G.C [15]. Thus, in order to carry out this plan and accelerate maternal mortality reduction, it is preferable to identify the causes and contributors to maternal mortality. Having every birth attended by skilled birth attendant, access to comprehensive emergency obstetric care, apply standard infection prevention techniques in the heath facility,and ensure effective referral system are the strategies of reducing maternal morbidity and mortality in relation to sepsis [15]. Knowledge of puerperal sepsis enables to practice the strategies towards this direct causes of maternal mortality and helps to achieve the sustainable developmental goals (SDGs) [15]. However, the effect of puerperal sepsis on maternal morbidity and mortality is minimally understood by different stake holders [15]. Assessing the prevalence of puerperal sepsis and its associated factors at the national level (Ethiopia) is helpful to challenge the fourth leading cause (sepsis) of maternal morbidity and mortality. Despite the fact that some studies have been conducted to assess the prevalence and associated factors of puerperal sepsis, there is a lack of data at the national level to show the prevalence and associated factors of puerperal sepsis.. Moreover, the prevalence and associated factors of puerperal sepsis were incoherent in primary studies. Therefore, this systematic review and meta-analysis aimed to estimate the overall prevalence of puerperal sepsis and its associated factors in Ethiopia. Methods This systematic review and meta-analysis were done by the methodology of preferred reporting items for systematic review and meta-analysis (PRISMA) cheek list (Additional file 1). It was conducted by systematic synthesis of the original studies about puerperal sepsis and associated factors in Ethiopia. Searching strategies International online databases (i.e. Pub Med, Web of Science, Science Direct, Embase, Google scholar, and HINARI), and Ethiopian university's online repositories were used to search articles. Studies published between October 1, 2000, and January 1, 2021, G. C were included in this systematic review and Meta-analysis. The adapted PECO format was utilized for this systematic review and Meta-analysis. This PICO was comprised of Population (P), Exposure (E), Comparison (C), and Outcome (O) as shown below. In each component of the adapted PECO, search terms are given. Using the above adapted PECO format, the following review questions were created to retrieve as many relevant primary studies as possible: Review questions1. What is the national prevalence of puerperal sepsis in Ethiopia? Review question 2. What are the factors associated with puerperal sepsis in Ethiopia? Each database was searched independently with some modifications of the search strategy. Human being and the English language was applied as limiters of the search. The type of searching strategy was line by line and it was done through the title (TI), abstract (Ab), and full-text categories. Boolean operators ("OR"and"AND") search operator was applied. Synonyms were also utilized to look for more main research. We broadened our search beyond systematic database searches to include retrieving reference lists of papers that met our criteria. In addition, thecited by' andrelated articles' capabilities of PubMed were used to conduct further literature searches. A literature search was conducted from February10/2021-March 10/2021.Finally, all studies that matched the title of the review were located and assessed for inclusion criteria. Two independent authors conducted the literature search, with differences handled by discussion and consensus. For PUBMED and Google scholar database searches, a sample of the primary search string has been provided as a supplementary file (Additional file 2). Outcome variable measurement Puerperal sepsis is a genital tract infection that can occur up to six weeks after delivery when the amniotic fluid ruptures, and a woman should have at least two of the following symptoms.: pelvic pain, oral tempra-ture≥38.5 C 0 , abnormal vaginal discharge, foul odours vaginal discharge, and delay in the involution of the uterus [13]. Independent variable definition Caesarean delivery Caesarean delivery is defined as the delivery of a fetus through surgical incisions made through the abdominal wall (laparotomy) and the uterine wall (hysterotomy) [16]. Rupture of membrane (ROM) ≥24 h Leakage of amniotic fluid for ≥24 h's duration of before giving birth. Multiparous mother Women who give birth ≥2 times after the age of viability regardless of the fetal outcome. Vaginal examination ≥5 When woman have ≥5 times vaginal examination during labor time. Anemia Hemoglobin level < 11 g/dl for postpartum mother. Criteria for inclusion and exclusion This systematic review and meta-analysis included primary studies of any design that reported magnitude of puerperal sepsis and/or associated factors in Ethiopia. However, primary studies were excluded due to any of the following reasons: (a) no report either prevalence or associated factors of puerperal sepsis, (b) articles without full text, (c) articles with poor quality score, (d) articles whose full text was not available within four weeks of email contact, and (e) narrative reviews, editorials, correspondence, abstracts, and methodological studies. Two authors (AM and ED) evaluated the eligibility of all searched studies independently, and any disagreement were resolved through discussion. Study screening and selection Findings were collected by using online databases and transferred from endnote version six manager to Microsoft excel to remove duplicated articles. There were two stages to the study selection process. First, the title and abstract were screened. Then there was full-text review. The titles and abstracts were checked by two authors independently (AM and ED). Studies that reported the prevalence and/or associated factors of puerperal sepsis were selected for full text review. Following full-text review, any study classified as potentially eligible by either author was considered as a full text and independently screened by both authors. Critical appraisal and reliability check After screening; the quality of the selected primary studies was assessed using the Newcastle -Ottawa quality assessment Scale (NOS) for prevalence and case-control studies. To evaluate cross-sectional studies, we used the following criteria: (1) The sample's representativeness; (2) Respondents who did not respond; (3) Determination of exposure (risk factor); (4) Based on the study design or analysis, the subjects in different outcome groups are comparable. Confounding variables are kept under control; (5) .Evaluation of the outcome; (6) Statistical test When articles received 7 points out of 9 for cross-sectional research, they were considered high-quality. We used the following criteria to appraisal case-control studies: (1) adequate case definition; (2) representativeness of the cases; (3) control selection; (4) control definition; (5) comparability of cases and controls based on design or analysis; (6) exposure determination; (7) Cases and controls were exposed using the same method; (8) the non-response rate was calculated. When articles scored 9 points out of 10 for case-control studies, they were considered high-quality. Two writers (AM and ED) scored the data, with disagreements handled by discussion and consensus. The quality scores that calculated the level of agreement for the independent reviews are reported (Additional file 3). During the quality assessment of each primary study, much stress was given to the suitability of the study aims, study design, sampling technique, data collection technique, statistical analysis, any sources of bias, and its management technique. Data extraction After collecting studies by using different online databases (AM) &(ED), autonomously extracted all required data with a standardized data extraction tool. We used two criteria for variables that are used for data extraction [1]. Clear and steady measurement of the variable in the included primary studies [2]. Statistically significant variable in the primary studies which is showed by AOR. The first author's name, year of publication, region, research area, study design, sample size, prevalence, and study quality were among the variables on which data was retrieved from the assessed primary studies. These crucial data were extracted utilizing a data extraction format created on a Microsoft Excel spreadsheet that was initially validated by extracting sample data from some suitable articles before making significant changes for the real data extraction. During extraction, we did not face inconsistent reporting of data for variables in the incorporated primary studies but if we got this inconsistent we would have used data transformation. Data analysis Data were entered into Microsoft Excel and then exported to the statistical software STATA Version 11 for analysis. The p-values of I 2 statistics were used to assess heterogeneity in reported prevalence. Random-effect model were used if heterogeneity within the studies observed. Fixedeffect model were used if homogeneity within the studies seen. While assessing the polled prevalence of puerperal sepsis in Ethiopia, heterogeneity was observed between studies (I 2 = 94.2%, P > 0.01). To assess the pooled prevalence of puerperal sepsis in Ethiopia, random-effect models were used. Subgroup analysis was done to identify possible source heterogeneity by using sample size categories. Eggers regression test was used to assess publication bias between the studies. Result Search result 645 articles were retrieved using a search strategy regarding the prevalence and associated factors of puerperal sepsis in Ethiopia at Pub Med, Web of Science, Science Direct, Embase, Google scholar and HINARI, and Ethiopian universities online repository. All articles found during the search were exported to Endnote, and 112 were removed due to duplication. Five hundred thirty-three studies were screened for eligibility, relevance, accessibility, and outcome of interest. Accordingly, 512 studies were excluded due to irrelevant titles, and 10 studies due to inaccessible full text. Five articles were removed due to different outcomes of interest. Finally, 6 studies were included in this systematic review and meta-analysis (Fig. 1). Study characteristics A total of seven studies with a total of 2222 sample sizes were included in this systematic review and metaanalysis. Based on study design; five studies were crosssectional [10][11][12][13][14] and two were case-control studies [1,17]. In this study, two regions and two administrative cities were included. Three studies were from the Amhara region [10,13,14], two study from the Oromia region [1,17], one study from Addis Ababa city [11], and one study from Dire Dawa city [12] (Table 1). Publication bias The egger's regression test was used to evaluate the likelihood of publication bias within the studies [18,19]. The test result revealed that publication bias was seen between the studies (egger's regression test p-value = 0.006). To eliminate publication bias across the studies, Duval and Tweedie's Trim and Fill analyses were used. The adjusted pooled prevalence of puerperal sepsis after adding two studies with fill and trim analysis was 8.539%(1.691,15.388). Hence, publication bias was solved when two studies were incorporated in the funnel plot by trim fill analysis (Fig. 2). Prevalence of puerperal sepsis The pooled prevalence of puerperal sepsis in Ethiopia was 14.811% (95%CI; 8.46: 21.16; I 2 = 94.2, P ≤ 0.001). This systematic review and meta-analysis revealed that marked heterogeneity was seen within the included studies (I 2 = 94.2, P ≤ 0.001). Therefore, random-effect models were applied to measure the pooled prevalence of puerperal sepsis in Ethiopia. In the included studies the maximum prevalence of puerperal sepsis was reported by Alemale etal. Which was 33.73% [14] and the lowest prevalence of puerperal sepsis was reported by Nigusie Abebaw which was 5.687% [10] (Fig. 3). Subgroup analysis Subgroup analysis was conducted to detect the potential source of heterogeneity within the included studies for this systematic review and meta-analysis. Subgroup analysis was performed with sample size category and by the region where the studies were done. Based on the sample size category, the maximum prevalence of puerperal sepsis was observed in studies that had a sample size of < 300 with 25.26% (95CI:9.06, 41.46). According to the region, the highest prevalence of puerperal sepsis was seen in the Amhara region with a prevalence of 18.54% (95CI: 3.69, 33.39) ( Table 2). The association between puerperal sepsis and CSD Three studies were incorporated in this class of systematic review and meta-analysis [1,10,13]. A significant association was observed between puerperal sepsis and CSD. The odds of developing puerperal sepsis among women who had CSD were 3.26 times higher than as compared to those who had a vaginal delivery (AOR =3.26, 95% CI: 1.90, 5.61). Heterogeneity within the studies was not observed (I 2 = 0). Hence, a fixed-effect model was applied to verify the association (Fig. 4). The association between puerperal sepsis and membrane rupture ≥24 h The association between puerperal sepsis and membrane rupture was assessed by using two studies [1,10]. The finding showed that a significant association was observed between puerperal sepsis and membrane rupture≥24 h. Accordingly, the likelihood of puerperal sepsis was 4.04 times higher in those mothers who had membrane rupture≥24 h as compared to their counterparts (AOR: 4.04, 95CI:2.54, 6.42). Heterogeneity was not observed across the studies (I 2 = 0). Hence, a fixedeffect model was applied to determine the association (Fig. 5). The association between puerperal sepsis and being multiparous mother The association between puerperal sepsis and multiparous mother was assessed by using two studies [13,14]. A significant association was observed between puerperal sepsis and being a multiparous mother. Accordingly, the likelihood of puerperal sepsis was 3.99 times higher in multiparous mothers as compared to their counterparts (AOR: 3.99, 95CI:1.82, 8.78). Heterogeneity was not observed within the studies (I 2 = 0). Hence, a fixed-effect the model was applied find out the association (Fig. 6). The association between puerperal sepsis and having vaginal examination ≥5 times The association between puerperal sepsis and vaginal examination ≥5 were assessed by using three studies [1,12,13]. The finding revealed that significant association was seen between puerperal sepsis and vaginal examination ≥5 times. Accordingly, the likelihood of puerperal sepsis was 3.15 times higher in those mothers who had vaginal examination ≥5 times as compared to their counterpart (AOR: 3.15, 95CI, 1.17, 8.52). Moderate heterogeneity across the studies was observed (I 2 = 74.3). Hence, random-effect the model was applied to find out the association (Fig. 7). The association between puerperal sepsis and anemia The association between puerperal sepsis and anemia were assessed by using two studies [11,20]. Significant association was observed between puerperal sepsis and anemia. Accordingly, the likelihood of puerperal sepsis was 5.68 times higher in those mothers who had anemia as compared to their counterpart (AOR: 5.68, 95CI:4.38, 7.36). Heterogeneity across the studies was not seen (I 2 = 0). Hence, fixed-effect the model was applied to determine the association (Fig. 8). Discussion This systematic review and meta-analysis was aimed to assess the pooled prevalence of puerperal sepsis and associated factors in Ethiopia. This study has showed that [7],Nigeria 11.4% [21]. The finding of the present study was higher than a study done in Osun State, Nigeria (0.78%) [6], and Sindh Pakistan (3.89%) [22] but lower than studies done in Sudan (72.9%) [9],Zambia (34.8%) [23], and India (68.65%) [8]. The difference could be the variation of sample size, Level of hygiene at the community and individual level, study area, causative agent for puerperal sepsis, institutional delivery coverage, and quality of maternity service. Good quality of maternity service and high rate of institutional delivery coverage decreases the rates of puerperal sepsis. Beside to this, good hygiene of the community and maternity women at an individual level might be helpful to prevent puerperal sepsis. The subgroup analysis of this study showed that the prevalence of puerperal sepsis among postpartum mothers was significantly varies across regions. The highest prevalence of puerperal sepsis was observed in Amhara region 18.54% (95CI:3.69, 33.39) and the lowest prevalence was seen in Self-administered cities (Addis Abeba and Dire Dawa) 10.58(95CI:6.13, 15.04). This could be the difference of awareness for maternal and child health services, distance of health institution which gives maternity services from their home, and availability health institutions and medical equipments. The awareness of postpartum mothers who lives in the town administrative (Addis Abeba and Dire Dawa) about the availability of maternity health services is high due to their exposure for radio, television, and mass media, and press. Beside to this, postpartum mothers who live in the town administrative (Addis Abeba and Dire Dawa) have good infrastructures such as road, and ambulance to rich timely and get maternity services in compare to who lives in Amhara region (Almost all part is rural and distant to get maternity health services). The current study revealed that CSD was significantly associated with puerperal sepsis. The odds of developing puerperal sepsis among women who had CSD were 3.26 times higher than as compared to those who had a vaginal delivery (AOR =3.26, 95% CI: 1.90, 5.61). These finding supported by studies done in Scotland [24], and Tanzania [25] which was reported that having CSD was associated with puerperal sepsis. The possible explanation could be due to poor infection prevention technique during operation procedure, Loss significant amount of blood due to poor homeostasis during CSD, and High load of obstetric of cases in the maternity ward. Ethiopia is one of the countries which have a high population with poor health service coverage especially maternal and child health service which might lead puerperal sepsis. Membrane rupture≥24 h was significantly associated with puerperal sepsis. Accordingly, the likelihood of puerperal sepsis was 4.04 times higher in those mothers who had membrane rupture≥24 h as compared to their counterparts (AOR: 4.04, 95CI:2.54, 6.42). This result is similar with studies done in Pakistan [22], and Kenya [26] which reported that membrane rupture ≥24 h is associated with puerperal sepsis. This could be once the membrane (which gives protection for upper reproductive tract from ascending infection) ruptured bacterial organisms which arises from external environment and vagina have got the chance to go through dilated cervix to the upper internal reproductive tract. Moreover, if a woman comes with ruptured membrane with dilated cervix and took long time to give birth the number of vaginal examination increased as time gooses and this might leads to puerperal sepsis. Being multiparous mother was significantly associated with puerperal sepsis. The likelihood of puerperal sepsis was 3.99 times higher in multiparous mothers as compared to their counterparts (AOR: 3.99, 95CI:1.82, 8.78). This finding is similar with the studies done in United States of America [27], and Pakistan [22] which reported that being multiparous mother is associated with puerperal sepsis. This could be due to when parity increases some obstetrical complications such as uterine rupture, Post partum hemorrhage, and retained placenta increases and this complication also increases the rate of puerperal sepsis due to numerous manipulation of the internal reproductive tract of the mother. Having vaginal examination ≥5 times was significantly associated with puerperal sepsis. Accordingly, the likelihood of puerperal sepsis was 3.15 times higher in those mothers who had vaginal examination ≥5 times as compared to their counterpart (AOR: 3.15, 95CI, 1. 17, 8.52). This result is similar with the studies done in Kenya [28] Egypt [29], South Asia [30]. This could be when mothers have unnecessary multiple vaginal examination during labor and delivery we inoculate bacteria from outside environment and vagina to the internal reproductive organs and this could leads to puerperal sepsis. The present study showed that anemia was significantly associated with puerperal sepsis. The likelihood of puerperal sepsis was 5.68 times higher in those mothers who had anemia as compared to their counterpart (AOR: 5.68, 95CI:4.38, 7.36). This study is similar to the study done in Scotland [24], and Nigeria [21]. The possible explanation could be anemic mothers lack natural tolerance for infection and this might leads to puerperal sepsis. Ethiopia is one of the countries which have poor socio-economic status; due to this pregnant and postpartum mothers face a lack of a balanced diet to challenge anemia and its associated complications like sepsis. Limitation This study is limited by the identifying of a few numbers of studies reporting puerperal sepsis, of which merely seven were deemed to be good procedural quality. This study might not represent the overall prevalence of puerperal sepsis at the country level due to the small number of studies and lack of representations of the different regions in Ethiopia. Conclusion The prevalence of puerperal sepsis was high in Ethiopia. CSD, Membrane rupture≥24 h, Being a multiparous mother, vaginal examination≥5, and anemia were factors significantly associated with puerperal sepsis. Appropriate standard infection prevention techniques during CSD shall be practiced to reduce the maternal burden of puerperal sepsis. The frequent vaginal examination should be discouraged during the intrapartum period. Besides this, routine Iron sulfate supplementation and counsel on iron reach foods during antepartum and postpartum shall be considered for all mothers.	2021-11-29T14:38:43.789Z	2021-11-29T00:00:00.000	{ "year": 2021, "sha1": "8fa17f493a22535cec49386bc46520a87f42048b", "oa_license": "CCBY", "oa_url": "https://archpublichealth.biomedcentral.com/track/pdf/10.1186/s13690-021-00732-y", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "8fa17f493a22535cec49386bc46520a87f42048b", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
15445305	pes2o/s2orc	v3-fos-license	New family medicine residency training programme: Residents’ perspectives from the University of Botswana Background Family Medicine (FM) training is new in Botswana. No previous evaluation of the experiences and opinions of residents of the University of Botswana (UB) Family Medicine training programme has been reported. Aims This study explored and assessed residents’ experiences and satisfaction with the FM training programme at the UB and solicited potential strategies for improvement from the residents. Methods A descriptive survey using a self-administered questionnaire based on a Likert-type scale and open-ended questions was used to collect data from FM residents at the UB. Results Eight out the 14 eligible residents participated to this study. Generally, residents were not satisfied with the FM training programme. Staff shortage, inadequate supervision and poor programme organisation by the faculty were the main reasons for this. However, the residents were satisfied with weekly training schedules and the diversity of patients in the current training sites. Residents’ potential solutions included an increase in staff, the acquisition of equipment at teaching sites and emphasis on FM core topics teachings. They had different views regarding how certain future career paths will be. Conclusions Despite the general dissatisfaction among residents because of challenges faced by the training programme, we have learnt that residents are capable of valuable inputs for improvement of their programme when engaged. There is need for the Department of Family Medicine to work with the Ministry of Health to set a clear career pathway for future graduates and to reflect on residents’ input for possible implementation. Introduction Residency training programmes in Family Medicine (FM) have been in existence in other parts of the world for a number of years as reported in studies carried out in the United States 1 and Saudi Arabia. 2 The development of FM residency programmes in Africa is now a reality with the setting of new FM centres in central, East, and West Africa. 3 The evaluation of those programmes in a SWOT (strength, weakness, opportunity and threats) analysis 4 reported weaknesses and strengths that will be worth looking at and comparing with other new training programmes such as the University of Botswana (UB) Family Medicine residency. The FM at the UB is one of the newly created programmes in Southern Africa 3 and is a 4-year programme with an additional provision for 2 years under the UB regulations for completion. We have not been able to access literature regarding challenges faced by newly created Southern African training programmes despite the existence of a twinning programme amongst countries in Southern Africa. 3 Therefore, the reasons for this lack of published data on experiences of new FM training programmes in Southern Africa are not known. The establishment of the first medical school in Botswana was motivated by a number of reasons. 5 These included the fact that shortage of medical doctors (MD) within the Botswana health system was high and inequitable distribution of doctors within the country existed with rural areas experiencing more shortage compared to urban areas. 5,6,7 FM was seen to have an important role in strengthening the health system, 8 and the recommendation of the World Health Organisation on primary care reform 9 attested to the need for FM training in Botswana. In addition, the overall effectiveness of family physicians (FPs) in some African countries 10,11 highlighted the need for such training in Botswana. To address the shortage of MDs in general and FPs in particular with the hope of addressing the health needs of the population, the government of Botswana through the UB started training for Master of Medicine (Internal Medicine and Paediatrics with Adolescent Health) in 2010, while other programmes for Master of Medicine (Family Medicine, Anaesthesia and Emergency Medicine) started in 2011. 5 The first graduates of Masters programmes were expected in 2014, while those for FM and other programmes started in 2011 were expected in 2015. Formative evaluation reported in studies done elsewhere using residents' opinions to adjust the FM training programme have proven to be helpful. 12,13 Therefore, it was important to identify the strengths and weakness of the UB FM residency programme similarly in order to help the UB Department of Family Medicine and other stakeholders to address issues and improve the training programme. The head of the Department of Family Medicine operates from the main campus in Gaborone; consequently, communication between the three teaching sites has been challenging because of the distance between the sites. These three sites communicate through Skype©, telephone and email, while the preferred face-to-face meetings are infrequent. At its inception in 2011, the FM residency programme had an initial resident-faculty ratio of 4:1, which deteriorated to 6:1 and 8:1 in Mahalapye and Maun, respectively, over time. The number of residents in the sites under study (Maun, Mahalapye) was four per each site in 2011, eight and six per site in 2012, respectively, in Maun and Mahalapye. These numbers remained unchanged till 2015 as there was no new intake of residents in 2013 because of acute staff shortage. The resident intakes from 2014 to 2015 were based in hospitals around Gaborone where the head of department was based. The number of lecturers in these sites was not stable as three of the appointed lecturers either resigned or did not apply for renewal of contract for personal reasons over time, while another member was enrolled to a 2-years master's programme abroad. This resulted in a drop from the best number of seven lecturers to the lowest number of three (one per site in Maun and Mahalapye where the study was conducted, and the head of department in Gaborone), with an unchanged number of residents in Mahalapye and Maun. Thus, there was an increase in faculty workload between end of 2013 to early 2015, with reduction of staff and arrival of continuing two streams of undergraduate students in Mahalapye and Maun. The undergraduates were Bachelor in Medicine, Bachelor in Surgery students in their third and fifth year, doing 8-week rural exposure rotations in groups of 6-10 students, and they required supervision and teaching by faculty in the Department of Family Medicine. Possible solutions for this acute staff shortage from the Department of Family Medicine's point of view were possible closure of existing separate training centres and relocation to Gaborone in order to optimise use of the few facility. However, this did not materialise. The faculty-resident ratio subsequently improved to the current 1:4, with the appointment of three new staff members from June 2015, and it is hoped that the growing residents' complaints regarding inadequate supervision will be partly addressed. The Department of Family Medicine uses a curriculum that was adapted from the Stellenbosch University (SU) training programme. In the context of Botswana, this consisted of the first 24 months training being based in district hospitals, and this consists of rotations in different specialties called 'clinical domains rotations' such as internal medicine, surgery, obstetrics and gynaecology, anaesthesia and paediatrics as indicated in Table 1 At the time of this study, the supervision during the hospitalbased rotations was mainly done by the specialists employed by the Ministry of Health (MOH), and the primary care/clinicbased placement was supervised by faculty members who were FPs. UB provides mandatory common core modules to all the residents regardless of discipline of specialisation within the Faculty of Medicine (FOM). These are communication skills, ethics and professionalism, public health and international health, research methods and introduction to the medical literature review. The FM academic activities include a monthly journal club and weekly tutorials consisting of case presentations, video viewing of consultations, clinical skills demonstrations, presentations of common problems encountered in primary healthcare and consultation skills discussions, which take place during tutorials and at the bedside. In the context of FM training in Botswana, continuing medical education presentations by residents, other health professionals and faculty members are also part of the teaching platform, conducted in the hospital meeting room for the benefit of the entire district hospital staff. To assure quality training in the new Department of Family Medicine, benchmarking was done with SU, and Limpopo (Now Sefako Makgatho University of Health Sciences), and accreditation with the College of Family Physicians of South Africa was sought and granted. Aims of the study This study assessed the level of satisfaction of residents with the FM training programme at UB, Botswana, and solicited from them possible solutions for improvement of the FM training programme. Contribution to field Findings of this study may be the first to be reported among new Southern African FM training programmes about the strengths, weaknesses and potential solutions that any new training programme is likely to face. This study may be considered as a contribution to the conversation about challenges and potential solutions of starting FM programmes and in newly created FM training sites in southern Africa. It is hoped that, this study will encourage a collaborative regional effort to overcome common problems in the teaching of FP through information sharing and similar multicentre studies for regional solutions to challenges in FM training programmes. Study design This was a descriptive survey using a structured selfadministered questionnaire. Setting The study was done at Maun and Mahalapye FM training sites where two district hospitals of about 260 beds each, including surroundings clinics are used for practical and theoretical training of residents in FM. Study population and sampling strategy All 14 eligible FM residents based in Maun and Mahalapye in December 2014 were invited to participate in the study. Six residents were based in Mahalapye and eight were based in Maun. During the study, the residents were either in their fourth or fifth year of training. The Department of Family Medicine did not have residents in the third year when the study was done, as during 2013 we did not recruit because of an acute shortage of staff. Data collection A presentation on the research project (information session) to all residents at both sites (Mahalapye and Maun) was done in December 2014. This was followed by a collective email, with the consent form and questionnaire attached, sent out to the residents. The self-administered graduate medical education (GME) survey questionnaire, 13 adapted to the Botswana setting, was used. The choice of the GME questionnaire was based on the availability of questions ready for use, and the ability of these questions to assess the context, inputs, process and output (CIPP). A more detailed questionnaire generated from a CIPP fomat 14 accessed after the design will be considered for use in a future evaluation of the FM programme. To complement the GME questionnaire, new questions using the same style were added to fit the Maun and Mahalapye settings. The questions were not considered to be major distortions to the validated questionnaire as none of the original questions was altered. Researchers who were lecturers of the participants in the two sites collected the filled questionnaires from labelled boxes at designated places in the two sites where it was agreed that forms were to be privately dropped. Data were collected through answers on a Likert-type scale, which assessed the level of satisfaction of residents regarding the content of the FM programme, the quality of teaching rendered by supervisors, the quality of feedback and assessment and their satisfaction with their work environment and teaching sites. The open-ended questions component captured participants' opinions on strengths and weaknesses of the FM programme, residents' opinions on the future career as FP in Botswana and recommendations for improvement. As a result of slow uptake, the initial two months planned for data collection were extended. A monthly reminder was sent out by email and verbally during the three months resulting in approximately a five -month data collection period, to ensure maximum participation of residents. Unfortunately, participation remained low regardless of efforts to get everyone to participate. Data analysis Quantitative data were captured in IBM SPSS 21 for statistical analysis. Frequencies were used to summarise the categorical variables. Answers to open-ended questions were categorised based on types of comments or solutions from the eight participants on a particular question. The two on-site researchers separately looked at data for possible themes and categorisation of comments. Final themes and categories were obtained after reconciling their findings, which were referred to as emerging themes. The process consisted in identifying and grouping similar ideas and answers into positive or negative categories with regard to strengths, weaknesses and solutions. Researcher triangulation was done by comparing findings from the two researchers after the review of same data set, and consensus was reached by discussion where divergent findings were noted. Of the eight respondents, the open-ended section of participant number seven was empty and was not included in the analysis. All eight questionnaires were completed in terms of the Likerttype scale section. The justification of the choice of training sites, the strengths and weakness of the programme, residents' opinions on their future career path and recommendation for programme improvement were analysed and reported. Results Of 14 eligible residents, 8 residents in their fourth or fifth years in the FM programme participated in this study. Three participants out of six were from Mahalapye and five out of eight were from Maun, making the total participation of more than half of eligible FM residents. Profiles of participants Five participants were from the Maun training site while three were from the Mahalapye site. There were five male (5/8) and three female (3/8) participants, aged between 31 and 37 years, of whom four were married (4/8) and four were single (4/8). Half of the participants (4/8) were in the fourth year of training. Residents' satisfaction with FM training programme All participants agreed that they were satisfied with the diversity of patients and diagnoses in the training sites (8/8). Over some aspects of the training programme, participants had divided views (4/8), with half of them reporting dissatisfaction while the other half were satisfied with the programme. These aspects are summarised in Figure 2 with the number of participants agreeing or disagreeing to have satisfaction with aspects of training programme, shown in the bar chart. Inpatient clinical teaching setting, the procedural surgical experiences, the formative feedback received for performance improvement, the scheduling of vacation and time off-duty and whether residents were to recommend the programme to friends are reported in the Figure. Most participants were not satisfied with other aspects of the training programme. They expressed particular dissatisfaction (number of residents dissatisfied/total of residents) with the following aspects of the programme: • the programme organisation (7/8); • the overall dissatisfaction with the programme (5/8); • the teaching sessions (6/8); • the guidance and mentorship (5/8); • the performance evaluation (5/8); and • the duty roster/work schedule (4/8). In-patient clinical trainings were satisfactory I will recommend this programme to a friend strongly disagree disagree agree strongly agree Source: Authors' own work Number of parƟcipants Aspect of training 5 Strengths of the programme Residents viewed strengths of the programme as the support from different stakeholders, staff commitment, regular tutorials and protected study time. Stakeholders' support Most participants considered the strength of the programme to be the support from regional universities, the support from private funders and the availability of infrastructure provided by the MOH: Regular tutorials and time to study Protected time and regular tutorial were considered by some as strengths of the programme as it allowed interaction with faculty and colleagues: 'Protected study time and Wednesday's classes [tutorials].'(P4) Weaknesses of the programme The shortage of staff, inadequate supervision, too little focus on FM topics, poor departmental administration and a lack of faculty experience were reported by the majority as weaknesses of the programme. Other weaknesses that were mentioned were non-competitive faculty remuneration that caused difficulty in attracting and retaining staff, current training sites not being ideal, too heavy workload for residents, the lack of differentiation of roles for different levels of residents and insufficient equipment for training. Staff shortage leading to inadequate supervision Most participants viewed the shortage of staff and the inadequate supervision to be weaknesses of the programme: 'Lack of human resources-lecturers.'(P3) 'Shortage of staff is the main problem.'(P5) The shortage of staff was differently expressed in terms of the clinical involvement of faculty and inadequate supervision: 'Because as it stands they are not actively involved in clinical medicine with residents unless if they come to assess, obviously that ties with a point I made above that there is serious shortage of staff that need to be increased.'(P2) Lack of supervision A participant expressed that the lack of supervision was the weakness of the programme while another considered the inability of recruiting more staff because of non-competitive salaries as a possible explanation for shortage of staff and inadequate supervision: 'The lack or inability to recruit staff due to money reasons.'(P3) 'Lack of supervision at inpatient and outpatient areas.'(P4) Departmental administration and faculty experience Not having a FM lecturer with previous teaching or administrative experience in a medical school was seen as another weakness of the programme: 'I also feel it is a blunder that amongst all our lecturers there is no one with prior teaching experience or having been part of administrative staff in running of a medical school that is why a lot of things are not going right.'(P2) The same participant referred to the failure of producing necessary documents for residents in time as an administrative weakness of the programme while another referred particularly to the inexperience and possible lack of awareness of UB regulation as the cause to the weakness of the programme: Ideal training sites Participants were divided as to whether Maun and Mahalapye were good training sites, with less than half of the participants favouring the sites. A participant thought that the current district hospital location of the training is part of the weakness of the programme, preferring a primary hospital location for training while a colleague was of the opposite opinion, favourable to district hospital: 'Training in a district site which I believe is not appropriate for family medicine training; personally I believe it will be better in a primary hospital.' (P2) 'Good sites locations [are] district hospital.' (P8) Work load, differentiation of tasks and equipment for training Limited medical equipment at training sites and overworking were considered to be weaknesses of the programme as they did not promote procedural clinical skills which should be residents' second nature. 'Patient overload, limited equipment and tools …, we need to acquire skills so that they become second nature …, we also need a skills lab with manikins to practice on.' (P1) http://www.phcfm.org Open Access Another resident pointed out that the programme failed to consider progression in seniority with number of years in the programme and there was no point at which a resident was considered senior to medical officers. Faculty were perceived to be not very concerned about defending residents' primary goal of learning as opposed to working in the hospital: 'We work as medical officers from first year to the last year and faculty has totally allowed the hospital to do as it wishes with residents, providing service at total compromise of our learning forgetting that our true mandate is learning.' (P2) Teaching and assessment Participants perceived the lack of focus in teaching FM concepts or topics and the preparation towards the final year examination as inadequate and weakening the programme: 'Lack of proper training in family medicine concepts.' (P4) 'No proper preparation for the exit exams.' (P4) Potential solutions Following were suggested: Increase of faculty number and monetary incentives Different participants viewed monetary incentives and staff increase as part of the solution to the current situation: 'Increase of staff, remuneration for part-time lecturers.' (P6) 'A joint lecturer appointment to improve lecturer-resident supervision and monitoring.' (P8) 'Increase supervision and staff so that the residents get exposed to the everyday environment of a family physician.' (P1) Yearly plan and mock examination preparation This comprises a yearly plan of activities, including mock preparation contrary to a 6-month plan that is being produced currently: 'Please plan the year properly, and have mini mock exams at least x2 per year [about], different procedural skills we are expected to know.' (P4) To help administer FM programme, it was suggested the alignment of the programme with other UB master's degree programme: 'Align program with other UB masters courses and the use of same resources.' (P8) Collaborative teaching effort within the University of Botswana From the collaborative teaching experience with public health, a participant thought that intensifying teaching collaborations with other UB departments will be beneficial for residents: 'Make use of other UB department in teaching us things like public health and research.' (P2) Acquisition of more equipment for skills training Some participants believed that acquiring on-site medical and simulation equipment will improve their skills which will help to provide quality care: 'Have a skills lab, make sure we have basic instruments to enable us to manage patients according to evidence based standards.' (P1) 'Avail medical equipment that may be useful in helping residents acquire certain skills.' (P2) Teaching more family medicine topics A participant suggested a possible solution that will help to have available adequate FM topics in the department: 'Draw a syllabus for a longer module on family medicine concepts.' (P8) Flexibility of the programme A participant thought that based on personal experience in the medical field residents should be allowed to spend more time in areas of weakness than in areas of strength, and therefore suggested: 'Letting residents spend more time in the disciplines they have more inadequacies in, rather than forcing everyone to do same length of time in each in each discipline.' (P2) Residents' opinions on the future career path as family physicians in Botswana Residents had mixed feeling about their future varying from uncertainty on career path to a bright future full of hope while two participants did not express their opinions on this. The uncertainty was supported by: 'I still currently have no confidence in Family Medicine concepts, and I still don't know where we will work when we finish.'(P4) 'A poor family physician deprived of opportunity to learn because of poor structure of programme …' (P2) 'Immediately after graduating will leave University and look for green pasture unless salary improves.'(P5) The optimistic views were supported by: 'I feel graduates are likely to improve the medical practice in Botswana by acting not only as doctors but also as mentors and managers to other health professionals … there is a bright future for graduate.'(P1) 'Vast opportunities.'(P8) 'Being able to function as medical manager.' (P3) Discussion In general, participants expressed dissatisfaction with the FM training programme. The shortage of staff was perceived to be a cause of many other unsatisfactory areas of FM training, while the only area of the programme with which all participants were satisfied was the exposure to a diversity of patients and diseases at both training sites. The dissatisfaction with the supervision found in this study may have been because of inadequate staffing as the facultyresidents ratio dropped to 1:8 and 1:6, respectively, in the Maun and Mahalapye sites for more than a year. This situation has fortunately been corrected during the second half of 2015. Adequate staffing may address many weaknesses http://www.phcfm.org Open Access of the Botswana FM programme such as inadequate supervision of residents and inadequate staff to facilitate examination preparation. The role of adequate supervision in FM residency is vital in learning facilitation as reported in a study in Saudi Arabia, 14 and this gives credence to participants' thoughts on this issue. However, inadequate supervision has been reported in different settings outside Africa, 12,15 including in other new FM programmes in Africa implied by the lack of FPs and local teachers. 4 In the case of UB, a FM programme which adopted a decentralised approach with inadequate staff in different training sites and difficulty in recruiting and retaining experienced staff necessitated consideration of growing our own staff, both the initial inexperienced staff and the graduates of the programme. Not doing so might endanger the development of FM in Botswana. However, the closure of the two rural sties contemplated by the UB FM programme in response to the acute staff shortage experienced was not supported during the first national FM conference, 16 which observed that a development of centres around Gaborone should be done keeping in mind staffing situation, while maintaining the existing two rural sites to ensure the spread of FM in Botswana. We have now four training centres in total, two of which are in villages near Gaborone. The administrative inexperience of the staff members was another aspect of dissatisfaction. At the time of this research, it was still difficult to recruit experienced staff. This is a common experience among low-income countries in the region but a high middle-income country like Botswana experienced similar challenges; this is why the solution may be for these new FM departments in the African region to grow from within and to develop regional collaboration for staff development. Poor organisation of new FM training programmes has been reported elsewhere 12,15,17 and this needs to be addressed in the UB programme. A suggestion that the Botswana FM programme should be aligned to other master's programmes at the UB is perhaps a good place to start. In fact, at the time this study took place, the FM programme and other FOM master's degree programmes were the only ones to start their year in January while other older masters' degree programmes in UB start in August, and this has implications on administrative issues. The concern that there should be more focus on FM-oriented topics was similar to the experiences of Turkish trainees 18 who missed FM topics during their hospital rotations. However, Japanese trainees 17 actually felt deprived of adequate clinical teaching during their FM programme. This shows the need of a balanced emphasis in the curriculum of the Botswana FM programme, namely between the clinical procedural skills in general and the FM concepts and special consultation skills that mould a good FP. Participants found the workload excessive and incompatible with their training. This experience is similar to findings in FM training programmes in some African countries. 4 This situation may be because of the inherent workload in primary care settings in Africa and in residency programmes in general. Balancing workload and protected learning opportunities in the FM residency programme is tricky because significant workload is itself a good preparation for future work as FP and a leader in the primary healthcare setting. The limitations of this study included the small sample size, the low response rate. This means that residents 'perceptions of the University of Botswana FM Master' programme requires further exploration. However, indications are that some of these findings could be transferable to other similar settings, 19 as shown by similarities with findings from other African countries following a SWOT analysis. 4 Because the study population has been subjected to training in an inadequately organised programme under inexperienced staff, this may have caused them not to want to participate in assessing a programme that had not met their expectations. However this study provides an important evaluation of the Botswana FM residency programme from the residents' perspective. Therefore, it contributes to the ongoing discussions about how the Botswana FM residency programme can be improved. Future evaluations of the programme may wish to address the extent to which the findings of this study influenced the development of the Botswana FM residency programme and the health of the population of Botswana. The programme seems to have educational management problem, faculty development issues and scarce resources. Lessons learned Following are lessons to learn from this programme evaluation: • There is a need for the MOH to define a clear career path for graduates to avoid residents' despair. This has since been addressed to a significant extent by the MOH. • Staff employment and development is crucial in new training programme of FM and this is being done. • Solutions or suggestions to improvement can emanate from residents in a training programme. • There is a need for involving all stakeholders in implementing suggested solutions, while the Department of Family Medicine should consider empowering willing graduates to join the department in teaching, as recruiting from outside has proved to be difficult. We recommend faculty development and regional collaboration, which may help the current faculty members to mature and gain required experience for quality teaching and supervision.	2018-05-08T17:41:34.199Z	2016-08-31T00:00:00.000	{ "year": 2016, "sha1": "d37bee20a691f50475bd97fc7a49d852cfda2f13", "oa_license": "CCBY", "oa_url": "https://phcfm.org/index.php/phcfm/article/download/959/1744", "oa_status": "GOLD", "pdf_src": "Grobid", "pdf_hash": "d9978e367e27aa7c7860ebe4f55397529fc0a406", "s2fieldsofstudy": [ "Medicine", "Political Science" ], "extfieldsofstudy": [] }
208548372	pes2o/s2orc	v3-fos-license	On the central levels problem The central levels problem asserts that the subgraph of the $(2m+1)$-dimensional hypercube induced by all bitstrings with at least $m+1-\ell$ many 1s and at most $m+\ell$ many 1s, i.e., the vertices in the middle $2\ell$ levels, has a Hamilton cycle for any $m\geq 1$ and $1\le \ell\le m+1$. This problem was raised independently by Buck and Wiedemann, Savage, by Gregor and \v{S}krekovski, and by Shen and Williams, and it is a common generalization of the well-known middle levels problem, namely the case $\ell=1$, and classical binary Gray codes, namely the case $\ell=m+1$. In this paper we present a general constructive solution of the central levels problem. Our results also imply the existence of optimal cycles through any sequence of $\ell$ consecutive levels in the $n$-dimensional hypercube for any $n\ge 1$ and $1\le \ell \le n+1$. Moreover, extending an earlier construction by Streib and Trotter, we construct a Hamilton cycle through the $n$-dimensional hypercube, $n\geq 2$, that contains the symmetric chain decomposition constructed by Greene and Kleitman in the 1970s, and we provide a loopless algorithm for computing the corresponding Gray code. Introduction The n-dimensional hypercube, or n-cube for short, is the graph Q n formed by all {0, 1}-strings of length n, with an edge between any two bitstrings that differ in exactly one bit. This family of graphs has numerous applications in computer science and discrete mathematics, many of which are tied to famous problems and conjectures, such as the sensitivity conjecture of Nisan and Szegedy [29], recently proved by Huang [23]; Erdős and Guys' crossing number Our results In this paper we consider the central levels problem, a broad generalization of the middle levels problem: Does the subgraph of the (2m + 1)-cube induced by the middle 2 levels, i.e., by levels m + 1 − , . . . , m + , have a Hamilton cycle for any m ≥ 1 and 1 ≤ ≤ m + 1? This problem was raised independently by Savage [34], Gregor and Škrekovski [20], and by Shen and Williams [38]. Clearly, the case = 1 of the central levels problem is the aforementioned middle levels problem (solved in [27]). Moreover, the case = 2 was solved affirmatively in a paper by Gregor, Jäger, Mütze, Sawada, and Wille [16] presented at ICALP 2018. Also, the case = m + 1 is established by the binary reflected Gray code Γ 2m+1 . Furthermore, the case = m was solved by El-Hashash and Hassan [7], and in a more general setting by Locke and Stong [26], and the case = m − 1 was settled in [20]. The main contribution of this paper is to solve the central levels problem affirmatively in full generality; see Figure 1 (a)-(d). Theorem 1. For any m ≥ 1 and 1 ≤ ≤ m + 1, the subgraph of the (2m + 1)-cube induced by the middle 2 levels has a Hamilton cycle. The most general question in this context is to ask for a Hamilton cycle in Q n that visits all vertices in any sequence of consecutive levels, i.e., the levels need not be symmetric around the middle, and the dimension n needs not be odd. These graphs are all bipartite, and to circumvent the imbalances that prevent the existence of a Hamilton cycle for general n and , we have to slightly generalize the notion of Hamilton cycles: Firstly, a saturating cycle in a bipartite graph is a cycle that visits all vertices in the smaller partition class (if it has P. Gregor, O. Mička, and T. Mütze 60:3 size 1, then a single edge is considered to be a cycle). Secondly, a tight enumeration in a (bipartite) subgraph of the cube is a cyclic listing of all its vertices where the total number of bits flipped is exactly the number of vertices plus the difference in size between the two partition classes. Clearly, if both partition classes have the same size, these two notions are equal to a Hamilton cycle. In fact, all cases of this more general problem, except the central levels problem, were solved already in [18], some of them conditional on a "yes" answer to the central levels problem. Combining Theorem 1 with these previous results, we now also obtain an unconditional result for this more general question. Corollary 2. For any n ≥ 1 and 1 ≤ ≤ n + 1, the subgraph of the n-cube induced by any sequence of consecutive levels has both a saturating cycle and a tight enumeration. An essential tool in our proof of Theorem 1 are symmetric chain decompositions. This is a well-known concept from the theory of posets, which we now define specifically for the n-cube using graph-theoretic language. A symmetric chain in Q n is a path (x k , x k+1 , . . . , x n−k ) in the n-cube where x i is from level i for all k ≤ i ≤ n − k, and a symmetric chain decomposition, or SCD for short, is a partition of the vertices of Q n into symmetric chains. It is well-known that the n-cube has an SCD for all n ≥ 1, and the simplest explicit construction was given by Greene and Kleitman [15] (see Section 2.2 below). Streib and Trotter [40] first investigated the interplay between SCDs and Hamilton cycles in the n-cube, and they described an SCD in Q n that can be extended to a Hamilton cycle; see Figure 1 (e). Their SCD, however, is different from the aforementioned Greene-Kleitman SCD. In this paper, we extend Streib and Trotter's result as follows; see Figure 1 (f). Theorem 3. For any n ≥ 2, the Greene-Kleitman SCD can be extended to a Hamilton cycle in Q n . The Greene-Kleitman SCD has found a large number of applications in the literature, e.g., to construct symmetric Venn diagrams [21,33], to solve the Littlewood-Offord problem [3,Chap. 4], or to learn monotone Boolean functions [25, Sec. 7.2.1.6] (see also [1,6,31,37,42]). Knowing that this SCD extends to a Hamilton cycle and that it is a crucial ingredient for solving the general central levels problem adds to this list of interesting properties and applications. Observe also that a Hamilton cycle that extends an SCD has the intriguing property that it minimizes the number of changes of direction from moving up to moving down, or vice versa, between consecutive levels in the cube. For comparison, the monotone paths constructed by Savage and Winkler [36] maximize these changes. Motivated by these results and by the aforementioned conjecture of Ruskey and Savage [32] that every matching in Q n extends to a Hamilton cycle, we raise the following conjecture: Conjecture 4. Every SCD can be extended to a Hamilton cycle in Q n . Although every SCD of Q n is the union of two matchings, there are matchings in Q n that do not extend to an SCD; take for example the two edges obtained by starting at the vertices 0 n and 1 n and flipping the same bit. Consequently, an affirmative answer to Conjecture 4 would cover only some cases of the Ruskey-Savage conjecture. Efficient algorithms Our proof of Theorem 1 is constructive and translates directly into an algorithm for computing the Hamilton cycle in time and space that are polynomial in the size of the graph (the middle 2 levels of Q n , n := 2m + 1), which is exponential in n. Often, it is desirable to have 60:4 On the Central Levels Problem a "local" algorithm that uses only time and space that are polynomial in n. Ideally, one might hope for O(n) space to store the current bitstring and some additional data structures, and O(1) time to compute the next bitstring on the cycle. Such algorithms are known for the binary reflected Gray code Γ n [2], and for the middle levels problem [28], i.e., for the extreme cases = m + 1 and = 1 of the central levels problem. There are fundamental obstacles that prevent us to obtain such a local algorithm from our proof, and it remains a challenging open problem to find such an algorithm. Our Theorem 3 on the other hand, can be translated into a simple algorithm that uses only O(n) space and O(1) time in every iteration to compute the next bitstring along the Hamilton cycle. A pseudocode description of this algorithm is available in [17]. We also implemented it in C++, available for download and for demonstration on the Combinatorial Object Server [5]. Proof ideas We first describe the ideas for proving Theorem 1. For any m ≥ 1 we define n := 2m + 1, and for 1 ≤ ≤ m + 1 we let Q n, denote the subgraph of Q n induced by the middle 2 levels. To prove that Q n, has a Hamilton cycle for general m and , we combine and generalize the tools and techniques developed for the cases = 1 and = 2 in [19] and [16], respectively. Our proof proceeds in two steps: In a first step, we construct a cycle factor in Q n, , i.e., a collection of disjoint cycles which together visit all vertices of Q n, . In a second step, we use local modifications to join the cycles in the factor to a single Hamilton cycle. Essentially, this technique reduces the Hamiltonicity problem in Q n, to proving that a suitably defined auxiliary graph is connected, which is much easier. In fact, the predecessor paper [16] already proved the existence of a cycle factor in Q n, , but this construction does not seem to yield a factor that would be amenable to analysis. In this paper, we therefore construct another cycle factor in Q n, , based on modifying the aforementioned Greene-Kleitman SCD of Q n by the lexical matchings introduced by Kierstead and Trotter [24]. The resulting cycle factor in Q n, has a rich structure, in particular the number of cycles and their lengths can be described combinatorially. The simplest way to join two cycles C and C from this factor to a single cycle is to consider a 4-cycle F that shares exactly one edge with each of the cycles C and C (the other two edges of F must then go between C and C ), and to take the symmetric difference of the edge sets of C ∪ C and of F , yielding a single cycle (C ∪ C ) F on the same vertex set as C ∪ C . We refer to such a cycle F as a flipping 4-cycle. For example, if we interpret the binary reflected Gray code Γ n as a cycle in Q n , we see that Γ n+1 = (0Γ n ∪ 1Γ R n ) F where F is the 4-cycle F = 0 n+1 , 010 n−1 , 110 n−1 , 10 n . In addition to flipping 4-cycles, we also use flipping 6-cycles, which intersect with the two cycles to be joined in a slightly more complicated way, albeit with the same effect of joining them to a single cycle. The most technical aspect of this part of the proof is to ensure that all flipping cycles used are edge-disjoint, so that the joining operations do not interfere with each other. To prove Theorem 3, we proceed by induction from dimension n to n + 2, treating the cases of even and odd n separately. We first specify a particular ordering of all chains of the Greene-Kleitman SCD, and then show that this ordering admits a matching that alternatingly joins the bottom or top vertices of any two consecutive chains in our ordering. In fact, there is a close relation between our proofs of Theorem 1 and 3: The aforementioned construction of a cycle factor in Q n, is particularly nice for = m + 1, i.e., for the case where we consider the entire cube. Specifically, in this case our cycle factor contains all chains from the Greene-Kleitman SCD. These cycles can be joined to a single Hamilton cycle in such a way, so as to give exactly the aforementioned Hamilton cycle constructed for proving Theorem 3. 60:6 On the Central Levels Problem Outline of this paper In Section 2 we discuss the Greene-Kleitman SCD and lexical matchings, and collect some other preliminaries. In Section 3 we describe our construction of a cycle factor in Q n, . Due to space constraints, in this extended abstract we are unable to provide the full details of the analysis of this cycle factor, and how to join its cycles to a Hamilton cycle. We rather give an informal high-level sketch of these steps in Section 4. In Section 5 we present our proof of Theorem 3. The omitted proof details, together with the pseudocode description of the corresponding loopless algorithm can be found in [17]. Preliminaries For the reader's convenience, important notations that are introduced in the following and used repeatedly in the paper are summarized in Table 1 at the end of this paper. Bitstrings and lattice paths For any string x and any integer k ≥ 0, we let x k denote the concatenation of k copies of x. We often interpret a bitstring x as a path in the integer lattice Z 2 starting at the origin (0, 0), where every 0-bit is interpreted as a -step that changes the current coordinate by (+1, −1) and every 1-bit is interpreted as an -step that changes the current coordinate by (+1, +1); see Figure 2. Figure 2 The correspondence between bitstrings (top) and lattice paths (bottom). Let D 2k denote the set of bitstrings with exactly k many 1s and k many 0s, such that in every prefix, the number of 0s is at least as large as the number of 1s. We also define D := k≥0 D 2k . Note that D 0 = {ε}, where ε denotes the empty bitstring. In terms of lattice paths, D corresponds to so-called Dyck paths that never move above the line y = 0 and end on this line. If a lattice path x contains a substring u ∈ D, then we refer to this substring u as a valley in x. The Greene-Kleitman SCD We now describe Greene and Kleitman's [15] construction of an SCD in the n-cube; see Figure 3. For any vertex x of the n-cube, we interpret the 0s in x as opening brackets and the 1s as closing brackets. By matching closest pairs of opening and closing brackets in the natural way, the chain containing x is obtained by flipping the leftmost unmatched 0 to ascend the chain, or the rightmost unmatched 1 to descend the chain, until no more unmatched bits can be flipped. It is easy to see that this indeed yields an SCD of the n-cube for any n ≥ 1. We always work with this SCD due to Greene and Kleitman, and whenever referring to a chain, we mean a chain from this decomposition. 1 1 1 1 1 0 1 1 0 1 1 0 1 0 0 1 1 1 1 1 0 1 1 1 1 1 1 0 1 1 0 1 1 0 1 0 0 1 1 1 1 0 0 1 1 1 1 1 1 0 1 1 0 1 1 0 1 0 0 1 1 1 0 0 0 1 1 1 1 1 1 0 1 1 0 1 1 0 1 0 0 1 1 0 0 0 0 1 1 1 1 1 1 0 1 1 0 1 0 0 1 0 0 1 1 0 0 0 0 1 1 1 1 1 1 0 1 0 0 1 0 0 1 0 0 1 1 0 0 0 0 1 1 1 1 1 0 0 1 0 0 1 0 0 1 0 0 1 1 0 0 0 0 1 1 1 1 0 0 0 1 0 0 1 0 0 1 0 0 1 1 Each chain C of length h in Q n can be encoded compactly as a string of length n over the alphabet {0, 1, * } in the form where u 0 , . . . , u h ∈ D. The symbols * represent unmatched positions, and the vertices along the chain are obtained by replacing the * s by 1s followed by 0s in all possible ways; see (2). For example, the chain shown in Figure 3 is C = * * * * * 01 * 01 * 010011 * * * 01, so we have Given a chain C of length h as in (1), the ith vertex of C from the bottom is where i = 0, . . . , h, and this vertex belongs to level k = n−h 2 + i. Note that every vertex x of Q n can be written uniquely in the form (2), and we refer to this as the chain factorization of x. For the following arguments, it will be crucial to consider the lattice path representation of x, with the valleys u 0 , . . . , u h that are separated by i many -steps, followed by h − i many -steps, i.e., the valley u i is the highest one on the lattice path. We Lexical matchings Lexical matchings in Q n were introduced by Kierstead and Trotter [24], and they are parametrized by some integer p ∈ {0, 1, . . . , n − 1}. These matchings are defined as follows; see Figure 4. We interpret a bitstring x as a lattice path, and we let x denote the lattice 60:8 On the Central Levels Problem path that is obtained by appending -steps to x until the resulting path ends at height −1. If x ends at a height less than −1, then x := x. Similarly, we let x denote the lattice path obtained by appending -steps to x until the resulting path ends at height +1. If x ends at a height more than +1, then x := x. We let L n,k denote the set of all vertices on level k of Q n , and we define a matching by two partial mappings M p,↑ n,k : L n,k → L n,k+1 and M p,↓ n,k : L n,k+1 → L n,k defined as follows: For any x ∈ L n,k we consider the lattice path x and scan it row-wise from top to bottom, and from right to left in each row. The partial mapping M p,↑ n,k (x) is obtained by flipping the pth -step encountered in this fashion, where counting starts with 0, 1, . . ., if this -step is part of the subpath x of x ; otherwise x is left unmatched. Similarly, for any x ∈ L n,k+1 we consider the lattice path x and scan it row-wise from top to bottom, and from left to right in each row. The partial mapping M p,↓ n,k (x) is obtained by flipping the pth -step encountered in this fashion if this -step is part of the subpath x of x ; otherwise x is left unmatched. It is straightforward to verify that these two partial mappings are inverse to each other, so they indeed define a matching between levels k and k + 1 of Q n , called the p-lexical matching, which we denote by M p n,k . We also define M p n := 0≤k<n M p n,k , where we omit the index n whenever it is clear from the context. In the following, we will only ever use p-lexical edges for p = 0, 1, 2. For instance, it is well-known that taking the union of all 0-lexical edges, i.e., the set M 0 , yields exactly the Greene-Kleitman SCD [24]. This property is captured by the following lemma, together with several other explicit perfect matchings, consisting of {0, 1, 2}-lexical edges between certain sets of vertices from our SCD; see Figure 5. To state the lemma, for a set M of edges of Q n and disjoint sets X, Y of vertices, we let M [X, Y ] denote the set of all edges of M between X and Y . Moreover, for any vertex x ∈ C − h,i , 1 < i < h ≤ n, we consider the chain factorization x = u 0 1 · · · u i−2 1 u i−1 1 0 u i+1 · · · 0 u h with u 0 , . . . , u h ∈ D, and we define a neighbor z(x) on the level below by Note that (x, z(x)) is a 0-lexical or 2-lexical edge in the first or second case, respectively. Lemma 5. For every n ≥ 3, the following sets of edges M [X, Y ] are perfect matchings in Q n between the vertex sets X and Y . ( The proof of Lemma 5 can be found in [17]. Cycle factor construction We now construct a cycle factor C n, in the graph Q n, , n = 2m + 1, i.e., in the subgraph of the n-cube induced by the middle 2 levels. Throughout this section we consider fixed m ≥ 1 and 2 ≤ ≤ m + 1. We construct the cycle factor incrementally, starting with chains from the Greene-Kleitman SCD and adding {0, 1, 2}-lexical edges between certain sets of vertices, see Figure 6. In the following, when referring to a subgraph given by a set of edges, we mean the subgraph of Q n, induced by those edges. Moreover, we say that a chain is short if its length is at most 2 − 3, i.e., if it does not span all levels of Q n, . Our construction starts by taking all those short chains, formally respectively. Note that the only vertices of short chains that have degree 1 in the set Next, between every pair of consecutive levels of Q n, we take all 0-lexical and 1-lexical edges that are not incident to a degree-2 vertex in X. Specifically, between these pairs of levels we take all 0-lexical edges from chains that are not short and all 1-lexical edges between chains that are not short. In addition, between the top two levels we take all 1- for 1 ≤ k ≤ . Note that Y 1 and Y contain all {0, 1}-lexical edges between the bottom two levels or the top two levels of Q n, , respectively. We also define As a consequence of these definitions and Lemma 5 (i) and (ii), the only vertices of Q n, that have degree 1 in the set X ∪ Y are exactly the vertices of C − 2 −1,i for 1 ≤ i ≤ 2 − 2. We thus add the edges defined in part (iii) of Lemma 5, which makes a cycle factor in the graph Q n, . Comparison with previous constructions Our cycle factor construction generalizes the construction for = 1 presented in [19,27], which simply consisted in taking the union of all 0-lexical and 1-lexical edges between the middle two levels. It also generalizes the construction for = 2 presented in [16], which also only used {0, 1, 2}-lexical matchings. In fact, all these earlier papers actually used {m, m − 1, m − 2}-lexical matching edges, but these are isomorphic to {0, 1, 2}-lexical edges by reversing bitstrings. The earlier construction for = 2 seemed rather arbitrary at the time, but now nicely fits into the general picture shown in Figure 6 1 . 1 As the picture of this construction resembles a rocket, with the tip on the left and the boosters on the right, one might be tempted to consider this rocket science. Figure 6 Illustration of the cycle factor C n, for = 2, 3, 4. Each bullet represents an entire set of vertices, as specified in the figure, lines between them specify perfect matchings between these sets. The {0, 1, 2}-lexical edges are drawn with solid, dashed, and dotted lines, respectively. In the bottom part, various sets of matching edges are highlighted. Sketch of the remaining proof steps It turns out that each cycle from the factor C n, defined in (7) visits vertices from an interval of 2r levels, where 2 ≤ r ≤ , around the middle. We refer to the number 2r as the range of the cycle, and we say the cycle is short if 2 ≤ r < , and long if r = . One can show that any short cycle with range r has length 8(r For long cycles, on the other hand, we are lacking such a detailed understanding of their structure. However, we are able to identify certain vertices on them, and to describe the operation of moving along one cycle from one such special vertex to the next one in terms of certain rotation operations on ordered rooted trees. Consequently, long cycles are obtained as equivalence classes of ordered rooted trees under such rotations. As outlined in Section 1.3, to join the cycles in our factor to a Hamilton cycle, we explicitly construct flipping 4-cycles and 6-cycles. The 4-cycles are used to join short cycles among each other and to long cycles, in such a way that every short cycle is joined to some long cycle, possibly via other short cycles. For this we exploit the fact that certain pairs of short chains from the Greene-Kleitman SCD are connected by many 4-cycles. Specifically, consider any short chain C of length h ≥ 3, and any chain C of length h − 2 obtained from C by replacing two consecutive s at positions a and b by 0 and 1, respectively. Using the definition of Greene-Kleitman chains, it is easy to check that C and C are connected by h − 2 many 4-cycles, each using a distinct edge of C and C , except the two consecutive edges of C that I C A L P 2 0 2 0 60:12 On the Central Levels Problem flip the coordinates a and b. The Greene-Kleitman SCD has an abundance of such pairs of heavily connected pairs of chains, and as our cycle factor contains all these short chains, we can exploit this to join short cycles to each other and to some long cycle in a tree-like fashion, by considering the short cycles by increasing range. Particular care must be taken to ensure that all selected flipping 4-cycles are edge-disjoint from each other, so that they do not interfere with each other in the joining process. The remaining task is to join long cycles to each other, and for this we use flipping 6-cycles between the topmost two levels of Q n, , ensuring that they are edge-disjoint from any flipping 4-cycles, which all live in the levels below. Such a flipping 6-cycle can be used to connect two long cycles with each other, and this operation can again be interpreted in terms of an operation on ordered rooted trees, which we call a pull operation. These 6-cycles have been described and used heavily already in the predecessor papers [16,19], where it was shown that they are all edge-disjoint. To complete the proof of Theorem 1, we show that all long cycles can be joined to each other by flipping 6-cycles, by showing that all equivalence classes of ordered rooted trees under the aforementioned rotations (which correspond to long cycles) can be transformed into each other by pull operations (which correspond to flipping 6-cycles). This step of the proof reduces the Hamiltonicity problem in Q n, to proving that a suitably defined auxiliary graph is connected, which turns out to be much easier. Proof of Theorem 3 In this section, we prove Theorem 3. All lemmas stated below follow from straightforward calculations; see [17] for details. For any chain C, we let \|C\| denotes its length, i.e., the number of s in C. For any chain C with \|C\| ≥ 2, we let f (C) and (C), respectively, denote the chains obtained by replacing the first two * s or the last two * s in C by 0 and 1. Note that if \|C\| ≥ 2, then we have f ( ( * C * )) = (f ( * C * )). Our goal is to order the chains of the Greene-Kleitman SCD in Q n , n ≥ 2, so that any consecutive pair of chains is joined at their top end vertices or bottom end vertices alternatingly, with the exception of any two consecutive chains of length 1 that are connected from the bottom end of one of them to the top end of the other, so as to form a Hamilton cycle. We call such an ordering of chains a cycle ordering. The following simple but powerful lemma, valid for arbitrary SCDs, shows that the direction in which each chain is traversed along the Hamilton cycle (upwards or downwards) is determined only by the chain length. Lemma 6. Let Λ n be a cycle ordering of chains of an SCD in Q n , n ≥ 2. In this Hamilton cycle, any two chains C and C with \|C\| ≡ \|C \| (mod 4) are traversed in the same direction. We now define a cycle ordering Λ n , n ≥ 2, for the Greene-Kleitman SCD. The corresponding Hamilton cycle is oriented so that it traverses the longest chain * n , which will be the first in the ordering Λ n , from bottom to top. Our construction works inductively, and the induction step goes from n to n + 2, with separate rules for even and odd n. The base cases are n = 0 and n = 1, for which the entire cube consists only of a single vertex and a single edge, respectively, so for these cases the notion of a cycle ordering is not defined. We call the chains of λ(C) arising from C the descendants of C. This rule replaces each chain C in Λ n by its descendants λ(C), where the order of descendants can be reversed, indicated by the superscript R, depending on the length of C modulo 4. For odd n, we define Λ 1 := * , and for n ≥ 1 and given Λ n we define Λ n+2 := ρ(Λ n ), where ρ is as before and To complete the proof of Theorem 3, it remains to show that any two consecutive chains in Λ n can be joined by an edge between their top ends or bottom ends alternatingly. For this we need the following simple lemmas that guarantee these connecting edges. All connecting edges between top and bottom ends among the descendants of a chain guaranteed by Lemma 8 are shown in Figure 7. The next two lemmas are illustrated in Figure 8. Proof of Theorem 3 (even n). We show that Λ n , n ≥ 2 even, defined in (9) is a cycle ordering of the Greene-Kleitman chains, by proving that any consecutive pair of chains is connected at their top or bottom ends alternatingly, starting with the first chain * n of length n that is traversed from bottom to top. We will also establish the following additional property P: For any two consecutive chains C and C connected at their top ends, we either have C = f (C ) or f (C) = C . These invariants can easily be checked for the induction base case n = 2, which is given by Λ 2 = * * , 01. For the induction step consider n ≥ 2 to be even, and assume that Λ n is a cycle ordering satisfying property P. By Lemma 8, the descendants λ(C) for any chain C from Λ n can be joined as shown on the left hand side of Figure 7, so we only need to check the connections between the first and last chains among consecutive groups of descendants. Indeed, if C and C are consecutive in Λ n and joined at their bottom ends, then C is traversed from top to bottom and C from bottom to top in the Hamilton cycle; see the left part of Figure 8. Consequently, by Lemma 6, we have \|C\| ≡ \| * n \| = n (mod 4) and \|C \| ≡ n (mod 4), i.e., by (8) the sequence Λ n+2 contains λ(C) R and λ(C ), and indeed, the bottom vertex of the last chain of λ(C) R , namely * C * , is connected to the bottom vertex of the first chain of λ(C ), namely * C * , by Lemma 9. Similarly, if C and C are consecutive in Λ n and joined at their top ends, then C is traversed from bottom to top and C from top to bottom in the Hamilton cycle; see the right part of Figure 8. Consequently, by Lemma 6, we have \|C\| ≡ n (mod 4) and \|C \| ≡ n (mod 4), i.e., by (8) the sequence Λ n+2 contains λ(C) and λ(C ) R , and indeed, the bottom vertex of the last chain of λ(C), namely ( * C * ), is connected to the bottom vertex of the first chain of λ(C ) R , namely ( * C * ), using that by property P we have either C = f (C ) or f (C) = C , so we can invoke Lemma 10. Moreover, property P still holds for Λ n+2 by the definition (9) (note that if \|C\| = 0, then we have 0C1 = f ( * C * )). The proof of Theorem 3 for odd n is very similar. In [17] we provide all details and a loopless algorithm for computing this Gray code. An implementation of this algorithm in C++ is available for download and for demonstration [5]. Table 1 A glossary for notation used in the paper. Qn n ≥ 1 the n-dimensional hypercube Q n, 1 ≤ ≤ m + 1 the subgraph of Qn induced by the middle 2 levels n = 2m + 1, m ≥ 1 D 2k k ≥ 0 the set of all Dyck paths (bitstrings) of length 2k D the set of all Dyck paths C a chain C = u0 * u1 * · · · * u h−1 * u h of length h ≥ 0 in the Greene-Kleitman decomposition, ui ∈ D for every i C h,i 0 ≤ i ≤ h the set of the ith vertices in all chains of length h C − h,i 0 ≤ i ≤ h as above but only in chains with ui = ε C + h,i 0 ≤ i ≤ h as above but only in chains with ui = ε L n,k 0 ≤ k ≤ n the set of vertices on level k in Qn M p n,k 0 ≤ k < n, 0 ≤ p < n the p-lexical matching between L n,k and L n,k+1 M p n , M p 0 ≤ p < n the set of all p-lexical edges in Qn \|C\| the length of a chain C, i.e., \|C\| = h for C as above f (C) \|C\| ≥ 2 the chain f (C) = u0 0 u1 1 u2 * · · · * u h for C as above l(C) \|C\| ≥ 2 the chain l(C) = u0 * · · · * u h−2 0 u h−1 1 u h for C as above λ(C) a sequence of descendant chains for a chain C, see (9), (10)	2019-12-03T18:01:30.000Z	2019-12-03T00:00:00.000	{ "year": 2019, "sha1": "af5ab47579ad72a585167ba7fe07b0f351d6549d", "oa_license": "CCBY", "oa_url": "https://doi.org/10.1016/j.jctb.2022.12.008", "oa_status": "HYBRID", "pdf_src": "MergedPDFExtraction", "pdf_hash": "b41bd0aa8fa2c1e7baed46ed3c93fd8a050967f3", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Computer Science", "Mathematics" ] }
27695087	pes2o/s2orc	v3-fos-license	Dry shear aligning: a simple and versatile method to smooth and align the surfaces of carbon nanotube thin films† The alignment of carbon nanotubes in thin films parallel to a surface is a topic of widespread research interest in the nanotube community. This is because, when carbon nanotubes are present as a randomly ordered bulk material, some of the often-touted electronic and optical properties of the individual nanotubes are suppressed, which confounds their full exploitation in a variety of devices and applications. We show here a new method of smoothing and aligning carbon nanotube thin films that is both inherently scalable and exceedingly simple. Depending on the type and purity of the nanotubes, the technique can also provide excellent surface alignment of the nanotubes in a dense and close packed array. A number of techniques of producing smooth and aligned carbon nanotube thin films over large areas have been reported with varying degrees of complexity, difficulty, and scalability, as well as resultant degree of nanotube alignment. These include the collapse of vertically aligned arrays or exfoliation of CVD grown forests, horizontal CVD growth, use of Langmuir-Blodgett or Langmuir-Schaeffer deposition, solution shearing from superacids or polyelectrolyte salt solutions at liquid crystal concentrations of nanotubes, shear alignment in a liquid film followed by filtration, evaporation-driven selfassembly of sidewall-functionalised or surfactant-stabilised suspensions, and floating evaporative self-assembly. Whilst successful, many of these techniques require very specific preparation and some have limited applicability beyond the laboratory environment. In contrast, this Communication reports a simple method of smoothing and aligning the surfaces of carbon nanotube thin films on substrates by applying lateral shear force to the films in the dry state. The new technique is fundamentally different to other methods such as the collapse or ‘pushing over’ of dense and ordered arrays which are already aligned in the vertical direction and are either reoriented by up to 90° to become horizontally aligned or are bent so that part of the length of each nanotube is more or less parallel to the surface. It is also different to the alignment of nanoparticles by drag forces in a thin liquid film followed by filtration, or of nanotube liquid crystals. As illustrated in Fig. 1, the process of dry shear aligning (DSA) is straightforward; involving the application of compressive force between an aligner and a substrate holding a nanotube film and then shearing of the aligner relative to the substrate. As exemplified in the SEM images shown in Fig. 2, the effects on nanotube film morphology can be dramatic. Before DSA, the films are composed of dense mats of randomly oriented and interwoven nanotube bundles, with bundle diameters and film roughness varying depending on the technique used to form the film. After DSA, the nanotubes on the surface of the films are uniformly oriented in the direction of shear and have been densified. So far, in our labs, we have applied the DSA technique to small and large diameter single walled nanotubes, double walled nanotubes and multiwalled nano- The alignment of carbon nanotubes in thin films parallel to a surface is a topic of widespread research interest in the nanotube community. This is because, when carbon nanotubes are present as a randomly ordered bulk material, some of the often-touted electronic and optical properties of the individual nanotubes are suppressed, which confounds their full exploitation in a variety of devices and applications. We show here a new method of smoothing and aligning carbon nanotube thin films that is both inherently scalable and exceedingly simple. Depending on the type and purity of the nanotubes, the technique can also provide excellent surface alignment of the nanotubes in a dense and close packed array. A number of techniques of producing smooth and aligned carbon nanotube thin films over large areas have been reported with varying degrees of complexity, difficulty, and scalability, as well as resultant degree of nanotube alignment. These include the collapse of vertically aligned arrays [1][2][3][4][5][6] or exfoliation of CVD grown forests, 7,8 horizontal CVD growth, 9 use of Langmuir-Blodgett 10 or Langmuir-Schaeffer 11 deposition, solution shearing from superacids 12,13 or polyelectrolyte salt solutions 14 at liquid crystal concentrations of nanotubes, shear alignment in a liquid film followed by filtration, 15 evaporation-driven selfassembly of sidewall-functionalised 16 or surfactant-stabilised suspensions, 17,18 and floating evaporative self-assembly. 19 Whilst successful, many of these techniques require very specific preparation and some have limited applicability beyond the laboratory environment. In contrast, this Communication reports a simple method of smoothing and aligning the surfaces of carbon nanotube thin films on substrates by applying lateral shear force to the films in the dry state. The new technique is fundamentally different to other methods such as the collapse or 'pushing over' of dense and ordered arrays 1-6 which are already aligned in the vertical direction and are either reoriented by up to 90°to become horizontally aligned or are bent so that part of the length of each nanotube is more or less parallel to the surface. It is also different to the alignment of nanoparticles by drag forces in a thin liquid film followed by filtration, 15 or of nanotube liquid crystals. [12][13][14] As illustrated in Fig. 1, the process of dry shear aligning (DSA) is straightforward; involving the application of compressive force between an aligner and a substrate holding a nanotube film and then shearing of the aligner relative to the substrate. As exemplified in the SEM images shown in Fig. 2, the effects on nanotube film morphology can be dramatic. Before DSA, the films are composed of dense mats of randomly oriented and interwoven nanotube bundles, with bundle diameters and film roughness varying depending on the technique used to form the film. After DSA, the nanotubes on the surface of the films are uniformly oriented in the direction of shear and have been densified. So far, in our labs, we have applied the DSA technique to small and large diameter single walled nanotubes, double walled nanotubes and multiwalled nano- Fig. 2 SEM images of (a, c, e) as-prepared nanotube films and (b, d, f ) the same films after dry shear aligning, where the film in (a) and (b) was produced by slide casting of HiPco nanotubes (SuperPureTubes, NanoIntegris) dissolved in sodium polyelectrolyte solution and had an order parameter after DSA of S 2D = 0.41, the film in (c) and (d) was produced by slide casting of gel sorted, small diameter metallic nanotubes made from raw HiPco material (NanoIntegris) and dissolved in sodium polyelectrolyte solution and had an order parameter after DSA of S 2D = 0.28, and the film in (e) and (f ) was produced by vacuum filtration onto a mixed cellulose ester membrane (HAWP, Merck Millipore) of raw HiPco material suspended in 1 wt% SDS solution and had an order parameter after DSA of S 2D = 0.22. In all cases DSA was conducted on the films mounted on glass slides. Absorption spectra of the three kinds of nanotube film are shown in Fig. S1. † tubes stabilised with surfactants, as well as those dissolved in chlorosulphonic acid or sodium polyelectrolyte solutions at concentrations below that required for liquid crystal ordering (if they were at liquid crystal concentration then the film formation process would already align the nanotubes, negating the need for DSA). The resultant level of order of the films follows the trend SW (small) > SW (large) > DW ≫ MW (Fig. S2 †). We have observed no difference in the response of semiconducting, metallic or mixed nanotubes although the more pure and free of catalyst particulates and other contaminants the starting material is, the cleaner the final film is. This can be readily seen in Fig. 2(f ) where many bright spots of high secondary electron emission are observed, corresponding to metal catalyst particles in the raw nanotube starting material, as well as some regions of blurriness which may correspond to amorphous carbon in the starting material, or perhaps to some residual surfactant. Also seen in Fig. 2(f ) are some shallow striations due to the aligner surface (Teflon in this case) not being atomically flat. Better flattening and alignment is observed with higher purity material, whereas extensive damage occurs to the films when particulates are present during shearing (Fig. S3 †). DSA can be applied to films created by vacuum filtration from aqueous or non-aqueous suspensions, or shearing/slide casting from isotropic solutions, with varying effects depending on the technique. In the case of vacuum filtration (Fig. S4 †), the DSA technique can be applied directly on the film after it has been transferred to a surface ( Fig. S4(e-h) †), or on the filtration membrane before transfer ( Fig. S4(i-l) †). Or, DSA could be applied before transfer to flatten/align one side of the film and provide an improved junction with the substrate, then after transfer to flatten and align the other side to provide a better junction with any additional material layers in the respective device. Comparing DSA of films made by vacuum filtration of single, double and multiwalled nanotubes (Fig. S4, S5 and S6, † respectively) it is clear that the degree of reorganisation of the nanotubes is heavily dependent on their type and purity. The smaller the diameter of the nanotubes, the easier they are to rearrange and hence the better the flattening and alignment, with the best results obtained from material such as high purity, gelsorted (6,5) nanotubes 20,21 (Fig. S7 †). For the aligner, we use Teflon for films still attached to the filtration membrane, although polycarbonate, ceramic, glass and steel all yield positive results, and latex for films on glass or silicon, though nitrile and rubber are also effective. Clearly, DSA is inherently scalable to nanotube films of arbitrary size and dimension without complication of the equipment setup since the essential elements are that pressure is applied to an aligner that is in contact with, and moving in relation to, a surface holding a nanotube thin film. In principle this could be applied in continuous roll-to-roll production processes. These characteristics are in stark contrast to some previous alternatives which could only be applied in batch production and which may require expensive tooling and/or add significantly to manufacturing complexity. [3][4][5][6][9][10][11]15 DSA does not require any specific preparation of the nano-tubes over and above that required to form the film by a chosen method. Importantly, and in contrast to other potentially industrial-scale techniques such as the collapse or drawing of CVD grown forests, this means that DSA can be applied to the whole range of nanotubes from raw mixtures of type and chirality through to very highly purified, chirality sorted material. In addition to the long range ordering apparent in Fig. 2, the other main effect of the DSA technique is to significantly reduce the film roughness. Fig. 3(a) and 3(b) show 3D AFM images of a vacuum filtered nanotube film before and after DSA. In this case the root mean squared roughness decreased substantially from 143 nm to just 3.3 nm, an outstanding improvement, and similar large decreases are observed for all the nanotube films we have studied, regardless of whether or not the nanotubes were aligned. The ability to create such smooth films is particularly advantageous in the context of using nanotube films in application where they are used in conjunction with thin layers of other materials. For example, where the nanotubes are used as electrodes or charge transport layers in organic photovoltaics, LEDs, capacitors, etc., in which the thickness of the material layer on top of the nanotubes could be well below 100 nm. As one would predict, the anisotropy induced by DSA causes the nanotube films to have a different response to polarised light depending on orientation. Fig. 3(c) shows the optical spectrum of the film in Fig. 3(a) and is invariant under polarisation. Polarised optical absorption spectra from the same film (the other half of the filtration membrane) after the application of DSA are shown in Fig. 3(d) and yield a 2D order parameter of 0.16, where the order parameter was calculated as per White and Taylor 22 and where This value is somewhat less than might be expected based on the SEM images however it must be noted that the alignment occurs only on the surface of thicker films, leaving the inner regions in their randomly oriented state, and this is particularly true when the films are still bound to the filtration membrane; with a proportion of the material penetrating into the pores and less exposed to shear. A comprehensive study of the effect of DSA on varied thicknesses of vacuum filtered films of large diameter single walled nanotubes was conducted (Fig. S8, S9 and S10 †). As well as the usual relationships between sheet resistance, thickness and doping which are well captured in the figure of merit ratio of DC electrical to optical conductivity (Fig. S10(a+c) †), 23 the data show both a small but distinct anisotropy in the conductance (Fig. S10(b+d) †) as well as a clear dependence of the extent of nanotube alignment on the film thickness, with a critical thickness corresponding to a transmittance of around T 550 = 80% (Fig. S11 †), above which the effects of DSA become more pronounced. This suggests that future fine tuning of the film thickness and DSA conditions may allow for the production of films composed only of the aligned surface region. Although the smoothing of the nanotube films is an intuitive process, the mechanism underlying alignment by DSA is not immediately clear. If the nanotubes were subjected to a flowing liquid, as in the case of nanotube fibre formation from chlorosulphonic acid in a faster flowing coagulant, 24 then the alignment could be explained as being due to the well-known effects of drag on the rotation/orientation of an anisotropic particle in a flow. Similarly for cellulose nanocrystals suspended in water, which can be aligned in thin liquid films subjected to doctor blading, as long as the volume concentration of the nanocrystals in the solvent is low enough to allow free movement, 25 and for nematic liquid crystals of carbon nanotubes dissolved by superacids 13 or via alkali metal reduction 14 and sheared in a thin liquid film. In such cases the alignment process can be understood in the context of well-known continuum theories modelling liquid crystal behaviour. 26 However, the situation is quite different in the case of a dry material. In determining the mechanism underlying DSA, considerable insight can be found in the work of Börzsönyi et al. who studied the shear induced alignment of various elongated particles and developed a numerical model of the experimental observations. 27 The process is shown to be very similar to that occurring in nematic liquid crystals, despite the completely different interparticle interactions involved. In Börzsönyi's model, the fundamental cause of the alignment is a reduction in friction between the material and the shearing plate by up to a third in the aligned state vs. the unaligned one. The degree of order scales with the aspect ratio of the individual particles up to 5 : 1 (the upper limit in the experiment). The fact that DSA appears so far to be a surface effect, unlike in the Börzsönyi et al. work, in which the degree of order was observed to be the same throughout the material, could be due to, (a) the much higher aspect ratio of the nanotubes (100 : 1 up to >1000 : 1) which, as discussed by Börzsönyi et al., leads to much greater levels of entanglement between neighbours and thus hinders movement of the nanotubes, (b) the extremely low friction that exists between nanotube sidewalls, 28 reducing the penetration depth of the shear force (and perhaps explaining why the order parameter is lower for nanotube films deposited from surfactant-stabilised suspensions vs. those deposited from true solutions in superacid, etc.the surface nanotubes are more free to slide past each other without the presence of residual surfactant) and, (c) the fact that in the Börzsönyi et al. experiments only one side of the bulk material was subject to a shearing surface whilst the other side was free to move, which is different to the situation in DSA where one side of the film is adhered to a stationary surface. Nevertheless, the model provides a strong foundation for understanding the current work. In summary, dry shear aligning is a simple post-fabrication treatment that can yield dramatic improvements in film roughness and homogeneity, along with excellent alignment of the surface nanotubes. Apart from the presentation of a facile and scalable technique to generate outstandingly smooth films from much rougher starting material, the main conclusion of this work is that, perhaps contrary to assumption, carbon nanotube films like the ones used in this work are not fixed structures, but are dynamic and malleable systems containing mobile elements that are capable of significant restructuring and reordering with appropriate mechanical intervention. The observation of realignment of the nanotube bundles reveals the fluidity and plasticity of such films and is a practical insight that may inform future work in the field. Considering the widespread use of thin nanotube films across a broad swath of fundamental and applied nanoscience, we expect that the dry shear aligning technique may be of benefit to many in the nanotube research community.	2017-06-18T11:26:53.309Z	2016-02-05T00:00:00.000	{ "year": 2016, "sha1": "a85943191324d28f7c158a3be765b3ccd45dd131", "oa_license": "CCBYNC", "oa_url": "https://pubs.rsc.org/en/content/articlepdf/2016/nr/c5nr08784h", "oa_status": "HYBRID", "pdf_src": "Anansi", "pdf_hash": "54dc1d014554eb4614d6297f45e8c851e6708987", "s2fieldsofstudy": [ "Engineering", "Materials Science", "Physics" ], "extfieldsofstudy": [ "Materials Science", "Medicine" ] }
248790201	pes2o/s2orc	v3-fos-license	The Spanish version of the short form of the Cyberchondria Severity Scale (CSS-12): Testing the factor structure and measurement invariance across genders Cyberchondria refers to excessive and repeated online health-related searching, which is associated with increased distress and anxiety. The Cyberchondria Severity Scale (CSS) is the most widely used measure for assessment of cyberchondria, and its shortened version (CSS-12) has recently been developed. The aim of the present study was to develop the Spanish version of the CSS-12 and test its psychometric properties. A community sample of 432 Spanish-speaking adults (67.6% women; mean age = 36.00 ± 15.22 years) completed the Spanish translation of CSS-12 along with measures of health anxiety, obsessive-compulsive, anxiety and depressive symptoms. The Spanish version of the CSS-12 comprises a general cyberchondria factor and four specific factors (‘excessiveness’, ‘compulsion’, ‘distress’, and ‘reassurance’). Multi-group confirmatory factor analysis indicated measurement invariance across gender groups. Internal consistency values for the total score and subscales were good to excellent. The CSS-12 showed strong correlations with health anxiety, and moderate to low correlations with anxiety, obsessive-compulsive and depressive symptoms, supporting the convergent and divergent validity of the CSS-12, respectively. In conclusion, these results show that the CSS-12 is a valid and reliable tool for measuring cyberchondria in both genders in the general Spanish population. The Internet and digital tools have had a great impact on how individuals access health information. In this sense, the Internet has gained ground over traditional sources of health information, such as medical books and encyclopedias or consults with physicians. In part because searching for medical information on the Internet has many advantages, the Internet is considered a low-cost tool for accessing information quickly, easily, and anonymously. These online searches can also empower individuals, allowing them to have more control over their personal health and their health care decisions (McManus et al., 2014;Starcevic et al., 2020). In some cases, however, individuals may feel distress or anxiety during or after online health research (OHR). This exacerbation of anxiety or distress about health because of excessive and repetitive health-related searches on the Internet is known as cyberchondria (Starcevic & Berle, 2013). Although the term "cyberchondria" has been criticized as potentially misleading and otherwise pejorative or stigmatizing because of its link with the concept of hypochondriasis (e.g., Brown, Skelly, & Chew-Graham, 2020), it has been widely used and generally accepted in the scientific literature (Starcevic, 2020). A recently proposed "working definition" of cyberchondria includes excessive online health searches that are compulsive, serve the purpose of seeking reassurance, worsen anxiety or distress, and continue despite their negative consequences (Vismara et al., 2020). The concept of cyberchondria has been related to different disorders and symptoms . In this sense, recent studies indicate that, although cyberchondria and health anxiety may share similar symptom presentations (e.g., an excessive and/or repetitive pattern of OHR may also be present in individuals with health anxiety), they can also reflect separate concepts (Fergus & Russell, 2016;Mathes et al., 2018;Starcevic et al., 2019). Likewise, cyberchondria has also been associated with obsessive-compulsive symptoms, given the repetitive and unwanted nature of online health-related searches (Fergus & Russell, 2016;Norr et al., 2015b;Starcevic et al., 2019). The Cyberchondria Severity Scale (CSS; McElroy & Shevlin, 2014) is a well-established and most commonly used measure of cyberchondria. McElroy and Shelvin developed the CSS based on the definition of cyberchondria as a multidimensional construct that reflects both anxiety and an element of compulsivity. Furthermore, the CSS was developed to measure the distress level associated with cyberchondria symptoms. The CSS consists of 33 items, which comprise five key domains (Compulsion, Distress, Excessiveness, Reassurance, and Mistrust of Medical Professionals) assessed on separate subscales and derived from exploratory factor analysis. As described by McElroy & Shevlin (2014), the 'Compulsion' factor refers to interference of OHR with different aspects of a person's life (e.g., Researching symptoms or perceived medical conditions online interrupts my work [e.g., writing emails, working on word documents or spreadsheets]). The 'Distress' factor refers to the negative emotional responses that result from OHR (e.g., I find it hard stop worrying about symptoms or perceived medical conditions that I have researched online). The 'Excessiveness' factor denotes the repetitive and time-consuming nature of OHR (e.g., I read different web pages about the same perceived condition). The 'Reassurance' factor reflects the extent to which OHR leads a person to consult with a medical professional (e.g., Discuss my online medical findings with my GP/health professional reassures me). Finally, the 'Mistrust of Medical Professionals' factor refers to the conflict that arises when the professional diagnosis does not match the individual's self-diagnosis from OHR (e.g., I take the opinion of my GP/medical professional more seriously than my online medical research [reverse-scored]). Although the original version of the CSS presents good psychometric properties, it has been suggested that it could be improved by shortening it and removing the items of the Mistrust of Medical Professionals factor because of its theoretical ambiguity (e.g., Barke et al., 2016;Fergus, 2014;Norr et al., 2015a). Several proposals have been made in this regard. Fergus (2014) observed that a second-order CSS model including all the CSS items fit the data well once the Mistrust of Medical Professionals factor was excluded and concluded that this factor did not measure the same construct as the other CSS factors. Similarly, Norr et al. (2015a) proposed a model of cyberchondria that comprised a separate Mistrust of Medical Professionals factor and a bifactorial model consisting of a General Cyberchondria factor and specific orthogonal factors (Compulsion, Distress, Excessiveness, and Reassurance). The first attempt to reduce the CSS was carried out by Barke et al. (2016) as part of the German validation of the CSS. They proposed a short version with 15 items (CSS-15), which retained the good psychometric properties of the original version. The CSS-15 included the original five CSS subscales to assess the same domains of cyberchondria. In contrast to CSS-15, Fergus and Spada (2018) proposed a 12-item version of the CSS that excludes items of the Mistrust of Medical Professionals factor. This scale presented better internal consistency and convergent validity than the CSS-15 (Fergus & Spada, 2018). The creators of the CSS also developed a 12-item version (CSS-12), excluding the Mistrust of Medical Professionals subscale, and selecting three items from each of the remaining four subscales (McElroy et al., 2019). The three items of each factor were chosen based on the following criteria: factor loadings, endorsement rates, impact on subscale internal consistency, length, and content. An analysis of the factor structure of these 12 items showed that the bifactorial model provided the best fit for the data, as Norr et al. (2015a) concluded after analyzing the full scale. Thus, McElroy et al. (2019) proposed that the structure of the CSS-12 consisted of a general and specific (orthogonal) cyberchondria factors (compulsion, distress, excessiveness, reassurance). The CSS-12 demonstrated good psychometric properties, including internal consistency and convergent validity. Besides the original validation study by McElroy et al. (2019), only the Italian version of the CSS-12 has been validated (Soraci et al., 2020). This version of the CSS-12 has demonstrated good psychometric properties, although the authors only tested a unidimensional model, without testing the originally proposed bifactorial model of the CSS-12. In summary, the CSS has presented with a number of issues. First, several abbreviated versions of the CSS have been proposed, and they all have different properties. However, the CSS-12 of McElroy et al. seems to be the most appropriate because the criteria used to reduce the number of items appear justified and most comprehensive. Second, there are different views about the most appropriate factor structure for the CSS (e.g., four factors, four first-order factors and one second-order factor, bifactor, etc.), but no study has compared the fit of all of these different factor structure proposals. In other words, there is a need to ascertain the most appropriate factor structure for the scale. Third, previous validation studies of the CSS, including both studies of the full version and those of the shorter versions, did not assess whether the structure of the instrument remained unchanged across genders. This is important because gender differences in the related construct of health anxiety have been observed (Bleichhardt & Hiller, 2007;Clarke et al., 2008;Fink et al., 2004), although no gender differences have been found with regard to searching for health information on the Internet (e.g., Berle et al., 2020;Muse et al., 2012). Furthermore, to the best of our knowledge, no version of the CSS has been translated into Spanish, limiting assessment of cyberchondria in the Spanish-speaking countries/ communities. Accordingly, the specific goals of the current study are as follows: (a) translating the CSS-12 from English to Spanish; (b) establishing the psychometric properties (factor structure, internal consistency, convergent and divergent validity) of the Spanish version of the CSS-12; and (c) analyzing measurement invariance according to gender (configural, metric, structural, and error variance invariance). Participants and Procedure Data were collected from a convenience sample between 2019 and 2020. Participants were recruited from the general community and university setting by snowball sampling, after providing relevant information via multiple channels, including face-to-face classes and social media, and inviting participants to share a link to the survey with their acquaintances. Participants completed all the measures through a secure survey platform (Limesurvey) and gave informed consent for their participation in this research. Approval for the study was obtained from the University of Valencia Human Research Ethics Committee. The inclusion criteria for participation in the study were as follows: 1) age between 18 and 65 years, and 2) absence of any mental disorder or significant medical illness in the previous year due to a possible effect of these conditions on the pattern of online health searches. Data initially obtained from the online platform were screened to exclude duplicates, inconsistent or obviously erroneous responses (e.g., current age of >100 years). The survey was completed by 459 individuals, but 27 individuals reported a current, diagnosed medical illness requiring treatment and were excluded from the analyses. Therefore, the final sample comprised 432 non-clinical community adults (67.6% females; 32.4% males), ranging in age from 18 to 63 years (Mean = 36.00; SD = 15.22). Most participants were single (49.8%), reported a medium socio-economic status (62.5%), and had a University-level education (68.1%). Instruments We administered four self-report instruments to assess cyberchondria, health anxiety, obsessive and compulsive symptoms and symptoms of depression and anxiety: CSS-12, Short Health Anxiety Inventory (SHAI), Obsessive-Compulsive Inventory-Revised (OCI-R), and Depression Anxiety Stress Scale 21 (DASS-21). Cyberchondria Severity of cyberchondria was assessed using the Short Form of the Cyberchondria Severity Scale (CSS-12; McElroy et al., 2019). It is a 12-item self-report instrument, with responses recorded on a 5-point scale (from 1 = 'never' to 5 = 'always') and total scores ranging between 12 and 60. The CSS-12 has demonstrated an appropriate reliability (Cronbach's α for its subscales ranging between .73 and .90), as well as convergent and divergent validity (McElroy et al., 2019). Psychometric properties of the Spanish version of the CSS-12 are reported in the Results section. The adaptation of the CSS-12 into Spanish was conducted according to the procedure described by Beaton et al. (2000). First, two members of the research team with experience in translation/validation of questionnaires and expertise in the area of health behaviors (SA and GGS) independently translated the scale into Spanish. These two versions were subsequently compared and adjusted to agree on a preliminary Spanish version of the scale. This preliminary version was then back-translated into English by a bilingual professional translator. There were minimal discrepancies that were discussed and considered until optimal agreement was reached. The final version is included in the Supplementary Information 2. Health Anxiety This construct was assessed using the Short Health Anxiety Inventory (SHAI; Salkovskis et al., 2002;Spanish version: Arnáez et al., 2019). The SHAI is an 18-item self-report measure that assesses health anxiety (i.e., concern for health, monitoring of changes in bodily sensations, and fear of the consequences of suffering from a serious illness) independently of actual physical health status. It is composed of two subscales: 'Likelihood of becoming ill' and 'Negative consequences of illnesses'. Responses on each item are rated on a 4-point Likert scale. The SHAI has demonstrated good reliability and validity in both clinical and non-clinical samples Salkovskis et al., 2002). In the present study, the internal consistency for the 'total score' (α = .90; ω = .90) and the 'Likelihood of becoming ill' subscale (α = .90; ω = .90) was excellent, and acceptable for the 'Negative consequences of illness' subscale (α = .67; ω = .70). Symptoms of Depression and Anxiety The Depression Anxiety Stress Scale-21 (DASS-21; Lovibond & Lovibond, 1995;Spanish version: Daza et al., 2002) is a 21-item self-report questionnaire that assesses symptoms of emotional distress. The DASS-21 comprises three subscales: (a) 'depression', measuring symptoms typically associated with dysphoric mood; (b) 'anxiety', assessing symptoms of physical arousal, panic attack, and fear; and (c) 'stress', measuring symptoms such as tension, irritability, or the tendency to overreact to stressful events. Each subscale is composed of seven items, and respondents rate each item on a 4-point Likert scale. The DASS-21 has shown high internal reliability and validity in both clinical (Osman et al., 2012) and non-clinical samples (Henry & Crawford, 2005). The Spanish version has also demonstrated strong internal consistency and good convergent and discriminant validity (Daza et al., 2002). In the current study, only the anxiety and depression subscales were used; their internal consistency was excellent (α = .90, ω = .91 for anxiety and α = .93, ω = .93 for depression). Statistical Analyses The online platform employed to collect the data required participants to answer all the questions before proceeding to the next survey section, in order to avoid missing data. In a first step, descriptive statistics concerning sociodemographic characteristics were calculated to compile a profile of the sample using the SPSS statistical package (version 24.0). Then, Confirmatory Factor Analyses (CFAs) were conducted in order to check the goodness of fit of different factorial solutions for the CSS-12. The software used to perform these analyses was the EQS. 6.4 (Bentler, 2006). Non-normal distribution of categorical data was addressed by applying robust estimation methods (robust Maximum Likelihood, ML) (Finney & DiStefano, 2013). Goodness of fit for the CFA models was assessed through the following indices: The Root Mean Square Error of Approximation (RMSEA), the Comparative and Incremental Fit Indexes (CFI and IFI, respectively), and the Standardized Root Mean Square Residual (SRMR). Satorra-Bentler Chi-Square (χ 2 ), general model significance (p), and Relative Chi-Square (χ 2 /df) are also reported. Excellent model fit was considered when χ 2 was not significant (p > .05), χ 2 /df was between 1 and 2, the CFI and the IFI were ≥ .95, the RMSEA ≤ .05, and the SRMR ≤ .05 (Bagozzi & Yi, 2011;Schermelleh-Engel & Müller, 2003). Using less restrictive criteria, values between 2 and 3 for χ 2 /df, ≥ .90 for the CFI and the IFI, ≤ .08 for the RMSEA, and ≤ .10 for the SRMR were considered acceptable (Hooper et al., 2008). To assess whether the factor structure of the CSS-12 was valid in both males and females, multi-group CFAs according to gender were carried out. Specifically, we tested four levels of measurement invariance: 1) configural (testing whether items load on the same factor across groups), 2) metric (testing whether item factorial loadings are equal across groups); 3) scalar (testing whether item intercepts are equal across groups) and 4) error variance invariance (testing whether item measurement errors are equal across groups). The adequacy of the increasingly constrained models was assessed through the difference between pairs of nested models (△) in the RMSEA, CFI and SRMR. A change ≥ .01 in the CFI, ≥ .015 in the RMSEA, and ≥ .03 in the SRMR indicates a significant decrease in the model fit when testing for measurement invariance (Chen, 2007). Internal consistency was assessed through the Ordinal Cronbach's alpha (α) and the McDonald's Omega (ω) (including both total and hierarchical ω). These indices were calculated using the R package "userfriendlyscience" (Peters, 2014). According to the criterion proposed by Hunsley & Mash (2008), an internal consistency between .70 and .79 was considered appropriate, between .80 and .89 good and ≥ .90 excellent. To test convergent validity of the CSS-12, we explored the relationships between cyberchondria and health anxiety; and in order to test the divergent validity we analyzed associations between cyberchondria and obsessive-compulsive symptoms, and anxiety and depressive symptoms. To address this aim, we calculated Pearson zero-order correlations between the CSS-12, the SHAI, the OCI-R, and the DASS-21. Finally, in order to test the robustness of these associations and to identify variables that predict cyberchondria, a hierarchical linear regression analysis was performed 1 3 using the stepwise method (for a detailed description of the method, see Hair et al. (2010)). Structural Analysis and Measurement Invariance According to Gender To assess whether the factor structure proposed by McElroy et al. (2019) was equivalent for the Spanish version of the CSS-12, we tested the adequacy of four factorial solutions: (a) the one-factor solution (i.e., all the items under a 1st order factor), (b) the four correlated 1st order-factor solution; (c) the bifactor model (i.e., a general cyberchondria factor together with four specific factors); and (d) a 2nd order model (i.e., grouping the four first-order factors under a second-order factor that explained the shared variance). Goodness-of-fit indices from all of the tested models are presented in Table 1. As Table 1 shows, the models with the most satisfactory fit indices were the bifactor model and the 2nd order model. Both models were almost indistinguishable in most of the goodness-of-fit indices assessed. As both models may be considered nested (Yung et al., 1999), a suitable approach to estimate their competing adequacy is to compute the difference in the χ 2 test (△χ 2 ). Given the non-normal distribution of our data and the use of a robust method for the estimation of our CFA models, the △χ 2 was calculated by using the formula for the "scaled difference χ 2 test" (i.e., a more complex approach that allows testing of significance of χ 2 changes in nested models not following a classical chi-square distribution) (Bryant & Satorra, 2012). When doing so, we observed a significant worsening of χ 2 in the 2nd order model compared to the bifactor model (scaled △χ 2 = 24.28; df = 7; p < .01), indicating the statistical superiority of the latter over the former. In the bifactor model, the level of significance of the Satorra-Bentler χ 2 did not exceed the .05 value necessary to consider it a satisfactory fit for the model. Nevertheless, it has been shown that this statistic is highly conditioned by sample size (Jöreskog & Sörbom, 1993;Markland, 2007). For this reason, it may be more appropriate to use other indices considered less sensitive to sample size to assess the adequacy of the factorial solutions. In this sense, the value of the relative χ 2 (χ 2 /df) was 2.86, with acceptable fit considered to be values below 3. The RMSEA was .066 which is lower than .08 (a threshold deemed to be indicative of an acceptable-fitting model). The SRMR was below the .05 value required by the strictest criteria to consider a perfect-fit model. Finally, the CFI and the IFI reached a value of .93, which is very close to the cut-off point established to consider it an excellent fit to the model. This model is comprised of a general cyberchondria factor (range of scores 12-60) and four specific factors (three items per factor; range of scores 3-15): 'excessiveness', 'compulsion', 'distress', and 'reassurance'. The resulting bifactorial model is presented in Fig. 1. To test measurement invariance of the CSS-12 according to gender, we conducted a series of model comparisons with multi-group CFA. As displayed in Table 2, gender configural invariance of the CSS-12 was supported (RMSEA = .040; CFI = .977; IFI = .979; SRMR = .040), and we subsequently estimated models with increasing levels of constraints to test higher levels of invariance. Regarding metric invariance, changes in the RMSEA, CFI, and SRMR did not show a significant worsening in the model fit for gender invariance (△RMSEA = .011; △CFI = .010; △SRMR = .023). Similarly, model fit did not significantly decrease when error invariance according to gender was tested (△RMSEA = .005; △CFI = .005; △SRMR = .006). However, when scalar invariance according to gender was tested, △CFI (.044) and △SRMR (.042) suggested the presence of differences at this level of measurement according to the gender. Internal Consistency Internal consistency for the general and specific cyberchondria scales ranges from .83 to .93 (Table 3). A slight difference was only observed between ordinal Cronbach's alpha (α) and McDonald's Omega (ω) in the reassurance subscale (α = .83 and ω = .84). Convergence between both indices was considered a good indicator of the scale internal consistency under different conditions (Zinbarg et al., 2005). Table 3 also shows the Pearson zero-order correlations between study measures. Supporting convergent validity of the CSS-12, correlations between the general cyberchondria factor and health anxiety symptoms as measured by the total score on the Short Health Anxiety Inventory (SHAI) were positive and significant (r between .42-.60). As for divergent validity, correlations between the CSS-12 and the Obsessive-Compulsive Inventory-Revised (OCI-R) (r between .16-.24) were positive, but weak. Similarly, the correlations between the CSS-12 and the subscales of 'anxiety' (r between .25-.39) and 'depression' (r between .21-.30) were positive, but moderate. Finally, a multiple linear regression was calculated to predict cyberchondria (CSS-12) based on health anxiety symptoms (SHAI), anxiety and depressive symptoms (DASS-21), and obsessive-compulsive symptoms (OCI-R). A significant regression equation was found (F (4, 427) = 64.9320, p < .001), with an R 2 of .378. The results of the multiple linear regression analysis revealed that depressive (β = .00, t = .043, p = .966) and OCD (β = .02, t = .466, * p = .641) symptoms were not statistically significant predictors of the model, whereas health anxiety symptoms (β = .54, t = 12.678, p < .001) and anxiety symptoms (β = .13, t = 2.341, p < .001) significantly predicted cyberchondria. Discussion The aim of this study was threefold: (a) to translate the CSS-12 (McElroy et al., 2019) into Spanish and validate it in a Spanish-speaking adult population; (b) to further analyze the factor structure of the scale by comparing the competing factorial solutions; and (c) to explore the applicability of the resulting factorial solution in both males and females (i.e., gender invariance). In this sense, the main conclusion derived from this study is that the Spanish version of CSS-12 is a reliable and valid measurement tool for assessment of cyberchondria in both genders, supporting the original bifactor model. We examined four models to determine the best-fitting factor structure of the CSS-12 (i.e., one-factor, four-factor, bifactor, second-order). The bifactor model had the best fit to the data, as proposed by McElroy et al. (2019) for the original CSS-12 and Norr et al. (2015a) for the full 33-item version of the scale. According to this factorial solution, the scale measures a unitary construct (i.e., a general cyberchondria factor), and contains meaningful specific dimensions (i.e., excessiveness, compulsion, distress, and reassurance factors) assessed via subscales. These dimensions are of relevance as they allow us to compare results with previous studies and analyze in detail various characteristics of the cyberchondria construct, which provides important information. However, as recommended by McElroy et al. (2019) and Norr et al. (2015a), the subscales should not be used in isolation due to a high degree of covariance with the general factor. Our results support convergent and divergent validity of the CSS-12. With regard to convergent validity, the CSS-12 total score and subscale scores showed moderate to high correlations with an established measure of health anxiety 1 3 (r from .42 to .60). Furthermore, health anxiety significantly predicted cyberchondria (total score). This strong relationship between cyberchondria and health anxiety is consistent with findings of the previous studies (e.g., Fergus & Russell, 2016;McElroy & Shevlin, 2014;McElroy et al., 2019;McMullan et al., 2019;Norr et al., 2015a;Starcevic et al., 2019). Therefore, individuals with health anxiety may be particularly prone to experiencing counterproductive outcomes from online health information seeking. However, although cyberchondria and health anxiety are closely related, they have also been shown to be distinct (Fergus & Russell, 2016;Mathes et al., 2018;Starcevic et al., 2019). The divergent validity of the CSS-12 was supported to the extent that OCD and depression, and to a lesser extent general anxiety, are considered conceptually distinct from cyberchondria. Specifically, the CSS-12 showed significant and positive correlations with anxiety (r from .25 to .39), depressive (r from .21 to .30) and OCD (r from .16 to .24) symptoms, although the correlation sizes were moderate to weak. These findings are somewhat in agreement with those reported by previous research. Thus, in contrast to the levels of health anxiety, levels of general anxiety were not found to predict the severity of cyberchondria (Arsenakis et al., 2021). Several studies showed a significant, but relatively weak relationship between depressive symptoms and cyberchondria (Barke et al., 2016;McElroy & Shevlin, 2014;Starcevic et al., 2019;Uzun & Zencir, 2018). A network analysis reported a significant, but weak relationship between OCD symptoms and cyberchondria , while other research reported this relationship to be weaker than that between health anxiety and cyberchondria (e.g., Fergus, 2014). With regard to the CSS-12 subscales, the Distress subscale showed the strongest associations with psychopathological measures (obsessive-compulsive, anxiety and depressive symptoms) compared to other CSS-12 subscales. This finding may have implications for further refinement of the construct of cyberchondria, given that Distress subscale assesses negative emotional responses to OHR. During the validation process of the Spanish version of the CSS-12, a particular attention was paid to its applicability to men and women because this issue has been neglected in research. Findings show that the factor structure of the CSS-12 (i.e., the bifactor structure) is equally applicable to both men and women (configural invariance). In addition, the saturation of each item in each factor (metric invariance) is also equivalent for both genders. However, the latent mean scores in men and women on the factors and items of the CSS-12 were different (scalar invariance). At a practical level, these findings suggest that the "factor structure" of the CSS-12 (i.e., the distribution of the items among the different subscales and the factorial loadings of the items on their corresponding subscale) is comparable in men and women. However, the clinical significance (or contribution) of certain items in the determination of cyberchondria differs according to gender. In this sense, increased scores in certain CSS items are related to increased levels of cyberchondria in one gender but not the other. Thus, gender differences in item contribution to the measurement of cyberchondria warrants further research. Limitations of the present study should be mentioned. First, due to the scarcity of measures in Spanish to assess cyberchondria, we did not use any other instruments that assess the same construct to further test the concurrent validity. Second, we evaluated gender only via two categories (male/female). This categorization does not represent the wide variety of gender expressions; for this reason, we encourage the use of measures that assess both cisgender and transgender identities (Tate, Ledbetter, & Youssef, 2013). Third, individuals with a serious or chronic illness were excluded from the sample, which could have affected the representativeness of the sample. However, we did it as we consider that the pattern of internet searches and anxiety associated could be different in those individuals and influenced by their diagnosis. Despite the limitations, this is the first study to report solid reliability and validity of the Spanish translation of the CSS-12, while supporting its bifactor model and use in both genders. We believe that this is an important contribution both in terms of making the most widely used cyberchondria instrument available to Spanish-speaking	2022-05-15T15:06:56.991Z	2022-05-13T00:00:00.000	{ "year": 2022, "sha1": "12c15b145a12097ecece3e6858ce4aeeb8f3837a", "oa_license": "CCBY", "oa_url": "https://link.springer.com/content/pdf/10.1007/s12144-022-03170-3.pdf", "oa_status": "HYBRID", "pdf_src": "Springer", "pdf_hash": "e22d6aded6b0fe2b2994278ea41589893a28005a", "s2fieldsofstudy": [ "Psychology" ], "extfieldsofstudy": [] }
218845564	pes2o/s2orc	v3-fos-license	Seed Priming with Salicylic Acid Improves Germination and Growth of Lathyrus sativus : Increasing the ability of crops to maintain growth and productivity under saline conditions is of paramount importance. The effect of salicylic acid (SA) priming on germination and physiological traits of Lathyrus sativus under salinity stress was studied in a factorial experiment based on a completely randomized design. The experimental treatments composed of SA at three levels (0, 0.1, and 0.2 mM) and NaCl salinity at three levels (0, 50, and 100 mM). The effect of salinity level and SA priming was significant on all recorded factors, except from final germination percentage. As salinity level increased, all germination and physiological traits declined compared with Introduction Lathyrus sativus L., commonly known as grass pea, is an annual species of Fabaceae family that is highly adapted to adverse environments. L. sativus is resistant to environmental stresses and produces good grain yields under adverse climates (Cocks et al., 2000). Therefore, it is commonly grown for human consumption and livestock feed in Asia and East Africa. This species is also an excellent candidate for green manure owing to its fast vegetative growth, succulent organs, dense foliage, low C/N ratio, low water requirement (Lazanyi, 2000). Salinity is a limiting factor of crop growth and yield in over 800 million ha of arable land (FAO, 2008) accounting for about 6% of the total global land. Salinity stress causes osmotic, ionic, and oxidative stresses, thereby generating several morphological, physiological, and chemical changes in plants and affecting photosynthesis, protein synthesis, lipid metabolism, and energy generation (Parida et al., 2005). Salinity stress can affect all physiological processes from germination to plant development. Photosynthesis is a key pathway in physiology of plants that is severely influenced by salinity. Previous research showed that NaCl significantly reduced growth parameters, Rubisco activity, photosynthetic efficiency and pigments, as well as sugar contents in maize while the effects of NaCl on the previous parameters were increased with NaCl concentrations (Khodary, 2004). Reduced activity of the Hill reaction was also observed in salt-stressed chloroplasts in wheat (El-Shintinawy, 2000), in cowpea (Vigna sinensis) (El-Shahaby et al., 2003), and in maize (El-Shahaby et al., 2003;Zeid, 2009). Salinity stress can also affect other plant physiological processes. Abscisic acid built in response to salinity induces stomatal closure and, thereby, limits CO 2 inflow to the plant (Leung et al., 1994). Salinity can decrease plant protein content by impairing protein synthesis and increasing the activity of protein hydrolyzing enzymes or may increase plant protein content by synthesizing new proteins and/or reducing proteolytic enzymes (Dubey, 1999). The harmful impacts of salinity stress on plants can be caused by osmotic and ionic stresses (Munns and Tester, 2008) that result in metabolic disorders in cells manifested in the build-up of reactive oxygen species (ROS), namely superoxide (O2), hydrogen peroxide (H2O2), and hydroxyl radical (OH -) (Halliwell, 2006). The main site of ROS generation in leaves under salinity stress conditions are chloroplasts (Cavalcanti et al., 2007). Under the influence of environmental stresses, the closure of the stomata, and the reduction of gas exchanges, as well as the continued absorption of light energy, result in disturbances in the electron transport chain in the reaction centers and the reduction of the quantum yield of photosystem II (Maxwell and Johnson, 2000). The Hill reaction is described as the photoreduction of an electron acceptor by the hydrogens of water, with the evolution of oxygen. The ultimate electron acceptor is NADP+. The Hill reaction can be measured in isolated chloroplasts. One of the most perceptible responses of plants to environmental stresses is the loss of photosynthesis due to impaired photosystem II activity (Andrews et al., 1995). One of the approaches to alleviate the impacts of salinity stress is seed priming. Priming refers to the pre-sowing treatment of seeds, by which seeds pass through initial stages of germination, but radicles do not emerge due to the low amount of imbibed water (Nascimento and Aragao, 2004;Ibrahim, 2016). This technique has a lot of advantages, such as fast and uniform emergence of seedlings, maturity progress, wider thermal range for germination, regeneration of damaged cells, reduction of barriers to embryo growth, quantitative and qualitative improvement of protein synthesis, seed dormancy breaking, improvement of environmental stress resistance during sowing, and eventually enhancement of plant growth and development (Ghasemi-Golazani et al., 2010;Ibrahim, 2016). Salicylic acid (SA) is a plant growth regulator that may have desirable effects on seedling growth and development (Kerantev et al., 2008;Khan et al., 2015). SA, or ortho hydroxyl benzoic acid, belongs to a group of phenolic compounds and is known as an important molecule that regulates plant reaction to environmental stresses (Senarajna et al., 2000). The significance of salicylic acid (SA) has been increasingly recognized in improved plant abiotic stress-tolerance via SA-mediated control of major plant-metabolic processes (Khan et al., 2015). In fact, SA alleviates salinity effects via increasing the level of hormones like auxins and cytokinins, e.g., it prevented any decrease in indoleacetic acid (IAA) and cytokinin contents and thus reduced stress-induced inhibition of plant growth (Shakirova et al., 2003), reducing the uptake of toxic ions, and contributing to membrane stability (El-Tayeb,2005;Samea-Andabjadid et al., 2018). The exogenous application of salicylic acid prevented the lowering of IAA and cytokinin levels in salinity stressed wheat plants resulting in the betterment of cell division in root apical meristem, thereby increasing growth and productivity of plants (Shakirova et al., 2003). When the seed is subjected to salt stress, the adsorbed SA (during the preparation) rapidly attaches to the glucose and converts to salicylic acid ß-glucoside (SAG). The enzyme that converts SA into SAG is acid salicylic glucosyl transferase. The produced compounds play an important role in expressing the genes associated with increasing seed resistance to salt stress. Among the RS20-RS19-RS17-RSS (genes that increase the tolerance to salinity by counteracting the toxicity of salt ions) and the PST1 gene increases salinity tolerance by scavenging the active oxygen species that causes oxidative stress (El-Tayeb, 2005).Exogenous application of SA to soybeans and corn (Khan et al., 2003) increased plant growth and caused positive changes under salinity conditions, such as increased stomatal conductance, transpiration, and photosynthetic rates in both soybean and corn. However, research data on the effect of seed priming with SA on seed germination and growth of L. sativus seedlings do not exist. Thus, the research question of this study was: can seed priming with SA improve germination and early growth of L. sativus under salt stress? Regarding the importance of Lathyrus sativus L in the field of medicine, soil improvement and green manure, this research was conducted to investigate the positive effect of salicylic acid for mitigating the salinity stress on germination, physiological traits and growth parameters of Lathyrus sativus L. Since salinity has been a major agriculture problem in West Azarbaijan Province in the region of Urmia Lake, the present study outcomes can be useful in combating with abiotic stresses, including salinity, towards the production of Lathyrus sativus L in this region. Germination and growth parameters The study was carried out in the laboratory of Agriculture and Biology Department of Urmia University in 2016 as a factorial experiment based on a completely randomized design with three replications. The treatments composed of seed priming with salicylic acid (SA) at three levels (0, 0.1, and 0.2 mM) and salinity with sodium chloride (NaCl) at three levels (0, 50, and 100 mM). The seeds of L. sativus were first disinfected with sodium hypochlorite 5% for 2 min, and then rinsed with distilled water. For priming, seeds were soaked in SA solution at pre-determined concentrations (0.1 and 0.2 mM) in darkness at 25°C for 8 h. Then, they were air-dried at room temperature for 24 h to reduce surplus moisture. For the germination assay, 100 seeds were placed between two filter papers in Petri dishes with a diameter of 9 cm. The Petri dishes were placed in a germinator at 25°C. In order to obtain the necessary moisture, seeds in Petri dish were watered every other day with distilled water and sodium chloride solution at concentrations of 0, 50 and 100 mM. To assess germination parameters (final germination percentage, mean germination time and germination speed index: as showed in formula), the seeds were counted at a certain hour every day until the number of germinated seeds reached a plateau for three consecutive days. The criterion for seed germination was the emergence of radicle at a length > 2 mm. On day seven, five seedlings were selected from each replication to measure the length of seedling, radicle, and plumule as well as seedling fresh and dry weight. After seedling fresh weight was recorded, the samples were oven-dried at 72°C for 48 h and the average of five samples was determined. The remaining seedlings in the Petri dishes were used to explore the physiological parameters of L. sativus, including leaf relative water content, Hill reaction, cell death, as well as post-harvest length and weight parameters. Four seedlings were selected from each replication. Seedlings were planted in perlite-containing pots and were placed in a growth chamber at light/dark regime of 16/8 h. To provide the required moisture for seedlings in the pot, also every other day, distilled water and Hoagland solution containing, 0, 50 and 100 mM sodium chloride was used. To measure the post-harvest length and weight parameters, root and stem length, root and stem fresh weight, and root and stem dry weight (oven-dried at 72°C for 48 hours) were recorded after 15 days. Relative water content To determine the relative water content of leaves, a quantity of 0.2 g was taken from a developed leaf in each replication, 1 cm 2 was detached from the middle part of its lamina, and then leaf discs were placed in a capped Petri dish containing distilled water, which was placed in darkness at 4°C for 16 h. Then, the leaves were taken out of the distilled water and after letting surplus moisture go, they were placed between two filter papers and their saturated weight was measured immediately. Next, leaves were oven-dried at 70°C for 48 hours to determine dry weight. Leaf relative water content (RWC) was calculated according to the following Formula 5. Hill reaction Hill reaction was measured according to Patsikka et al. (2001). A quantity of 0.2 g of fresh leaf tissue was weighed. Then, it was crushed with 3 mL of 50 mM phosphate buffer with pH 7 cooled down by freezing. The filtrated extract was centrifuged at 10,000 rpm for 2 min and the supernatant was removed. Then, 3 mL of cold phosphate buffer (50 mM, pH 7) was added to the deposit of the centrifuge and the deposit was suspended with a paintbrush. After that, 0.5 mL of the solution was taken and was added with 2 mL of cold phosphate buffer (50 mM, pH 7) and 0.2 mL of dichlorophenolindophenol (DCPIP). Immediately, its absorption was measured at 550 nm with a spectrophotometer. Then, the tubes were exposed to a 150-W lamp for 20 seconds and absorption was measured again. This practice was reiterated for 5 min until we had T = 100. The extent of DCPIP reduction was measured as a percentage of the non-SA-treated control. 4. Cell death measurement Cell death is a criterion showing the extent of damage to the cell membrane and was measured according to Baker and Monck (1994) using the absorption of the Evans blue reagent. To measure cell death, three 1-cm pieces of root ends were placed in Evans blue reagent 0.025% in water for 30 min. Then, the pieces were rinsed with water for 15 min. After that, the samples were crushed in 1 mL of 50% methanol solution. The extract was placed in a bain-marie at 50 °C for 15 min, and then, it was centrifuged at 14,000 rpm for 15 min. Afterwards, the absorption was measured at 600 nm with a spectrophotometer and finally, cell death was stated as a percentage of control. Mean germination time was obtained according to Ellis and Roberts (1981) Relative water content was estimated by Weatherley (1950) Data were analyzed with the MSTAT-C software package. Treatment means were compared using Duncan's multiple range test at P < 0.05. Also, all graphs were drawn in MS-Excel software package. Germination and growth parameters Final germination percentage (FGP) was not affected by salinity level and SA priming. However, germination speed index (GSI) was significantly influenced by salinity level, SA, and their interaction. The salinity levels of 50 and 100 mM reduced GSI significantly (16.65 and 18.55 (% /day) respectively) compared with control (28.00 % /day) (Figure 1). Priming with 0.2 mM SA improved GSI both under control and salinity conditions, but with 0.1 mM SA only under stress conditions. Salt stress effect led to the increase of MGT from 2.173 (day) in control to 3.229 and 2.773 (day) in 100 and 50 mM of NaCl respectively. The priming with both SA concentrations reduced this parameter significantly, but in a similar extent (Figure 2). Salinity and SA priming affected radicle, plumule, and seedling length, without significant interaction. Both levels of salinity resulted in reduction of radicle, plumule, and seedling growth. The reduction of plumule and seedling growth was higher at higher salinity levels. However, growth reduction of radicle was the same at both salinity levels. SA-primed seeds had longer radicles, plumules, and seedlings than non-primed seeds. However, the observed effect did not depend of SA concentration (Table 3). Salinity stress reduced seedling vigor significantly in a dose dependent manner. Higher loss in seedling vigor occurred at higher salinity level (100 mM). Seeds primed with both SA concentrations caused similar improvement of seedling vigor (Table 3). Both salinity level reduced seedling fresh weight as compared with control. Higher reduction was shown at 100 mM than 50 mM NaCl. In contrast, SA priming enhanced seedling fresh weight as compared with non-primed seeds. The highest effect was observed when 0.2 mM SA was applied (Table 3). However, the low rates of SA tested in this study were not sufficient to improve fresh weight under salinity stress. Salinity stress and SA priming influenced seedling dry weight significantly, without significant interaction. With the increase in salinity level, seedling dry weight was reduced. NaCl level of 100 mM resulted in loss of seedling dry weight from 16.2 mg in control to 12.76 mg in stress combination. The application of SA (0.2 and 0.1 mM) improved seedling dry weight (15.67 and 14.96 mg, respectively) versus control (Table 3), but the effect was significant only for SA at 0.2 mM. Relative water content Analysis of variance revealed the significant effect of salinity, SA priming, and their interaction on leaf relative water content (RWC) at P < 0.01 (Table 1). Under control and under stress conditions both SA concentrations caused the increase of RWC (Figure 3). Hill reaction and cell death There were significant differences in the Hill reaction between salinity stress levels and SA rates. As salinity was intensified, the Hill reaction was decreased significantly. The lowest rate of 54.84% was obtained at 100 mM of salinity. The application of SA improved the Hill reaction rate. The highest effect of improvement was shown in plants grown from seeds primed with 0.2 mM SA ( Figure 4). Increased salinity level resulted in a significant increase of cell death percentage. The application of SA resulted in a decrease of cell death percentage ( Figure 5). Post germination test Salinity level and SA priming influenced root and stem length, root fresh and dry weight significantly (P < 0.01), while the interaction of salinity with SA was insignificant for these traits ( Table 2). The smallest root length (22.22 cm) and stem length (31.46 cm) were obtained from 100 mM salinity, while the greatest root length (34.35 cm) and the greatest stem length (52.82 cm) was obtained from SA 0.2 mM (Table 3). Different SA levels varied significantly in their effects, so that SA concentration of 0.2 mM had the greatest impact considering both these traits (root and stem length) (Table 3). Salinity resulted in significant loss of root fresh weight, and consequently its dry weight, as compared with control (Table 3). Salinity level of 100 mM caused root fresh weight to decrease from 0.1848 g to 0.1087 g in control. Also, root dry weight at this salinity level was decreased from 0.04956 g to 0.01844 g in control. On the other hand, SA primed seeds favored root fresh and dry weight. Root fresh weight differed significantly in plants treated with 0.1 mM SA from those treated with 0.2 mM SA. Indeed, plants treated with 0.2 mM SA exhibited the greatest root fresh weight. Plants treated with 0.1 mM SA and those treated with 0.2 mM SA had similar root dry weights (0.03333 and 0.04467 g, respectively) ( Table 3). Stem fresh and dry weights were influenced significantly by salinity level, SA priming, and their interaction (Table 2). When there was no salinity stress, SA application improved stem fresh and dry weight. This increase was the greatest at SA rate of 0.2 mM, resulting in stem fresh and dry weights of 1.207 and 0.1750 g, respectively. At salinity level of 50 mM NaCl, while 0.2 mM SA improved these traits (stem fresh and dry weight), it did not significantly differ from SA concentration of 0.1 mM. As salinity level was increased to 100 mM NaCl, only the higher SA concentration (0.2 mM) improved stem fresh and dry weight to 0.4067 and 0.03867 g, respectively (Figure 6 & 7). Figure 7. Stem dry weight among treatments (different letters indicate significant differences at P < 0.05). Discussion and Conclusion The study showed SA has significant effect on germination indices, growth and physiological parameters under salinity stress. SA increased GSI and decreased MGT on salt stress. The loss of GSI under salt stress could be related to the negative impact of low water potential on water uptake as well as toxic effect of ions (Na and Cl) on biochemical processes and catabolic (enzymatic hydrolysis of seed storage materials) and anabolic (generation of new tissues by materials hydrolyzed at the first step) stages of germination (Shamsadin Saeid et al., 2008). However, the positive effect of SA on GSI could be due to reducing oxidative damage under high salinity (Lee et al., 2010). It is possible that SA stimulates the seed germination via bio-synthesis of gibberellic acid and acts as thermogene inducers (Shah, 2003). The reduced MGT in SA primed seeds may be attributed to the increased water uptake and promotion of the biological processes during germination provoked by SA in those seeds (Debez et al., 2018), and the reduced accumulation of Na + and Clˉ ions by SA application (Jini and Joseph, 2017). Entesari et al. (2012) reported the same effect on the mung bean grown under salinity stress and primed with SA. Hamid et al. (2010) reported that SA priming of wheat seeds under salinity stress resulted in the production of more vigorous and larger seedlings and enhanced chlorophyll, dissolved sugars and proteins content of the plant. The positive effect of SA treatment on seedling growth under salinity stress could probably be caused by the involvement of this growth regulator in cell division (Shakirova et al., 2003;Dolatabadian et al., 2009). Increased cell division of apical meristem of initial roots, which in turn resulted in an increased level of elongation was shown in SA treated wheat (Shakirova et al., 2003). In this study priming with SA was decreased negative effects of salinity on root and shoot length, fresh and dry weight. Delavari et al. (2014) founded germination, length of root and shoot, fresh and dry weight decreased under salinity but pre-treatment with SA improved them.SA treatment alleviates osmotic stress and allows better water uptake. The mechanism by which SA increases the growth of the root and shoots in some plants are still unknown, but it is possible that SA adjusts elongation and cell division with other substances such as auxin (Nourafcan, 2015). The mechanism by which SA improves root and shoot growth of some plants is not well-understood, but SA is likely to regulate cell elongation and division through the aid of other compounds, like auxin (Shakirova et al., 2003). It was also shown that SA hinders the oxidation of auxin (Farkhonded et al., 2012). So, it seems that the increased level of seedling dry weight is related to the SA-induced increase in root and stem length through different ways. It has been reported that the desirable effect of SA on seedling vigor under no stress conditions is accompanied with increased level of IAA and ABA (Shakirova et al., 2003). As mentioned above, the low effect of SA on seedling vigor under salinity stress could be attributed to the low rates of SA tested in this study. The mechanism by which SA improves root and shoot growth is not well-understood. However, Fariduddin et al. (2003) stated that SA inhibits auxin oxidation, whose elevated content increases net photosynthetic rate in leaf. Since salinity stress reduces cell division, it seems that the increase in seedling fresh weight associated with increasing root and shoots length that is affected by salicylic acid. According to our results salinity reduced relative water content and SA improved that. Likewise, Agarwal et al. (2005) concluded that foliar application of SA improved RWC of leaves in wheat plants. Singh et al. (2015) reported the exogenous application of SA reduced the growth inhibition of plants caused by NaCl, and increased leaf relative water contents. The increased level of leaf RWC by SA can be related to the role of SA in increasing the capability of the antioxidant defensive system, alleviating stress, and increasing membrane stability and cohesion as well as osmotic adjustment through the NaCl 50mM NaCl 100mM Stem Dry Weight (g) increased level of potassium as a crucial element in maintaining cell turgor (Bandurska and Stroinski, 2005;Korkmaz et al., 2007). Reduced activity of the Hill reaction was also observed in salt-stressed chloroplasts of various plant species (El-Shintinawy, 2000;El-Shahaby et al., 2003;Zeid, 2009). Proteins D1 and D2 of the photosystem II are also damaged under stress conditions. These proteins are the main components of the photosystem II and their degradation results in photoinhibition (Bissati et al., 2000;Kruk et al., 2005). SA by retention and accelerating the repair of protein D1 and D2, also induction of protein kinases and reversible phosphorylation of proteins, reduce the severity of damage under stress conditions (Hui-Jie et al., 2011). The enhancement of Hill reaction activity with SA priming may be due to increased synthesis of chlorophyll content along with acceleration of photosynthesis performance and carbohydrate metabolism (Khodary, 2004). Ervin et al. (2005) Observed increased activity of superoxide dismutase after treatment of plants with SA and argued that SA activates the antioxidant system and transmits the message to enhance the efficiency of the photosystems II. Bhattacharjee and Mukherjee (2002) reported that salinity induces oxidative stress causing membrane degradation. Salinity stress induces oxidative stress and, thereby, escalates the generation and accumulation of active radicals. This effect, in turn, oxidizes proteins and lipids and ruins membrane structure (Molassiotis et al., 2006). SA contributes to membrane maintenance by influencing polyamines, like putrescine, spermine, and spermidine, and generating membrane-stable complexes (Nemeth et al., 2002). This response may act through reducing the amount of hydrogen peroxide (Borsani et al., 2001), which is a toxic molecule in germinating seeds (Wojtyla et al., 2016). This study assessed the effect of salinity stress and SA seed priming on L. sativus germination parameters and early growth, for which research data do not exist in the literature. Salinity stress (50 mM and 100 mM NaCl) resulted in significant decline of germination parameters, seedling vigor, and seedling growth, whereas the application of SA priming alleviated some negative effects of salinity on germination and related traits and improved most growth and physiological traits of L. sativus. According to the results, it can be concluded that the priming of L. sativus seeds with SA can alleviate the effect of salinity and improve the resistance of seedlings to salinity stress.	2020-04-27T20:37:39.241Z	2020-03-31T00:00:00.000	{ "year": 2020, "sha1": "abeb1a7ea085c0ed428aeea9d45904aa62fa869c", "oa_license": "CCBY", "oa_url": "https://dergipark.org.tr/en/download/article-file/1028251", "oa_status": "GOLD", "pdf_src": "Adhoc", "pdf_hash": "22885e9b026a9cebd2818963448179e4490a0caf", "s2fieldsofstudy": [ "Agricultural And Food Sciences" ], "extfieldsofstudy": [ "Biology" ] }
2952376	pes2o/s2orc	v3-fos-license	Gene Expression and Cytokine Profile Correlate With Mycobacterial Growth in a Human BCG Challenge Model Background. Bacillus Calmette-Guerin (BCG) vaccine is the most widely administered vaccine in the world, yet its mechanism of action remains unclear. We hypothesize that certain immune pathways are associated with reduced mycobacterial growth following BCG challenge in human volunteers. Methods. We used samples from a mycobacterial challenge in which previously BCG-vaccinated or BCG-naive adults in the United Kingdom were challenged intradermally with a standard dose of BCG. Any remaining BCG was quantified in a skin biopsy specimen obtained 2 weeks after challenge and used as a measure of BCG growth and functional antimycobacterial immunity. We measured the immune response over the 2-week challenge, using DNA microarrays and flow cytometry, and correlated this with mycobacterial growth. Results. The magnitude of the immune response to BCG is greater in previously vaccinated volunteers, and this correlates with reduced mycobacterial growth but increased scarring at the vaccination site. In particular, the interferon γ and interleukin 17 pathways are strongly induced in previously vaccinated volunteers and correlate with reduced mycobacterial growth in this population. Conclusion. This study identifies pathways associated with control of mycobacterial growth in vivo in human volunteers and supports the use of BCG challenge as a tool for evaluating vaccine efficacy and identifying mechanisms of antimycobacterial immunity. Tuberculosis is a major global health problem, with an estimated 8.6 million cases and 1.3 million deaths in 2012 [1]. Effective vaccination is likely to be necessary for the long-term control of the tuberculosis epidemic. However, bacillus Calmette-Guerin (BCG) vaccine, the only currently licensed vaccine for the prevention of tuberculosis, provides variable protection against pulmonary disease [2], and in tuberculosis-endemic countries, the incidence of tuberculosis remains high despite widespread BCG coverage. Research efforts into new tuberculosis vaccines have focused largely on 2 strategies: (1) modify BCG or replace it with an attenuated strain of Mycobacterium tuberculosis, or (2) improve on the protection of BCG through prime-boost regimens, often using viral vectors expressing M. tuberculosis antigens, to enhance the memory cells primed by vaccination with BCG [3]. Twelve novel tuberculosis vaccines are currently in clinical trials [1]. The results of the first efficacy trial of a novel vaccine, modified vaccinia virus expressing antigen 85A (MVA85A), were published in early 2013 [4] and showed no enhanced protection, compared with BCG alone, in South African infants. Despite these advances, the development of new vaccines against tuberculosis remains hampered by the difficulty in evaluating efficacy. The frequency of new M. tuberculosis infection is very low, even in high-burden settings, making efficacy trials long and expensive. Although animal models exist, none exhibit all stages of human disease, and the extent to which they accurately predict protection in humans is unclear. Because of the tissue damage caused by tuberculosis and the difficulty in ensuring complete clearance of infection, human challenge with M. tuberculosis is currently not ethically possible. The clinical trial from which samples in this study were collected was conducted as part of an effort to create a human model of mycobacterial growth and its control, using BCG as a challenge organism. The trial included 4 groups who received the following vaccination regimes before BCG challenge: A, none; B, MVA85A; C, BCG (≥6 months prior to the trial); and D, BCG (≥6 months prior to the trial) followed by MVA85A 4 weeks before challenge. Groups B and D, who received MVA85A, were not included in this study, but the original group names have been retained here. The primary analysis of the trial showed a reduction in BCG growth in the previously BCG-vaccinated groups, compared with the BCG-naive groups, and that BCG growth was inversely correlated to the interferon γ (IFN-γ) enzyme-linked immunosorbent assay (ELISPOT) response to purified protein derivative tuberculin (PPD-T) [5]. In this study, we used flow cytometry and gene expression analysis to identify biological correlates of mycobacterial control in this setting, using stored samples from the trial. Study Design Samples used in this study were obtained from a phase 1 trial (clinical trials registration: NCT01194180), which was approved by the Medicines and Healthcare Products Regulatory Agency (EudraCT 2010-018425-19) and the Oxfordshire Research Ethics Committee A (reference 10/H0505/31). The study design was described in detail by Harris et al [5]. Groups included in this study are group A (BCG naive) and group C (BCG vaccinated; median time since vaccination, 10 years). All volunteers were intradermally challenged with a standard vaccine dose of BCG (SSI, Statens Serum Institut); 0.1 mL containing 2 × 10 5 -8 × 10 5 colony-forming units [CFU]) as previously described [5]. A single operator performed skin biopsies on the BCG challenge site of all volunteers 2 weeks after challenge, as previously described [6]. All biopsy specimens were processed, DNA was extracted, and quantitative polymerase chain reaction (qPCR) was performed as previously detailed [5] and described below. Peripheral blood mononuclear cells (PBMCs) for gene expression analysis were collected and cryopreserved as previously described [7] on the day of challenge (day 0) and days 2, 7, and 14 after challenge. Whole blood specimens for cytokine analysis were collected on days 0, 2, and 14 and processed as described below. BCG Quantification by PCR Biopsy specimens were snap frozen on the day of challenge and later thawed and homogenized in 1 mL of sterile phosphate-buffered saline (PBS). Homogenate was thawed, and BCG DNA from 200 µL of homogenate was released using the tough microorganism lysing kit (Precellys) in a Precellys 24 machine by shaking 3 times at 6500 rpm for 30 seconds each. Homogenate was transferred to a separate tube, and 50 µL of PBS was used to wash the remaining homogenate from the beads. Next, 180 µL of animal tissue lysis buffer and 20 µL of proteinase K (Qiagen) were added, vortexed, and incubated at 56°C for 4 hours. From this point, the extractions were performed as previously described [5,6]. qPCR primers ET 1 and ET 3 were used for detection of BCG DNA. The sequences used are given in the article by Harris et al [5]. PCR reactions were performed as previously described [5,6], using BCG-naive macaque tissue homogenate as a negative control. A standard curve was obtained by extracting BCG DNA from 1 in 10 serial dilutions of 5 pooled vaccine vials in PBS and correcting for live BCG from the corresponding CFU on solid agar. Gene Expression Analysis Cryopreserved PBMCs were rapidly thawed in a 37°C water bath and transferred to a 15-mL Falcon tube containing 10 mL of R10 (Roswell Park Memorial Institute medium with 10% fetal calf serum, 1% L-glutamine, 1% Pen-Strep, and 1% sodium pyruvate). PBMCs were pelleted, and supernatants were discarded and resuspended in 10 mL of R10 with 20 µL of Benzonase (Merck Chemicals) and rested overnight at 37°C in 5% CO 2 . PBMCs were counted on a Casy Counter (Roche), and 2 × 10 6 cells were stimulated for 12 hours with either R10 medium alone or containing 1 × 10 6 CFU of BCG (Statens Serum Institute). After 12 hours, supernatant was removed, and the PBMCs were resuspended in 350 µL of RLT buffer (Qiagen) containing 10 µL/mL β-mercaptoethanol and frozen at −20C. RNA was extracted using the RNeasy kit (Qiagen) according to manufacturer's instructions, including the optional protocol for DNA digestion (RNase-free DNase kit, Qiagen). Messenger RNA was amplified from the total RNA, using the Illumina Totalprep kit (Ambion) according to manufacturer's instructions. RNA quantity and quality was assessed using a Nanodrop ND-1000 Spectrophotometer and an Agilent Bioanalyzer (Agilent RNA 6000 Nano Kit). A total of 750 ng of amplified complementary RNA was labeled and hybridized to Illumina Human HT-12 v4 beadchips as specified in the manufacturer's instructions. Beadchips were scanned on an Illumina iScan machine, and data were extracted using the GenomeStudio software. Stimulation of Whole Blood Mycobacteria-specific intracellular cytokines (ICS) were measured in whole-blood samples as previously described [8]. Briefly, blood samples were incubated with 1 µg/mL αCD28 and 1 µg/mL αCD49d (BD) and stimulated with 20 µg/mL PPD (SSI, Denmark) and 5 µg/mL staphylococcal enterotoxin B (Sigma Aldrich); unstimulated blood samples served as negative controls . Stimulated and unstimulated blood samples were incubated at 37°C in 5% CO 2 for 6 hours, 3 µg/mL Brefeldin A (Sigma Aldrich) was added, and cells were incubated for another 6 hours in a timed water bath. Whole-blood samples were then treated with 2 mM ethylenediaminetetraacetic acid (Gibco), and red blood cells were lysed using FACS Lysing solution (BD). Samples were frozen for batched ICS analysis. Microarray Analysis Raw Illumina probe data were exported from Beadstudio and screened for quality, using the R package arrayQualityMetrics [9]. Gene expression data were analyzed using the bioconductor platform in R [10]. Genes not expressed above background levels in any sample were removed (P < .05). In limma [11,12], background correction and quantile normalization were performed using the neqc function [13]. Probes with an interquartile intensity range of < 0.3 (log 2 transformed) across all samples were filtered using bioconductor's genefilter package. Lists of differentially expressed genes were generated using limma (P value cutoff, .05 after Benjamini-Hochberg correction [11][12][13]). Pathway analysis was performed using the Internetbased tool DAVID (Database for Annotation, Visualisation and Integrated Discovery) [14]. Heat maps were created in R, using the gplots package [15]. Genes selected for inclusion in Figure 1 were selected as they contributed most highly to the enrichment of the gene ontology categories shown in Table 1. The genes in Table 2 were selected to include those from each group (A and C) with the highest fold changes (and equivalent values for the other group, if it was only highly differentially expressed in one group). A few additional genes, such as the one encoding TNF, are included for interest because of their importance to the field. Statistical Analysis Statistical analysis was performed using Prism (GraphPad) software. The Mann-Whitney U test was used to compare cytokine responses between the 2 study groups. Differences were considered statistically significant at P values of < .05. Determination of the BCG Scar Severity Rank Photographs of the BCG injection challenge site were taken on day 14, immediately before biopsy, to document each participant's reaction to BCG. A high degree of heterogeneity in local reactions to BCG was noted. The photographs were ranked Differentially expressed genes over the 2-week challenge period were determined using linear modeling in limma. The lists of differentially expressed genes for each group were analyzed using DAVID to identify significantly enriched gene ontology terms. a By the Benjamini-Hochberg method. continuously, from least to most severe, by the trial physician subsequently through the end of the trial but before the quantification of BCG in biopsy specimens. The ranking took account of size, redness, and swelling of the inflammatory reaction at the site of injection and is specified in Supplementary Figure 1. Immune Response to BCG Is Weaker in BCG-Naive Volunteers BCG induces changes in gene expression of circulating PBMCs over the 2-week period following BCG challenge. Twenty-four volunteers, 13 with a prior history of BCG vaccination, were challenged with BCG, and blood specimens were collected immediately before challenge (day 0) and 2, 7, and 14 days later. We determined gene expression in unstimulated PBMCs and identified genes within the groups at each time point that were differentially expressed relative to baseline. Both groups showed changes in expression of genes related to the immune system during the challenge, but the fold change and the number of significantly differentially expressed genes were higher in the previously vaccinated group (group C). A total of 1500 genes were differentially expressed, compared with baseline, over the challenge period in group C, whereas 500 were differentially expressed in group A. In both groups, differentially expressed genes showed enrichment of genes associated with the immune response, T-cell activation, glycolysis, and apoptosis, but these changes were stronger in group C than in group A ( Table 1 and Figure 1). The greatest number of differentially expressed genes was seen 2 days after challenge, although for genes involved in T-cell activation the peak occurred later. For all volunteers, we also determined gene expression profiles of PBMCs stimulated for 12 hours with BCG. As in the unstimulated samples, differentially expressed genes in both groups reflected a strong innate component in the immune response to BCG. However, fold changes were much higher in group C, showing a role for memory responses in increasing the magnitude of the immune response to BCG. Fold changes for BCG-stimulated PBMCs compared with unstimulated PBMCs, are shown for a subset of genes on days 0 and 14 (the times of BCG challenge and biopsy, respectively) for the 2 groups in Table 2. Gene expression was determined for each group, and at each time point, between BCG-stimulated and unstimulated peripheral blood mononuclear cells. Changes in gene expression were determined using linear modeling in limma, including the volunteer as a factor. We determined PPD-specific cytokine responses in wholeblood samples collected from volunteers in groups A and C at days 0, 2, and 14 after BCG challenge (Figure 2A and 2G). Frequencies of CD14 + cells producing TNF-α were comparable at baseline, indicating preexisting innate immune response to BCG in the 2 study groups. At day 14, there were more CD14 + TNF-α-expressing cells in group C, compared with group A. Study of the lymphocyte cytokine profile in the 2 groups revealed that levels of CD4 + T cells producing IFN-γ and TNF-α and CD8 + T cells producing IFN-γ were significantly higher in group C, compared with group A, at all 3 time points. Additionally, levels of CD4 + T cells producing IL-2 and IL-17 were significantly higher in group C volunteers at days 0 and 14. As monocytes are considered an important population of cells in which mycobacteria reside, while lymphocytes are known to be the major effector cells in tuberculosis immunity, we investigated the effect of BCG challenge on the ratio of monocytes to lymphocytes (defined as the ratio of CD14 + cells to CD3 + T cells in the unstimulated samples). This ratio was significantly higher in group C at days 2 and 14 after challenge than in group A ( Figure 2H). Finally, polyfunctional CD4 + T cells were detected in both groups, with group C showing significantly higher frequencies of CD4 + T cells making multiple cytokines simultaneously ( Figure 2I). We next correlated cytokine production quantified by flow cytometry of PPD-stimulated whole blood with expression of the same cytokines measured by microarray analysis of BCGstimulated PBMCs (Figure 3). The percentage of CD4 + T cells expressing IL-2, IFN-γ, and IL-17 correlated with the microarray values obtained for these genes (IFN-γ: Pearson r = 0.68, P = .001; IL-2: Pearson r = 0.68, P = .01; and IL-17: Pearson r = 0.51, P = .02). By contrast, the values for TNF-α production did not correlate with either TNF-α-expressing CD4 + T cells or TNF-α-expressing CD14 + cells (data not shown), perhaps reflecting production of TNF by a greater variety of cell types. BCG Growth Correlates Inversely With Scar Severity and Cytokine Production BCG growth was measured by qPCR of the biopsy specimen, and these data have been previously reported [5]. Photos of the BCG vaccination site were taken immediately before biopsy, and these were ranked to give a measure of severity of the local reaction (termed "scar severity"). The photos were ranked by the trial clinician before determination of the BCG burden in the biopsy specimen by qPCR. Ranked photos are shown in Supplementary Figure 1. There was an inverse correlation observed between scar severity and BCG growth (Spearman ρ = −0.71, P < .001; Figure 4). Expression of cytokines and related genes, as measured by both flow cytometry and DNA microarray, were correlated to BCG growth and scar severity values (Table 3) , and CXCL3 showed an inverse correlation with BCG growth and a positive correlation with scar severity. By contrast, the pattern-recognition receptor NOD2 and members of the leukocyte immunoglobulin-like receptor (LILR) family correlated in the opposite direction. Additionally, in polyfunctional T cells, the production of the following combinations of cytokines at day 14 showed a correlation with inhibition of BCG growth: IFN-γ, TNF-α, and IL-2 (P = .0007); TNF-α, IL-2, and IL-17 (P = .0127); IFN-γ and TNF-α (P = .0125); and TNF-α and IL-17 (P = .0091). The response to BCG/PPD stimulation changed over the course of the challenge, with the fewest correlations seen 2 days after BCG, reflecting the evolving immune response. DISCUSSION BCG has been administered to >3 billion people since its introduction >90 years ago. During this time, the prevalence of tuberculosis has fallen dramatically in some countries, but in others it is higher than ever. The use of a human BCG challenge model as a surrogate of protection against which to test novel vaccines may prove a valuable tool in early selection of promising vaccine candidates [5,6]. BCG is known to be highly effective in the United Kingdom, giving around 80% protection [16]. The aims of the study were to characterize the kinetics of the immune response to BCG in naive and previously vaccinated volunteers and to look for correlations between these immune parameters and the number of mycobacteria recovered from the site of injection at the end of the challenge period. The immune response in unstimulated and stimulated cells in both groups showed activation of innate immunity to BCG. In the previously vaccinated volunteers, however (group C), the fold changes were much higher, indicating that prior exposure to BCG increases the magnitude of the immune response. These observations are consistent with previous microarray studies done in naive and BCG-vaccinated mice challenged with BCG [17]. Although the 2 groups showed a degree of overlap in differentially expressed genes, fold changes were much higher in the vaccinated mice. Additionally, similar pathways were modulated in both human volunteers and mice. BCG challenge in previously BCG-vaccinated humans caused the monocyte to lymphocyte ratio to increase, compared with that in BCGnaive subjects, which is likely to affect the gene expression measured by microarray. The significance of this change is not clear and could be caused, for example, by proliferation of monocytes following BCG vaccination. The BCG challenge model allows associations to be made between different immune parameters and BCG growth. In this trial, previously BCG-vaccinated volunteers had significantly lower amounts of BCG recovered from the challenge site, compared with BCG-naive volunteers, consistent with the protective effect of BCG in this population. This was therefore a good opportunity to identify potential correlates of mycobacterial control. Genes in which a change in expression in the stimulated as compared to unstimulated samples was associated with BCG growth included IFNG and IL17F, together with other genes associated with these 2 cytokines, such as NOD2, IL22, IL23A, and FCGR1B. Several recent studies have reported important roles for these cytokines in protection from M. tuberculosis infection. IFN-γ is known to be necessary, although not sufficient, for protection, and recent studies also suggest an important role for T-helper type 17 (Th17) cells and the IL-23/IL-17 pathway. The latter can provide partial protection from M. tuberculosis challenge and have been shown to be necessary drivers of Th1 immunity and IFN-γ responses in the face of interleukin 10 production during infection [18][19][20]. In cattle, IL-22 and IFN-γ production by PPD-stimulated PBMCs were identified as the primary predictors of vaccine induced protection in a Mycobacterium bovis challenge model [21]. Additionally, a recent article identified NOD2 as a crucial component of epigenetic reprogramming of monocytes following BCG vaccination, which led to nonspecific protective effects [22]. In this study, levels of polyfunctional CD4 + T cells were significantly higher in BCG-vaccinated volunteers 2 weeks after BCG challenge, and cells producing multiple cytokines have previously been shown to be associated with protection in a Leishmania major model [23]. Here, we show a negative correlation between BCG growth and frequencies of CD4 + T cells producing combined cytokines (IFN-γ, TNF-α, and IL-2; TNF-α, IL-2, and IL-17; IFN-γ and TNF-α; and TNF-α and IL-17) 14 days after challenge. In this study, the identification of T cells producing IL-2 and IL-17 in previously BCG-vaccinated volunteers before BCG challenge may suggest the presence of a pool of central memory T cells, a response that is then boosted following BCG challenge. Other studies have previously shown that BCG can induce central memory T-cell responses in different populations [24,25]. Unfortunately, markers to identify memory populations of T cells were not included in these experiments, and exploring this would require further testing. It is unlikely that time since BCG vaccination can explain the data, because we have not seen a correlation between response to BCG by IFN-γ ELI-SPOT and time since BCG vaccination in this or any other previous studies. Finally, although our data show an inverse correlation between cytokine production and BCG growth, in a previously published efficacy trial in South African infants, increased cytokine production in response to PPD stimulation in BCG-and MVA85A-vaccinated infants was not associated with protection [4]. However, this may be because the magnitude of the response was much lower in South African infants. Furthermore, differences in age and population make it difficult to draw parallels between the 2 studies. The increase in local adverse events following a second BCG vaccination is well documented and was also seen in this study [6,26,27]. The severity of local inflammation correlated with the immune response (IL17F, IFNG, FCGR1B) and increased control of mycobacterial growth. The balance between bacterial killing and excessive inflammation is at the core of the relationship between humans and M. tuberculosis. These data show that too little inflammation fails to control mycobacterial growth, whereas a protective immune response comes at the cost of collateral damage. BCG has been shown to be effective in protecting the United Kingdom population from tuberculosis, suggesting that the spectrum seen here in group C may indicate an optimal balance. In Malawi, where BCG is not protective, vaccinated infants develop smaller scars, weaker Th1 responses, and stronger Th2 and regulatory responses in response to PPD-T stimulation, compared with their United Kingdom counterparts [28]. Likewise, laboratory mice in Brazil and Mexico show less susceptibility to infection with M. tuberculosis and also less protection from BCG [29]. Although the data shown here and recent studies suggest a role for the IL-17 pathway in the development of a protective response, excessive activation of this pathway has also been associated with detrimental tissue damage in this and other diseases [18,30,31], and the level of this cytokine's importance in vaccine-induced immunity is presently unknown. The human BCG challenge model may become a powerful tool in future vaccine research, both in terms of vaccine evaluation and for the identification of potential immune correlates of protection. An important next step will be to test this model and characterize the response to BCG in populations where BCG is not effective, which may yield insight as to why these differences exist and how they relate to control of mycobacterial growth. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.	2017-04-20T19:25:03.655Z	2014-11-07T00:00:00.000	{ "year": 2014, "sha1": "ced5ae7719f323dfc5bdcc4dd3dc0afb015b22d6", "oa_license": "CCBY", "oa_url": "https://europepmc.org/articles/pmc4392868?pdf=render", "oa_status": "GREEN", "pdf_src": "PubMedCentral", "pdf_hash": "39492ab1a3180981f7507ed828f2959960868e29", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
244918653	pes2o/s2orc	v3-fos-license	Study on modeling of the air ionization process in the technology of long-term storage of fruit and grape This article presents the results of modeling the process of air ionization in the technology of long-term storage of fruits and grapes in fruit storage facilities.Also was determined the main forces acting on ions in the ionization zone, in the volume of the fruit storage and on the surface of the processed product in order to establish the ionization modes and design the discharge gap of the ionizer. Based on the results of the research, the issues of the ionizer placement in the volume of the fruit storage have been resolved. The results of theoretical studies have been verified experimentally and the corresponding dependencies of the parameters of ionized air was obtained. Introduction In the Republic, special attention is paid to the development of infrastructure for processing and storage of fruit and vegetable products with the aim of uninterrupted supply of the population with fresh fruit all year round and reducing losses during long-term storage. For example, in 2017, more than 250 enterprises for the processing of agricultural products were built and modernized, more than 130 refrigerators with a total capacity of 85,700 tons of product [1,2]. Various methods of storing fruits and vegetables are being established, the peculiarities of product storage technology using electrotechnological methods are being studied. To ensure the required quality of product processing, it is necessary to create certain parameters for the storage regimes of fruits directly on the surface of the product.Based on the results of theoretical and experimental studies of ionization technology, optimal modes of room ionization, parameters of ionized air, a scheme and device for ionization of air, and principles of control over the parameters of ionized air have been proposed [3,4].The main parameters of ionized air and the characteristics of the air environment of fruit storage have been analytically determined. The results obtained have been studied experimentally, verified and analyzed [5]. For air ionization in fruit storage technology, an electric ionizer with pointed electrodes has been developed, the main parameters of the device have been determined taking into account the technological requirements [6] The model of the air ionization process consists of three fundamentally different elements: the ionization process in the discharge gap of the ionizer, the process of dispersion of air ions in the room, and the process of deposition of air ions on the surface of the processed product. Each of these processes requires a separate analysis. The process of air ionization in the discharge gap was studied experimentally and analytical expressions were obtained, however, the dispersion of air ions in the room and the process of deposition of air ions on the surface of the processed product were considered superficially or severally. Therefore, in our research, the ionization process was analyzed from the ion source to the processed product. Research Methodology Corona discharge electro ionizers operate on the basis of excitation and ionization in a highly inhomogeneous electric field on the surface of the electrodes of neutral molecules and air atoms at a sufficient electric field strength.A highly inhomogeneous electric field is obtained by two electrodes with different curvatures of the surface. One of the electrodes has a small curvature of the surface in the form of a tip, where an increased voltage is applied and is a source of ions and is called a discharge electrode. The second electrode is in the form of a plane or a large curvature of the surface and is grounded. To obtain a small size of the ionizer, the discharge and grounded electrodes are located in a sufficiently close distance. With sufficient voltage on the corona electrode (2.5-3 kV), the process of air ionization occurs in the discharge gap [5,6].When storing fruit in an ionized air environment, electromagnetic forces directly affect biological organisms directly, without conversion to another type of energy, therefore, the technological process is performed with low energy consumption. Corona-discharge air ionizers are rationally used in the technology of long-term storage of fruits and vegetables. They are cheap, the processing technology is simple, easy to use and performed in various versions. Discharge electrodes can be installed on top of stacks of food crates in the form of antennas. At the same time, the quality of product processing will not be high, since air ions are distributed unevenly in the volume. For uniform ionization of air in large rooms, ionizers are installed in the ventilation system [7]. In this case, the ions are dissipated not only by electrostatic forces but also by ventilation. With the optimal distance between the ionizers, it is possible to ensure a uniform distribution of air ions and high-quality processing of the product throughout the entire volume [8,9]. In large rooms, for example in industrial fruit storage facilities, the processed product is located at great distances from the ionizer. In order to obtain the effect of processing the product, air ions must reach the surface of the product. In this case, air ions pass through three different characteristic zones: the ionization zone on the discharge electrode, the zone of dispersion of space charges in the room, the zone of deposition and formation of an ion layer on the surface of the processed product.In the zone of ionization under the influence of an electric field of high intensity, air molecules acquire a certain charge. In this zone, the electric field is highly non-uniform and mainly electric forces act and the process will be very unstable. Along the lines of force of the electric field an electric wind formed under the influence of which ions are carried out of the ionization zoneIn large rooms, for example in industrial fruit storage facilities, the processed product is located at great distances from the ionizer. In order to obtain the effect of processing the product, air ions must reach the surface of the product. In this case, air ions pass through three different characteristic zones: the ionization zone on the discharge electrode, the zone of dispersion of space charges in the room, the zone of deposition and formation of an ion layer on the surface of the processed product.In the zone of ionization under the influence of an electric field of high intensity, air molecules acquire a certain charge. In this zone, the electric field is highly non-uniform and mainly electric forces act and the process will be very unstable. An electric wind is formed along the lines of force of the electric field under the influence of which ions are carried out of the ionization zone. The forces acting on the ions are determined by the strength of the electric field and the number of charges of the particles and are determined from the expression which is called the Coulomb force [10]: The particle is also influenced by the force of gravity: (2) Due to the uneven distribution of ions in the volume, a particle with a dielectric constant is acted upon by an electric field force with an electric field strength E, which is determined from the expression: Depending on the size and shape of the particles, the resistance force of the medium acts on the ions. If we assume that the particle has a spherical shape, this force is determined from the following expression: It was noted that in order to evenly distribute ions in the volume of the fruit storage, the ionization system is combined with the ventilation system. The intensity of ionization and the distribution of ions in an enclosed space depends on discharge electrode voltage and the air flow rate When determining the dependence of the quality of ionization on the operating and design parameters of the ionizer, preliminary information was given to the potential of the discharge electrodes and the dimensions of the discharge gap of the ionizer. The parameters of the outer zone of the corona discharge are determined by the joint solution of the differential form of the Gaussian equation and the dependences of the continuity of the current and the potential of the electric field and the current density and density of the space charge [11]. In modeling, the potential of the discharge electrodes is taken to be equal to the voltage of the electrodes, and the potential of the grounded electrodes is taken to be equal to zero, i.e.: (5) Figure 1. The design of the discharge gap between the needle and the ring: 1-needle, 2-ring, d-diameter of the pointed electrode (needle); D-is the diameter of the ring; ri -are the radii of the circles inscribed in the needle profile; qi-is the amount of a point charge located along the tip; k-is the amount of linear charge located along the ring; x, y-coordinates of calculated points in the electric field Usually, when calculating the electric field of tip electrodes, the method of equivalent charges is used [12]. The discharge gap will be located between the needle and the ring cutout in the grounded plane, with the needle being perpendicular to the center line of the ring. The length of the pointed electrodes L, the distance between the tips , the radius of curvature of the tip's point , the distance from the point of the tip h (Figure 1).The potential distribution in the discharge gap is represented as the sum of the potentials of point charges (q) located along the tip and the potentials of linear charges located along the ring (τ) ( Figure 1). The equivalent of the potential of point charges is as follows: here: q i -the amount of point charge r i -radius of the circumference inscribed in the profile of the needle h-distance from the tip's point to the plane of the ring; х,уcoordinates of the calculated points, where the potential of the electric field is determined. Taking into account the condition , we compose a system of equations of the first order using Maxwell's formulas and obtain the following system: Results Having solved this system of equations, we obtain the distribution of the space charge density along the tip (2-fig). In this case, the parameters of the electric field increase sharply when the control point approaches the tip's point. Now we are placing the equivalent linear charges along the circumference of the ring and equate its potential to zero: . Since the radius of the ring is much larger than its thickness (R >> δ), the rings can be taken as a "circular torus". In this case, the potential of linear charges is represented as the potential of the charged axis: (8) here: τ -the number of linear charges along the ring. The potential of several concentric linear charges with a radius can be expressed as follows: The potential of an arbitrary point of the discharge gap is determined as the sum of the potentials of point and linear charges: (10) The electric field strength in the discharge gap can be determined from the following ratio: E = The distribution of the electric field strength in the discharge gap along the axial line of the tip has the form: (11) We find the space charge distribution in the outer zone of the corona discharge electric field using the differential form of the Gauss equation: (12) Also, taking into account the expression of the formula, in the final form we can write an expression for the concentration of the space charge of the room in the following form: here, е -electron charge. The density of the pointed electrodes is one of the factors of the intensity of air ionization. The most effective ionization mode is obtained when their density is 150-474 pcs / m2. In this case, the length of the tip also depends on the distance between them. In turn, the distance between the needles depends on the length of the discharge gap [13]. The density of the tip electrodes is limited by the effect of mutual shielding of the electric fields of the tips. The mutual screening of the electric fields of the points, apart from the weakening of the ionization intensity, also affects the ion flux in the volume. [14]. Sh. Muzafarov was engaged in the determination of the design parameters of the tip discharge electrodes and their arrangement at our institute; he used ionizers for air purification and for ozonation [15,16]. It has been proven that the electric corona charge effectively cleans the air from pollution, especially from fine dust. In the studies, various power sources were used: a positive and negative direct current source, a pulsed alternating current source of industrial and increased, for each case the optimal parameters were determined. Based on the results of the analysis, we studied the features of the technology of long-term storage of fruits, the processes occurring in storage products, sources of product loss, the effect of air ions on fruits, optimal air ionization modes, the main parameters of the corona-discharge ionizer which provides the requirements of the air ionization technology of fruit storage. The results of theoretical and experimental studies were verified directly in production conditions. The analytically obtained results ρ ,, E, were verified experimentally, which are shown in Figures 3 and 4. Thus, with a voltage across the discharge electrodes of 2.8-6 kV, with a discharge gap of 25-40 mm in the volume of the room, we obtain the space charge density within k /, and the volume concentration of ions within ion / [17]. Based on the research results, the optimal design parameters of the ionizer and their arrangement along the room were determined. If the ionizers are at a distance of 2 -2.5 meters from each other and the ionization process is performed together with ventilation, the uniformity of the distribution of ions in the volume is within 85 -87% [18]. Based on the results of experimental studies, the distribution of the potential and the electric field strength and the concentration of ions in the 6-volume were obtained ( Figure 5). The distribution of the main parameters of the electric field of the corona discharge in the outer zone of air ionization is obtained by the same method.In this case, the parameters of the electric field increase sharply when the control point approaches the discharge electrode, with the coordinate x = 0; y = 10. The results obtained are the basis for the development of an air ionization device for closed rooms. As a result of the research, the following conclusions were gotton 4. Conclusions a) Application of ionization using a corona discharge in various technological processes is due to the simplicity and cheapness of the method, it is performed by low energy consumption and control versatility. The operating parameters of air ionization depend on the design parameters of the ionizer and the characteristics of the power source. With analytical and experimental determination of the parameters of the electric field of the corona discharge and ionization of air, the errors of the results do not exceed 3 ÷ 5%. At a density of space charges in the outer zone k / the volume concentration of ions is within the limits of ion /. b) Ionized air in a closed room moving from the ionizer to the surface of the processed product passes through three characteristic zones: the ionization zone, the dispersion of the volume charge in the volume and the zone of coverage of the product surface with an ionic layer. The parameters of these zones differ sharply from each other and require separate consideration. c) In the technological process of ionization, air ions are affected by the electric field strength of the corona discharge, the own weight of the ionized particle, the electric field strength of the space charge, the medium resistance force and the electric field force of the ionic layer of the product surface. d) The parameters of the electric field of the corona discharge have a sharply variable character of change and have a non-stationary form; therefore, all results should be confirmed by the data of experimental studies.	2021-12-07T20:09:09.730Z	2021-12-01T00:00:00.000	{ "year": 2021, "sha1": "8b739659976569dcb42a9563fcf1533d23556af2", "oa_license": null, "oa_url": "https://doi.org/10.1088/1755-1315/939/1/012012", "oa_status": "GOLD", "pdf_src": "IOP", "pdf_hash": "8b739659976569dcb42a9563fcf1533d23556af2", "s2fieldsofstudy": [ "Agricultural And Food Sciences" ], "extfieldsofstudy": [ "Physics" ] }
229351366	pes2o/s2orc	v3-fos-license	The Effects of Oxygen Functional Groups on Graphene Oxide on the Efficient Adsorption of Radioactive Iodine Oxygen-containing functional groups tend to induce a strong interaction between solid adsorbents and iodine molecules, yet have not been systematically investigated. Herein, on the basis of a series of nitric acid-treated graphene oxide (GO) with different contents of oxygen functional groups for iodine adsorption, it was found that the iodine uptake capacity is proportionate to the oxygen content and the diversities of oxygen-containing groups. The density functional theory (DFT) calculation results also suggest that oxygen-containing groups result in strong interactions between iodine molecules and the adsorbents through a covalent bond-forming process, among which -OH groups possess a higher adsorption energy averagely. Such theoretical and experimental work deepens our understanding of the effects of oxygen functional groups on iodine adsorption and provides novel ideas for future design and synthesis of high-performance solid adsorbents for radioactive iodine. Introduction Nuclear power has made great contributions to the society due to the high-power density and low greenhouse-gas emission [1,2]. However, the nuclear industry is always accompanied by the production of nuclear waste (e.g., spent nuclear fuel) or hazardous environmental release [3,4]. Several radioactive gases ( 129 I, 131 I, 85 Kr, and 127 Xe) are formed during the fission process [5]. Among them, 129 I and 131 I (half-lives being~1.57 × 10 7 years and 8.02 days, respectively) will be involved in the metabolic process of human and eventually cause thyroid injury [6,7]. Therefore, these two iodine radioisotopes must be captured efficiently and stored securely. Up to date, loads of adsorbents including silica [8], chalcogen-based aerogels [9], activated carbon [10], zeolites [11,12] and porous frameworks [13][14][15][16] have been studied during the last few decades. In order to improve the surface chemistry of solid adsorbents for higher iodine uptake, heteroatom doping (e.g., nitrogen doping) has drawn great attention since their adsorption capacity can be effectively strengthened through efficient surface modification [17][18][19][20]. Recently, Kangxin Xiao et al. has successfully synthesized N-doped porous carbons from orange peel waste, the iodine adsorption capacity of which was up to 2252 mg·g −1 [10]. Meanwhile, we noticed that oxygen doping might have similar effects as well. For example, Deng et al. studied the surface features and adsorption behaviors of adsorbents from sludge to methylene blue and iodine, and the X-ray photoelectron spectroscopy (XPS) suggested that the functional groups with high contents of oxygen on the surface of adsorbent served as active sites for the adsorption process [21]. S. S. Zyryanov et al. attributed the most intense line at 529.6 eV in the XPS spectra to a chemical bond between oxygen and iodine during the investigation of a stainless-steel surface irradiated with protons in an iodine medium. They believe that one of the forms of iodine is also due to the O-I bond [22]. During the systematic investigation of the adsorption characteristics of radioiodine molecules on three kinds of low-index surfaces of Cu 2 O, the calculation was carried out using first principles density functional with periodic slab models. For the Cu 2 O (100) surface, it is unusually found that I 2 molecule moves to the nearby O s position (denoted as O s1 ) after being optimized in the case of Cu s site, indicating that the adsorption of I 2 molecule by surface oxygen atom is prior to that of copper ones [23]. Debasis Banerjee et al. reported two microporous metal organic frameworks for I 2 adsorption. Both the single-crystal X-ray diffraction and Raman spectroscopy reveal distinct sorption sites of molecular I 2 to the phenyl-and phenol-based linkers stabilized by the I···π and I···O interactions, which favors the selective uptake of iodine over water vapor [24]. With the characteristics of easy preparation, good chemical and thermal stabilities, porous carbon possessing large surface area and high porosity has drawn a great deal of attention as an ideal adsorbent for iodine capture. Moreover, carbon materials have shown the possibility of overcoming the weak interaction with the iodine gases through surface functionalization. Thus, a series of oxygen-rich microporous activated carbons were synthesized in our previous work [25]. A positive correlation was found between the iodine uptake value and the oxygen content in the iodine adsorption experiments. The sample with the highest oxygen content displayed an iodine capture capacity up to 6.44 g·g −1 , preliminarily suggesting the effects of oxygen. However, apart from oxygen-containing functional groups (-OH, -COC, C=O and -COOH), there are several influencing factors including the specific surface areas (SSAs), pore size and pore volume, related to the iodine adsorption performance of porous carbon materials. More complicatedly, the textural properties such as SSA and pore volume vary according to the different oxygen content on the carbon materials [25,26]. Variability in both oxygen content and pore size is not ideal for studies aimed at determining the effect of the former. As a result, experiments and calculations in this field have not been done or reported before. Therefore, in this study we developed a series of nitric acid-treated samples based on graphene oxide, which were used to investigate the impact of oxygen content since the previously mentioned factors have negligible effects on them. To further reveal the interactions between oxygen-containing functional groups and iodine molecules, density functional theory (DFT) calculations were also performed, which prove the experimental results theoretically. Both the experiment and calculation results offer a new insight for the future design of solid adsorbents for iodine adsorption. Material Synthesis Graphene oxide (GO) was prepared by chemical exfoliation of graphite according to an improved Hummers method, which was then dispersed by sonochemical irradiation. Then, 1 g of 2000 mesh natural graphite, 34 mL of H 2 SO 4 and 0.74 g NaNO 3 were mixed together, which was then stirred constantly in an ice bath. Next, 5 g KMnO 4 was added gradually while stirring. The temperature of the mixture was kept below 20 • C throughout the whole process by cooling. Subsequently, the mixture was stirred at 35 • C for 3 h and diluted with deionized water (250 mL). The color of the mixture changed into bright yellow after 4 mL of 30% H 2 O 2 was added. The mixture was then ultrasonically treated for 1 h. The possible residual metal ions were removed by filtering and washing with 1 L of 1:10 HCl aqueous solution, which was then removed by 1 L of deionized water. The GO suspension was freeze-dried before being used. The frozen GO (400 mg) were dissolved in 40 mL of nitric acid (HNO 3 68%), and a homogeneous mixture was obtained after 30 min sonication. After stirring for 1 h intensely, the mixture was transferred into a 50 mL Teflon-lined autoclave, sealed and heated at 100 • C, 110 • C and 120 • C for 6 h and the samples were labeled as GO-100, GO-110 and GO-120, respectively. The suspend solution was cooled to room temperature naturally. The final product was collected by centrifuging the mixture, and then dried in vacuum for further use after being washed with deionized water for multiple times. Characterization A CHN elemental analysis was performed using a vario EL cube elemental analyzer (Elementar, Langenselbold, Germany). The surface structure of the sample and energy dispersive spectroscopy (EDS) was obtained by a GeminiSEM 500 instrument (Carl Zeiss, Oberkochen, Germany). Transmission electron microscopy (TEM) was performed on a JEOL JEM-F200 (HR) instrument (JEOL LTD, Tokyo, Japan). The chemical state of the samples was analyzed by X-ray photoelectron spectroscopy using an AXIS ULtrabld instrument (Kratos, Manchester, UK). High-resolution spectra were charge corrected to the C 1s peak at 284.6 eV. Iodine Adsorption Measurements Approximately 20 mg of GO/GO-100/GO-110/GO-120 and excess amounts of iodine crystals were placed in two 5 mL beakers, respectively. The beakers were then transferred into a sealed glass vessel. The adsorption experiment was carried out at 75 • C under normal pressure. The adsorption amount was monitored by recording the sample mass as a function of time. The uptake capacity Q t was calculated using Equation (1), in which m 0 is the initial mass of GO/GO-100/GO-110/GO120, and m t is the mass after t minutes of adsorption. Calculation Method DFT calculations were carried out using Gaussian09 program (Gaussian, Wallingford, CT, USA). The B3LYP functional is one of the most popular and accurate functionals. The 6-311G (d, p) basis sets were used for H, C and O atoms. For I atoms, the LANL2DZ, i.e., Los Alamos effective core potential together with double valence set (D95V) was employed. The Morphology of GO after HNO 3 Treatment The morphology of the samples was first studied via SEM. As demonstrated in Figure 1a,d, the surface of the original graphene oxide is relatively smooth. After HNO 3 treatment by a simple hydrothermal reaction, the surfaces of all the samples obtained at different temperatures (100 • C, 110 • C and 120 • C) become rough (Figure 1b-d,f-h). A large number of wrinkles and cracks are distributed unevenly on the observed surfaces, which may be due to the strong oxidation effect of HNO 3 . With the temperature further increasing to higher levels, the cracks and wrinkles on the surface (e.g., GO-120) became more evident. In addition, transmission electron microscopy (TEM images in Figure 2b-d) reveals that the HNO 3 -treated graphene oxide remains the same as untreated graphene oxide nanosheets (Figure 2a) in the microscopic morphology despite the change in macroscopic morphology after HNO 3 treatment. Oxygen Functional Groups The elemental compositions of the samples were further measured by CHN analysis. Table 1 shows the elemental content of all the samples. According to the results, the oxygen content of GO was the lowest (48.86 wt.%) and showed a slight upward trend after HNO3 treatment. More specifically, the oxygen content increased from 50.60 wt.% to 52.04 wt.% as the temperature increased from 100 °C to 120 °C. Similarly, the atomic ratio of C/O decreased from 1.51 to 1.23, indicating the existence of rich oxygen content in the HNO3-treated GOs. To further verify the intrinsic properties of the oxygen, the X-ray photoelectron spectroscopy was performed. The spectra were charge corrected (C 1s at 284.6 eV) and peak fitting of the high-resolution XPS spectra was also carried out, making it possible to estimate the bonding state of carbon and oxygen. The C 1s peaks of GO (Figure 3a), GO-100 ( Figure 3b) and GO-110 ( Figure 3c) can be assigned to three peaks being C-C (284.6 eV), C-O (286.6 eV) and COOH (289.0 eV), respectively [27][28][29]. For the sample GO-120 (Figure 3d), in addition to the three peaks mentioned above, one more peak appears at the binding energy of 285.2 eV, which can be attributed to C-OH groups [30][31][32]. Figure 4a shows the contents of different oxygen-containing surface functional groups on GO, GO-100, GO-110 and GO-120 calculated by fitting the C 1s peaks in Figure 3. There are two types of functional groups on the surface of GO/GO-100/GO-110, COOH and C-O. After HNO3 treatment, the content of C-O group shows a slight increase. When the temperature of hydrothermal reaction further reaches 120 °C, both the COOH and C-O group content decreased moderately, while the C-OH group accounts for approximately 40%. Oxygen Functional Groups The elemental compositions of the samples were further measured by CHN analysis. Table 1 shows the elemental content of all the samples. According to the results, the oxygen content of GO was the lowest (48.86 wt.%) and showed a slight upward trend after HNO3 treatment. More specifically, the oxygen content increased from 50.60 wt.% to 52.04 wt.% as the temperature increased from 100 °C to 120 °C. Similarly, the atomic ratio of C/O decreased from 1.51 to 1.23, indicating the existence of rich oxygen content in the HNO3-treated GOs. To further verify the intrinsic properties of the oxygen, the X-ray photoelectron spectroscopy was performed. The spectra were charge corrected (C 1s at 284.6 eV) and peak fitting of the high-resolution XPS spectra was also carried out, making it possible to estimate the bonding state of carbon and oxygen. The C 1s peaks of GO (Figure 3a), GO-100 ( Figure 3b) and GO-110 ( Figure 3c) can be assigned to three peaks being C-C (284.6 eV), C-O (286.6 eV) and COOH (289.0 eV), respectively [27][28][29]. For the sample GO-120 (Figure 3d), in addition to the three peaks mentioned above, one more peak appears at the binding energy of 285.2 eV, which can be attributed to C-OH groups [30][31][32]. Figure 4a shows the contents of different oxygen-containing surface functional groups on GO, GO-100, GO-110 and GO-120 calculated by fitting the C 1s peaks in Figure 3. There are two types of functional groups on the surface of GO/GO-100/GO-110, COOH and C-O. After HNO3 treatment, the content of C-O group shows a slight increase. When the temperature of hydrothermal reaction further reaches 120 °C, both the COOH and C-O group content decreased moderately, while the C-OH group accounts for approximately 40%. Oxygen Functional Groups The elemental compositions of the samples were further measured by CHN analysis. Table 1 shows the elemental content of all the samples. According to the results, the oxygen content of GO was the lowest (48.86 wt.%) and showed a slight upward trend after HNO 3 treatment. More specifically, the oxygen content increased from 50.60 wt.% to 52.04 wt.% as the temperature increased from 100 • C to 120 • C. Similarly, the atomic ratio of C/O decreased from 1.51 to 1.23, indicating the existence of rich oxygen content in the HNO 3 -treated GOs. To further verify the intrinsic properties of the oxygen, the X-ray photoelectron spectroscopy was performed. The spectra were charge corrected (C 1s at 284.6 eV) and peak fitting of the high-resolution XPS spectra was also carried out, making it possible to estimate the bonding state of carbon and oxygen. The C 1s peaks of GO (Figure 3a), GO-100 ( Figure 3b) and GO-110 (Figure 3c) can be assigned to three peaks being C-C (284.6 eV), C-O (286.6 eV) and COOH (289.0 eV), respectively [27][28][29]. For the sample GO-120 (Figure 3d), in addition to the three peaks mentioned above, one more peak appears at the binding energy of 285.2 eV, which can be attributed to C-OH groups [30][31][32]. Figure 4a shows the contents of different oxygen-containing surface functional groups on GO, GO-100, GO-110 and GO-120 calculated by fitting the C 1s peaks in Figure 3. There are two types of functional groups on the surface of GO/GO-100/GO-110, COOH and C-O. After HNO 3 treatment, the content of C-O group shows a slight increase. When the temperature of hydrothermal reaction further reaches 120 • C, both the COOH and C-O group content decreased moderately, while the C-OH group accounts for approximately 40%. The Iodine Adsorption Performance Considering the high oxygen content and the diversity of surface functional groups, the obtained sample was used as a solid adsorbent for iodine vapor capture. The adsorption measurements were performed in a sealed vessel at 75 • C under atmospheric pressure [27,33]. The adsorption amount was monitored by recording the sample mass as a function of time. Figure 4b shows the iodine adsorption performance of the four samples. Although the saturated iodine adsorption capacity of the HNO 3 -treated GOs was not as high as that of the oxygen-rich activated carbon with ultra-high surface area used as adsorbent in our previous work (6.44 g·g −1 ) [25], the relationship between the capture value and the oxygen content is evident in Figure 4b. The iodine capture capacity increased from 0.17 g·g −1 to 0.44 g·g −1 proportionally with increasing oxygen content ( Table 1). The GO-120 shows the highest saturation value. Figure 5 shows the EDS and XPS results of GO-120 after adsorption of iodine. The elemental mapping displayed in Figure 5c,d proves its oxygen-rich nature and confirms the successful adsorption of iodine on it. Figure 5e shows the wide scanning XPS spectrum of GO-120 after iodine adsorption. It can be seen that in addition to the two peaks corresponding to C 1s and O 1s, I 3d peaks are also present in the spectrum, again illustrating the same point. The Iodine Adsorption Performance Considering the high oxygen content and the diversity of surface functional groups, the obtained sample was used as a solid adsorbent for iodine vapor capture. The adsorption measurements were performed in a sealed vessel at 75 °C under atmospheric pressure [27,33]. The adsorption amount was monitored by recording the sample mass as a function of time. Figure 4b shows the iodine adsorption performance of the four samples. Although the saturated iodine adsorption capacity of the HNO3-treated GOs was not as high as that of the oxygen-rich activated carbon with ultra-high surface area used as adsorbent in our previous work (6.44 g·g −1 ) [25], the relationship between the capture value and the oxygen content is evident in Figure 4b. The iodine capture capacity increased from 0.17 g·g −1 to 0.44 g·g −1 proportionally with increasing oxygen content ( Table 1). The GO-120 shows the highest saturation value. Figure 5 shows the EDS and XPS results of GO-120 after adsorption of iodine. The elemental mapping displayed in Figure 5c,d proves its oxygen-rich nature and confirms the successful adsorption of iodine on it. Figure 5e shows the wide scanning XPS spectrum of GO-120 after iodine adsorption. It can be seen that in addition to the two peaks corresponding to C 1s and O 1s, I 3d peaks are also present in the spectrum, again illustrating the same point. DFT Calculation Based on the above experimental results, the positive influence of high oxygen content deriving from the abundant oxygen-containing surface functional groups for the iodine adsorption performance can be confirmed. To further deeply reveal the effects of different oxygen functional groups, the interactions between the carboxyl group (-COOH), hydroxyl group (-OH), epoxy group (-COC) and carbonyl group (-C=O) on the carbon surface and iodine molecules were studied via density functional theory calculations. In the calculations, two models (the perfect model and the defected model) were performed. Firstly, perfect graphene surfaces were considered. All the oxygen-containing functional groups were placed on the edged and central position over the graphene surface ( Figure S1, Supplementary Materials). On the perfect graphene surface, -COOH group and -C=O were only placed on edged position (perfect-edged-COOH and perfect-edged-C=O) and -COC group was only placed on central position (perfect-central-COC) due to the instability issue. Due to the impossibility of centrally located structure of COOH, the addition of -COOH group around perfect-central-OH group and around perfect-central-COC was not considered [34]. Figure 6 shows the B3LYP functional-optimized structures for graphene-I 2 (C 24 H 12 -I 2 ), perfect-edged-COOH-I 2 , perfect-edged-OH-I 2 , perfect-edged-C=O-I 2 , perfect-central-OH-I 2 and perfect-central-COC-I 2 systems. The corresponding adsorption energies are summarized in Table S1 and plotted in Figure 7. Figure 6a shows the interaction between I 2 and pure graphene (the C 24 H 12 model) without any oxygen functional groups. As a result, the I-C distance is very long (8.431 Å), and the I-I bond distance is unchanged compared with the I-I distance in an isolated I 2 molecule (2.863 Å). Accordingly, the adsorption energy is estimated to be +0.3 kJ·mol −1 (unstable), suggesting a weak interaction between iodine molecule and graphene. In comparison, when iodine molecules are exposed to the oxygen-containing graphene surfaces, the I-I bond distances become longer in all cases (Figure 6b-f and Table S2), indicating the iodine molecules were attracted to the oxygen-containing groups. Interestingly, it's found that iodine molecules would bind not only with oxygen atoms but also with carbon atoms activated by nearby oxygen functional groups (within red circles). For example, three carbons atoms were calculated to coordinate with the iodine molecule with the bond distances of 3.202 Å in perfect-edged-COOH-I 2 system, 2.970 Å in perfect-edged-OH-I 2 system and 3.297 Å in perfect-edged-C=O-I 2 system, which are much shorter than that of I-C distance between I 2 and pure graphene. It means that the introduction of the oxygen functional groups on the graphene surface would activate the nearby carbon atoms, enhancing the interaction between I 2 and graphene. Meanwhile, the bond distances between iodine molecules and two oxygen atoms in perfect-central-OH-I 2 system and perfect-central-COC-I 2 system were calculated to be 2.107 Å and 2.720 Å, respectively. Accordingly, the binding energy of I 2 on surface oxygen functionalized graphene became negative approximately from −9.2 to −81.4 kJ·mol −1 (Figure 7), indicating the enhanced adsorption of I 2 on the oxygen functionalized graphene compared to that on the graphene (C 24 H 12 model). Notably, the binding energy of I 2 on perfect-central-OH reached a high of −81.4 kJ·mol −1 , which is much larger than other oxygen functional groups, showing the strongest interaction between I 2 and OH-terminated graphene. This calculation result is well consistent with the experimental results of the highest iodine capture for GO-120, where GO-120 also contains more -OH than the other samples. The aforementioned perfect-edged and perfect-central structures are constructed based on perfect graphene structures. However, due to the strong oxidation of the concentrated HNO 3 , the structures of HNO 3 -treated GO (GO-100, GO-110 and GO-120) are more or less damaged. Therefore, other models with different degrees of defects on graphene were considered as well ( Figure S2). Here, three typical types (single defect (D1), double defects (D2) and triple defects (D3) were constructed in their initial structures) in Figure 8. The initial structures for the above three typical defects were optimized with B3LYP functional. On the edge of central defect over defected graphene surface, edged -COOH (defected-edged-COOH), -OH (defected-edged-OH), -C=O (defected-edged-C=O) group and -COC (defected-central-COC) group near the edge of the defect were constructed. However, it was found that the defected-edged-C=O group tends to transfer into -COC group automatically, thereby the iodine interaction on this group was not considered [29,35]. The aforementioned perfect-edged and perfect-central structures are constructed based on perfect graphene structures. However, due to the strong oxidation of the concentrated HNO3, the structures of HNO3-treated GO (GO-100, GO-110 and GO-120) are more or less damaged. Therefore, other models with different degrees of defects on graphene were considered as well ( Figure S2). Here, three typical types (single defect (D1), double defects (D2) and triple defects (D3) were constructed in their initial structures) in Figure 8. The initial structures for the above three typical defects were optimized with B3LYP functional. On the edge of central defect over defected graphene surface, edged -COOH (defected-edged-COOH), -OH (defected-edged-OH), -C=O (defected-edged-C=O) group and -COC (defected-central-COC) group near the edge of the defect The aforementioned perfect-edged and perfect-central structures are constructed based on perfect graphene structures. However, due to the strong oxidation of the concentrated HNO3, the structures of HNO3-treated GO (GO-100, GO-110 and GO-120) are more or less damaged. Therefore, other models with different degrees of defects on graphene were considered as well ( Figure S2). Here, three typical types (single defect (D1), double defects (D2) and triple defects (D3) were constructed in their initial structures) in Figure 8. The initial structures for the above three typical defects were optimized with B3LYP functional. On the edge of central defect over defected graphene surface, edged -COOH (defected-edged-COOH), -OH (defected-edged-OH), -C=O (defected-edged-C=O) group and -COC (defected-central-COC) group near the edge of the defect The adsorption behavior of iodine by oxygen-containing groups on the graphene surface under different types of defect conditions was investigated next (Figure 9). Compared to the I-I bond lengths in iodine molecules alone (Figure 6a), it is evident that when iodine molecules interact with oxygen-containing groups on the surface of graphene of different defect types, the I-I bond lengths in iodine molecules are also increased to some extent, which is similar to what happens in the perfect model. This also indicates that the presence of oxygen-containing groups has a slight pull on the iodine molecule. Similarly, in the defected models, the oxygen functional groups on the graphene surface, such as the carboxyl groups in double defects and triple defects structures, can activate the nearby carbon and hydrogen atoms (in red circles) to form adsorption active sites. In addition to this, the average bond length between -OH and iodine molecules was found to be the shortest regardless of the defected structure. This is also in agreement with the experimental results in GO-120 sample. The adsorption energies of different oxygen-containing functional groups on the surface of different defected graphene for iodine molecules were further calculated. As shown in Figure 10, the adsorption energies of all the oxygen-containing defected graphene for iodine molecules are located in the range of −5.3 kJ·mol −1 (double defects-COC) to −42.2 kJ·mol −1 (single defect-OH). In general, the -OH group tend to have a larger adsorption energy averagely ( Figure 11). This explains why GO-120 displays the highest iodine uptake capacity. Such results further indicate that the introduction of oxygen-containing functional groups on the surface of both the perfect and defective models allows the oxygen-containing functional groups to interact directly with the iodine molecules, as well as to activate the surrounding carbon atoms. Due to the synergistic promotion of these two interactions, the adsorption capacity of graphene to iodine molecules is increased. Materials 2020, 13, x 9 of 13 were constructed. However, it was found that the defected-edged-C=O group tends to transfer into -COC group automatically, thereby the iodine interaction on this group was not considered [29,35]. The adsorption behavior of iodine by oxygen-containing groups on the graphene surface under different types of defect conditions was investigated next (Figure 9). Compared to the I-I bond lengths in iodine molecules alone (Figure 6a), it is evident that when iodine molecules interact with oxygen-containing groups on the surface of graphene of different defect types, the I-I bond lengths in iodine molecules are also increased to some extent, which is similar to what happens in the perfect model. This also indicates that the presence of oxygen-containing groups has a slight pull on the iodine molecule. Similarly, in the defected models, the oxygen functional groups on the graphene surface, such as the carboxyl groups in double defects and triple defects structures, can activate the nearby carbon and hydrogen atoms (in red circles) to form adsorption active sites. In addition to this, the average bond length between -OH and iodine molecules was found to be the shortest regardless of the defected structure. This is also in agreement with the experimental results in GO-120 sample. The adsorption energies of different oxygen-containing functional groups on the surface of different defected graphene for iodine molecules were further calculated. As shown in Figure 10, the adsorption energies of all the oxygen-containing defected graphene for iodine molecules are located in the range of −5.3 kJ·mol −1 (double defects-COC) to −42.2 kJ·mol −1 (single defect-OH). In general, the -OH group tend to have a larger adsorption energy averagely ( Figure 11). This explains why GO-120 displays the highest iodine uptake capacity. Such results further indicate that the introduction of oxygen-containing functional groups on the surface of both the perfect and defective models allows the oxygen-containing functional groups to interact directly with the iodine molecules, as well as to activate the surrounding carbon atoms. Due to the synergistic promotion of these two interactions, the adsorption capacity of graphene to iodine molecules is increased. Optimized structures for single-defect structure (a-c), double-defects structure (d-f) and triple-defects structure (g-i) with B3LYP functional. Conclusions In conclusion, a series of graphene oxide nanosheets with rich surface oxygen functional groups were prepared as solid adsorbents for radioactive iodine adsorption after a simple nitric acid treatment. A positive correlation was observed between the iodine uptake capacity and the oxygen content and the diversities of oxygen functional groups according to the experimental results. The sample GO-120, which possesses an ultrahigh oxygen content of up to 52.04 wt.% and most abundant oxygen functional groups, displays the highest iodine uptake capacity. To further prove the effects of oxygen on iodine adsorption, the density functional theory calculation was also carried out. Both perfect and defected surfaces were considered on the surface of graphene to match the real experimental material. The DFT calculation results show that the presence of oxygen functional groups enhances the interaction between the iodine molecules and adsorbents. There is a covalent bond forming during the interaction between iodine molecule and O atoms or C/H atoms activated by nearby oxygen functional groups. Among the four kinds of oxygen functional groups, -OH-I2 system displays the highest adsorption energy averagely (−34.88 kJ·mol −1 ) while the adsorption energy of -COOH and -COC-groups is relatively lower (−23.28 kJ·mol −1 and −17.87 kJ·mol −1 , respectively). The -C=O group has the least obvious influence for the average adsorption energy is only −5.6 kJ·mol −1 . The effects of different oxygen functional groups are systematically investigated in this work. which opens up a new research direction for the design of carbon materials used as iodine adsorbents in the future. Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Table S1: Adsorption Conclusions In conclusion, a series of graphene oxide nanosheets with rich surface oxygen functional groups were prepared as solid adsorbents for radioactive iodine adsorption after a simple nitric acid treatment. A positive correlation was observed between the iodine uptake capacity and the oxygen content and the diversities of oxygen functional groups according to the experimental results. The sample GO-120, which possesses an ultrahigh oxygen content of up to 52.04 wt.% and most abundant oxygen functional groups, displays the highest iodine uptake capacity. To further prove the effects of oxygen on iodine adsorption, the density functional theory calculation was also carried out. Both perfect and defected surfaces were considered on the surface of graphene to match the real experimental material. The DFT calculation results show that the presence of oxygen functional groups enhances the interaction between the iodine molecules and adsorbents. There is a covalent bond forming during the interaction between iodine molecule and O atoms or C/H atoms activated by nearby oxygen functional groups. Among the four kinds of oxygen functional groups, -OH-I 2 system displays the highest adsorption energy averagely (−34.88 kJ·mol −1 ) while the adsorption energy of -COOH and -COC-groups is relatively lower (−23.28 kJ·mol −1 and −17.87 kJ·mol −1 , respectively). The -C=O group has the least obvious influence for the average adsorption energy is only −5.6 kJ·mol −1 . The effects of different oxygen functional groups are systematically investigated in this work. which opens up a new research direction for the design of carbon materials used as iodine adsorbents in the future. Supplementary Materials: The following are available online at http://www.mdpi.com/1996-1944/13/24/5770/s1, Table S1: Adsorption energy values of different oxygen-containing group, Table S2: Bond distances of different oxygen functional groups, Figure S1: Distinct structures of oxygen functional groups on perfect surface of graphene. The carbon atoms activated by neighboring oxygen functional groups are within red circles, Figure S2: Distinct structures of oxygen functional groups on three kinds of defected surfaces of graphene. The carbon atoms activated by neighboring oxygen functional groups are within red circles.	2020-12-23T06:16:38.443Z	2020-12-01T00:00:00.000	{ "year": 2020, "sha1": "f2f4c6fb78cfbe909245f48cc75860f7a819ee85", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/1996-1944/13/24/5770/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "79cae308e9d18b352f19d2f3bcace2ff43d55109", "s2fieldsofstudy": [ "Chemistry" ], "extfieldsofstudy": [ "Medicine", "Chemistry" ] }
12399826	pes2o/s2orc	v3-fos-license	Predisposing Factors for Hypoglycemia and Its Relation With Mortality in Critically Ill Patients Undergoing Insulin Therapy in an Intensive Care Unit Background: Hypoglycemia is a common and the most important complication of intensive insulin therapy in critically ill patients. Because of hypoglycemia’s impact on the cardinal organs as a fuel, if untreated it could results in permanent brain damage and increased mortality. Objectives: In this study, we aim to evaluate the incidence of hypoglycemia, its risk factors, and its relationship with mortality in critically ill patients. Patients and Methods: Five hundred adult patients who admitted to an intensive care unit (ICU) were enrolled in this study. A program of glycemic control with a target of 100 - 140 mg/dL was instituted. We used the threshold of 150 mg/dL for septic patients, which were monitored by point of care devices for capillary blood measurement. We detected hypoglycemia with a blood sugar of less than 50 mg/dL and with the detection of each episode of hypoglycemia, blood glucose measurement was performed every 30 minutes. Results: Five hundred patients experienced at least one episode of hypoglycemia, almost always on the third day. Of 15 expired patients who had one hypoglycemia episode, the most common causes were multiple trauma and sepsis. Increases in the sequential organ failure assessment (SOFA) number augmented the hypoglycemia risk to 52% (P < 0.001). Moreover, in patients with acute kidney injury (AKI), the risk of hypoglycemia is 10 times greater than in those without AKI (RR: 10.3, CI: 3.16 - 33.6, P < 0.001). ICU admission blood sugar has a significant relationship with mortality (RR: 1.01, CI: 1.004 - 1.02, P < 0.006). Hypoglycemia increased the mortality rate twofold, but it was not significant (RR: 1.2, CI: 0.927 - 1.58, P = 0.221). Conclusions: Our results showed that the SOFA score, AKI, and hemoglobin A1c are the independent risk factors for the development of hypoglycemia and demonstrated that ICU admission blood glucose, Hba1c, and hypoglycemia increased the risk of death, but only ICU admission blood glucose is significantly related to increased mortality. Background Dysglycemia is common in critically ill patients. Hyperglycemia is associated with adverse outcomes, including increased mortality, so insulin therapy and glucose control had been recommended to improve patient outcomes (1)(2)(3)(4)(5)(6)(7)(8)(9). However, intensive insulin therapy is associated with an increased risk of hypoglycemia, which is a possible predictor of morbidity and mortality in critical ill patients (10,11) and a limiting factor for intensive insulin therapy. Over the past two decades, many studies have been conducted on intensive insulin therapy in critically ill patients, demonstrating controversial results. Spontaneous episodes of severe hypoglycemia are rare during the management of critical ill patients, and many factors contribute to its occurrence, such as underlying disease, malnutrition, infection, different glucose measurement methods, and chronic liver or kidney diseases (12). Since the introduction of intensive insulin therapy strategies in intensive care units (ICUs), hypoglycemia has become a daily concern during the management of critically ill patients (13). An absolute or relative insulin excess with inadequate energy intake together with limited exogenous glucose production and increased glucose utilization are the fundamental causes of hypoglycemia in ICUs. Several studies have shown that the number of hypoglycemic episodes may not be increased by intensive insulin therapy (14,15). There is some evidence that hypoglycemic episodes are directly responsible for an increased mortality rate in critical ill patients (10,16). However, another case control study in critically ill patients receiving insulin therapy showed that the occurrence of hypoglycemia was not associated with an increased risk of mortality (17,18). As a result of several interventional clinical trials performed in critically ill patients, intensive monitoring and treatment of glucose levels in critically ill patients is emerging as a standard of care for these patients. So there are some concerns about which patients should be treated, the target level of hyperglycemia, and the incidence and risk factors for hypoglycemia, as hypoglycemia is a major limiting factor for the implementation of intensive insulin therapy and related mortality and was a reason for the early termination of the multicenter Glucontrol and VISEP trials (14). The typical manifestations of hypoglycemia is not routinely seen in critical ill patients, mostly because of the masking of their clinical pictures and physiologically blunted response. Little is known about the pathophysiology and consequences of hypoglycemia in ICUs, in contrast with an extensive body of literature on the pathophysiology and consequences of hypoglycemia in diabetes mellitus. Therefore, recognizing the risk factors for hypoglycemia would help to identify patients who are at increased risk for hypoglycemia. Objectives In this study, we aim to evaluate the incidence of hypoglycemia, its risk factors, and its relationship with mortality in critically ill patients. ICU Setting Five hundred critically ill patients from Feb 2011 to Sept 2013 were enrolled in this study. Two ICUs of Tabriz University of Medical Sciences (Shohada hospital and the ICU general of Imam Reza hospital) with mixed surgical and medical patients were included in this study. Cardiac surgeries were not performed at these two hospitals. All routine managements guided by protocols and a team of intensivists as directors of a multidisciplinary approach. The nurse patient ratio was 1:2 and full-time respiratory therapists accompanied the team. Inclusion criteria were all patients who admitted to theses ICUs. The study was approved by the ethics committee of Tabriz University of Medical Sciences. Glucose Control Protocol A program of glycemic control with a target of 100 -140 mg/dL was instituted. We used the threshold of 150 mg/dL for septic patients. This protocol was monitored by point of care devices for capillary blood measurement, and central laboratory values of venous blood were gathered to detect blood glucose at the specific time schedules and the accuracy of glucometers. Insulin therapy was performed by the frequent use of subcutaneous regular insulin as well as continuous intravenous regular insulin. We did not use any other type of insulin in our protocol. Blood glucose measurements were performed every hour, and if 4 consecutive measure were in the target range, the intervals were increased to 2 hours. If 3 consecutive measurements were in the target range, we performed measurements every 4 hours. If glucose was not in the target range, the measurement intervals were reduced to every hour. We detected hypoglycemia with blood sugar of less than 50 mg/dL, and with the detection of each hypoglycemia episode, blood glucose was measured every 30 minutes. All patients received energy from the enteral rout, except when they had contraindications, in which case we started parenteral nutrition. We calculated patients' daily caloric needs based on 25 kcal/kg. Statistical Analysis We used SPSS version 16 for statistical analysis. Data were presented as mean ± standard deviation. We used Student's t-test to compare two quantitative parameters. The chi-square test was used for the analysis of qualitative variables. To identify the predictors of hypoglycemia, we performed a stepwise logistic regression model for the mentioned variables. A p-value of less than 0.05 was considered significant. To assess the association between hypoglycemia and ICU mortality, we carried out a multivariate stepwise Cox proportional hazard regression model, adjusting for the abovementioned variables. Because the occurrence of hypoglycemia is also time-dependent, the time until the first occurrence of hypoglycemia was included. The results were expressed as adjusted hazard ratios (AHRs) and 95% CIs. Additionally, we carried out the same analyses stratified by selected variables. Continuous variables were categorized into two groups based on the median values. Results A total of 500 critically ill patients who admitted to the ICUs of Shohada and Imam Reza hospitals (Tabriz Uni-versity of Medical Sciences) from Feb 2011 to Sept 2013 were enrolled in this study. Patients' demographic characteristic are shown in Table 1. As shown in Table 1, their most common diagnoses were multiple traumas, brain tumors, and lower limb fractures. Almost half the patients received one of the following drugs: aspirin, corticosteroid, or beta blocker. All patients except 10 received enteral nutrition. Forty-six patients died during the study, of which 26 were men and 20 were female. Fifty patients experienced at least one episode of hypoglycemia, which was almost always on the third day. Of 15 expired patients who experienced one episode of hypoglycemia, the most common causes were multiple trauma and sepsis. The effects of age, sex, APACHE, sequential organ failure assessment (SOFA), admission blood sugar, blood urea nitrogen, acute kidney injury (AKI), and HbA1C on hypoglycemia occurrence were analyzed using the logistic regression method and after 8 times modeling SOFA, AKI, and hemoglobin A1c were recognized as effective (independent) variables on hypoglycemia. The analysis showed that increases in the SOFA number augmented the risk of hypoglycemia to 52% (P < 0.001) ( Table 2). In addition, in patients with AKI, the risk of hypoglycemia is 10 times greater than in patients without AKI (RR: 10.3, CI: 3.16 -33.6, P < 0.001). HbA1c has a direct correlation with the occurrence of hypoglycemia, as with increasing HbA1c the risk of hypoglycemia is increased threefold ( Table 2). Discussion Since the first leuven study intensive insulin therapy has led to improved inflammation and infection (19) which is associated with improved patient survival (20)(21)(22)(23)(24). Although numerous studies have concluded that tight glycemic control can positively impact the clinical outcomes in ICU patients (25), the apparent benefit of narrowly regulated tight glycemic control may come at the expense of an increased rate of hypoglycemia (26,27). We determined that hypoglycemia is common in critically ill patients, and in this study hypoglycemic patients were significantly older and had higher HbA1c and APACHE scores, which was similar to the results of previous (28,29) studies. Patients with type 1 diabetes and patients with longstanding type 2 diabetes may have an impaired counter-regulatory response. This may help to explain why patients who used insulin before ICU admittance were at a higher risk of developing hypoglycemia (30). But in our study, logistic regression modeling after matching patients based on age, sex, and APACHE showed that only SOFA, AKI, and HbA1c are independent variables related to hypoglycemia. In Van den Bergh's study (19), the most important risk factor for developing hypoglycemia was a discontinuation of nutrition or a reduction in glucose intake. ICU admission blood glucose, HbA1c, and hypoglycemia in our study increased the risk of death, but only ICU admission blood glucose is significantly related to increased mortality. As the lowering or discontinuation of nutrition without adjusting insulin therapy was associated with hypoglycemia, after any changes in the nutrition protocol, we decreased the blood sampling to 30 minutes to detect hypoglycemia more rapidly, which did not lead to higher mortality. The incidence of hypoglycemia was almost 10%, which differs from other studies (10,15,19). The difference in hypoglycemia incidence might be related to the type of patients, the intensity of protocols, the sampling methods (arterial vs venous), and the measurement frequency. Our study results showed that there is no association between the first episode of hypoglycemia and mortality, but Egi et al. (31) showed that an early onset of hypoglycemia following ICU admission is related to higher mortality levels. There are three explanations for the association between hypoglycemia and outcomes: first, the severity of hypoglycemia may be associated with the severity of the illness. Second, hypoglycemia may be a biomarker of imminent death. Third, hypoglycemia might have a deleterious biological effect on critically ill patients. This study showed that hypoglycemia did not have a significant effect on mortality, which was similar to the results of NICE SUGAR (32) and Arabi (28), but inconsistent with the results of Egi et al.'s study, which showed that the severity of hypoglycemia was significantly related to mortality (31). Our study showed that there were no gender differences for hypoglycemia, which contradicts Merimee et al.'s finding of a lower counter-regulatory threshold in women compared to men (33). Several studies have shown that severe hypoglycemia is independently associated with a higher risk of death with a greater duration of hospital stay (14,34,35). These researchers have suggested that every hypoglycemic event may increase the mortality rate, which is in contrast with our results, possibly due to the delayed recognition and impaired counter-regulatory responses in critically ill patients, which leads to poor clinical outcomes. Our results suggest that the duration of hypoglycemia episodes may be short, largely due to intensive monitoring. A limitation of this study was that we did not examine the permanent neurologic dysfunction after hypoglycemia, secondary outcomes (i.e., renal replacement therapy, critical illness neuromuscular complications, nosocomial infections), or data on the blood glucose variability on outcomes. Moreover, we did not mention mechanical ventilation as a predisposing risk factor for hypoglycemia via a direct or indirect effect by sedation. Our results showed that the SOFA score, AKI, and HbA1c are the independent risk factors for the development of hypoglycemia and demonstrated that ICU admission blood glucose, HbA1c, and hypoglycemia increased the risk of death, but only ICU admission blood glucose is significantly related to increased mortality.	2016-08-09T08:50:54.084Z	2016-01-31T00:00:00.000	{ "year": 2016, "sha1": "e7f119207895799534bd81ba326e8d6fe99c1e61", "oa_license": "CCBYNC", "oa_url": "https://europepmc.org/articles/pmc4835586?pdf=render", "oa_status": "GREEN", "pdf_src": "PubMedCentral", "pdf_hash": "e7f119207895799534bd81ba326e8d6fe99c1e61", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
225798513	pes2o/s2orc	v3-fos-license	The Allelopathic Effects Characteristics of Fresh Jatropha (Jatropha curcas L.) Leaves Extract on the Germination and Growth of Bok Choy (Brassica rapa L.) Sprouts *Correspondence Address: dianputrisani123@gmail.com The purpose of this study was to determine the concentration of Jatropha (Jatropha curcas L.) leaves extract on the germination and growth of Bok Choy (Brassica rapa L.). This study employed the completely randomized design with Jatropha (Jatropha curcas L.) leaves extract as the primary factor within 5 levels of concentration, namely 0% v/v, 5% v/v, 10% v/v, 15% v/v, and 20% v/v. The treatments were repeated 5 times. The variables measured in this study were root length, fresh and dry weight (aerial part and root), the length of aerial part, relative water content, and the total count of chlorophyll a,b. The homogeneity of the data was tested using the Levene statistics. The results showed that allelopathy of the Jatropha caused a stimulatory effect on the fresh weight and the dry weight of the sprouts with a maximum concentration of 10% v/V. The fresh weight increased from 38.22 to 49.16 or 22.25% while the dry weight increased from 3.40 to 4.42 or 23.07%. However, Jatropha plant can be consumed as table salt, and as substitutes for fuel, dyes, wounds healer, dysentery and jaundice, as well as flower extract can be used as medicine (Sarimole dkk., 2014;Setyaningsih dkk., 2013;Ulung & Studi, 2014). Jatropha has very short and broad rods, making them almost invisible. This stem functions as a leaf shaper and support growth or is often called heavy feeders. Bok Choy sprouts, which originated from China, can grow well in the highlands and the lowlands (Firmansyah dkk., 2009). Bok Choy has smooth leaves, no hair, and no head shape. Its stems are wide and sturdy. The spines of the leaves and the leaves are similar to green mustard, but the leaves are thicker. Bok Choy also contains many nutrients including protein, vegetable fat, carbohydrates, fiber, Ca, Mg, sodium, vitamin A, and Vitamin C (Efriyadi, 2018;Perwitasari dkk., 2012;Tiya dkk., 2019). Jatropha leaves water extract showed inhibitory effects on seed germination, shoot length, and root length of green chili (Capsicum anum L.) and Bok Choy (Brassica rapa L.) (Rejila & Vijayakumar, 2011). Bok Choy (Brassica rapa L.) is a type of vegetables belonging to the Brassicaceae (Kasmiyati dkk., 2018;Wiryono & Nurliana, 2019). The Bok Choy originates from China and was introduced to Japan. It is in the same family as Chinese vegetables (Harahap & Sari, 2019). Bok Choy requires more nitrogen for its growth is often called heavy feeders (Barokah, R., Sumarsono, S., & Darmawati, A., 2017). This study was done because there has been no previous study that examines the characteristics of Jatropha extracts on Bok Choy. METHOD This research was conducted in January 2019 at the Botany I Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, University of Lampung. The tools used in this study were beaker glass, Erlenmeyer flask, measuring cups, trays, test tubes, and racks, pipette drop, plastic cups, labels, blenders, rubber bands, UV spectrophotometers, digital scales, rulers, ovens, funnels, scissors, knife, and centrifuge. The materials used in this study included the seeds of Bok Choy of Nauli F1 varieties produced by PT. East-West Indonesia, 96% alcohol, tissue, distilled water, Jatropha leaves taken from Pringsewu area, and Whatman filter paper No. 1. The experiment was carried by employing the Completely Randomized Design (CRD) with Jatropha leaves water extract as the main factor consisting of 5 levels of concentration: 0% v/v, 5% v/v, 10% v/v, 15% v/v, 20 % v/v as the treatments. Each treatment was replicated 5 times. The dependent variable in this study was the root length, the length of the aerial parts, dry weight, fresh weight, dry weight of the aerial part, fresh weight of the aerial part, relative water content, and the total count of chlorophyll a,b. The independent variable in this study was the concentration of Jatropha leaves water extract. The parameter of this study was the mean (µ) variable of germination growth. The Jatropha leaves water extract stock solution was made by smoothing 100 grams of Jatropha leaves by using a blender and then added 500 ml of water. After that, it was filtered into beaker glass which produced 20% v/v. The Bok Choy seeds were selected to obtain good seeds by soaking them in water for 24 hours. The seeds were germinated using a tray that had been coated with distilled water-soaked tissue paper. The trays consisted of 5 cups and each cup was given 32 seeds. The selected seeds were then sprayed with distilled water for 7 days to germinate. The plastic cups used as a place seeds growth were washed first. 25 plastic cups were labeled with treatment and repetition notation. Each germinated seed contained in the trays was then transferred to a plastic cup to be observed for 7 days. On the second day, the germinated seed was treated by giving 10 ml of Jatropha fresh leaves water extract with a concentration of 5%, 10%, 15%, and 20%. The observation on the sprouts was carried out after 7 days of planting. The observed variables were the root length, the length of the aerial parts, dry weight, fresh weight, dry weight of the aerial part, fresh weight of the aerial part, relative water content, and the total count of chlorophyll a,b. By using a UV spectrophotometer at wavelengths of 648 nm and 664 nm, the absorbance of the chlorophyll extract was measured. Levene test used to determine the homogeneity of the data. After that, a variety of analysis was carried out at the 5% significance level. If there were differences between treatments, then a further test should have been performed with the Tukey method. The relationship between the concentrations of Jatropha leaves water extract with the germination growth was determined based on linear regression. RESULTS AND DISCUSSION The germination growth was evaluated based on the changes in the length of root and the length of aerial parts and the fresh and dry weight of the sprouts. The Tukey test at a 5% significance level showed that the control's fresh weight was not significantly different from the treatments. Then the fresh weight of 10% treatment was significantly different from the fresh weight of 20% treatment. The control's dry weight was significantly different from the dry weight of 10% treatment. The equation of y = -0.007x 2 + 0.138x + 3.293 with the coefficient of determination (R²) of 0.522 and the correlation coefficient (r) of 0.72 showed a strong relationship between the extract and the dry weight. The optimum concentration of Jatropha extract can stimulate dry weight by 9.2% with a maximum dry weight of 3.93 mg. The Levene test at 5% on the relative water content level showed that the sample was homogeneous (attachment 2 p-value > 0.05). The analysis of variance at a 5% significance level did not affect the relative water content of Bok Choy sprouts. Sprouts' Dry Weight (mg) Based on Table 3, the chlorophyll content on each concentration changed. The chlorophyll content was not only increased but some were stated to decrease. Therefore, the extract concentration affected certain chlorophyll content. Figure5. The Relationship between the Concentration of Jatropha Fresh Leaves Water Extract and Chlorophyll Content a The equation of = −0.0021 2 + 0.0279 + 0.591 with the coefficient of determination (R²) of 0.961 and the correlation coefficient (r) of 0.98 showed a strong relationship between the extract and the dry weight. The optimum concentration of the extract can stimulate chlorophyll-a levels by 6.6% with maximum chlorophyll-a of 0.68 mg. Table 1 shows that the allelopathic action of Jatropha curcas has a stimulatory effect on the fresh weight and the dry weight of the sprouts with a maximum concentration of 10% v/v. Fresh weight increased from 38.22 to 49.16 or 22.25% while the dry weight increased from 3.40 to 4.42 or 23.07%. Based on the results of this study, at the concentration of 15% and 20%, the growth of aerial parts began to decline. There were no allelopathy effects of Jatropha leaves water extract on the length of aerial and the length of the root. The results showed that Jatropha leaves water extract at a concentration of 20% tended to reduce chlorophyll content a, b and the total numbers of Bok Choy leave. However, there was no effect on relative water content. The intensity of the allelopathic effect depends on the concentration of the substance contained in the extract. Allelopathic compounds may act synergistically which causes effects that might activate or inhibit the growth of other plants depending on the tested concentration. Previous research by (Sanderson dkk., 2013) proved that Jatropha leaves extract did not provide an allelopathic effect on lettuce seeds germination, however, a concentration of 15% significantly inhibited the development of aerial and radicular parts. Meanwhile, (Rejila & Vijayakumar, 2011) state that Jatropha extract (Jatropha curcas L.) showed a stimulatory effect on seed germination and shoot length in Sesamum indicum L. The stimulation effect was directly proportional to the increase in concentration (5%, 10%, 15%, 20%). However, this did not occur in root growth where all treatments were inhibited and the control was not inhibited. CONCLUSIONS AND SUGGESTIONS Based on the results of the study, the Jatropha fresh leaves water extract serves as a stimulant at low concentration and inhibit at high concentrations on the growth of Bok Choy. The optimum concentration to serve as a stimulant is 15% v/v. The writers suggest the future researchers research the effect of Jatropha fresh leaves water extract on the growth of other plants. It is expected that this research can be used as a reference for further research.	2020-10-30T09:02:05.041Z	2020-06-29T00:00:00.000	{ "year": 2020, "sha1": "57b270fa3695fccf0cf9d914e50d032febfe775f", "oa_license": "CCBYSA", "oa_url": "http://ejournal.radenintan.ac.id/index.php/biosfer/article/download/4205/3685", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "ffb169d857c1628fb79de7ec1b87b96e4de25699", "s2fieldsofstudy": [], "extfieldsofstudy": [ "Biology" ] }
234050036	pes2o/s2orc	v3-fos-license	The Measurement of Impulse Buying Toward Emotional Responses . The objective of this research aims to know the influence of sales promotions and store atmosphere towards impulse buying with emotional responses as an intervening variable. The research method explores the primary data using quantitative approach for analysis. The research data of 310 respondents, come from the questionnaire disbursement by google form. The data analysis using SPSS 23.0 and SEM methods (Structural Equation Modeling) 88.0 LISREL. The research results of the analysis stated that sales promotions has no effect towards impulse buying with t value 1.28 (< 1.967); store atmosphere has no effect towards impulse buying with a t value -2.25 (< 1.967); emotional responses affect the impulse buying with t value 4.56 (> 1.967); sales promotion affect to emotional responses with t value 7.68 (> 1.96) and store atmospheric affect on impulse buying with value t of 10.12 (>1.967). INTRODUCTION The turnover of national retail sales in Indonesia in 2016 reached the highest point along its growth history, where in that year Indonesia occupied the fifth rank in the Global Retail Development Index (GRDI) according to A.T Kearney (2016), a consultant company for global management with total turnover of national retail sales amounting USD 324 billion. In the previous years, Indonesia occupied the 12th rank in 2015 and the 15th rank in 2014. The growth of retail business in Indonesia illustrates Indonesian people's high consumption of goods offered by retailers. This makes Indonesia a potential market for such a business model to continuously grow, creating a tight competition in this country among retailers, both foreign and local. Therefore, retailers need an appropriate strategy to run their business, responding people's needs and wants, and to build sustainable competitive advantages. One of the strategies commonly implemented by retailers is retail mix strategy which consists of some or all elements, namely location, purchase/service procedures, merchandising (goods/services being offered), price, store atmosphere, customer services, and promotion method. These elements may become the attractiveness for retail business customers. One of the industries in Indonesia which consistently increases its sales is leather and footwear industry. This shows an interesting opportunity for leather goods and footwear retailers from both domestic and foreign countries to grow and develop in Indonesia. This business opportunity is also realized by the biggest shoe retailer in the Western Hemisphere, namely Payless Shoesource which came to Indonesia in 2011. Based on the data of the growth of non-oil and gas processing industry issued by Statistical Data Center, the footwear sales in Indonesia in 2011 reached IDR 21.8 billion (Indonesia Government Statistical Berau, 2017). Payless Shoesource confidently enters shoe retail market in Indonesia by cooperating with Mitra Adiperkasa Group, a franchise company, to develop the retail network of Payless in several places in Indonesia. In running its business, Payless Shoesource has implemented some strategies to create competitive advantages, one of them is retail mix strategy. Payless exists in Indonesia offering a concept of store which differs from common shoestore, where Payless displays its products based on the foot size, aiming to provide the visitors with easiness to find the product suitable with their foot size. This concept surely makes the store atmosphere of Payless different. In addition to different store atmosphere that Payless Shoesource creates, it also implements the strategies commonly implemented by other retailers, for example promotion activities, which one of them is sales promotion. The implementation of these strategies are expected to enhance the possibility of purchase which will give a short term benefit in the form of sales increase and long term benefits in the forms of value creation and competitive advantages. That is why the author is interested and focuses this study on two aspects of Payless Shoesource's retail mix, namely store atmosphere and promotion method, i.e. sales promotion. Through strategy implementation in these two aspects, Payless expects consumers' response, in this case emotional response to the shopping environment so that it will affect their purchase decision makings, one of them is impulse buying decision. II. METHODS Analysis unit is the smallest unit of a research object desired by the researcher as the classification of data collecting. The research method in this study is quantitative approach; data collecting is carried out by distributing questionnaires through Google form; data analysis is quantitative or using statistics to test the hypothesis (Efferin, 2004:55). The Population of this research are Payless Shoesource visitors in Jakarta and suburb area. Sampling was done by Nonprobability Sampling Design technique with purposive sampling. It was found that the sample of this research was 310 respondents. The respondents in this study are visitors and consumers who have known, visited, or bought a product in Payless Shoesource. Respondent Profile Anslysis by Sex From the data respondent distribution by sex, it is found that the percentage of female respondents is 75% and male respondents is 25%. This indicates that the visitors and consumers of Payless Shoesources are dominated by female people. Respondent Profile Analysis by Age It is found that the respondents by age group have the following percentages 10-15 years as many as 2%, 16-25 years as many as 94%, 26-35 years as many as 2%, 36-45 years as many as 1%, and >45 years as many as 1%. Thus, it seems that people in the age of adolescent to adult (16-25 years) become the target market of Payless Shoesources and the dominant respondents in this study. Respondent Profile Analysis by Occupation The respondents in this study are also categorized based on the criterion of occupation. The distribution of respondents by occupation is found as follows: Student 18%, College Student 54%, Employee 20% and Entrepreneur 8%. This indicates that the visitors and consumers of Payless Shoesource are dominated by college students, followed by employees and students, where those people are really the market segment of Payless Shoesources. III. RESULT AND DISCUSSION Result of Structural Equation Model (SEM) After carrying out validity test, reliability test and classic assumption test and all the instruments of this study are stated as valid and the data is free from the symptoms of classic assumption, then the next test is to know the influence degree of sales promotion and store atmosphere variables on impulse buying with emotional responses as the intervening variable. The test is done using structural equation model (SEM) method and the outputs of structural equation are obtained: The first structural equation indicates that sales promotion (PP), store atmosphere (SA) and emotional responses (ER) variables simultaneously influence impulse buying (IB) with the coefficient of determination value 0.23. It means the simultaneous influence degree of sales promotion, store atmosphere and emotional responses variables on impulse buying is 23%, and the rest of 77% is influenced by other factors not examined in this study. The small coefficient of determination shows that sales promotion, store atmosphere and emotional responses variables are simultaneously not effective in affecting the impulse buying of Payless Shoesources visitors. The result of the second structural equation shows the influence of sales promotion and store atmosphere variables on emotional responses variable with the coefficient of determination value 0.70. It means the simultaneous influence degree of sales promotion and store atmosphere variables on emotional responses variable is 70%, while the rest of 30% is influenced by other factors not examined in this study. The big coefficient of determination value indicates that sales promotion and store atmosphere are simultaneously effective in affecting the emotional responses of Payless Shoesource visitors, where emotional responses in this study act as the intervening variable. From those two structural equations above, it can be known the direct influence, indirect influence and total influence of each variable tested as follows: ∑ Analysis on Direct Influence The direct influence of exogenous latent variables (KSI), namely sales promotion and store atmosphere, on the endogenous latent variables (ETA), namely emotional responses and impulse buying, can be seen from the values of Gamma (γ) and Beta (β) in the structural equation, where β is the coefficient of endogenous variable influence on endogenous variable whereas γ is the coefficient of exogenous variable influence on endogenous variable. So, the direct influence of sales promotion on impulse buying (γPP,IB) in this study is 0.10 and the direct influence of store atmosphere on impulse buying (γSA,IB) is -0.21. Whereas the direct influence of emotional responses on impulse buying (βER,IB) is 0.54. ∑ Analysis on Indirect Influence In addition to direct influence, this study also examines the indirect influence of exogenous latent variables (KSI), namely sales promotion and store atmosphere on endogenous latent variable (ETA), namely impulse buying, through another endogenous latent variable (ETA), namely emotional responses, through the following calculation: ∑ Analysis on Total Influence Total influence can be known by summing up the direct influence and indirect influence that have been obtained. So, the total influence of each exogenous latent variable (KSI) on endogenous latent variable (ETA) in this study is as follows: ß PP on IB is 0.31. ß SA on IB is 0.09. IV. CONCLUSION From the series of tests that have been done to know the influence of sales promotion and store atmosphere on impulse buying with emotional responses as the intervening variable in Payless Shoesource Jakarta and suburb area using the tools of statistical analysis as well as the above discussion and analysis, then conclusion can be made as follows: 1. Sales promotion positively but unsignificantly influences the impulse buying of Payless Shoesources visitors with the value of beta coefficient 0.10 and tstatistics (1.28) < ttable (1.967). Managerial Implication Sales and store atmosphere promotions do not directly influence the impulse buying of Payless visitors, but sales promotions and store atmosphere will have more influence on impulse buying when passing emotional responses. Therefore, it is important for Payless to improve situations that will build up the emotional response of visitors to be able to lead them to buying actions in this case impulse buying. In terms of sales promotion, for the discounted dimension, Payless needs to increase or expand the delivery media related to the ongoing promotion. From the shopping coupon dimension, Payless needs to increase the intensity of giving coupons more often. As for the direct sales dimension, Payless needs to approach sales directly to consumers more effectively, not only through offers made by cashiers at the point of payment. In terms of store atmosphere, for the uniqueness dimension, Payless needs to improve the unique design of the room. From the dimensions of the fixture (Paying), Payless needs to reorganize the supporting tools in the store to make it more pleasing to the eye. While from the side of ensemble display (product categorization), Payless needs to increase the attractiveness of the categorization that has been done. In terms of emotional responses, for the dimension of pleasure, Payless needs to create an atmosphere that can provide a calm feeling while in the store. While for the arousal dimensions of Payless visitors, Payless needs to improve its ability to foster a feeling of enthusiasm when visitors are at Payless. And for the dominance dimension, Payless needs to provide attractive offers delivered using loudspeakers to attract the public to visit Payless. In terms of impulse buying, for the dimension of spontaneity, Payless needs to increase the attractiveness of the display, so it doesn't seem monotonous. From the dimensions of compulsion strength, Payless needs to improve its ability to make visitors reckless in buying products by improving product quality, creating a good atmosphere and attractive offers. Whereas from the dimensions of excitement and stimulation, Payless needs to increase external stimulation Such as seduction or complete information provided by a salesperson related to a product that will convince prospective buyers to take action to purchase. Researchers have poured substantial theories into each variable, but there are still limitations in various things so that things that need to be considered and improved and improved for future researchers are find, including: 1. Specify the population so that the sampling can be more directed. 2. Manually distributing questionnaires (not only using google form). 3. Looking for variable dimensions that are more precise and in accordance with the characteristics of the research object. 4. Confirming the indicators formed. 5. Test the same respondent with different time periods. 6. Take data during peak season. 7. Looking for intermediate variables other than emotional responses or changing management performance measurements other than impulse buying, because after being tested together with the variable x, namely sales promotion and store atmosphere, the value of the determination coefficient (R2) is low, which is only 0.23. 8. Using the same model but with different research objects.	2021-05-10T00:04:34.584Z	2021-01-27T00:00:00.000	{ "year": 2021, "sha1": "5f77a2b1fb6b02116b47f87466e40e6dea136d40", "oa_license": "CCBYSA", "oa_url": "https://ijstm.inarah.co.id/index.php/ijstm/article/download/144/94", "oa_status": "GOLD", "pdf_src": "Adhoc", "pdf_hash": "f942f95b7c520def058ca9fd18993ef47ca9e527", "s2fieldsofstudy": [ "Business" ], "extfieldsofstudy": [ "Psychology" ] }
54052125	pes2o/s2orc	v3-fos-license	New Bound State Energies for Spherical Quantum Dots in Presence of a Confining Potential Model at Nano and Plank ’ s Scales In present work, the exact analytical bound-state solutions of modified Schrödinger equation with Modified central potential consisting of a Cornellmodified plus pseudoharmonic harmonic potential (MCMpH) have been presented using both Boopp’s shift method and standard perturbation theory, we have also constructed the corresponding noncommutative Hamiltonian which containing two new terms, the first one is modified Zeeman effect and the second is new spin-orbital interaction. The theoretical results show that the automatically appearance for both spin-orbital interaction and modified Zeeman Effect leads to the degenerate to energy levels to ( ) 1 2 2 + l sub states. Introduction It is well known that to study any quantum chemical-physical model, in different fields of sciences like atomic, nuclear, molecular, harmonic and harmonic spectroscopy, we need to solve the non relativistic Schrödinger equation and relativistic two equations: Klein-Gordon and Dirac .To obtain profound interpretation in Nano and plank's scales, much work in case of the noncommutative space-phase at two, three and N generalized dimensions has been done for solving the three fundamental previously equations .The notions of noncommutativity of space and phase developed on based to the Seiberg-Witten map, Boopp's shift method and the star product, defined on the first order of two infinitesimal parameters antisymmetric as : ..… (1) Which allow us to obtaining the two new non nulls commutators and [ ] * j i p p , ˆ, respectively as: ………….…..…….. (2) It is important to notice, that the Boopp's shift method will be applying in this paper instead of solving the (NC-3D: RSP) with star product, the Schrödinger equation will be treated by using directly usual commutators on quantum mechanics, in addition to the two commutators [29][30][31][32][33][34][35][36][37][38][39][40][41][42][43]: rewritten the radial wave function ( ) r l n, ψ to the form [48]: , , 1 φ ψ = …………….………..….…..…….. (8) Then, the equation (7) reduces to following form [48]: Where, ( ) ( ) = ε and then, the complete normalized wave functions and corresponding energies for the ground state, the first existed states, and th n excited state, respectively [48]: and the two normalized constants ( l N 0 , l N 1 ) are given by [48]: and . Noncommutative Phase-space Hamiltonian for (MCMpH) Formalism of Boopp's shift Based on the previous works [31][32][33][34][35][36][37][38][39][40][41][42][43], we give a brief review to the fundamental principles of modified Schrödinger equation in (NC-3D: RSP), to achieve this goal we apply the important 4-steps on the ordinary (SE): The main goal of this work is to extend our study in reference [41] for the potential (MCMpH) including new term into noncommutative three dimensional spaces and phases on based to the principal reference [48] to discover the new spectrum and possibility to obtain new applications for the modified potential in different fields.The rest of present search is organized as follows: In next section, we give briefly review to the Schrödinger equation with (CMpH) in three dimensional spaces.In section 3, we shall briefly introduce the fundamental concepts of Boopp's shift method and then we apply this method to derive the deformed potential and noncommutative spin-orbital Hamiltonian.In section 4, we apply perturbation theory to find the spectrum for ground stat and first excited states and then we deduce the spectrum produced automatically by the external magnetic field.In section 5, we conclude the global noncommutative Hamiltonian and we resume the global spectrum for (MCMpH) in first order of two infinitesimal parameters' Θ and θ in (NC-3D: RSP). Finally, the important found results and the conclusions are discussed in the last section. …….. (19) It is clear that, the first 4-terms in eq. ( 19) represent the ordinary potential while the rest term is produced by the deformation of space and phase.The global perturbative potential operators for studied potential (MCMpH) in both (NC-3D: RSP) will be written as: E for spin up and spin down, respectively, at first order of two parameters Θ andθ .In order to achieve this goal, we apply the standard perturbation theory: Where, a m 0 2 = β and 1 ' Where, the five terms ( ) are given by: In order to obtain the above integrals, we applying the . Where the two factors and are given by: It's important to notice that the above two terms and are represent the noncommutative geometry of space and phase, respectively. The Exact Spectrum of First Excited States ( 1) following special integration [50]: New Bound State Energies for Spherical Quantum Dots in Presence of a Confining Potential Model at Nano and Plank's Scales Maireche. The aim of this subsection is to obtain the new modifications to the energy levels for first excited states and corresponding spin up and spin down, respectively at first order of two parameters Θ and θ for (MCMpH) which are obtained by applying the standard perturbation theory as: Where, ( ' ) and then a direct simplification to the above equations (32.1) and (32.2) gives: ............... (33) .............. (34) Where, the 15-terms are given by: In order to obtain the results of above equations, we apply the special integral which represents by eq. ( 28): The explicit results obtained above allow us to get the exact modifications for (MCMpH) in (NC: 3D-RSP) On the other hand, it is possible to found another automatically symmetry for (CMpH) related to the influence of an external uniform magnetic field, generated from the effect of the new geometry of space and phase, it is deduced by the two following two replacements: Here χ and σ are infinitesimal real two proportional's constants and to simplify the calculations we choose the magnetic field k B B = and then we can make the following translation: 43.2) of eigenvalues of energies are reels and then the noncommutative diagonal Hamiltonian operator will be Hermitian operator.Furthermore, we can obtain the explicit physical form of this operator based on the results ( 23) and ( 42) for (CMpH), its represent by diagonal noncommutative matrix of order ( ) and and As it's mentioned in our previous works [31][32][33][34][35][36][37][38][39][40][41][42][43], the atomic quantum number m can be taken ( and we have also two values for 2 1 ± = l j , thus every state in usually three dimensional space of (MCMpH) will be in (NC-3D: RSP): ( ) It is important to notice that the appearance of the polarization states of a fermionic particle indicates the validity of the results in the field of high energy where Dirac equation is applied, which allowing to the validity to results of present search on the Plank's and Nano scales level.If we make the limits ( ) ( ) we can obtain all the results of ordinary quantum mechanics. Conclusion In this article, we have investigated the solutions of the Schrödinger equation for modified (MCMpH) potential.We showed that the obtained degenerated spectrum for the modified studied potential depended by new discrete atomic quantum numbers: basis on quantum mechanics, then the operator E ≡ ˆ denote to the ordinary operator of Hamiltonian for of Zeeman Effect in quantum mechanics.To obtain the exact noncommutative magnetic modifications of energy ( ) for (MCMpH) we just replaced the ....................(39) 3-parameters: + k , Θ and θ in the Eqs.(30.1) and (38.1) by the following new parameters: m with ( magnetic modifications of spectrum corresponding the ground states and first excited states, respectively.The new global exact spectrum of lowest excited states for (MCMpH) in (NC-3D: RSP) produced by the diagonal elements of noncommutative Hamiltonian operator .It is clearly, that the obtained previous results which are presented by Eqs.(30.1), (30.2), (38.1), (38.2), (43.1) and ( r 2 p New Bound State Energies for Spherical Quantum Dots in Presence of a Confining Potential Model at Nano and Plank's Scales Maireche. Now, the aim of this subsection is to obtain the modifications to the energy levels for ground states ...............(37.3) New Bound State Energies for Spherical Quantum Dots in Presence of a Confining Potential Model at Nano and Plank's Scales Maireche. i L i = ......(37.4)	2018-12-01T20:45:12.755Z	2016-01-22T00:00:00.000	{ "year": 2016, "sha1": "7b47e145d8d09c6732eab87316e76962d68ab7a1", "oa_license": "CCBY", "oa_url": "http://www.jnanoworld.com/articles/v1n4/nwj-016-abdelmadjid-maireche.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "7b47e145d8d09c6732eab87316e76962d68ab7a1", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
17253013	pes2o/s2orc	v3-fos-license	Identification of differential gene expression in in vitro FSH treated pig granulosa cells using suppression subtractive hybridization FSH, which binds to specific receptors on granulosa cells in mammals, plays a key role in folliculogenesis. Its biological activity involves stimulation of intercellular communication and upregulation of steroidogenesis, but the entire spectrum of the genes regulated by FSH has yet to be fully characterized. In order to find new regulated transcripts, however rare, we have used a Suppression Subtractive Hybridization approach (SSH) on pig granulosa cells in primary culture treated or not with FSH. Two SSH libraries were generated and 76 clones were sequenced after selection by differential screening. Sixty four different sequences were identified, including 3 novel sequences. Experiments demonstrated the presence of 25 regulated transcripts. A gene ontology analysis of these 25 genes revealed (1) catalytic; (2) transport; (3) signal transducer; (4) binding; (5) anti-oxidant and (6) structural activities. These findings may deepen our understanding of FSH's effects. Particularly, they suggest that FSH is involved in the modulation of peroxidase activity and remodelling of chromatin. In order to find new regulated transcripts, however rare, we have used a Suppression Subtractive Hybridization approach (SSH) on pig granulosa cells in primary culture treated or not with FSH. Two SSH libraries were generated and 76 clones were sequenced after selection by differential screening. Sixty four different sequences were identified, including 3 novel sequences. Experiments demonstrated the presence of 25 regulated transcripts. A gene ontology analysis of these 25 genes revealed (1) catalytic; (2) transport; (3) signal transducer; (4) binding; (5) anti-oxidant and (6) structural activities. These findings may deepen our understanding of FSH's effects. Particularly, they suggest that FSH is involved in the modulation of peroxidase activity and remodelling of chromatin. Background The development of ovarian follicles leading to ovulation requires endocrine regulation by the gonadotropins FSH and LH as the main actors. The complex regulatory network also includes steroids and peptides (e.g. growth factors, inhibins) acting via the autocrine and paracrine pathways. Recent studies have highlighted the importance of FSH in ovarian follicle maturation: in FSH-deficient mice the folliculogenesis is blocked prior to antral formation [1,2]. In order to obtain functional gametes, granulosa cell (GC) communication with the oocyte also seems essential [2,3]. GCs constitute an important compartment in the mammalian ovarian follicle, contributing to follicle development. They actively participate in the endocrine function of the ovaries by secreting oestradiol or progesterone under FSH stimulation. Besides their functional importance, GCs have been intensively studied for their convenient isolation. In murine, porcine, and bovine species they constitute a well-standardized model for the in vitro study of GC function, including hormonal regulation. Even if much data has been accumulated on the action of gonadotropins on GCs, the entire spectrum of genes regulated by FSH is not known. Besides recent advances in the generation of normalized cDNA libraries [4,5] and expres-sion analysis with differential display PCR and microarrays [6,7], SSH approach [8] was more efficient in accessing low-level expressed transcripts. We have therefore used the SSH method to isolate FSH-regulated genes in pig primary GC. These results increase our understanding of the physiological processes involved in the response of GC to FSH. In particular, FSH may play a role in the modulation of peroxidase activities and the remodelling of chromatin. Cell cultures Pig granulosa cells were isolated and cultured as described previously by Hatey et al [9]. Briefly, the granulosa cells were isolated from medium (around 3 mm in diameter) healthy follicles from immature swine ovaries. Cells were plated and grown to confluence in a serum-containing medium, which was replaced after 5 days of culture by a serum-free medium with or without FSH (Gonal-F™ 0.5 UI/ml, Serono laboratory) and incubated for a 48-h period before RNA extraction. FSH stimulation efficiency was tested both by measuring progesterone secretion in the culture medium using HPLC analysis [10] and by controlling the increase in P450scc and IGF1 genes expression using Northern blot analysis. RNA and polyA extraction Total RNA extraction was performed according to Chomczynski and Sacchi [11] with minor modifications [12]. Poly(A)-containing RNA was selected with Dynabeads mRNA Purification Kit (Dynal) following the manufacturer's instructions. For quality control, total RNA and mRNA were denatured with formaldehyde and size-fractionated through a 1% agarose gel according to standard methods. The integrity of each RNA sample was checked by ethidium bromide staining of the gel. Before reverse transcription, DNase I treatment of RNA was performed as described [13]. Reverse-Transcription Total DNase I treated RNA (2 μg) from control and induced cells were used for reverse transcription (RT) using Superscript™ II Reverse Transcriptase (Invitrogen) and oligo-dT15 primers (Roche) according to the manufacturer's recommendations. Suppression subtractive hybridization (SSH) SSH was performed with 1 μg of mRNA using the Clontech PCR-Select cDNA Subtraction Kit (Clontech) with minor modifications. The primary PCR amplification was achieved through 30 PCR cycles starting with 1 μl of 6 fold diluted second hybridization reaction. The secondary PCR amplification was achieved through 12 PCR cycles starting with 2 μl of 10 fold diluted primary PCR amplification. The PCR products were cloned using the TOPO™ TA Clon-ing R Kit (Invitrogen). Agarose gel analysis allowed the exclusion of empty clones or those containing more than one product. Forward subtraction H1 (FSH/C) used mRNA of control cells as a driver to select genes induced by FSH and reverse subtraction H2 (C/FSH) used mRNA of FSH-induced cells as a driver to select genes repressed by FSH. Differential screening As the final SSH products were enriched, but not strictly composed of differentially expressed cDNAs, a screening procedure was set up to sort out the false positive clones. Inserts were amplified by PCR starting from each colony. Each cDNA was amplified by PCRusing Invitrogen Taq polymerase: initiation with one cycle of 7 min at 94°C and amplification with 20 cycles (94°C for 30 s, 68°C for 30 s and 72°C for 1.5 min). Primers used were nested PCR primer1 (5'-TCGAGCGGCCGCCCGGGCAGGT-3') and nested PCR primer2 (5'-AGGGCGTGGTGCGGAG-GGCGGT-3'). PCR products were checked by electrophoresis on agarose gel. PCR products were denaturated in NaOH 0.5 M, EDTA 25 mM and 5% bromophenol blue, vacuum transferred onto two identical Hybond N membranes (Millipore) and UV crossed-linked. Macroarrays were subsequently hybridized with the different probes (cDNA of control and treated cells). The screening was performed visually. was added to each reverse transcription to monitor the transcription. First strand cDNAs were purified by G50 chromatography and quantified by measuring the incorporated radioactivity. For each cDNA sample (control and FSH-treated cells) the same dilutions were made starting from the same amount of material. The PCR conditions were: denaturation 5 min at 94°C, followed by (22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40) cycles of (30 s, 94°C, 30 s, 56°C, 1.5 min, 72°C) in presence of 1.5 mM MgCl 2 . The primers are listed in table 4. Ten μl of PCR products were analysed by electrophoresis on 1.5% agarose gel. Each experiment was performed at least three times. An external standard (plant mRNA: I11a accession number: y10291) was added to each RNA sample (200 fg for 2 μg total RNA sample) before reverse transcription and allowed the control of the cDNAs quantifications and dilutions using specific primers (Figure 1). Regional assignment Genomic localisation of the PCR products was done using the pig somatic cell hybrid panel (SCHP) as described previously by Tosser-Klopp et al. [13]. Each hybrid was scored for the presence of a pig specific DNA fragment and the assignments were performed using online software [14]. We also used the IMpRH panel 7000 rads [15] as described by Lahbib-Mansais et al. [16]. The results of radiation hybrid PCR products were analysed using the IMpRH mapping tool [17,18]. Functional Gene Ontology annotation The functional annotation analysis was performed on the 25 genes found to be regulated by Northern or comparative RT-PCR. Since our sequences are rather short, and mainly belong to 3'UTR portions of genes, available public tools to assign GO molecular function to nucleic acid sequences lack sensitivity. So we set up the following workflow: -Each sequence was compared to EMBL database via a NCBI blastn with an e-value limit set to 1e-3 -Blast results were parsed and filtered to keep hits having a) at least a coverage of 50% of the query sequence, b) 70% of identities, c) its coordinates inside a gene/mRNA/ CDS location on the subject sequence. -For each query sequence, candidate hits were sorted by a) their closeness to the pig genome, and b) their coverage and percent of identity. -Annotations present in the EMBL record of the hit were parsed to look for cross references to proteins from Uni-Prot/SwissProt databases -Finally, using cross reference tables provided by GOA http://www.ebi.ac.uk/GOA/, molecular function GO was validation of comparative RT-PCR inferred from these best hits and associated to the sequences. Suppression subtractive hybridization The SSH approach was performed on primary granulosa cells from cultures treated or not treated by FSH. mRNA from control granulosa cells was subtracted from the mRNA from FSH treated granulosa cells and vice versa. We isolated 226 clones from the forward hybridization H1 (FSH/C, FSH-induced genes) and 275 from the reverse subtraction H2 (C/FSH, FSH-repressed genes). In order to eliminate false positive clones, differential screening was used. The clones were spotted on two sets of macro-arrays and differential screening was performed using cDNA from the control and the treated cells as probes. We identified 28 (12.4%) induced cDNA clones and 19 (6.9%) repressed cDNA clones (Table 1). We also randomly selected 29 cDNA clones in both forward and reverse subtraction which provided no differential signals or no signal at all. Sequence analysis These 76 selected clones (28+19+29) were sequenced and compared against public sequence databases. We identified 64 different sequences of which 3 were novel and 14 corresponded to different regions of the same 5 genes. We finally dealt with 55 genes. The results are summarized in Table 1. The complete list of genes is given in Table 2. Regulation study Northern and comparative RT-PCR were used to analyse the differential expression of the selected genes. Out of the 55 genes, 18 could not be analysed according to their short length. The results of the 37 genes left are presented in Table 3. Some genes could not be analysed by Northern (no signal or non interpretable results) and were thus analysed by RT-PCR. Also, 9 genes which gave a good signal by Northern analysis were also analysed by RT-PCR to compare the results (Fig 1). Finally, out of the 37 genes, 25 were FSH-regulated (Table 3). In agreement with literature, HSD3B1 and CYP11A expression was induced by FSH in granulosa cells and ACTA2 expression was repressed by FSH (Fig 2). Chromosomal localisation Among the same 37 genes, 11 had already been located on pig chromosomes. Using the SCHP and the IMpRH hybrid panel, and the same primer pairs as for regulation studies, we tried to localise the 26 genes left. We successfully localised 10 genes, for the other 16 genes, the primers used amplified rodent DNA and stopped the analysis of the results (Table 4). Functional gene ontology analysis The 25 regulated genes were analysed using gene ontology annotation in order to document the processes involved in FSH effect on granulosa cells from medium sized follicles. For each sequence, we tried to assign molecular function GO identifiers using the methodology explained previously. We limited the analysis to the first levels of the molecular function ontology tree, just counting genes present in each node. The donut chart of figure 3 shows the distribution of the regulated genes according to their molecular function (see Additional file: supplementaldata-fig 3 for the data used to draw the chart). As genes can be assigned to more than one molecular function, some categories are drawn as outer partial rings in the donut chart (see also supplemental data). Numbers in this chart represent the number of genes observed for each category. Discussion Our objective has been to identify the genes regulated in granulosa cells in response to FSH stimulation. The identification of such genes will give valuable information about the molecular processes associated with follicular growth. An increasing number of studies are being undertaken on candidate genes that regulate folliculogenesis, e.g. IGF [18], EGF [19], TGF-β [20] and other members of the transforming growth factor superfamily, like BMPs [21,22] and GDF-9 [23,24]. In parallel with such individual studies, we now have access to technologies that allow us to study a large number of genes simultaneously. We applied the SSH technique coupled with a differential screening procedure, to GCs treated with FSH compared with untreated controls. Among 501 clones initially obtained, 76 were sequenced and were found to correspond to 55 different genes ( Table 2). Sequence analysis revealed redundancy from 2 different mechanisms: 1) gene redundancy, and 2) clone redundancy. This phenomenon is observed for highly expressed genes like-HSD3B1 and alpha-actin. High level of expression can also explain why two genes (alpha-actin and complement cytosolic inhibitor) are present in both the forward and reverse libraries (data not shown): the subtraction process may not be efficient for them. Regulation studies Thirty seven genes were analysed for regulation. Ten genes did not give a signal with Northern blot or macroarrays (table 3) and were successfully analysed by RT-PCR, underlining the low level of expression of some genes identified through SSH: they probably correspond to rare transcripts. The Cox2 gene and H2-170 gave discordant results when using subtraction or Northern analysis, possibly due to the high abundance of their mRNAs [25,26]. However, Northern experiments demonstrated clearly their high expression and regulation in granulosa cells. We found some genes already known to be regulated by FSH in pigs e.g. HSD3B and CYP11A which are upregulated by FSH (Figure 2). We corroborated in pigs that alpha and gamma actin were downregulated by FSH, whereas vimentin was not affected, as observed in rats [27]. These genes validate the biological model and the analytical methods, as well as reinforcing previous studies [28]. Localisation of cDNA and comparative mapping This paper describes 10 localisations of genes or ESTs on the pig genome using the somatic or IMpRH hybrid panel. Three genes and 4 ESTs were assigned with a LOD score of >10. Localisations of these genes were in accordance with the data from the human genome. In spite of a relatively low LOD score of 5.95 (significance limit 6), THBS1 was assigned on chromosome 1 in agreement with the expected localisation, using a comparative map between humans and pigs [29,30]. The aim of these localisations was to identify candidate genes for reproductive Quantitative Trait Loci (QTL). This could be the case for col4A5 which is located on chromosome X at the same place as a plasma FSH concentration QTL [31]. Col4A5 was up regulated by FSH and is a positional candidate for this QTL. This result obviously deserves further investigation. Functional gene ontology analysis In order to understand the pathways involved in the GC response to FSH, GO was used to cluster regulated genes according to their molecular function. Most of the tools used for automatic GO annotation of EST sequences, such as GOst [32] or OntoBlast [33], are mainly based on homology searches against well annotated protein databases. In our case, the sequence information obtained from the SSH clones is relatively short, and is biased towards 3' UTR; thus these tools are not convenient. Some other tools, such as Goblet [34], GoFigure [35] or Blast2GO [36] allow homology searches against nucleic databases, but once again with low sensitivity for short sequences. We thus used an in-house procedure to browse the GO classification and extract valuable information. The resulting classification (Fig 3 and Additional file) brings to light mainly 5 functional activities: "catalytic" and "signal transducer" activities directly linked with the steroid activity then "binding", "antioxidant" and "structural molecule" activities predominantly linked with differentiation pathways. On the other hand, our data demonstrate for the first time the involvement and/or the regulation of different genes in: Catalytic activities COX-2 was upregulated by FSH in our study and has been implicated as an important factor in female fertility [37]. In response to FSH, COX-2 induction could then stimulate progesterone production via the PGE2/EP2 pathway and play a role in ovulation by supporting cumulus expansion [38,39]. Indeed, mice deficient for COX-2 failed to ovulate, showing that COX-2 is necessary for ovulation [40]. 1) The upregulation of the HP1-BP74 gene (histone h1/h5 family), has never been shown before to be involved in granulosa cells development. Histones are highly conserved proteins involved in the package of chromosomes by interacting with DNA. Particularly, the HP1 family plays an important role in chromosomal biology and gene silencing. The HP1-BP74 gene was also shown to be involved in neuronal functional maturation [41]. This Table 1 : Differential screening by macro-arrays. This table summarises the results of differential screening by SSH and the results of sequence analysis: Forward subtraction corresponds to H1 SSH and up-regulated genes by FSH (FSH/C). Reverse one corresponds to H2 SSH and down-regulated genes by FSH (C/FSH). Random selection corresponds to clones that provided no differential signals or no signal at all in both forward and reverse subtraction. Forward 2) This study shows also the FSH up regulation of the CCT3 gene (chaperonin containing TCP1) known to mediate the folding of alpha-and beta-tubulin. FSH could thus intensify the remodelling of the microtubule cytoskeleton. Such a modification has been shown to be related to the maintenance and remodelling of heterochromatin during mammalian spermiogenesis [42]. Clone 3) We demonstrated the downregulation of annexin V: its binding to the cell membrane corresponds to the earlier events of apoptosis and is used to detect healthy live cells (annexin V negative) [43,44]. It is also a protein kinase C inhibitor which plays a potential role in cellular signal transduction [45]. This downregulation by FSH in GC may prevent apoptosis. 4) Finally, we found the downregulation of matrin by FSH. This protein that is localized in the nuclear matrix may have a role in RNA transcription thanks to its acidic region [46] and could be phosphorylated by nuclear PKC epsilon [47]. Signal transducer activity Among the genes involved in signal transducer activity, our study showed the upregulation by FSH of the Scavenger Receptor Class B Type I (SCARB1). This receptor is involved in both cholesterol delivery for steroid hormone production and in the recognition of apoptotic granulosa cells. We underline here the relationship with steroid hormone production, according to the downregulation of annexin V by FSH that suggests a differentiation process rather than an atretic one [48]. Anti-oxidant activities During follicle growth, swine granulosa cells are physiologically exposed to a progressive oxygen shortage. In vitro reactive oxygen species (ROS), such as hydrogen peroxide (H2O2) and lipid hydroperoxide (LOOHs) provided by oxido-reduction reactions, can either negatively or positively affect the differentiation of the gametes nota-bly during spermatogenesis [49,50]. Among the anti-oxidant activities, we noticed the upregulation by FSH of genes like glutathione peroxydase 3 (GPX3). GPX3 seems to regulate free hydoxyperoxides. The upregulation of this peroxidase activity by FSH could prevent atresia by protecting the membrane lipids and allowing differentiation of the follicle. Structural molecule activity We identified several interesting regulated genes in the structural molecule activity because they intervene in the extracellular matrix and cytoskeleton. Adhesion proteins, such as type IV collagen, increase the connections between cells and have also been shown to increase FSH receptors and progesterone production of GCs from immature porcine ovarian follicles [51]. The stimulation by FSH of type IV collagen gene expression could thus reinforce the effect of FSH and play a role in the local control of ovarian follicular dynamics [52]. Conclusion These results demonstrate the validity of both our cellular model and the SSH approach in identifying genes involved in response to FSH. In this way, and in addition to the regulation of the steroidogenesis and morphological changes already described, our data suggests that there is a role of FSH in the chromatin remodelling and protection against peroxides leading the follicle into a differentiation process rather than into atresia. Interestingly, we have been able to demonstrate the involvement and/or regulation of new genes such as HP1-BP74, cox-2, CCT3, SCARB1, GPX3, and also of unidentified genes. These new or unidentified genes will require further studies. Particularly, expression studies associated with histological techniques (in situ hybridization, immunohistochemistry) will allow a better understanding of the involvement of these genes. SSH has demonstrated its efficiency in our hands. We will now use it to further improve our knowledge of folliculogenesis in pigs by analysing fresh GC from ovarian follicles at different developmental stages. Figure 1 and 2. In the Table, C = FSH corresponds to non regulated genes by FSH, FSH>C corresponds to up-regulated genes and C>FSH corresponds to down-regulated genes. NI corresponds to Non Interpretable, 0 corresponds to no signal. * these genes gave no signals during macro-array screening and thus were not controlled by Northern. (Continued) Functional annotation: Molecular function Figure 3 Functional annotation: Molecular function. The donut chart shows the distribution of the 25 regulated genes according to their molecular function (inner circle). Genes that can be assigned to more than one molecular function are indicated by outer partial rings in the donut chart. Numbers in this chart represent the number of sequences for each category.	2014-10-01T00:00:00.000Z	2006-01-01T00:00:00.000	{ "year": 2006, "sha1": "a4f0fb56d68b574dbe78041d2a3cbb4c67776941", "oa_license": "CCBY", "oa_url": "https://rbej.biomedcentral.com/track/pdf/10.1186/1477-7827-4-35", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "a4f0fb56d68b574dbe78041d2a3cbb4c67776941", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [] }
212779915	pes2o/s2orc	v3-fos-license	Systematic Coarse-Grained Models for Molecular Systems Using Entropy † : The development of systematic coarse-grained mesoscopic models for complex molecular systems is an intense research area. Here we ﬁrst give an overview of different methods for obtaining optimal parametrized coarse-grained models, starting from detailed atomistic representation for high dimensional molecular systems. We focus on methods based on information theory, such as relative entropy, showing that they provide parameterizations of coarse-grained models at equilibrium by minimizing a ﬁtting functional over a parameter space. We also connect them with structural-based (inverse Boltzmann) and force matching methods. All the methods mentioned in principle are employed to approximate a many-body potential, the (n-body) potential of mean force, describing the equilibrium distribution of coarse-grained sites observed in simulations of atomically detailed models. We also present in a mathematically consistent way the entropy and force matching methods and their equivalence, which we derive for general nonlinear coarse-graining maps. We apply, and compare, the above-described methodologies in several molecular systems: A simple ﬂuid (methane), water and a polymer (polyethylene) bulk system. Finally, for the latter we also provide reliable conﬁdence intervals using a statistical analysis resampling technique, the bootstrap method. Introduction The enormous range of length and time scales involved in complex materials presents a challenging computational task, mainly, due to a wide range of relaxation times. A standard methodology to overcome problems of long relaxation times is to abandon the chemical detail and describe the molecular system by fewer degrees of freedom. Thus, systematic coarse-grained (CG) models are developed by averaging out the details at the molecular level, and by representing groups of atoms by a single CG particle. The challenge is to derive reliable coarse models both for reproducing the structural and the dynamical properties of systems. That is, to identify and effective approximate force field, approximating the potential of mean force (PMF), and then approximations to kinetic coefficients such as the friction. Methods to approximate the PMF are well studied in the literature. Examples include: (a) The Boltzmann inversion methods, also known as structural-based, which rely on matching the radial distribution function [1][2][3][4][5][6]. (b) The information theory based variational inference method relies on the minimization of the relative entropy (RE) between the configurational distributions of the system and the approximate one, [7][8][9][10]. (c) The Force Matching (FM) relies on minimizing the distance between the forces exerted on the CG particles and the approximate ones [11][12][13]. Recently, we have introduced a path-space variational inference methods were introduced, capable of inferring dynamical models of coarse-grained systems, [9,14]. There the Relative Entropy Rate (RER) is defined as the appropriate quantity to infer the coarse dynamics for stationary system, while the path space force matching is introduced. The purpose of the current work is to present a short review of the information theoretic methodologies ( relative entropy, and relative entropy rate) and their relation to the force matching and path-space force matching methodologies, through the application to different molecular systems. Methodology Let a prototypical problem of N classical atoms in a box of volume V at temperature T. We denote q = (q 1 , ..., q N ) ∈ R 3N the position vector and p = (p 1 , ..., p N ) ∈ R 3N the momentum vector of the N atoms. The probability of an elementary configuration q is given by the Gibbs probability, where U(q) is potential energy of a state q, Zis the normalization constant (partition function), and β = 1 k B T with k B the Boltzmann constant and T the temperature. In the above relation the kinetic part of the Hamiltonian has been integrated out. Coarse-graining (CG) is a standard methodology to overcomes the large range of length and time scales by averaging out the details of the atomistic level at the molecular level through representing groups of atoms by a single particle. The CG map Π : R 3N → R 3M determines the position vectors of M CG particles (or beads)q = (q 1 , ...,q M ) ∈ R 3M . Note that M < N but still M >> 1. From now on, we will use the bar "¯" notation for objects related to the CG model. The probability that the CG system has configurationq is given bȳ The quantityŪ is the M−body potential of mean force (PMF). The corresponding conservative force is thusF PMF (q) = −∇Ū PMF (q). While the above formula is exact, the accurate calculation of the PMF for a realistic model of a complex molecular system is a challenging task. This challenge is due to the high dimensionality of the integral, and the M vector as well. Therefore, we develop methods to find an effective potential in a parameterized form, U e f f (q; θ), θ ∈ Θ, which best approximates the PMF, i.e.,: Moreover, we assume that the evolution of the particles is described by a continuous time process {X t } t≥0 = (q t , p t ) t≥0 , with path space distribution P [0,t] , and invariant measure the Gibbs probability, Equation (1). The approximate coarse space dynamics we adopt are described by a Markov process {X t } t≥0 in R m with a parametric path space distributionQ θ [0,t] , θ ∈Θ. Information Theoretic Variational Inference: The Relative Entropy Here we adopt the information theoretic variational inference approach as the methodology to derive optimal approximate coarse models bot at equilibrium and dynamical regimes. This variational approach encompasses the minimization of the Relative Entropy (RE) between probability measures. The relative entropy (Kullback-Leibler divergence), [15], of two probability measures P(dω) and Q(dω) on a common measurable space (Ω, B) is given by provided P Q, i.e., P is absolutely continuous with respect to Q, and R (P\|Q) = +∞ otherwise. The functional R (P\|Q) defines a pseudo-distance between two measures as R (P\|Q) ≥ 0 and R (P\|Q) = 0 if and only if P = Q, P-a.s. In the case these probability measures have corresponding probability densities p(ω) and q(ω) Equation (2) where Π * µ denotes the push-forward of the microscopic measure µ. When the system is at equilibrium the optimization principle is When considering continuous time observations, in work [14] we prove that the path-space minimization principle (3) reduces to the path-space force matching (PSFM). In stationary dynamics the Relative Entropy Rate (RER) is defined by where P and Q denote the corresponding stationary processes. For discrete time observations (a) from the microscopic Gibbs density D n t = {X 1 , . . . , X n t } , or (b) the path-space distribution P [0,T] at dynamical regimes, D n s ,n t = {X k 1 , . . . , X k n t } n s k=1 , consideringthe estimator for the RE, the optimal parameter estimate is given by [14], . p andq θ are the microscopic and coarse space transition probability densities of the Markov processes X t andX t , respectively. Note that if the time series are stationary, the RER optimization iŝ Relative Entropy and Force-Matching The Force-Matching (FM) method estimates an effective CG potential that reproduces best the potential at the reference all-atom system, by solving the optimization problem i.e., we minimize the average difference between the atomistic F(q) forces and the corresponding CG forcesF(Π(q); θ), where · denotes the Euclidean norm in R 3M and E µ [·] denotes the expectation with respect to the probability Gibbs measure µ(dq). The minimization problem for the discrete observations, and the linear parametric representation of the forceF(·; θ) = G(·)θ, is The path-space force matching optimization problem is, [14], for which the discrete optimization problem becomeŝ Relative Entropy and Structural-Based Methods The structural-based methods, such as the direct inverse Boltzmann (DBI), iterative Boltzmann inversion (IBI), and inverse Monte Carlo (IMC) methods, use the pair correlation function g (2) (q) and the assumption that the interactions depend only on the distance R between particles, that is g (2) (q) =ḡ(R).ḡ(R) is called the radial distribution function. Thus the CG effective interaction is given byŪ that is the average density of finding the CG particle 1 at a distance R from the particle 2. The structural methods are thus based on the pair correlation function between CG particles, in contrast to the RE which is considering the joint probability distribution of the CG particles. In case the PMF can be exactly described by pair functions then the RE and structural methods coincide. Results and Discussion In the current section, we present the application of the variational inference methods, the RE, and the FM, for representative molecular systems: A simple fluid (bulk methane), a system of water molecules, and a polyethylene melt, at equilibrium conditions. We moreover study the bulk methane system out-of equilibrium, specifically we apply the PSFM at a transient time regime. Bulk Methane The molecular system consists of 666 methane molecules at temperature T = 100 K, and T = 80K. We employed molecular dynamics simulations to generate the microscopic space data based on which we applied the inference methods. Details on the atomistic simulations are given in [16]. For the coarse-grained representation of methane we have used a one-site representation with a pair potential. The pair potentials we have tested are (a) expansions with linear and cubic B-splines (with 48 parameters) and (b) the Lennard-Jones parametric form (with two parameters). A comparison of the FM, and IBI methods is depicted in Figure 1 [17]. The result depicts slight difference of the FM method to the RE and IBI. Figure 2 presents the performance of the FM and PSFM methods at equilibrium verifying the validity of the PSFM and its reduction to the FM method. A study at transient time regimes is presented in work [16]. Water The model system consists of 1192 molecules at ambient conditions (T = 300 K, P = 1 atm). Details on the atomistic simulations are given in [17]. For the coarse-grained representation of H 2 O, we have also used a one-site representation with a pair potential. Figure 3a depicts the resulting pair potential obtained with the RE and FM methods. The RE and FM potentials have a very similar structure with two minima, though the actual values of the potential are different. Figure 3b shows that the pair correlation function derived by CG simulations with the RE potential and the target one (from atomistic simulations) are very close. That is, the RE potential can reproduce with sufficient accuracy the pair correlation. Polyethylene Melt The model system consists of 96 polyethylene chains of 99 monomer units (−CH 2 − ), i.e., N = 9504. The simulations were performed under NVT conditions at temperature T = 450 K. For the coarse-grained representation we consider a 3:1 mapping representation, i.e., three monomer units form one CG particle. With this application we study the effect of the size of the available observations (system configurations), and quantify uncertainties due to the small number of observations. Figure 4 depicts the derived FM potential for a large set of observations. In addition, shows the 95% confidence set obtained with a statistical analysis resampling technique (bootstrap method) of a small observations set, which captures the large-set outcome. Conclusions In the current work we presented a short review of the information theoretic variational inference method for coarse-graining molecular systems, for systems at-and out-of-equilibrium. Moreover, we presented the connection to the Force Matching method and its relation to the structural based methods. The application of all methods to the methane system shows that the RE and IBI methods give similar results while the FM differs slightly. While for the water model the RE and FM resulting potentials differ substantially, which is not surprising as we know that the two methods are equivalent only asymptotically. We verify the validity of the PSFM, i.e., deriving the piar potential using time-series data, since it produces the same results to the FM, i.e., with identically distributed data. Finally, with the application to the polyethylene system, we show that when the availability of observations is limited the bootstrapping method can provide reliable confidence intervals to the pair potential.	2020-01-09T09:17:41.853Z	2019-11-22T00:00:00.000	{ "year": 2019, "sha1": "5622e4123d75ba64c1f1161c178911b8cf63c6de", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2504-3900/46/1/27/pdf", "oa_status": "GOLD", "pdf_src": "ScienceParsePlus", "pdf_hash": "e59e90088f42640ecce22fc84de430ee970d6b96", "s2fieldsofstudy": [ "Chemistry" ], "extfieldsofstudy": [ "Mathematics" ] }
23232318	pes2o/s2orc	v3-fos-license	Counterion correlations and attraction between like-charged macromolecules A simple model is presented for the appearance of attraction between two like charged polyions inside a polyelectrolyte solution. The polyions are modeled as rigid cylinders in a continuum dielectric solvent. The strong electrostatic interactions between the polyions and the counterions results in counterion condensation. If the two polyions are sufficiently close to each other their layers of condensed counterions can become correlated resulting in attraction between the macromolecules. To explore the counterion induced attraction we calculate the correlation functions for the condensed counterions. It is found that the correlations are of very short range. For the parameters specific to the double stranded DNA, the correlations and the attraction appear only when the surface-to-surface separation is less than 7 /AA. I. INTRODUCTION In the last few years a new phenomenon has attracted attention of the community of soft condensed matter physicists -appearance of attraction between like charged macromolecules in solutions containing multivalent ions. The problem is particularly fascinating because it contradicts our well established intuition that like charged entities should repel [1][2][3]. The fundamental point, however, is that the electrolyte solutions are intrinsically complex systems for which many body interactions play a fundamental role. The attraction between like charged macromolecules is important for many biological systems. One particularly striking example is provided by the condensation of DNA by multivalent ions such as Mn 2+ , Cd 2+ and various polyamines [4][5][6]. This condensation provides an answer to the long standing puzzle of how a highly charged macromolecule, such as the DNA, can be confined to a small volume of viral head or nuclear zone in procaryotic cell. Evidently, the multivalent ions serve as a glue which keeps the otherwise repelling like-charged monomers in close proximity [7]. In eukaryotic cells, the cytosol is traversed by a network of microtubules and microfilaments -rigid chains of highly charged protein (F-actin)which in spite of large negative charge agglomerate to form filaments of cytoskeleton [8]. The actin fibers are also an important part of the muscle tissue, providing a rail track for the motion of molecular motor myosin. Although the nature of attraction between like charged macromolecules is still not fully understood, it seems clear that the attractive force is mediated by the multivalent counterions [9][10][11][12][13][14][15]. A strong electrostatic attraction between the polyions and the oppositely charged multiva-lent counterions produces a sheath of counterions around each macromolecule. The condensed counterions can become highly correlated resulting in an overall attraction. It is important to note that the complex formed by a polyion and its associated counterions does not need to be neutral for the attraction to arise. Under some conditions the correlation induced attraction can overcome the monopolar repulsion coming from the net charge of the complexes. Recently a simple model was presented to account for the attraction between two lines of charges [16][17][18]. Each line had Z discrete uniformly spaced monomers of charge −q, and n ≤ Z/α condensed counterions of charge αq free to move along the rod. The net charge of such a polyioncounterion complex is Q eff = −(Z − αn)q ≤ 0. Nevertheless, it was found that if n > Z/2α and α ≥ 2, at sufficiently short distances, the two like-charged rods would attract [17]. It was argued that the attraction resulted from the correlations between the condensed counterions and reached maximum at zero temperature. If n < Z/2α the force was always found to be repulsive. Clearly, a one dimensional line of charge is a dramatic oversimplification of the physical reality. If we are interested in studying the correlation induced forces between real macromolecules their finite radius must be taken into account [19,20,22]. Thus, a much more realistic model of a polyion is a cylinder with a uniformly charged backbone [19,20] or with an intrinsic charge pattern [21,22] as, e.g., the helix structure of DNA molecule. Furthermore, the condensed counterions do not move along the line, but on the surface of the cylinder. Unfortunately, these extended models are much harder to study analytically. In this paper we explore the effects of finite polyion di-1 ameter on the electrostatic interactions between the two polyions using Monte Carlo simulations. We find that the finite diameter and the associated angular degrees of freedom of condensed counterions significantly modify the nature of attraction. Thus, although there is still a minimum charge which must be neutralized by the counterions in order for the attraction to appear, this fraction is no longer equal to 50% as was the case for the line of charge model. We find that the critical fraction depends on the valence of counterions and is less than 50% for α ≥ 2. For monovalent counterions no attraction is found. The crystalline structure of the condensed counterions, as first suggested by simulations of Gronbech-Jensen et al. [19] and Refs. [12,16,18,20], is also not very obvious. In particular we find very similar distributions of condensed counterions in the regime of attractive and repulsive interactions. The structure of this paper is as follows. The model and the method of calculation are described in section II. In section III, we present the results of the simulations. The conclusions are summarized in section IV. A. The model The DNA model considered here is an extension of the one proposed earlier by Arenzon, Stilck and Levin [16,17]. A similar model has been recently discussed by Solis and Olvera de la Cruz [20]. The polyions are treated as parallel rigid cylinders of radius R and Z ionized groups, each of charge −q, uniformly spaced -with separation b -along the principle axis, Fig. 1. Besides the fixed monomers, each polyion has n ≤ Z/α condensed counterions with valence α and charge αq, which are constrained to move on the surface of the cylinder. To locate a condensed counterion it is necessary to provide its longitudinal position, z (0 ≤ z < Z), and the transversal angle, θ (0 ≤ θ ≤ 2π). To simplify the calculations, the angular and the longitudinal degrees of freedom are discretized, see Fig. 1. The surface of the cylinder is subdivided into Z parallel rings with a charged monomer at the center of each ring. Each ring has m sites available to the condensed counterions, see Figs. 1 and 2. The hardcore repulsion between the particles requires that a site is occupied by at most one condensed counterion. The two polyions are parallel, with the intermolecular space treated as a uniform medium of dielectric constant ǫ. We introduce occupation variables n k i for the two polyions, so that i = 1, 2, . . . , Z(m + 1) and k = 1, 2. Thus, n k i = 1 if the i'th site of the k'th polyion is occupied by a particle of valence α i = {−1, α} (negative core charge or counterion of valence α, respectively), otherwise n k i = 0. Note that the core charge is always "occupied", while the counterions are free to move between the Zm ring sites of each polyion. The Hamiltonian for the interaction between the two polyions is, with the i = j. All lengths are measured in units of b (for DNA, b = 1.7Å). The dimensionless quantity, ξ = βq 2 /bǫ, is the Manning parameter, which for DNA is ξ = 4.17. The partition function is obtained by tracing over all the possible values of n k i consistent with the constraint of fixed number of counterions per polyion, Clearly, this is a very crude model of the interaction between two macromolecules in a polyelectrolyte solution. 2 The molecular nature of the solvent is ignored. Also the number of condensed counterions is fixed instead of being dependent on the separation between the particles. Nevertheless, we believe that this simple model can provide some useful insights for the mechanism of attraction in real polyelectrolyte solutions. B. The observables We are interested in statistical averages of observables such as the energy and the force between the two polyions. Furthermore, to understand the nature of the interaction between the two macromolecules it is essential to study the correlations between the condensed counterions on the two polyions. The force is obtained from the partition function Eq. (2), bβF = −∇(ln Q). From symmetry, only the y-component is different from zero. For finite macromolecules the symmetry between the two polyions can not be broken [18]. Hence it is impossible to produce a true crystalline order in a finite system at non-zero temperature. Since within our simplified model the two polyions have exactly the same number of condensed counterions, the average angular counterion distribution n k i (z, θ k i ) must be symmetric with respect to the mid-plane y = d/2, see Fig. 1. The angle θ k i labels the site i on polyion k, see Fig. 2. Thus, n 2 3 (z, θ 2 3 ) denotes the occupation variable for the site 3 on the ring z located on polyion 2, with an angle of 3(2π/m). Indeed, Fig. 3 shows that the density profiles are completely symmetric (up to fluctuations). In spite of this symmetry it is possible for the counterions on the two polyions to become highly correlated. Clearly, the strength of these correlations will depend on the product ξα 2 and the separation between the two macromolecules. Considering Fig. 2, it is evident that if the site 2 on the first polyion is occupied, the likelihood of occupation of the site 8 on the second polyion will be reduced. To explore the nature of electrostatic correlations, we define a counterion-hole correlation function between the adjacent rings on the two polyions, Here · · · denotes the ensemble average. This function should be non-zero when sites on the two polyions are correlated, that is if one is occupied by a condensed counterion there is an increased probability of the second being empty. C. Simulations To calculate the force between the two polyions, we have performed a standard Monte-Carlo (MC) simulation with the usual Metropolis algorithm [23]. First, one counterion on polyion 1 is randomly chosen and displaced to a vacant position on the same polyion. This move is accepted or rejected according to the standard detailed balance criterion [23]. We do not permit exchange of particles between the polyions. Next, the same is done for polyion 2. In one Monte-Carlo step (MCS) all 2n condensed counterions on the two polyions are permitted to attempt a move. The long-ranged nature of the Coulomb interaction requires evaluation of all the pair interactions in Eq. (1) at every MCS. Due to the limited computational power available to us, we have confined our attention to relatively small systems with Z < 100 and m = 10. We have checked, however, that for m = 10 the force has already reached the continuum limit and did not vary further with increase of m. Also we note that the "thermodynamic limit" is reached reasonably quickly, so that there is a good collapse of data already for Z > 50, see Fig. 4. 2000 MCS served to equilibrate the system, after which 500 samples were used to calculate the basic observables, namely, the mean force and energy. To obtain the correlation functions, 5000 samples were used with 5000 MCS for equilibration. III. RESULTS AND DISCUSSION The simulations were performed for ξ = 4.17 and R/b = 8.2, relevant for DNA. For monovalent counterions the simulation results indicate that the force is purely repulsive. This is in complete agreement with the experiments [1], which do not find any indication of DNA condensation for monovalent counterions. 3 For divalent counterions the force between the two complexes can becomes negative, indicating appearance of an effective attraction, Fig. 4. The range of attraction is larger than was found for the one dimensional line of charge model, Ref. [17]. Within the Manning theory [24] 88% of the DNA's charge is neutralized by the divalent counterions. However, there are indications that even a larger fraction of DNA's charge can become neutralized by the multivalent ions if the counterion correlations are taken into account [14]. In this case the interaction is purely attractive, with the range of about d/b ≈ 20 or 34Å (7Å surface-to-surface), Fig. 4. A minimum number of condensed counterions is necessary for attraction to appear. In Fig. 5 we present the surface-to-surface separation, (d 0 − 2R), below which the force between the two complexes becomes negative (attractive), as a function of the number of multivalent counterions. For the case of DNA with divalent counterions α = 2, the attraction appears only if 40% of the core charge is neutralized. For α = 3 this fraction decrease to 30%. Furthermore, decrease in the value of the Manning parameter, ξ, increases the minimum number of condensed counterions necessary for the attraction to appear. This is fully consistent with the fact that the attraction is mediated by the correlations between the condensed counterions. Since raise in temperature tends to disorganize the system, the state of highest correlation between the condensed counterions corresponds to T = 0 or ξ = ∞. The surface-to-surface distance at which the attraction first appears tends to zero as the number of condensed counterions is diminished. We find d 0 − 2R ∼ (µ − µ c (α)) ν , where the average counterion concentration is µ = n/Z and the critical fraction µ c depends on the valence of condensed counterions α. From Fig. 5 it is evident that ν = 1. This should be contrasted with the line of charge model Ref. [17], for which ν = 1/3. Fig. 4 with n = 7 and the separation between the two macromolecules is d/b = 32.8. The graph shows that at this distance there are almost no correlations between the condensed counterions. From Fig. 4 we also see that the effective force is repulsive. In Fig. 6 we show two snapshots of the characteristic equilibrium configurations for (a) d/b = 32.8 and (b) d/b = 16.8. Looking at this figures it is difficult to see something that would distinguishes between them, both appear about the same. There is no obvious crystallization or transversal polarization suggested in previous studies [19,20]. Yet, the case (a) corresponds to the repulsive, while the case (b) corresponds to the attractive interaction between the polyions. To further explore this point, in Figs. 7 and 8 we present the site-site correlation function, Eq. (3), for macromolecules with Z = 20 and n = 7. For d/b = 32.8 the surface-to-surface distance between the two polyions is sufficiently large for their condensed counterions to be practically uncorrelated, Fig. 7. On the other hand, for d/b = 16.65 strong correlations between the condensed counterions are evident, Fig. 8. The Fig. 8 shows that the sites two and three on the first polyion are strongly correlated with the sites seven and eight on the second polyion, respectively. It is these correlations between the adjacent sites on the two polyions which are responsible to the appearance of attraction between the two macromolecules when they are approximated, Fig. 4. IV. SUMMARY We have presented a simple model for polyion-polyion attraction inside a polyelectrolyte solution. It is clear from our calculations that the attraction results from the correlations between the condensed counterions and reaches maximum for T = 0. The thermal fluctuations tend to diminish the correlations, decreasing the amplitude of the attractive force. Consistent with the experimental evidence, the attraction exists only in the presence of multivalent counterions. Our simulations demonstrate that a critical number of condensed counterions is necessary for the appearance of attraction. The fraction of bare charge that must be neutralized for the attraction to arise depends on the valence of counterions. The larger the valence, the smaller the fraction of the bare polyion charger that must be neutralized for the attraction to appear. This result should be contrasted with the line of charge model [17] for which the critical fraction was found to be equal to 50%, independent of the counterion charge. 5	2018-04-03T01:20:59.490Z	2000-11-17T00:00:00.000	{ "year": 2000, "sha1": "e837c66b1a6b83b73ea8ae973bbd2483204e3cc5", "oa_license": "CCBYNCSA", "oa_url": "https://lume.ufrgs.br/bitstream/10183/103715/1/000308514.pdf", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "e837c66b1a6b83b73ea8ae973bbd2483204e3cc5", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Chemistry", "Physics", "Medicine" ] }
960125	pes2o/s2orc	v3-fos-license	A Cloud-Based X73 Ubiquitous Mobile Healthcare System: Design and Implementation Based on the user-centric paradigm for next generation networks, this paper describes a ubiquitous mobile healthcare (uHealth) system based on the ISO/IEEE 11073 personal health data (PHD) standards (X73) and cloud computing techniques. A number of design issues associated with the system implementation are outlined. The system includes a middleware on the user side, providing a plug-and-play environment for heterogeneous wireless sensors and mobile terminals utilizing different communication protocols and a distributed “big data” processing subsystem in the cloud. The design and implementation of this system are envisaged as an efficient solution for the next generation of uHealth systems. Introduction As wireless medical sensors are becoming more popular and cloud computing technology is growing up, they provide the condition for the development of a body area sensor network (BASN) component and a cloud environment for the ubiquitous mobile healthcare (uHealth) systems. BASNs are considered to be an important means to relieve the pressure of insufficient medical resources in an aging era and are becoming a strategic direction of the uHealth research. At present, BASN are still at an early developing stage and facing a series of challenges, such as the existence of heterogeneous sensor protocols and "big data" computing and mining. BASN is defined as a wireless network, which is formed by sensors located on, and/or biosensors transplanted into, the human body [1,2] and a data collector (Sink) used for medical data collection in real time. BASN can gather medical data, perform classified learning, and analyze data in real time, thus realizing an early medical warning [3,4]. Being a network type of huge significance and demand, BASNs become a strategic direction of the uHealth research. Researchers show that, at present, there are 1 billion people worldwide with underweight and at least 0.3 billion people with a morbid obesity. By 2015, the number of subhealth world population will reach 1.5 billion, and by 2020, the expense on chronic diseases will reach 10 thousand billion dollars [5]. By 2025, the aging population will reach 1.2 billion and this is a giant challenge to the provision of sufficient medical resources. Aiming at these global problems, changing the way of providing medical services becomes quite important. As long as the unnecessary links in the medical service system are wiped off, current technology can improve the health condition and life quality of the mankind, while at the same time medical resources will be utilized fully and in an optimal way [6]. BASN can solve the problem of shortage of both material and manpower resources in the medical sector effectively by classifying, learning, analyzing, and storing the patients' medical data and by providing the patients with early medical warnings, as a supplement to the traditional medical systems. The uHealth systems are based on the Internet of Things concept [7], whereby the medical treatment emphasizes the object management. This change of the concept urges the mobile medical technology to adapt. "Things" in the uHealth systems include doctors, patients, medical devices, and sensors. "Join" refers to all things being appreciable, interactive, and controllable. "Network" is the working flow of medical treatment and health management. The uHealth systems are taking shape to a brand-new ecosphere. At this "big data" era, researchers show that the data generated by sensors will reach petabyte (PB) level annually [8], if electronic health recording is set up only for 0.1 billion people. The medical cloud provided by uHealth systems can reduce the cost of medical informatization by providing a foundation for storing and mining of "big data" with high availability, expansibility, and performance of the data storage system; a "big data" management and processing platform which adapts to different needs; a data cleaning and loading mechanism; a real-time data search and complex data analysis package, and so forth. All of these can be efficiently used for disease prevention, health condition monitoring, and timely remedy, as a strategy and a core content of the future uHealth development. Related Research At present, in both industry and academia, a lot of significant work is done in this area. For example, in the CodeBlue medical care project, conducted by the U.S. Harvard Sensor Networks Lab [9], an ad hoc sensor network infrastructure was proposed and developed for emergency medical care, by providing a flexible naming and discovery algorithms to ensure seamless transfer of data in the system. The MiThril NGN wearable research platform, developed by MIT Media Lab [10], is a next generation context-aware system providing human-computer interaction with body worn applications. The MyHeart telemedicine EU FP6 project [11], led by Philips, has developed an intelligent system for the prevention of cardiovascular diseases by periodic monitoring on the vital signs and establishing the health status in real time. Current research in the BASN area is mainly going in two directions-power saving and communication protocols. In [12], an ultralow-power wireless sensor network solution was proposed. The authors stated that the design of a low-power sensor is very important as the battery lifetime is expected to increase only by 20% in the next ten years. Medical sensors were not mentioned, but from research it is clear that reducing the power consumption is one of the main goals when designing wireless healthcare middleware. In [13], Intel provided an evaluation software kit for the development of embedded devices, which are used to interact with medical devices. It is based on the Windows Mobile operating system, and the devices must be certified by Continua. Libresoft (http://openhealth.libresoft.es/) reported on an open-source ISO/IEEE 11703 Personal Health Data (hereafter it is called X73 [14]) system with its own Bluetooth application programming interface (API). The authors stated that it is not easy to develop a uHealth system operating in a cloud environment. In [15], a uHealth system based on the X73 standards was proposed, but the system seems as not being able to provide a fully ubiquitous solution. Although the literature reports on a number of valuable results, BASNs are still at an early developing stage. There are many important issues which need to be solved and researched further. As the low-powered processing chips, smart mobile terminals, cloud computing, and NGN develop rapidly, there is a significant opportunity for healthcare system development. Researching on BASN systems upon cloud computing is of important significance to medical informatization. This paper focuses on the development of a cloud-based X73 uHealth system, consisting of a healthcare middleware in the BASN domain and a distributed data processing system in the cloud. Figure 1 depicts the high-level view of a common BASNbased uHealth system. Operating as part of the system, sensor Figure 2: The X73 uHealth system architecture. nodes obtain and send medical data to a Sink node. After some aggregation in the Sink node, the data is pushed to a medical server for further processing and mining. The main problem in the BASN domain is related to sensors compatibility. Different vendors may implement different communication protocols into their medical sensors, such as ISO, IEEE, and HL7, each one with its own communication mechanism, data format, and so forth. To solve this problem, the IEEE approved the X73 standard [16], which provides an efficient real-time exchange method for plug-and-play operation. Due to the complexity of the X73 protocols, in the industry, currently only Continua Health Alliance (http://www.continuaalliance.org/) produces a hardware which is fully compatible with this standard. In the academia, Korean scholars have reported that a sensor could be adapted to the X73 protocols by means of an extra hardware [17], but this will increase the cost of the sensor. Developing of software middleware as an adapter is proposed in this paper. As cloud computing offers a low-cost solution to "big data" storage and processing, a kind of healthcare system enterprise solutions upon cloud computing emerged recently, for example, proposed by IBM, Microsoft, Cisco, and so forth. Most of them, however, are only concerned with the infrastructure, that is, the informatization of hospitals, medical treatment clouds, and so forth. Only few companies are doing research on the BASN-oriented cloud. In the academia, Laleci et al. have proposed an electronic health record system based on cloud computing [18]. Sobhy et al. have put forward the theory of MedCloud [19], but did not propose the application of BASN. Even though these are valuable examples in the area of medical data collection and storage based on cloud computing, BASN, especially the data acquisition technology upon X73 BASN, was not involved in these systems. The problem of processing a massive amount of medical data with higher throughput and lower delay needs to be studied more extensively. From the view of applications, BASN is with wide prospect. As the aging society is short of medical resources, BASN could offer an effective solution to this problem, which will surely be the core one in the development of future uHealth systems. Further fusion with IoT will lay down the application direction of BASN in the future. For example, BASN-based system can help monitor the eruption of epidemic situation, mass outbreak disease, or common disease in the community, and as such can provide prevention and/or early warning on emergency events. BASN will continue being developed toward smart, micro, and wearable orientation. In the meantime, the establishment of large-scale BASNbased health monitoring platforms utilizing ubiquitous wireless communication techniques is envisaged to take place. With the X73 standard and cloud computing technology, this paper reports the development of a distributed uHealth system for medical data collection, storage, analysis, and mining with high performance and high reliability. In the Medical Sensors tier, a number of wireless medical sensors (S)-such as pulse oximeter, heart rate monitor, blood pressure monitor, thermometer, weighting scale, and glucose meter-operate in a plug-and-play manner. They use Bluetooth or ZigBee for communication with the ISO/IEEE 11073 based middleware (Sink) in the Personal Gateway tier. The mobile phone of the patient acts usually as such a gateway. A software agent, operating on the phone, periodically collects data from the sensors and sends a personalized message to a log data node and/or emergency system under the Always Best Connected and best Served (ABC&S) communication paradigm [20]. The message contains medical parameters' values, the user's location, medical device's information, IP address, and so forth. In the cloud, a relevant cloud ant collects all such messages by using the defined scheduling algorithms and generates medicalcare advices/instructions/statements which are sent back to patients. The medical-care contents are personalized and customized to best suit each mobile phone and patient. In the cloud, a set of data mining applications run for log data collecting, analyzing, processing, and advising. Different patients may receive different medical-care advices and statements based on their personal profiles and current medical condition. Also, the cloud provides medical experts' and doctors' manual diagnosis when needed. The X73 uHealth system, described in this paper, proposes a brand new environment based on an X73 PHD BASN and a cloud architecture. BASN. The developed X73 healthcare middleware supports the domain information model (DIM), medical device encoding rules (MDER) and decoding library, and the communication model (CM). DIM defines details of the health device object, medical device system (MDS), and metrics. MDER expresses DIM objects by means of the abstract syntax notation-one (ASN.1) specifications [21]. The service model (SM) defines the object access methods and event reporting. CM states the finite state machine (FSM) for the ISO/IEEE 11073 agent (sensor node) and the ISO/IEEE 11073 manager (middleware). A rule engine is used for separating the business logic from the middleware. A gateway agent acts as a bridge between the personal mobile server and the rest of the uHealth system. The middleware uses a multiagent technology and a sensor adapter software to realize protocol conversion for non-X73 sensors. The middleware architecture of the sensor node is depicted in Figure 3. The communication support layer is at the bottom of this architecture. It is provided by the operating system of the user's mobile phone. The middle layer includes the ISO/IEEE 11073 PHD device specializations. The DIM, SM, CM, rule engine, and gateway agent operate at the top layer. Cloud Architecture. Based on the cloud computing technology, the designed and developed X73 cloud architecture includes an efficient distributed message system used for medical data collection, a fault-tolerant real-time computing system for data storage, and a Hive system for data mining. The distributed framework Apache Hadoop is suitable for the design of the X73 uHealth cloud as it provides an efficient Hadoop Distributed File System (HDFS) [22] for data storing and a Hadoop MapReduce [23] for "big data" processing. In order to increase the performance of the cloud platform, this paper introduced a set of components for efficient collection, storage, and analysis of "big data" describing the user's medical condition. As regards the medical data collection and storage, the traditional data storage way of the O (lg n) B + tree algorithm is not suitable for this system due to its low efficiency. A Publish/Subscribe based distributed message system, supporting two methods of synchronous collecting and asynchronous subscription, is proposed in this paper. As regards medical data storage and analysis, the high fault-tolerance HDFS provides possibility to store data on a low-cost computer with high aggregate bandwidth across the Hadoop cluster. The Facebook's Hive [24], acting as data warehouse, provides data summarization, query, and analysis in the Hadoop distributed environment. Based on the Hadoop ecosystem, the X73 uHealth cloud application architecture is shown in Figure 4 System Design 4.1. Middleware of X73 uHealth Sensor Node. The X73 standard defines the concept of Agent and Manager. The Agent represents the personal healthcare equipment (i.e., sensors), whereas the Manager represents the smart terminal equipment (i.e., the Sink in our system). The X73 healthcare middleware is mainly concerned with the communication interface and data exchange between the sensors and the Sink node. In the process of data transmission, an X73 software adapter is needed for the conversion of data formats, The Scientific World Journal HFile · · · · · · · · · · · · · · · · · · exchange protocols, and interface protocols. The X73 protocol stack of a sensor node is shown in Figure 5. Domain Information Model (DIM). In the X73 standard, the Agent is expressed as a set of objects. Each object has one or more attributes. Attributes are used to describe medical data sent to the Manager by the Agent and to represent the Agent's various states, behaviors, and so forth. In addition to the attributes, the objects can also have a series of methods, such as GET and SET. The Manager communicates with Agents mainly through these methods. In addition, the Agent can produce a series of Events. When data in the Agent changes, it can invoke the corresponding Event to send the new data. Service Model (SM) . SM defines a flexible and efficient way for an Agent to send configuration information to the Manager. This way the Manager can create an Agent object. The Agent can report standard or nonstandard configuration information, which can be identified by the Manager during the state of association. In the process of configuration and information processing, the Manager will ask the Agent to describe all objects. Depending on the requirements of different applications, the Agent may vary from a very simple to a very complicated one. The Manager can save all the objects of an Agent through the Map object. This way it provides a plug-and-play functionality for the Agent. SM also defines the protocol used by an Agent to send measurement data to the Manager. Communication Model (CM) . CM defines the topological structure to enable one or more Agents to connect to a single Manager in a point-to-point (P2P) fashion. The IEEE 11073 standard is independent of the transport-layer protocol and assumes that an Agent and the Manager establish a connection by means of the Bluetooth or ZigBee standards. For each P2P connection, the internal system is defined by a Finite State Machine (FSM). The FSM defines the state and substate of the interaction between an Agent and the Manager, for example, the state of connection, association, and operation. CM also defines the entry, exit, and error information that may occur during the process of data transmission. Taking into account that Java provides an efficient and hardware-independent platform for building both enterprise applications and portable-devices applications [25], it was selected for the implementation of the X73 protocol stack in order to provide system uniformity, distribution, and portability. The main modules include an ASN.1 codec library, Medical Device Encoding Rules (MDER), a message sequence mechanism, and FSM. In addition to the Adapter used for non-X73 sensors, a Gateway Agent sensor adapter was implemented by using the rules engine, which supplements the X73 uHealth system with a distributed processing ability and personalized features through embedding a lightweight Java-based Multiagent System (MAS). uHealth Data Processing Subsystem Based on Cloud Architecture. In order to process the high volume of medical message flows in the cloud, an efficient storage strategy has been developed and implemented with a O(1) disk storage cost. The proposed cloud architecture consists of a distributed message module, a distributed real-time computing module, and a distributed log collection module, which are implemented based on Kafka [26], Storm [27], FlumeNG [28], and Hadoop [22]. Figure 6 shows the architecture of the distributed data processing subsystem in the X73 uHealth system. Message Queues Module. In the X73 uHealth system, it is necessary to develop a high-throughput distributed message queues module to satisfy the high volume of user's requests. Kafka [26] is an open-source software, which improves the processing performance and extensibility by lowering the complexity of the message queue system and thus meets the requirements of the X73 uHealth system. Kafka uses ZooKeeper [29] to coordinate and manage the relationship between the producer, broker, and consumer. Besides, Kafka implements an automatic load balancing technology based on the Zookeeper. Real-Time Computing Module. In the X73 uHealth system, an efficient computation of a complex medical data is often needed in a timely manner. For instance, the system should be able to process messages in the range of one million per second. Storm [27] can meet this demand. It is an open-source, distributed, and fault-tolerant real-time computing system. Storm receives real-time medical data from the message queue, compares it with various predefined health parameter thresholds, and monitors the patient's state of health in real time. When one of the medical parameter values continues to deviate abnormally, the system can intelligently send a health-warning information to the patient and concerned family members for the purposes of disease prevention and illness treatment in real time. Log Collection Module. In order to minimize the potentially large amount of log data in the X73 uHealth system, a robust and reliable fault-tolerant distributed log collection module is required. FlumeNG [28] is a reliable open-source, log collection system, which can satisfy this system requirement. FlumeNG serves as a link between the distributed data collecting component and Hadoop data center in this system. Hadoop Data Center. The X73 uHealth Hadoop cluster includes one master node and a number of slave nodes [30]. The master node consists of a Name Node and a JobTracker; the slave node consists of a Data Node and a TaskTracker. Figure 7 shows the structure of the X73 uHealth Hadoop cluster. X73 uHealth Sensor Node Implementation. As explained before, the X73 standard is mainly composed of three parts-DIM, SM, and CM. The following subsections provide implementation details of each of these. Domain Information Model (DIM). DIM defines the object model of the X73 standard. In our implementation, DIM is an abstract class, which includes MDS, Metric, PM Store, PM Segment, and Scanner subclasses. Figure 8 shows the UML diagram of DIM. MDS represents specific medical equipment. For instance, its subclass MDS 10404 represents a Pulse-oximeter. One MDS uniquely identifies an Agent in the entire system. The Agent provides information to the Manager by the MDS internal attributes. MDS contains some Metrics, Persistent Metric Store (PM Store), and other objects. The Metric class is the base class which is used to represent all the measured values, Agent status, and contextual data object. PM Store is the data collected by the Agent, which is composed of metadata objects and PM-Segment objects. Persistent Metric Segment (PM Segment) contains metadata and zero or more entity objects. Each entity object consists of one or more measurement elements. The Scanner observes the measurement data, encapsulates it to an event, and sends it to the Manager. Service Model (SM) . SM is used to exchange data between an Agent and the Manager. Message exchange and command execution mechanisms are provided, that is, the connecting and disconnecting messages, behavior message, data message, event, and so forth. Communication Model (CM) . CM defines the communication between an Agent and the Manager, which is an important part in the X73 system. A FSM is used to describe the state transitions of the Agent and Manager (Figure 9). Another role of CM is to change the DIM's abstract data model to transfer syntax. With the Medical Device Encoding Rules (MDER), mapping of DIM Java objects to an ASN.1 binary format (or vice-versa) is achieved. The state class is the parent class of the Disconnected and Connected states. When an Agent is initiated, it is in Disconnected state. This state identifies that the connection between the Agent and the Manager has not been established yet. After connecting, the Agent will receive a Transport Connection Indication from the transport layer and the state changes to Connected. As long as there is an established transport connection, the Agent would be in Connected state all the time. This state includes four subclasses-Associating, Disassociating, Unassociated, and Associated. When the Agent wants to release the current association, it would send an Association Release Request to the Manager, and then will enter the Disassociating state. In the case of timeout, the Agent will send an Abort Request to the Manager, and then will enter Unassociated state. If operating normally, the Agent would always be in Associated state, which includes two subclasses-Operating and Configuring. If the Manager recognizes the configuration information sent by the Agent, it will send an Accept Response back to the Agent. Then the Agent enters the Operating state. When the Manager cannot identify the configuration information, it will send back an Accepted-Unknown-Config Response, and the Agent will go to the Configuring state. The relationship between DIM, SM, and CM is depicted in Figure 10. [32] is an open-source Java rule engine platform developed by JBOSS (http:// www.jboss.org/overview/) and enhanced by im-plementing the Rete algorithm for object-oriented systems with the forward chaining excitation method. It is well suited to act as a rule engine API in this middleware. Figure 11 shows the Drools operation in the X73 Sink node. With this interface The Scientific World Journal design pattern, when a new rule is introduced in the system, the latter does not need to be recompiled. This ensures loose-coupling of the system. Gateway Agent. The Java Agent Development framework (JADE) [33]-a FIPA specification compatible open- source software framework-is adopted to implement the X73 uHealth system as a MAS with a distributed ability. An agent platform is running on the log data node (c.f. Figure 2). It provides an agent management system (AMS), a directory facilitator (DF), and message transport services. The gateway agent operates in mediator mode, that is, the JADE container is split into a front-end (running on the mobile personal server) and a back-end (running on the log data node). Figure 12 shows the JADE architecture of the X73 sink node. BASN Data Processing Subsystem Implementation Based on Cloud Architecture. The main goal of using a distributed message queue in this architecture is to free up the web server from the analytical data processing and objects creating, which are both time-and resource consuming. The traditional Message Queue system is mainly a mutual integration among enterprise applications, but the amount of data produced is not particularly big. In the system described here, the Message Queue system consumes the "big data" sent by (Android) clients in a timely fashion, in order to achieve very high performance and throughput. Kafka and FlumeNG are the main components of the system. Kafka. Kafka performs a Buffer role between the web server and FlumeNG. The Message Producer is written in Java and deployed on a Tomcat server. The Producer processes data in an asynchronous nonblocking way. After receiving data from the Android client, the Producer would first cache it in the memory, and then when the triggering moment occurs or the number of messages reaches the preconfigured threshold, it would send all messages in a batch. The adoption of the asynchronous processing and the batch sending mechanism can greatly improve the Kafka system's throughput and network utilization. Figure 13 illustrates the use of Kafka in the X73 uHealth system. FlumeNG. To guarantee reliability and fault tolerance of the whole system, the FlumeNG is built with a FailOver architecture. Every Kafka consumer runs a flumeMainClient Agent on a machine; at the same time another flumeBackup-Client Agent runs on a backup machine. When flumeMain-Client Agent fails, the client's data stream can be redirected to flumeBackupClient Agent transparently. The data collected from the FlumeMainClient or flumeBackupClient would be sent to the remote flumeMainCollector Agent. Similarly to the flumeMainClient Agent, the flumeMainCollector Agent also has a flumeBackupCollector Agent, which runs on a backup machine. In case of abnormal operation of the flumeMainCollector program, the flumeMainClient's or flumeBackupClient's data flow would also be automatically redirected to the flumeBackupCollector Agent. In addition, each Channel in the Agent is implemented with a FileChannel, so as to ensure that a message lost will not happen when the Agent operates abnormally. Figure 14 shows the use of FlumeNG in the X73 uHealth system. The UML diagram of the X73 uHealth cloud architecture is shown in Figure 15. The operation of the HBase is encapsulated into a HealthDao Java object, which provides APIs for the whole system thus making the HBase easy to use. In the Kafka part, the healthSpout is the key object. The consumer is initialed by this object with predefined properties. The output of Kafka is directed to Storm for real-time computing and to FlumeNG for saving. In the Storm part, a HealthTopology uses Map/Reduce to operate on the Bolt object and saves the results on the HBase database. X73 uHealth-Based Application. On the client side, various medical sensor nodes (such as pulse oximeter, blood pressure monitor, weighing scale, glucose meter, activity hub, etc.) send data to an intelligent terminal equipment, such as a mobile phone, a set-top box, and so forth, which provides an IEEE 11073 software adapter service. The gathered data is then transmitted to a remote cloud computing center. On the cloud side, the uHealth service provider may provide APIs for corresponding data mining, that is, searching for all kinds of potential medical threats or diseases and sending analysis information to doctors, community service staff, health Customer service layer Figure 16: The architecture of the X73 uHealth application. Figure 17: The X73 uHealth system testbed prototype. and fitness service system, and so forth. The X73 uHealth based application consists of five layers ( Figure 16). The designed ISO/IEEE 11073 based healthcare middleware was tested on an Android mobile phone. A pulse oximeter was used as a medical sensor device ( Figure 17). For communication with the middleware, an X73 adapter was used to ensure compatibility. In the future, a pure HTML5compatible UI application will be developed to replace the Android application. Performance Evaluation of X73 uHealth Cloud. To evaluate the performance of the X73 uHealth cloud component, a client generation of data was simulated and the results were collected through a script. The whole setup included two Kafka distributed message queue servers, two FlumeNG Agent servers, three Storm clusters composed of six servers, and a Hadoop cluster composed of six servers. Each server was installed on an Intel XEON PC with E5620 CPU and 8 GB memory. The message size was set to 300 bytes. Test 1 shows that when the number of concurrent threads grows from 10 to 75, the throughput of the system is increasing. The maximum throughput reached is 165800 messages Number of threads Number of processed messages per second per second. However, for the number of threads more than 75, the system throughput gradually decreases to 103240 messages per second (with 200 threads). In test 2, the number of Kafka Brokers and Storm Supervisors is two times that of test 1. The average system throughput reached is about twice the throughput of test 1, and the throughput slightly fluctuates with increasing the number of threads. Compared with test 2, test 3 only uses one extra Storm Supervisor. The increase of the system throughput is not significant, but the fluctuation of the throughput becomes very obvious with increasing the number of threads. In test 4, the number of Kafka Brokers is increased by one, compared to test 3. When the number of threads increases from 10 to 100, the system throughput shows an increasing trend. The throughput reaches a maximum of 334700 messages per second when the number of threads is 100. However, as the number of threads continues to increase, the throughput sharply decreases. In test 5, the number of Kafka Brokers and Storm Supervisors is two times that of test 2. The system throughput, however, is not significantly increased compared with test 2, and the peak of throughput does not exceed that of test 4. The above test results clearly demonstrate that the number of Kafka Brokers and Storm Supervisors used in the X73 cloud will influence the performance of the system. The optimal number of Kafka Brokers and Storm Supervisors will depend on the size of the clusters and messages used, and as such can be found for each particular case. Conclusion The design of a cloud-based X73 uHealth system has been presented in this paper. The system consists of a "big data" producer part and a "big data" consumer part. In the producer part, a body sensor area network (BASN) produces a set of medical data for its corresponding patient. Each such network contains a number of small wireless medical sensors and a Sink node. To provide a plug-andplay environment for heterogeneous medical sensors which use different communication protocols, the system was developed based on the ISO/IEEE 11073 personal health data (PHD) standards. It works as a multiagent system (MAS) to provide intelligent collection of medical data from wireless sensors attached to the human body and subsequently to send the gathered data to a log data node based on the Always Best Connected and best Served (ABC&S) communication paradigm. In the consumer part, a cloud-based architecture consumes the "big data" generated by the BASNs. It includes a Kafka-based distributed message module for caching of data by providing a load balancer and a high-throughput mechanism. A Storm-based distributed real-time computing	2018-04-03T03:09:54.622Z	2014-03-10T00:00:00.000	{ "year": 2014, "sha1": "1ca2bdbc85e148b958aa9a787d6ebce10bcb80ab", "oa_license": "CCBY", "oa_url": "http://downloads.hindawi.com/journals/tswj/2014/145803.pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "53b11139cec8785ff3d5a0d179ea02785def3156", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Computer Science", "Medicine" ] }
7720488	pes2o/s2orc	v3-fos-license	Antioxidant activity and hepatoprotective potential of agaro-oligosaccharides in vitro and in vivo Background Agaro-oligosaccharides derived from red seaweed polysaccharide have been reported to possess antioxidant activity. In order to assess the live protective effects of agar-oligosaccharides, we did both in vitro and in vivo studies based on own-made agaro-oligosaccharides, and the structural information of this oligosaccharide was also determined. Method Structure of agaro-oligosaccharides prepared with acid hydrolysis on agar was confirmed by matrix-assisted ultraviolet laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and NMR. The antioxidant effect of agaro-oligosaccharides on intracellular reactive oxygen species (ROS) was assessed by 2', 7'-dichlorofluorescin diacetate. Carbon tetrachloride was used to induce liver injury, some index including SOD, GSH-Px, MDA, AST, ALT were examined to determine the hepatoprotective effect of agaro-oligosaccharides. Results Agaro-oligosaccharides we got were composed of odd polymerizations with molecular weights ranged from 500 to 2500. Results from intracellular test indicated that agaro-oligosaccharides could significantly scavenge the level of oxidants in the hepatocytes, more beneficially, also associated with the improvement of cell viability In vivo studies of the antioxidant effects on tissue peroxidative damage induced by carbon tetrachloride in rat model indicated that agaro-oligosaccharides could elevate the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and decrease the level of malondialdehyde (MDA), glutamate oxaloacetate transaminase (AST), glutamic pyruvic transaminase (ALT) significantly. At 400 mg/kg, MDA level reduced 44 % and 21 % in liver and heart, SOD and GSH-Px increased to highest in liver and serum, while ALT level decreased 22.16 % in serum. Conclusion Overall, the results of the present study indicate that agaro-oligosaccharides can exert their in vitro and in vivo hepatoprotective effect through scavenging oxidative damage induced by ROS. icteric hepatitis to necroinflammatory hepatitis, cirrhosis, and carcinoma have been proved to associate with the redox imbalance and OS (oxidative stress) [2]. Therefore, a potential novel approach, namely developing antioxidant drugs to treat and protect liver injury and liver disease, has been proposed [3]. This strategy is aimed to devise and incorporate antioxidants into the therapeutic for control of viral infections or protecting body from alcohol or other toxin damage. We think antioxidants are able to reduce hepatic inflammation and fibrosis, thus slowing or even preventing progression to cirrhosis. One of such candidates is agaro-oligosaccharides prepared from agar, which was chosen in the present study. Agar was easily extracted from red algae and widely be used as food and gelling agent with historic record of more than a thousand years in China and Japan. In recent years, agaro-oligosaccharides which derived from agarose have been widely investigated in structures and bioactivities [4][5][6][7][8]. Many beneficial health properties of agaro-oligosaccharides are attributed to their antioxidant activities. For example, agaro-oligosaccharides have been proved to possess antioxidative activities in scavenging hydroxyl free radical, scavenging superoxide anion radical and inhibiting lipid peroxidation in various chemical assays [9][10][11]. Enoki et al. [12] also reported that the agarobiose shows the ability to suppress the expression of iNOS (inducible nitric oxide synthase), an enzyme associated with the production of NO. In our previous work, we also discussed the indirect attenuate effect of agaro-oligosaccharides towards oxidation of human liver cells induced by antimycin A [13]. These reports exhibited the potential prospects of agaro-oligosaccharides as functional ingredient to prevent the ROS related diseases. However, no researches have been done about their antioxidant effect in the in vivo system. Therefore, in order to evaluate the ROS scavenging activity of agaro-oligosaccharides as well as possible liver injury protection from OS with the respects of degree of polymerization, we firstly prepared agaro-oligosaccharides with different degrees of polymerizations, then use the compounds to examine the in vitro and in vivo antioxidant effects depending on hepatocyte cellular assay of H 2 O 2 induced damage and experimental rat model of carbon tetrachloride (CCl 4 ) induced toxic hepatitis. Preparation of agaro-oligosaccharides Agaro-oliogsaccharides were prepared by acid hydrolysis. In order to evaluate the difference of DP of oligosaccharides on bioactivity, hydrolysis solution was fractionated by activated carbon column. After loading the hydrolysate onto column, the column was washed with 2 liters water to remove salts and monosaccharides. Followed this step, the agaro-oligosaccharides fraction was eluted sequen-tially with 8 %, 15 % and 25 % hydroalcoholic solution. Each fraction from the column was concentrated under reduced pressure and lyophilized. Structural information of agaro-oligosaccharides The average molecular weight of three fractions was measured as described by Somogyi et al. [14]. The nuclear magnetic resonance (NMR) spectra were acquired on an AVANCEDMX-500-NMR spectrometer. Samples were dissolved in D 2 O. 13 C NMR spectra of 4% (w/v) solutions were recorded at 35°C under 100.69 MHz. Proton decoupled 13 C NMR chemical shifts were measured in parts per million. For 1 H-NMR, samples (7-10 mg) were dissolved in D 2 O (0.5 ml), and spectra were recorded at room temperature using a spectral width of 5.7 kHz, 90° pulse, an acquisition time of 4.4 s for 144 scans. Mass spectrometry analysis was performed on a Bruker Reflex III MALDI-TOF-MS (Bruker-Daltonik, Germany) in the delayed extraction and positive mode. An accelerating voltage and a reflectron voltage were set at 20 kV of 22.8 kV, respectively, during the measurements. 2, 5-Dihydroxybenzoic acid was used as matrix (20 mg/ml; 3:2 water/MeCN) and approximately 10-100 pg of the DP-H agaro-oligosaccharide mixture was deposited as a mixture together with the matrix on a stainless steel target, and subsequently dried under reduced pressure. During the experiments, the laser power was adjusted to a level just above the threshold for formation of observable ions. The results from 20 to 100 laser shots were summed for sample. Measurement of intracellular ROS generation Intracellular oxidant stress was monitored by measuring changes in fluorescence resulting from intracellular probe oxidation. Human hepatocyte L-02 purchased from Chinese Institute of Biochemistry and Cell Biology was cultured in RPMl-1640 medium with 20 % fetal bovine serum. Viable cells (10 5 /ml) were plated into a 96-well for 1 day. On the day of the experiments, after removing the medium, the cells were washed with PBS for three times and then incubated with different doses of agaro-oligosaccharides in 5 % CO 2 at 37°C for 2 h. After incubation, 20 μM DCFH-DA was added for another 45 min. The DCFH-DA was removed by washing the cells with PBS. 100 μM H 2 O 2 were added into cells for 45 min and the fluorescence change was monitored by fluorescence spectorphotometer at λ ex = 475 nm, λ em = 525 nm [15]. Cell viability and cytotoxicity assessment The cell viability was quantified using MTT assay. Briefly, 1 × 10 4 cells were seeded in each well of microtiter plate and allowed to attach overnight. Cells were treated with various doses of agaro-oligosaccharides for different period according to the experiment purpose. For cytotoxicity test, the hepatocyte L-02 was treated for 48 h. But for the detection of protective effect of agaro-oligosaccharides on H 2 O 2 damage, the L-02 was only treated for 2 h, and then 100 μM H 2 O 2 was added for another 2 h. MTT in PBS was added to each well, followed by incubation for 4 h at 37°C. The formazan crystals were dissolved in DMSO. The optical density was determined with a microculture plate reader at 492 nm [16]. Animals model Mature Wistar rats weighing 150 ± 20 g were supplied by the animal center of Hangzhou, China. The animals were housed in a room with a 12 h light/dark cycle at about 22°C and fed on standard diet with ad libitum access to drinking water. All treatments were conducted between 9:00 am and 10:00 am to minimize variations. In this study, rats were randomly divided into six groups. Group 1 (control, n = 8): water for 10 days followed by administration of liquid paraffin only; group 2 (CCl 4 , n = 8): water for 10 days followed by administration of CCl 4 on the final day; group 3 (positive control, n = 8): vitamin C (200 mg/kg) + CCl 4 ; group 4 to 6 (n = 8): agaro-oligosaccharides (200, 400, 600 mg/kg, respectively) + CCl 4 . Rats were injected i.p. with vitamin C or agaro-oligosaccharides for ten consecutive days. On the final day, all animal except control group were administered with 20 % CCl 4 in liquid paraffin at a dose 5 ml/kg to induce hepatotoxicity. Previous studies demonstrated that the OS indexes could reach a maximum at 48 h after CCl 4 i.p. administration [17], therefore, in this work rats were sacrificed by collecting the blood from the carotid artery after 48 h of administration. Two organs (liver and heart) were excised immediately. Biochemical assays Serum was separated by centrifugation at 1000 × g at 4°C for 10 min. 10 % organ homogenates including liver and heart were prepared in ice-cold isotonic physiological saline. The GSH-Px, MDA, SOD, AST and ALT levels of tissue and serum were measured by spectrophotometric methods as described in the assay kits. Statistical analysis All data are expressed as mean ± SD. In cell based assay, the control and agaro-oligosaccharides treated cells were compared by student t-test. In animal assay, the statistical tests were one-way ANOVA followed by post-hoc Newman-Keuls multiple comparisons test. A probability level of 0.05 was considered statistically significant. Preparation and structure analysis of agarooligosaccharides Activated charcoal column has been performed as saccharide isolation tool for decades. Depending on this technology, we successfully achieved to isolate three fractions of agaro-oligosaccharides with average molecular weight of 619, 1126 and 1631, respectively, eluted by 8 %, 15 % and 25 % aqueous alcoholic solution. We use these three fractions for the following experiments, designated as DP-L, DP-M and DP-H, according to their differences in molecular weight. Since agar is a linear copolymer of galactose (G), alternated with 3, 6-anhydrogalactose (A), the structural difference of agaro-oligosaccharides are mainly related with the degree of polymerization. In this report, the 1 H-NMR and 13 C-NMR spectra of agaro-oligosaccharides was studied using acid hydrolyzed fragments, and typical deshielded 1 H-NMR and 13 C-NMR signals corresponding to the anomeric hydrogens and carbons were obtained and presented in Fig. 1. Assignments were based on the close similarity with literature values, and the interpretation of these signals was indicated in Table 1. The spectra give out twelve distinctive major anomeric carbon signals which were expected for the major disaccharide repeat unit. The presence of these signals demonstrates the presence of floridean starch in this fraction, because all of the signals illustrated the galactose ring structures present in seaweed galactans [18,19]. The 13 C NMR spectrum of oligosaccharide are very consistent with those previously published for neoagarose series, with chemical shifts of carbons of unit G'-1α and G'-1β appeared at identically 92.4 and 96.4 ppm, having intensities in the ratio of 1 : 2. While, from the result, we didn't observe any signal for 3, 6-anhydrogalactose at the non reducing ending (a peak at 91.4 ppm) [20][21][22]. These results reflect the presence of galactose units at the reducing ends of the reaction products, but no 3, 6-anhydrogalactose at non-reducing end. We applied MALDI-TOF-MS in order to know the structural information of composition and DP in the agaro-oligosaccharide fractions which were obtained from hydrolysis. The results shown in Fig. 2 indicated that a large number of well-regulated peaks are present, and these agaro-oligosaccharide ions could be identified as series of sodium molecular ions with relatively high intensities corresponded to 509, 815, 1121, and so on. It can be seen, by comparing these ions, that the molecular mass difference between every two adjacent ion is the same as 306 Da. This molecular mass difference of 306 Da is the exact molecular mass of agaro-biose (GA), the basic structural unit, which is 324 Da, minus 18, which is the number of H 2 O's molecular weight, therefore, it is clearly observed that the agaro-oligosaccharides had very regular molecular structures with gradient increase of its chain length with the polymerization unit of agarobiose. Further calculation for m/z 509, the lowest high intensity ion observed in the mass spectrum, found that m/z 509 corresponds to the sodium adduct of agarotriose (GAG) [M tri +Na] + . Based on this information, the ion at m/z 815, 1121, 1427.... corresponds to agaro-oligosaccharides for n = 5, 7, 9..., respectively with galactose at both reducing end and non-reducing end. For agaro-oligosaccharides, two forms of saccharides exist depending on the end sugar moiety, namely, neoagaro-series with 3, 6-anhydro-galactose at the non-reducing end and agaro-series with galactose at the non-reducing end. The results obtained here indicated that our sample obtained belong to agaro-series with odd numbers of sugar unit. The antioxidant action of agaro-oligosaccharides in cell based assay We firstly investigated the antioxidant activities of agarooligosaccharides in the cellular system. DCFH-DA, which can be conversed from non-fluorescence into fluorescence through oxidation, was used as fluorescent probe to monitor the changes of oxidative stress in hepatocyte L-02 induced by addition of H 2 O 2 . In our experiment, all the measurements were carried out at the steady stage (incubation time, 60 min) in order to minimize variations, because it has been reported that treatment of H 2 O 2 will lead to the abruption of ROS in few minutes, and then decrease to a steady stage [23]. Fig. 3 showed that addition of agaro-oligosaccharides caused concentration-dependent attenuation of DCF fluorescence. Three groups of agaro-oligosaccharides showed almost no inhibitory activity at the 125 μg/ml. When the concentration increased, DP-H expressed highest activity, followed by DP-M, much weaker for DP-L group, which indicating that the antioxidant bioactivity in vitro improves with the higher degree of polymerization of agaro-oligosaccharide. Fig. 4 is a typical fluorescent microscopic picture of the DCF fluorescence in hepatocyte L-02 1 H-NMR and 13 C-NMR spectra of solid acid hydrolysate http://www.nutritionj.com/content/5/1/31 treated with DP-H. It is obvious that H 2 O 2 lead to the production of ROS, which transformed the DCFH into DCF (Fig. 4D), showing more fluorescent cells than untreated cells (Fig. 4A). DP-H additions decreased the free radical formation. Fig. 4B clearly illustrated that DP-H at concentration of 1 mg/ml could inhibit the oxidation of DCFH significantly. While with the concentration decreased to 125 μg/ml (Fig. 4C), the number of fluorescent cells was also increased, and which means that the antioxidant activity of agaro-oligosaccharides acts in a concentrationdependent manner. Protective effect of agaro-oligosaccharides on oxidative stress injury Oxidative stress is an important factor to induce the cell death. Cell viability assay showed that the presence of H 2 O 2 (100 μM) resulted in cell death ratio increasing to 60 % after 2 h of treatment (Fig. 5). Compared to H 2 O 2 alone, cell death was reduced obviously when exposed to each agaro-oligosaccharide group at the higher concentrations (from 500 μg/ml to 1 mg/ml). The cell viability significantly increased to 64.26 % for DP-M treated cells at the concentration of 1 mg/ml (Fig. 5). At low concentrations (125 μg/ml to 500 μg/ml), there was almost no variation observed between the agaro-oligosaccharide treated cells and the control, except DP-M treated group showing some weak cell protective effect. These results demonstrated that the antioxidant activities of agaro-oligosaccharides were positively correlated with the improvement of the cell viability. In order to test whether agaro-oligosaccharides affected the growth of human hepatocyte L-02 without H 2 O 2 treatment, cell proliferation was assessed by direct MTT assay. The cells were incubated with various amounts of agarooligosaccharides for 48 h, and the change in cell number was determined by analyzing the values of cells treated with agaro-oligosaccharide versus that of control (Fig. 6). From result, we found that compared with control group, the agaro-oligosaccharides exhibited very slight effects on the cell growth. After 48 h of treatment, the growth is slightly inhibited as of 14.18 % for DP-H at 1 mM, while for DP-M and DP-H, the corresponding cell proliferation ratio was >100 % with concentration ≤ 250 μM, which means that, at proper concentration, the agaro-oligosaccharides can promote the proliferation of L-02 cells. Therefore, the cell survival effect of agaro-oligosaccharide alone in the antioxidation cellular assay can be considered almost naught because the cells were only treated for 2 h, so we concluded that agaro-oligosaccharides can effectively protect the cells from oxidation induced death through scavenging intracellular oxidative damage induced by ROS. Effect of agaro-oligosaccharides on an acute CCl 4 oxidative damage We further studied the in vivo antioxidant effects of agarooligosaccharides. It was not uncommon that compounds possessing in vitro activity, however, fail to maintain the activity when administrated into body. We established an oxidative animal model by CCl 4 injection. Considering the proliferation and antioxidant effects of agaro-oligosaccharides on hepatocyte, we used the mixture of DP-M and DP-H as our sample for animal test. The effects of agarooligosaccharides on oxidative stress in rats were estimated by determining the activities of MDA, SOD, GSH-Px, ALT and AST in serum and tissues. MDA level is a main marker of endogenous lipid peroxidation [24]. In CCl 4 treated group, the MDA level increased significantly in liver (F = 2.087, P < 0.05), but little difference was observed in serum, which confirmed that the toxicity of CCl 4 is focused in the liver. By contrast, MDA level in the agaro-oligosaccharides treated groups decreased significantly compared with CCl 4 treated group. At 400 mg/kg, the MDA level reduced at least 44 % and 21 % in liver (F = 4.274, P < 0.05) and heart, respectively, versus the CCl 4 treated group. Actually, the MDA level of agaro-oligosaccharides treated groups showed almost the same as the blank control group (Table 2). It provided the SOD and GSH-Px are intracellular antioxidant enzymes that protect against oxidative process [25]. As show in Table 3 and 4, a single high dose injection of CCl 4 induced severe oxidative damage and the SOD and GSH-Px level decreased markedly. While various concentrations of agaro-oligosaccharides could effectively normalize the enzyme activities and the two indexes were even higher than Vitamin C group. In liver and serum, the SOD level reached to highest at 400 mg/kg (F = 3.878, P < 0.05; F = 9.363, P < 0.05). Similar results were obtained in case of the GSH-Px activities. Serum levels of transaminases (ALT, AST) were used as indicators to evaluate the attribution of agaro-oligosaccharides to the structure damage of the liver [26,27]. In this experiment, the enzyme assays of serum transaminases showed that a toxic dose of CCl 4 significantly raised the levels of ALT and AST to 687 (F = 3.761, P < 0.05) and 415 U/l (F = 4.204, P < 0.05). Agaro-oligosaccharides could inhibit the enzyme activities effectively. The ALT level reached to minimum when the sample concentration was 400 mg/kg (22.16 % less than the control group). For AST, the agaro-oligosaccharides reduce it in a dose dependent manner. At the highest concentration (600 mg/kg), AST level decreased to 222 U/l. However, it is strange to find that Vitamin C didn't reduce AST but raised it to 32 % versus the control without CCl 4 treatment (Fig. 7). Discussion Among therapeutics for liver diseases, protective drugs have been attracted more and more attentions, such as antioxidant prevention approaches. In this paper, we MALDI-TOF mass spectrum of agar hydrolysate focused on the in vitro and in vivo antixoidative activities of agaro-oligosaccharides with the model related with liver disease. Agaro-oligosaccharides are linear oligomers cleaved from agar which is built of 1, 4-linked 3, 6-anhydro-α-L-galactose alternating with 1, 3-linked β-D-galactopyranose. When agar is attacked by degradation reagents, such as hydrolysis enzyme, acid or alkali, numerous possibilities for combination, viz., the repetition of AG, GA, AGA, or GAG, etc will exist. In this research, depending on NMR and MALDI-TOF-MS analysis, we detected the precise structural features of our hydrolysate. NMR results give us information that our product is agarose structure, furthermore, there was no signal of A at reducing end. In the spectrum of MALDI-TOF-MS, the first high intensity peak observed at m/z 509 was assigned to (M tri +Na) + containing two galactopyranose (Galp) residues and one 3,6anhydrogalactopyranose (AnGalp) residues, followed by a series of agaro-oligosaccharides: agaropentaose, agaroheptanose, agarononaose, and so forth. In our case, the agaro-oligosaccharides with odd polymerization degree were dominant. For the in vitro antioxidant studies, we noticed that agarooligosaccharides expressed different antioxidant abilities with different ranges of DPs. In them, the fraction of DP-H with average MW of 1631 showed highest free radical scavenging activity which agrees well with the result obtained by Zhao et al. [10]. However, Enoki et al. [12] found, in a different assay system, that agarobiose possessed the highest ability to inhibit the expression of Effect of agaro-oligosaccharides on DCF fluorescence in hepatocytes It is quite significant that the in vivo animal experiment for agaro-oligosaccharides is quite consistent with the in vitro assays. Besides successful protection of liver damage by efficiently inhibiting MDA formation and decreasing AST and ALT, agaro-oligosaccharides enhance the activities of antioxidant enzyme system of the host, including SOD, GSH-Px. We also notice that vitamin C only slightly reduced AST and ALT level in rats in our experiment, although it prevented MDA formation effectively (Fig. 7). The result indicates that agaro-oligosaccharides have bet-ter impact to improve the hepatoprotective ability. Since antioxidant enzymes such as SOD and GSH-Px are considered to be a primary defense system for oxidative damage prevention, agaro-oligosaccharides exert antioxidant not only through its own radical scavenging activity, but also, by boost the host antioxidant enzyme system. On the other hand, we found that when the sample concentration increased from 400 mg/kg to 600 mg/kg, several indexes showed a different change. At concentration of 600 mg/ kg, the MDA level increased slightly and SOD, GSH-Px and AST activities reduced a little. This result implied that excessive administration of agaro-oligosaccharides will decrease their antioxidant ability with unknown reasons. In conclusion, by carefully examining the antioxidant protective effects of agaro-oligosaccharides both in vitro and in vivo, the agaro-oligosaccharides prepared via solid acid hydrolysis showed consistent and concentration-dependent antioxidation activities, as well as significant protection against liver injury. Conclusion These results support a beneficial relationship between antioxidant activity and hepatoprotective effect of agarooligosaccharides which belong to agaro-series with odd numbers of sugar unit as their dominant composition. Effect of agaro-oligosaccharides on cell survival during H 2 O 2 exposure XJY have made substantial contributions to conception and design. Effect of agaro-oligosaccharides on AST and ALT activity in serum LJ participated in the cell biology research.	2017-06-23T00:17:31.639Z	2006-12-02T00:00:00.000	{ "year": 2006, "sha1": "f2212f3a5292d7427787e85089a68353a72948f9", "oa_license": "CCBY", "oa_url": "https://nutritionj.biomedcentral.com/counter/pdf/10.1186/1475-2891-5-31", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "39f6f1eb7e2e5d4d587bb3990c7d579a198dd932", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [] }
245292386	pes2o/s2orc	v3-fos-license	Genome Features of a New Double-Stranded RNA Helper Virus (LBCbarr) from Wine Torulaspora delbrueckii Killer Strains The killer phenotype of Torulaspora delbrueckii (Td) and Saccharomyces cerevisiae (Sc) is encoded in the genome of medium-size dsRNA viruses (V-M). Killer strains also contain a helper large size (4.6 kb) dsRNA virus (V-LA) which is required for maintenance and replication of V-M. Another large-size (4.6 kb) dsRNA virus (V-LBC), without known helper activity to date, may join V-LA and V-M in the same yeast. T. delbrueckii Kbarr1 killer strain contains the killer virus Mbarr1 in addition to two L viruses, TdV-LAbarr1 and TdV-LBCbarr1. In contrast, the T. delbrueckii Kbarr2 killer strain contains two M killer viruses (Mbarr1 and M1) and a LBC virus (TdV-LBCbarr2), which has helper capability to maintain both M viruses. The genomes of TdV-LBCbarr1 and TdV-LBCbarr2 were characterized by high-throughput sequencing (HTS). Both RNA genomes share sequence identity and similar organization with their ScV-LBC counterparts. They contain all conserved motifs required for translation, packaging, and replication of viral RNA. Their Gag-Pol amino-acid sequences also contain the features required for cap-snatching and RNA polymerase activity. However, some of these motifs and features are similar to those of LA viruses, which may explain that at least TdV-LBCbarr2 has a helper ability to maintain M killer viruses. Newly sequenced ScV-LBC genomes contained the same motifs and features previously found in LBC viruses, with the same genome location and secondary structure. Sequence comparison showed that LBC viruses belong to two clusters related to each species of yeast. No evidence for associated co-evolution of specific LBC with specific M virus was found. The presence of the same M1 virus in S. cerevisiae and T. delbrueckii raises the possibility of cross-species transmission of M viruses. Introduction Killer yeast strains can kill non-killer strains because they secrete proteins that are killer toxins. A certain type of killer yeast can also kill other types of killer strains that belong to the same yeast species. Some killer yeast strains can also kill yeasts (killer and non-killer) that belong to other species. Each killer yeast is immune to its own toxin and also to toxins secreted by other yeast strains with the same type of killer phenotype [1][2][3]. Best known killer phenotype of Torulaspora delbrueckii (Td) and Saccharomyces cerevisiae (Sc) is encoded by cytoplasmic medium-size dsRNA viruses (V-M). The killer yeast strains also contain a helper large-size (4.6 kb) dsRNA virus (V-LA) that is required for maintenance and replication of V-M. The helper V-LA provides the capsid and polymerase required for V-M maintenance. A specific LA virus may support different types of satellite M viruses but only one type of V-M has been discovered in each yeast killer type so far [1,2]. Another large-size (4.6 kb) dsRNA virus (V-LBC) may join V-LA and V-M in the cytoplasm of the same S. cerevisiae strain, although LBC viruses have not been described to date as having are not typical LBC viruses because they share some structural motifs with the (+)RNA of LA viruses. Analysis of the dsRNA Genomes from TdV-LBCbarr1 and TdV-LBCbarr2 Two different sequences were obtained from the L dsRNA (agarose electrophoresis band of 4.6 kb) present in the Kbarr1 EX1180 strain, one similar to that of LA virus genomes (named TdV-LAbarr1) that was previously characterized [12], and another sequence similar to that of LBC viruses (named TdV-LBCbarr1). However, only one sequence was obtained from the L dsRNA present in Kbarr2 EX1257 strain (named TdV-LBCbarr2). This strain contains two killer viruses, Mbarr1 and M1 [3], which indicates that TdV-LBCbarr2 is the helper virus required for replication and maintenance of both M viruses. The complete sequence found for TdV-LBCbarr1 cDNA was 4763 nucleotides in length, and 5115 nt for TdV-LBCbarr2, which are slightly larger than the size estimated by agarose-gel electrophoresis ( Table 1). Most of the sequence of both LBCbarr genomes (4565 nt central stretch) showed about 52% nucleotide identity with the previously known ScV-LBC1-original and ScV-LBClus4 genomes ( Figure 1, Figures S1 and S3), while it only shared 39% identity with Saccharomyces and Torulaspora LA genomes. We considered this central stretch as the canonical sequence of LBCbarr genomes, and sequences upstream and downstream as 5 -and 3 -extra sequences, respectively; as it was previously considered for V-LA and V-M genomes [3,12]. The canonical sequences of TdV-LBCbarr1 and TdV-LBCbarr2 genomes share great identity (94%), which means that both are the same virus with some nt-sequence differences. Therefore, we mainly focused on the TdV-LBCbarr2 sequence for genome comparison with other L viruses. TdV-LBCbarr2 canonical genome length (4565 bp) is slightly shorter than that of ScV-LA (4580 bp), TdV-LA (4591 bp), and ScV-LBC (4615 bp). Its genome organization is similar to that described for ScV-LA1-original, containing two ORFs [11]. The first ORF (from nt 28 to nt 2124) belongs to the coat (Gag) protein of the virion, and the second ORF (from nt 2157 to nt 4489) belongs to the viral RNA-dependent RNA polymerase (RdRp). This polymerase is expected to be synthetized as a Gag-Pol fusion protein by a −1 ribosomal frameshift at the conserved frameshifting site (or slippery site) located upstream of the Gag ORF stop codon (1977GGAUUUU1983) (Figure 1 and Figure S1), as previously described for ScV-LA1-original and ScV-LBC1-original [4,7,11,12]. These different ORF assignments were done by also considering the amino-acid sequence homology of the Gag-Pol fusion protein of both TdV-LBCbarr to those of L viruses from Saccharomyces (see below). Figure 1. Partial multiple sequence alignment between ScV-LBC1-original, ScV-LBClus4, and TdV-LBCbarr2 (+) strand nucleotide sequences (cDNA). The full sequence alignment is presented in Supplemental Material ( Figure S1). 5'GAA(A/T)TT conserved motif (5' conserved), translation initiation (start of Gag and Gag-Pol), termination (stop of Gag or Gag-Pol) codons, ribosome frameshifting site (−1 frameshift site), frameshifting associated sequence (stem-loop for frameshift), packaging signal (stem-loop for packaging), and replication signal (stem-loop for replication) are indicated, shaded and/or underlined in the nucleotide sequence. Different residue in LBCbarr2 with respect to LBCbarr1 virus is yellow shaded. , ribosomal frameshift. Asterisks () indicate identical nucleotide positions. The secondary structures of the putative cis signals for frameshifting, packaging, and replication of TdV-LBCbarr2 are displayed at the right of the sequence panel. Stop of ScV-LBC Gag-Pol Stem-loop for replication LBCbarr2 ACGGC------------4565 LBC1-ori ACATACGATACTACGCA 4615 LBClus4 ACATACGATACTACGCC 4615 * Figure 1. Partial multiple sequence alignment between ScV-LBC1-original, ScV-LBClus4, and TdV-LBCbarr2 (+) strand nucleotide sequences (cDNA). The full sequence alignment is presented in Supplemental Material ( Figure S1). 5 GAA(A/T)TT conserved motif (5 conserved), translation initiation (start of Gag and Gag-Pol), termination (stop of Gag or Gag-Pol) codons, ribosome frameshifting site (−1 frameshift site), frameshifting associated sequence (stem-loop for frameshift), packaging signal (stem-loop for packaging), and replication signal (stem-loop for replication) are indicated, shaded and/or underlined in the nucleotide sequence. Different residue in LBCbarr2 with respect to LBCbarr1 virus is yellow shaded. Figure S1). 5'GAA(A/T)TT con (start of Gag and Gag-Pol), termination (stop of G site (−1 frameshift site), frameshifting associated seq nal (stem-loop for packaging), and replication s shaded and/or underlined in the nucleotide sequen LBCbarr1 virus is yellow shaded. , ribosomal fram positions. The secondary structures of the putative lication of TdV-LBCbarr2 are displayed at the righ TdV-LBCbarr2 canonical genome length LA (4580 bp), TdV-LA (4591 bp), and ScV-LBC ilar to that described for ScV-LA1-original, con nt 28 to nt 2124) belongs to the coat (Gag) prot nt 2157 to nt 4489) belongs to the viral RNA polymerase is expected to be synthetized as a frameshift at the conserved frameshifting sit Gag ORF stop codon (1977GGAUUUU1983) for ScV-LA1-original and ScV-LBC1-original [ were done by also considering the amino-acid protein of both TdV-LBCbarr to those of L vir There are other important regions in TdV highly conserved in ScV-LA and ScV-LBC g There are other important regions in TdV-LBCbarr genomes that are similar to those highly conserved in ScV-LA and ScV-LBC genomes [4,6,12,13,16]: (i) a 18-nt stemloop region involved in frameshifting (2003TCCCGGGTGTGTCGGGGA2020), that is located 19 nt downstream from the slippery site; (ii) a 18-nt stem-loop (4152TACGCA-GATATTGACGTG4169), that is located 174 nt downstream from the RdRp domain and is responsible for binding to and packaging of the (+)RNA strand of L viruses; and (iii) a 20-nt stem-loop (4543GTTGTGACCTATATGACAAC4562), that is located 3 nt upstream from the 3 end and is responsible for RNA replication (Figure 1 and Figure S1). These motifs are found in both TdV-LBCbarr genomes in equivalent positions with respect to the genome sequence of ScV-LA1-original, which is the best known among the yeast L viruses [11]. In ScV-LA1-original, the stem-loop for frameshifting is located 4 nt downstream from the slippery site, the stem-loop for (+)RNA packaging is 191 nt downstream from RdRp domain, and the stem-loop for RNA replication is 2-4 nt upstream from the 3 end. These locations are very similar to that found in both TdV-LBCbarr genomes: 19 nt, 174 nt, and 3 nt, respectively. The only difference between both TdV-LBCbarr genomes is one nt change in the 18-nt stem-loop required for virus packaging, that is 4152TACGCA-GATATTGATGTG4169 in LBCbarr1 and 4152TACGCAGATATTGACGTG4169 in LBCbarr2. As a consequence, only the secondary structure of LBCbarr2 stem-loop is similar to that found in ScV-LA1-original in the sense of this stem-loop shows a critical "A" residue protruding from the stem in both cases [14,20]. However, this "A" is protruding from the 3 side of the stem in TdV-LBCbarr2, as in ScV-LBC1-original, instead from the 5 side of the stem as found in ScV-LA1-original and TdV-LAbarr1 ( Figure 2). 19 nt downstream from the slippery site; (ii) a 18-nt stem-loop (4152TACGCAGA-TATTGACGTG4169), that is located 174 nt downstream from the RdRp domain and is responsible for binding to and packaging of the (+)RNA strand of L viruses; and (iii) a 20nt stem-loop (4543GTTGTGACCTATATGACAAC4562), that is located 3 nt upstream from the 3′ end and is responsible for RNA replication (Figures 1 and S1). These motifs are found in both TdV-LBCbarr genomes in equivalent positions with respect to the genome sequence of ScV-LA1-original, which is the best known among the yeast L viruses [11]. In ScV-LA1-original, the stem-loop for frameshifting is located 4 nt downstream from the slippery site, the stem-loop for (+)RNA packaging is 191 nt downstream from RdRp domain, and the stem-loop for RNA replication is 2-4 nt upstream from the 3′ end. These locations are very similar to that found in both TdV-LBCbarr genomes: 19 nt, 174 nt, and 3 nt, respectively. The only difference between both TdV-LBCbarr genomes is one nt change in the 18-nt stem-loop required for virus packaging, that is 4152TACGCAGA-TATTGATGTG4169 in LBCbarr1 and 4152TACGCAGATATTGACGTG4169 in LBCbarr2. As a consequence, only the secondary structure of LBCbarr2 stem-loop is similar to that found in ScV-LA1-original in the sense of this stem-loop shows a critical "A" residue protruding from the stem in both cases [14,20]. However, this "A" is protruding from the 3′ side of the stem in TdV-LBCbarr2, as in ScV-LBC1-original, instead from the 5′ side of the stem as found in ScV-LA1-original and TdV-LAbarr1 ( Figure 2). Although these three stem-loops are also assumed to be present in the genome of S. cerevisiae LBC viruses, they are not easily identified in equivalent positions with respect ********* * ** * * ******* * Although these three stem-loops are also assumed to be present in the genome of S. cerevisiae LBC viruses, they are not easily identified in equivalent positions with respect to the nucleotide sequence of ScV-LA1-original. Some stem-loops have been suggested as required for frameshifting, RNA packaging, and RNA replication of ScV-LBC1-original ( Figure 1 and Figure S1) [4,21]. We found these proposed signals in all the newly sequenced ScV-LBC genomes, in the same location. As previously described, the stem-loop proposed for frameshifting in ScV-LBC genomes is overlapping 1 nt with the ribosome slippery site [4], the stem-loop for (+)RNA packaging is 349 nt downstream from RdRp domain (which is 158 nt downstream from the expected location in LA viruses [12,13], and the two possible stem-loops for RNA replication (separated 9 nt of distance between them, and located 4-6 nt upstream from the 3 end [21]) are weak stem-loop structures (∆G = −7.5 and −2.9 kJ/mol, respectively) ( Figure 1, Figures S1 and S2). TdV-LBCbarr genomes also contain the 5 AU-rich regions of L and M dsRNA viruses that facilitate "melting" of the molecule and the access of the RNA polymerase to the (−)RNA template for conservative transcription [3,5,17,22]. In this case, this motif (5 GAAATT) is more similar to that found in ScV-LBC genomes (5 GAATTT) than to that found in ScV-LA genomes (5 GAAAAA) [12]. Analysis of the Gag-Pol Sequences from TdV-LBCbarr1 and TdV-LBCbarr2 No relevant identity was found between the putative Gag-Pol amino acid sequences of both TdV-LBCbarr and those of LA viruses (24-24.8%), similarly to that found between Gag-Pol of ScV-LBC1-original and ScV-LA1-original (24.6%). TdV-LBCbarr Gag-Pol sequences showed 44% global identity with that of ScV-LBC1-original (Figure 3, Figure 5 and Figure S2). Despite this modest identity, both LBCbarr Gag-Pol sequences contain most of the key motifs for Gag and Pol functions previously found in Saccharomyces LBC viruses. This identity is much lower than that found among Gag-Pol sequences of known S. cerevisiae LBC-viruses, which is greater than 95% ( [4] and see below). This modest identity increases to 64-66% when comparing only the highly-conserved RdRp-domain of TdV-LBCbarr and ScV-LBC polymerases. This domain contains the five motifs (3, A, B, C, and D) conserved among RdRps, which are essential for RNA polymerase activity [23,24]. The most conserved amino acids of these five motifs are identical in all RdRps of yeast L viruses except one amino acid in motif A, which is "DYDDFNS" in all known Saccharomyces and T. delbrueckii LA viruses, and "DFDDFNS" in all S. cerevisiae LBC viruses. Surprisingly, motif A of both TdV-LBCbarr polymerases is identical to that found in LA viruses. However, surrounding residues of these five conserved amino acids of LBCbarr polymerases are in most cases coincident with those of S. cerevisiae LBC viruses (Figure 4). The identity between Gag sequences of TdV-LBCbarr and those of ScV-LBC viruses also is low, about 24-37.8%. However, the Gag His154 residue of ScV-LA1-original (that is His155 in ScV-LBC1-original, and His156 in TdV-LBCbarr) required for the cap-snatching mechanism in the virion [25], and the four putative crucial residues for cap recognition (Tyr-150, Asp-152, Tyr-452, and Tyr-538 in ScV-LA1-original; that are Tyr-152, Asp-154, Tyr-449 and Trp-543 in ScV-LBC1-original [26]) are also present in the Gag protein of both LBCbarr viruses (Tyr-152, Asn-154, Tyr-452 and Trp-545; Figure 3 and Figure S2). In addition to the slightly different location for each residue in the Gag sequence, the main difference among these viruses is that the forth residue is Trp in LBC1-original and LBCbarr viruses, while it is Tyr in ScV-LA1-original. The same difference was found when comparing all known LA and LBC viruses from Saccharomyces and T. delbrueckii yeasts (data not shown). The identity between Gag sequences of TdV-LBCbarr and those of ScV-LBC viruses also is low, about 24-37.8%. However, the Gag His154 residue of ScV-LA1-original (that is His155 in ScV-LBC1-original, and His156 in TdV-LBCbarr) required for the cap-snatching mechanism in the virion [25], and the four putative crucial residues for cap recognition (Tyr-150, Asp-152, Tyr-452, and Tyr-538 in ScV-LA1-original; that are Tyr-152, Asp-154, Tyr-449 and Trp-543 in ScV-LBC1-original [26]) are also present in the Gag protein of both LBCbarr viruses (Tyr-152, Asn-154, Tyr-452 and Trp-545; Figures 3 and S2). In addition to the slightly different location for each residue in the Gag sequence, the main difference among these viruses is that the forth residue is Trp in LBC1-original and LBCbarr viruses, while it is Tyr in ScV-LA1-original. The same difference was found when comparing all known LA and LBC viruses from Saccharomyces and T. delbrueckii yeasts (data not shown). Comparison of TdV-LBCbarr with LBC Viruses from Saccharomyces Yeasts The dsRNA and Gag-Pol sequences of both TdV-LBCbarr were compared with their counterparts from S. cerevisiae to analyze their phylogenetic relationship. The genomic sequence of ScV-LBC1-original, ScV-LBClus-EX229, and ScV-LBC2-S3920 were already known (Tables 1 and 2). The genomes of ScV-LBC1 from strain EX231, ScV-LBC2 from EX1125, and ScV-LAlusA from EX1160 were de novo sequenced by HTS techniques for this study. ScV-LBClus4 from EX229, previously sequenced by Sanger procedure and named ScV-L-BC-lus [4], was also de novo sequenced to assess the accuracy of our HTS procedure. Sequences obtained by both procedures share 99.9% identity. Only six nucleotide changes were found in the genome of ScV-LBClus4 with respect to ScV-L-BC-lus: C548A, T704C, T2667G, G2668C, G2669T, and G2671A. As nucleotides of ScV-LBClus4 (HTS) in these positions coincided best with the rest of the S. cerevisiae LBC viruses, this sequence was the one used for further comparison. Comparison of TdV-LBCbarr with LBC Viruses from Saccharomyces Yeasts The dsRNA and Gag-Pol sequences of both TdV-LBCbarr were compared with their counterparts from S. cerevisiae to analyze their phylogenetic relationship. The genomic sequence of ScV-LBC1-original, ScV-LBClus-EX229, and ScV-LBC2-S3920 were already known (Tables 1 and 2). The genomes of ScV-LBC1 from strain EX231, ScV-LBC2 from EX1125, and ScV-LAlusA from EX1160 were de novo sequenced by HTS techniques for this study. ScV-LBClus4 from EX229, previously sequenced by Sanger procedure and named ScV-L-BC-lus [4], was also de novo sequenced to assess the accuracy of our HTS procedure. Sequences obtained by both procedures share 99.9% identity. Only six nucleotide changes were found in the genome of ScV-LBClus4 with respect to ScV-L-BC-lus: C548A, T704C, T2667G, G2668C, G2669T, and G2671A. As nucleotides of ScV-LBClus4 (HTS) in these positions coincided best with the rest of the S. cerevisiae LBC viruses, this sequence was the one used for further comparison. Identity among Sc-LBC viruses was similar to that previously found [4], above 95% for amino acid sequences. Two clusters were found to include all LBC viruses: S. cerevisiae cluster, in which ScV-LBClusA-EX1160, ScV-LBC1-EX231 and ScV-LBC2-EX1125 isolated from Extremadura seem to be the same virus (99.9-100% identity of Gag-Pol); and T. delbrueckii cluster grouping TdV-LBCbarr1-EX1180 and TdV-LBCbarr2-EX1257, that were also isolated from Extremadura, and also appear to be the same virus (98.3%). Viruses from different clusters share a fairly low identity rate (43.5-44.2%), despite that most viruses were isolated from the same region. Given the high similarity of ScV-LBClus4-EX229, ScV-LBC2-S3920 and ScV-LBC1-original with the rest Sc-LBC virus (95-100%), they appear to be just variants of the same virus ( Figure 5). It is noteworthy that the same LBC virus was found can coexist with different killer M viruses in different yeast strains (Table 1). Identity among Sc-LBC viruses was similar to that previously found [4], above 95% for amino acid sequences. Two clusters were found to include all LBC viruses: S. cerevisiae cluster, in which ScV-LBClusA-EX1160, ScV-LBC1-EX231 and ScV-LBC2-EX1125 isolated from Extremadura seem to be the same virus (99.9-100% identity of Gag-Pol); and T. delbrueckii cluster grouping TdV-LBCbarr1-EX1180 and TdV-LBCbarr2-EX1257, that were also isolated from Extremadura, and also appear to be the same virus (98.3%). Viruses from different clusters share a fairly low identity rate (43.5-44.2%), despite that most viruses were isolated from the same region. Given the high similarity of ScV-LBClus4-EX229, ScV-LBC2-S3920 and ScV-LBC1-original with the rest Sc-LBC virus (95-100%), they appear to be just variants of the same virus ( Figure 5). It is noteworthy that the same LBC virus was found can coexist with different killer M viruses in different yeast strains (Table 1). Analysis of TdV-LBCbarr Genomes The average nucleotide identity between both T. delbrueckii LBCbarr viruses and S. cerevisiae LBC viruses (52%) was lower than that found between LA viruses of the same yeast species (60%) [12]. Moreover, this nucleotide identity was much lower than that found between Sc-LBC viruses (89-100%). This can be expected because these two yeast species are usually found in different ecological niches [12,28,29]. This finding raises the possibility that LBCbarr viruses are a new type of virus different from the already known LA and LBC viruses. Notwithstanding this, the organization of both TdV-LBCbarr genomes is similar to that of ScV-LA and ScV-LBC. They contain the same two ORFs, Gag and Gag-Pol. However, while TdV-LA and ScV-LA share 87.5-100% identity in some regions considered important for the virus replication (such as the frameshifting region required for translation of Gag-Pol fusion protein, or the virus packaging signal), TdV-LBC and ScV-LBC only share the ribosome slippery site located upstream of the Gag ORF stop codon (GGAUUUU). Interestingly, the three stem-loops involved in frameshifting, RNA binding and packaging, and RNA replication of TdV-LBCbarr genomes resemble those secondary structures found in similar positions in ScV-LA genomes, especially those of TdV-LBCbarr2 ( Figure 2). This may explain why Sc-LA and Td-LBCbarr viruses have capability to maintain killer M viruses; whereas Sc-LBC viruses that have these stem-loops located in other positions do not have not this capability. TdV-LBCbarr2 contains a stem-loop for packaging and encapsidation (ES signal) similar to the ES of S. cerevisiae LA and M1 viruses, and also similar to the ES of TdV-Mbarr1. This may explain why TdV-LBCbarr2 has capability to maintain both M1 and Mbarr1 viruses in the same cell. However, a difference of only one nucleotide in LBCbarr1 ES with respect to LBCbarr2 ES (C4166T) changes its secondary structure, making LBCbarr1 probably unable to maintain the M1 virus. Optionally, the ES secondary structure of LBCbarr1 is similar to a newly identified possible ES in the Mbarr1 genome, raising the possibility that LBCbarr1 may maintain Mbarr1 at the same time that TdV-LAbarr1 does ( Figure 2). I.e., Mbarr1 could be maintained by two different helper viruses in EX1180 strain, Td-LAbarr1 and Td-LBCbarr1. We confirmed the presence of all motifs and features previously proposed as required for viral replication of ScV-LBC1-original in all the newly sequenced S. cerevisiae LBC genomes; and the particular location of the stem-loop proposed for frameshifting, that overlaps 1 nt with the ribosome slippery site [4] (Figure 2). The ribosome frame shift in L viruses happens with low frequency, in about 1% of ribosomes [30]. The overlapping of the slippery site and the frameshifting stem-loop may imply a steric effect that decreases the frame shift frequency, reducing the amount of Cap-Pol required for viral replication. This may explain, at least in part, the low relative amount of LBC virus with respect to LA virus in S. cerevisiae [9,31]. In addition to this, the genome location and secondary structure of the stem-loops involved in viral replication, that is different to those of Sc-LA and Td-LBCbarr viruses, may account for the inability of Sc-LBC viruses to behave as helper virus to maintain killer M viruses. Analysis of TdV-LBCbarr Gag-Pol Sequences Gag-Pol sequence of LBCbarr contained most of the key motifs for Gag and Pol functions previously described in ScV-LBC1-original, as well as similar surrounding residues. However, motif A of RdRp domain is identical in TdV-LBCbarr and LA viruses. This motif A is highly conserved in viruses of the family Totiviridae, and is clearly of functional importance for the polymerase. Moreover, in addition, motifs 3, B, C and D, is required for the support of M1 killer virus by ScV-LA1-original [24]. This finding, together with the similarity found between relevant motifs in the genome of LBCbarr and LA viruses (stem-loops for frameshifting, RNA packaging, and RNA replication), again suggests that TdV-LBCbarr (but not ScV-LBC) may have similar capability than ScV-LA for replication and helping other satellite M killer viruses to replicate in the same yeast cell. I.e., Td-LBCbarr viruses, and especially TdV-LBCbarr2 do not seem to be typical LBC viruses because they share several functional motifs with LA viruses. This contrasts with what was previously found for TdV-LAbarr1, which appears to be a typical LA virus similar to those already found in S. cerevisiae, although it belongs to a different genus of yeast [12]. Phylogenetic Relationship and Transmission of LBC Viruses Only two clusters were found when comparing the nucleotide or amino acid sequences of all LBC viruses, both directly related to the host yeast species of the viruses. The S. cerevisiae cluster includes all ScV-LBC isolated from Extremadura, as well as two viruses isolated from elsewhere (ScV-LBC2-S3920 and ScV-LBC1-original); and the T. delbrueckii cluster includes the only two TdV-LBCbarr available, which are also isolated from of Extremadura although from locations sited about 70 km away. It appears that genome analysis of new LBC viruses, isolated from different habitats and locations in the world, is required in order to acquire comprehensive knowledge of the phylogenetic relationship of these viruses. Despite this, we did achieve some interesting findings, such as: (i) the same LBC virus can coexist with different killer M viruses in different S. cerevisiae strains, (ii) LBCbarr2 can support two different viruses (M1 and Mbarr1) in the same T. delbrueckii strain, and (iii) the same M virus can be supported by different L viruses (M1 by LA in S. cerevisiae or by LBCbarr2 in T. delbrueckii). This indicates that there is not an association or co-evolution of specific LBC with specific M viruses, contrary to that previously suggested [4]. Similar results were previously found for LA and M viruses [12]. Furthermore, the presence of the same M virus (M1) in different yeast species (S. cerevisiae and T. delbrueckii) raises the possibility of cross-species transmission among different yeast species, which eventually may coincide in the same habitat as wine fermentation [4,12]. However, the contrary may be suggested for LBC viruses, since Td-LBCbarr viruses are the most distant from the rest of Sc-LBC viruses (44% Gag-Pol identity), even from those of S. cerevisiae strains isolated from the same location ( Figure 5B). Besides this, as previously suggested for LA viruses [12], a co-evolution of a specific LBC virus with its specific yeast host can be hypothesized. The percentage of identity between Gag sequences of T. delbrueckii and Saccharomyces LBC viruses was lower than that found for full Gag-Pol or RdRp-domain sequences. These results are similar to that previously found for comparison between T. delbrueckii and Saccharomyces LA viruses. As a consequence, a comparison of Gag aminoacid sequence can be considered the best option for grouping LA and LBC viruses that are closely related [12]. Yeast Strains and Culture Media T. delbrueckii killer Kbarr1(EX1180) and Kbarr2 (EX1257) yeasts are prototrophic strains isolated from the spontaneous fermentation of grapes from vines located in the Albarregas river valley of Mérida and southern Badajoz (both in Extremadura, southwestern Spain), respectively [3,15]. S. cerevisiae killer strains EX231, EX1125, EX229, EX231, and EX1160 were also isolated from wine spontaneous fermentations in close locations of the Ribera del Guadiana region in Extremadura. These strains show different mtDNA RFLP profiles and contain different M dsRNA isotypes [3,32]. All these yeasts are also prototrophic strains. Table 3 summarizes the yeasts used in this study. The killer phenotype and presence of viral dsRNA (L and M) in these yeasts was analyzed previously [3,12,15]. Standard culture media were used for yeast growth [33]. Purification of V-LBC dsRNA from Killer Yeasts Nucleic acid Samples from killer yeast strains were obtained as described previously [12,34]. CF-11 cellulose chromatography was used to obtain the dsRNA from each yeast strain [35]. Agarose (1%) gel electrophoresis was used for separation of L and M dsRNA of each sample. The slower-moving dsRNA band (4.6 kb) was cut out of the gel and purified with RNaid ® Kit (MP Biomedicals, LLC, Illkrich, France). This procedure was repeated for each L virus to obtain at least 20 µg of dsRNA. Preparation of cDNA Libraries from Purified V-LBC dsRNA and DNA Sequencing Library preparation of cDNA and high-throughput sequencing (HTS) were done at the Unidad de Genómica Cantoblanco (Fundación Parque Científico de Madrid, Spain) as described previously [12]. Very briefly, the first strand of cDNA was synthesized using random primers dTVN and dABN (from Isogen Life Science, De Meern, The Netherlands) and SuperScriptIII retrotranscriptase. Thereafter, the second cDNA strand synthesis, end repair, 3 -end adenylation, and ligation of the TruSeq adaptors were done (Illumina). The adaptor oligonucleotides include signals for further amplification and sequencing, and also short sequences referred to as indices which allow multiplexing in the sequencing run. An enrichment procedure by PCR was performed to amplify the library, ensuring that all the molecules in the library included the desired adaptors at both ends. The final libraries were denatured prior to seeding on a flow cell, and sequenced on a MiSeq instrument using 2 × 80-2 × 150 sequencing runs. Sequence Assembly of Virus Genomes The analysis and assembling of cDNA sequences were done by the company Biotechvana (Technological Park of Valencia, Spain) as previously described [12]. SOAP deN-OVO2 [36] was used to obtain a de novo assembly based on two Illumina libraries for each virus, trying multiple assembly attempts with scaffolding and insert size of 200, and varying the Kmer value (being 47 the most effective). Contigs shorter than 300 nucleotides were removed from the contig file. The remaining contigs were used as input to the NR database of the NCBI via the BLASTX search protocol [37] implemented in the GPRO 1.1 software [38]. Highly significant similarity was found between several contigs/scaffolds and some known viral RNA sequences (LA, LBC, and others) or host transcripts. Supposed contaminating sequences non-homologous to previously known LBC genomes were filtered from the assembly. Each virus was sequenced at least three times using independent samples and different dates during a period of several years. Full coverage of the canonical genome sequence was obtained at least twice for each virus, and 100% identity was found for all sequences obtained from the same yeast strain. Only full coverage sequences were used for comparison of viral genomes from different yeasts. Sequence Analysis Tools The sequence identity and phylogenetic relationship (phylogram) among LA genomes were obtained by the ClustalW(2.1) program for comparing nucleotide sequences [39], and MUSCLE(3.8) program for comparing amino-acid sequences [40]. The MFOLD program (http://unafold.rna.albany.edu/?q=mfold/RNA-Folding-Form, accessed on 10 November 2021) was used to predict the folding of ssRNA [41]. The parameters used were: folding temperature fixed at 37 • C; ionic conditions, 1M NaCl, no divalent ions; percent suboptimality number, 5; upper bound on the number of computed foldings, 50; maximum interior/bulge loop size, 30; maximum asymmetry of an interior/bulge loop, 30; maximum distance between paired bases, no limit. Conclusions Td-LBCbarr viruses share some genome features (such as nucleotide sequence identity, ribosome frame-shift slippery site, and conserved 5 GAAATT) and most Gag-Pol functional motifs (involved in cap snatching and RdRp activity) with Sc-LBC viruses. However, TdV-LBCbarr2 resembles LA viruses in the genomic signals related to RNA encapsidation and replication, and in the motif A of RdRp domain; which provide it with a helper capability to maintain up to two M viruses in the same yeast cell. This raises the possibility of LBCbarr viruses being a new type of virus different to the already known LA and LBC viruses. The discovering of new viruses similar to LBCbarr will be required to examine this question. The co-evolution of the LBC and M viruses seems unlikely, although the co-evolution of LBCbarr viruses with a given yeast host may occur in a specific habitat or location. Funding: This research was funded by Extremadura Regional Government (Consejería de Economía, Ciencia y Agenda Digital), grant number GR18117 and the Spanish Ministry of Education and Science, and the European Regional Development Fund (ERDF-European Union), grant number AGL2017-87635-R. Rocío Velázquez gratefully acknowledges the support of a studentship from the Extremadura Regional Government. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable.	2021-12-19T16:51:43.373Z	2021-12-01T00:00:00.000	{ "year": 2021, "sha1": "b63e45d0a50875cdb49d24c8b65eef25888b72b0", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/1422-0067/22/24/13492/pdf?version=1639638389", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "aee1062edad12aba90900bbeaf1de2f1f5afdefb", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
233282004	pes2o/s2orc	v3-fos-license	Image Fusion based on Cross Bilateral and Rolling Guidance Filter through Weight Normalization Introduction: Image Fusion is the method which conglomerates complimentary information from the source images to a single fused image . There are numerous applications of image fusion in the current scenario such as in remote sensing, medical diagnosis, machine vision system, astronomy, robotics, military units, biometrics, and surveillance. Objective: In this case multi-sensor or multi-focus devices capture images of the particular scene which are complementary in the context of information content to each other. The details from complementary images are combined through the process of fusion into a single image by applying the algorithmic formulas. The main goal of image fusion is to fetch more and proper information from the primary or source images to the fused image by minimizing the loss of details of the images and by doing so to decrease the artifacts in the final image. Methodology: In this paper, we proposed a new method to fuse the images by applying a cross bilateral filter for gray level similarities and geometric closeness of the neighboring pixels without smoothing edges. Then, the detailed images obtained by subtracting the cross bilateral filter image output from original images are being filtered through the rolling guidance filter for scale aware operation. In particular, it removes the small-scale structures while preserving the other contents of the image and successfully recovers the edges of the detailed images. Finally, the images have been fused using a weighted computed algorithm and weight normalization. Results: The results have been validated and compared with various existing state-of-the-art methods both subjectively and quantitatively. Conclusion: It was observed that the proposed method outperforms the existing methods of image fusion. INTRODUCTION The multi-sensor images captured through multiple sensors systems have very limited information and are not capable of providing the complete information of the particular scene due to the principle physical limitations constraints in the sensor systems. It is difficult to get all the information about a particular scene when captured through a single sensor. Hence, image fusion techniques are employed to combine this multi sensory information together while avoiding the loss of information. Fusion algorithms combine two images captured through different sensors in a single image so that the adequate information of the particular scene is transferred to a single image that contains comprehensive visual data about the subject under study. Digital photography [1], military for weapon detection [2], helicopter navigation [3], medical sector, navigation, remote sensing, and military [4 -9] are the current areas of application in image fusion. In the field of medical imaging, the CT (Computed Tomography) images give the information related to the bone structures and in the case of the MRI image, it shows brain tissue anatomy and soft tissue detail. Image fusion techniques find widespread applications in surgical aspects in order to fetch adequate information corresponding to the specific target areas. In the literature section of this manuscript, we have discussed the different existing methods [10]. The main objective of the proposed method is to enhance the performance of the image fusion technique by combining two images captured from different sensors into a single image. By applying detailed images obtained by subtracting cross bilateral filter output and then a scale aware operation using rolling guidance filter, an improved fusion performance is demonstrated. The paper has been summarized as follows: Section 2 presents the literature survey. Section 3 presents the proposed method. Section 4 presents the discussion of the experimental framework including results and discussion and Section 5 describes the conclusion and future scope. LITERATURE SURVEY Numerous image fusion techniques have been implemented so far in the literature. Image fusion is performed at three processing levels, i.e., signal, feature, and decision. The signal level fusion of an image is also termed as the image fusion at the pixel level, which fuses visual information corresponding to each pixel from a number of registered images into a single fused image and is termed as fusion at the lowest level. The object-level fusion of image is also known as feature level fusion of image fusing feature descriptor information and object details which have been extracted from the individual input images. Finally, symbol or decision level fusion of image represents probabilistic decision information retrieved by local decision-makers using applying results of feature level processing on each image. Among a large number of image fusion methods proposed in the literature, the multi-scale decompositions approach (wavelet, pyramid) [11,12] and the data-driven techniques [13,14] are the most popular. However, these approaches are more prone to artifacts into a fused image. To overcome these issues, optimization-based image fusion techniques were proposed. However, these techniques exhibited a large number of iterations to obtain an appropriate fused image. Sometimes these techniques tend to do over smoothing of the fused images due to multiple iterations. Subsequently, the edge-preserving techniques are currently popular in the field of image fusion. These processes of image fusion use edge-preserving smoothing filters for the motive of fusion. Over a decade, researchers and scientists have implemented plenty of studies in the field of image fusion [15 -18]. The lifting-based wavelet decomposition [19] is proposed to minimize the mathematical complexity with a smaller memory requirement for image fusion. An image fusion technique for the satellite images using multi-scale wavelet analysis is presented [20]. Also, the pixel level multi-focus fusion of image was introduced on the basis of the image blocks and artificial neural network [21]. Further, the image fusion at pixel level was presented by decomposing the input images by applying wavelet, curvelet, bandalet, and contourlet transform [22 -24]. An image fusion method [25] employs Discrete Cosine Transform (DCT) based fusion of images instead of pyramids or wavelets and the performance of these techniques is similar to convolution and lifting based wavelets. Further, Discrete Cosine Harmonic Wavelet Transform (DCHWT) [26] has been introduced to reduce the mathematical complexity. A variant of standard bilateral filter, known as the cross bilateral filter was introduced with pixel significance which employs a second image to shape the filter kernel [27,28]. In a study [29], a Bilateral filter is used for multiscale decomposition of multilight image collections for detail and shape enhancement. Also, temporal joint bilateral filter and dual bilateral filter were introduced [30] for multispectral video fusion in which the former employs infrared video to find filter coefficients and filters the visible video with respect to these coefficients. Further, the application of bilateral filter for the hyperspectral fusion of images was introduced [31], which separates weak edges and fine textures after subtracting bilateral filter results from the input image. The magnitude of these different images is used to detect the weights directly. Another version of a bilateral filter, which employs center pixel from the infrared image and the neighborhood pixels from the visual image to locate the filter kernel and works on the infrared image, was introduced for human detection through the fusing of infrared and visible images [32]. Further, multiscale directional Bilateral Filter (BF), which is the combination of the bilateral filter and Directional Filter Bank (DFB) was introduced for a multisensor fusion of images to accomplish the edgepreserving capability of the bilateral filter and directional detail capturing capability of Directional Filter Bank (DFB) [33]. METHODOLOGY The proposed method addresses the shortcomings of the previously existing image fusion methodologies. The proposed method fuses two medical images taken from two different sensors. Firstly, the proposed algorithm uses a cross bilateral filter that uses one image for finding the kernels and the other for the filtering. The detailed images are obtained after subtracting cross bilateral filter output from the respective input images. Then details image are input to the rolling guidance filter for scale aware operation. It removes the smallscale structures while successfully recovering the edges of the detail images. Here, the weights are computed by calculating the strength of the detail images obtained after scale aware operation. Finally, the computed weights are multiplied with the source images followed by weight normalization (Fig. 1). Cross Bilateral Filter The bilateral filter is a local, nonlinear, and non-iterative method which integrates the low pass filter and edge stopping mechanism that reduces the filter kernel when the intensity difference between the pixels is more. As both levels of gray similarities and geometric closeness of neighborhood pixels are taken into considerations, the weights of the filter depend on both Euclidian distance and the distance in gray or color space. It smoothens the image by preserving the edges applying neighborhood pixels. Mathematically, for an image X, bilateral filter output at a location of the pixel m is estimated below as follows [34]: (1) where G δs (\|\|m -n\|\|) is a geometric closeness function, and G δr (\|X(m) −X(n)\|) is the level of gray similarities or edge stopping function is known as normalization constant \|\|m-n\|\| is Euclidean distance between m & n as well as S is a spatial neighborhood of m. The values of δs and δr check the behavior of the bilateral filter and the dependency of δ r /δ s values and the derivative of the original signal on the behavior of the bilateral filter is studied elaboratively [35]. The appropriate δs value is chosen based on the optimal amount of LPF (Low pass filter) and blurs more for higher δs, since it integrates values from the large distance locations [36]. Also, if an image is scaled up or scaled down, δ s must be adjusted in order to retrieve desired results. It seems that an optimal range for the δ s value is (1.5-2.1); likewise, the ideal value for the δ r relies on the amount of the edge to be preserved accordingly. If the images get attenuated or amplified, δ r has to be adjusted in order to obtain the optimal result. Cross bilateral filter considers both levels of gray similarities and the geometric closeness of neighborhood pixels in image X to adjust the filter kernel and filters the image Y. Cross Bilateral Filter output of image Y at a pixel location m is estimated as [37]: (2) Where G δs (\|\|m -n\|\|) is a geometric closeness function G δr (\|X(m)−X(n)\|) is the level of gray similarities or edgestopping function is known as normalization constant. Here, the detailed images retrieved after subtracting cross bilateral filter output from the respective source images, for image X and Y are given by X D = X-X CBF and Y D = Y-Y CBF, respectively. In the Fig. (2) represents stimulated input Dataset 3 images and corresponding Cross Bilateral Filter output along with detail images of the same. Rolling Guidance Filter The method involves two pivotal steps, i.e., small structure removal and edge recovery. Small Structure Removal The first step is to remove small structures. As mentioned, the Gaussian filter is related to structure scale determination. Therefore, we illustrate this operator in the weighted average form, which uses the input image as X D and Y D and output image X RGF and Y RGF . Let m and n are index pixel coordinates in the images and δs is the standard deviation. Then, we express the filters as: (3) (4) and N(m) is a set of the neighboring pixels of m. This filter removes the structures whose scale is less than the δs according to the scale space theory. It is implemented competently by separating kernels in the perpendicular directions. Edge Recovery The iterative edge recovery process forms the essential contribution in this method. Here, images I RGF and J RGF are iteratively updated. We represent and as the result in the t-th iteration. In the beginning, and are set as X RGF and Y RGF in equation (3) and (4) which is the outcome of the Gaussian filtering. The value of the I RGF t+1 and J RGF t+1 in the tth iteration is retrieved in a joint bilateral filtering form given the source X D and Y D and the value as per earlier iteration and . X D and Y D are the same input images used in equation (3) and (4), δs and δr check the spatial and range weights accordingly. The expression can be understood as a filter that smoothes the source images X D and Y D guided by the structure of X RGF t and Y RGF t . The process differs in nature from how earlier methods engage a joint lateral filter that iteratively changes the guidance image and obtains illuminating effects. PIXEL BASED FUSION RULE The fusion rule proposed in the paper [38] is discussed here for completing the comparison of the performance of the proposed method. The weights are calculated by applying the statistical properties of a neighborhood of detail coefficients instead of wavelet coefficients [38]. The window of the size w x w around a detail coefficient X RGF (p,q) or Y RGF (p,q) was observed as a neighborhood to calculate its weight. The neighborhood is represented as matrix S. Each row of the matrix S is considered as an observation and column as a variable to calculate unbiased estimate C h p,q of its covariance matrix [39], here p & q are spatial coordinates of the detail coefficientsX RGF (p,q) or Y RGF (p,q) Where a k is the k th observation of w dimension variable and is the mean of the observations. It observed that the diagonal of matrix C h p,q gives the vector of variances for each column of matrix S. The eigenvalue of matrix C h p,q is calculated and the number of the eigenvalues directly depends on the size of C h p,q . The sum of these eigenvalues is directly equivalent to the horizontal detail strength of the neighborhood and is represented as HdetailStrength. Likewise, the non-biased covariance C v p,q is calculated by treating each column of S as an observation and row as a variable (opposite of that of C h p,q ), and the sum of eigenvalues of C v p,q gives vertical detail strength VdetailStrength. It is described as: Where eigen k is the k th eigenvalue of the unbiased estimate of the covariance matrix. The weight assigned to a particular detail coefficient is calculated by adding these two details strengths. Hence, the weight depends only on the strength of the details and not on the actual intensity values: After calculating the weights for all detail coefficients relating to both source images, the weighted average of the source images will represent in the fused image. Here, WT x and WT y are the weights of detail coefficients X RGF and Y RGF which belong to the respective input images X and Y, then the weighted average of both is calculated in the fused image using Equation 9. (9) PARAMETERS FOR EVALUATION OF THE FUSION PERFORMANCE Evaluation of image fusion performance is a difficult task since it does not possess any ground truth for comparison. In literature, numerous parameters have been presented to measure the performance of the image fusion. Some of the parameters are considered to evaluate the performance of our study. They are as follows [22,38]: Average pixel intensity (API) or mean ( ) measure an index of contrast and is represented by: (10) Where f(a,b) is the pixel intensity at (a,b) and pxq is the size of the image 2. Standard Deviation is the square root of the variance, which considers the spread in data and is, given by: 3. Average Gradient estimates a degree of clarity and sharpness and is represented by: (12) 4. Entropy measures the amount of information constitute in the image and represented by: (13) where Z k is the probability of the intensity k in an 8-bit image. Mutual Information quantifies the overall mutual information between original images to the fused image, which is represented by: Where is the mutual information between input image X and fused image Z And is the mutual information between the input image Y and fuse image Z. Fusion Symmetry or Information Symmetry (FS or IS) represents how much symmetric the fused image with regards to input images and is represented by: Covariance(S)=E [(S-E[S]) (S-E[S] T ] where And 8. Spatial Frequency calculates the entire information level in the areas of an image and is calculated as: where And In addition, object fusion of image performance characteristics [18,40] based on gradient information is taken into consideration. This gives an in-depth analysis of fusion performance by total fusion performance, fusion loss, and the fusion artifacts or artificial details [18,40], and the symbolic representation is given as: (17) In most of the cases, it observed that the addition of these parameters may not lead to unity. Hence, these parameters are revised and the modification has been proposed for calculating the fusion artifacts [26]. The equation is given as follows for completeness: (18) Where indicates positions of fusion artifacts in which fused gradients are stronger than input. RESULTS AND DISCUSSION The experiments were carried out on a different set of medical images [43,44]. In this paper, image fusion performance analysis and comparison are executed for three standard test pairs of medical images namely medical (Dataset 1), medical (Dataset 2), and medical (Dataset 3). A fused image of the proposed method is compared with different methodologies discussed [5,6,41,42] with the simulation parameters specified in the respective techniques. The parameters applied for the proposed method are δs = 1.8, δr =4, and kernel size=7. The conventional performance parameters as follows: API, AG, CC, SF, Entropy H, FS, MI, and SD are shown in Table 1, and the objective performance parameters Q XY/Z , L XY/Z , N XY/Z and N m XY/Z along with their respective sum are presented in Table 2. The quality of the fused image is better in case of the higher Q XY/Z factor and lower values for L XY/Z , N XY/Z and N m XY/Z for better performance. Since the aim of image fusion is to transfer maximum information from source to the fused image for better performance and enhance accurate, stable, and comprehensive details such that the fused image is more suitable for human perception, visual analysis and qualitative analysis. The competent image fusion techniques should possess the following three characteristics for better visual analysis; (1) It must transfer the basic or adequate information from the primary image to the fused image. (2) It must not lose the information of the primary image during the process of image fusion. (3) It must not produce any noise or artifacts to the fused image. In order to compute the performance visually, the fused images of Dataset 1, Dataset 2, and Dataset 3 are shown in Figs. (3-8). Fig. (3) represents the source images, output image represented by the proposed method in R, and images of other methods presented in the S [5], T [6], U [41], and V [42], respectively. From Tables 1 and 2 it can be observed that the image quality of the proposed method outperformed other methods due to less losses of information and good visual performance. The proposed method was able to extract more appropriate information from source images to the fused image as compared to other methods. Tables 1 and 2 indicate that the method [5] is better compared to other methods but the proposed method is distinctly better than the method [5]. The proposed fused image has shown better performance in the case of FS, CC, N XY/Z and N m XY/Z . Our method has high Q XY/Z (appropriate contents transferred from original images) and low L XY/Z (loss of information is less) and also the results were verified by the visual perception of the fused images. Figs. (6, 7 and 8) show the actual visual images of the medical images compared with different methodologies. It was observed that the fused images obtained through the proposed method are comparatively better than the fused images of T ( [6], U [41], and V [42]. It was also observed that the image quality of the fused images of S [5]and proposed method R shows more similarities and the visual as well as the experimental information obtained from both shows more similarities, but the proposed method has shown improvements in all the factors necessary for quality and transfers of the information from source images to the fused images. Images of fusion results of S [5] 1 and 2, we can observe that the proposed method comparatively performed better than the existing method in terms of the Q XY/Z factor. In the case of all the medical images, L XY/Z and N XY/Z factors of the proposed method were comparatively less than the other previous method, which is good for the performance of image fusion. It is clear from the image fusion visual qualities, simulations and experimental results that the proposed method performs well as compared to other methods in terms of the qualities and quantities parameters. CONCLUSION In this manuscript, source images are processed with cross bilateral filter and detailed images are obtained after subtracting the cross bilateral output images from source images. The detailed images so obtained are input to the rolling guidance filter for scale aware edge preservation operation. This filtering process removes the small-scale structures while preserving the textures of the image and recovers the edges of the detailed images. The output of the rolling guidance filter is used for the computation of the weights. The weights are computed by measuring the strength of horizontal and vertical information in the source images. The computed weights are multiplied with the source images followed by weight normalization to obtained a fused image. A pair of multi-sensor medical images was tested in the proposed method to see the performance of the proposed as well as existing image fusion methods. It was observed that the proposed method performed better in almost all objective parameters compared to other methods. The application of the other type of filters instead of applying cross bilateral filter for obtaining the detail layers as well as rolling guidance filter for performing scale aware operation can be the potential future scope of the research work. ETHICS APPROVAL AND CONSENT TO PARTICIPATE Not applicable. HUMAN AND ANIMAL RIGHTS No human and animals were used in this study. CONSENT FOR PUBLICATION Not applicable. AVAILABILITY OF DATA AND MATERIALS Not applicable. FUNDING None.	2021-04-17T20:37:21.691Z	2020-12-31T00:00:00.000	{ "year": 2020, "sha1": "716d759f8fd7984d754b5c14ccb36fb327782f87", "oa_license": null, "oa_url": "https://doi.org/10.2174/1874440002013010051", "oa_status": "GOLD", "pdf_src": "MergedPDFExtraction", "pdf_hash": "716d759f8fd7984d754b5c14ccb36fb327782f87", "s2fieldsofstudy": [ "Computer Science", "Engineering" ], "extfieldsofstudy": [ "Computer Science" ] }
265352812	pes2o/s2orc	v3-fos-license	Determinants of oral health status: an ecological study in Iran Objectives The aim of this ecological study was to assess the association between behavioral, social position, circumstance factors, and caries experience in 35- to 44-year-old adults in Iran at a provincial level. Materials and methods The data from the 2011 Iranian Oral Health Survey were obtained from all 31 provinces across Iran on the population level. Oral health status was measured as the number of decayed, missing (MT), and filled (FT) teeth and the percentage of the population who were edentulous. Data were also gathered from each province on the percentage of smokers (Non-Communicable Diseases Risk Factors Surveillance Provincial Report 2009), per capita consumption of free sugars, concentration of fluoride in the drinking water (National and Sub-national Burden of Disease (NASBOD) Survey), number of dentists per 10,000 people, mean years of schooling of adults, expected years of schooling of children, life expectancy at birth and Gross National Income (Integrated Public Use Microdata Series, Global Data Lab). The data were analyzed using simple and multiple linear regression (α = 0.05). Results Mean DMFT was positively associated with the percentage of smokers (B = 0.01 95%CI 0.01–0.14), and negatively with fluoride concentration (B =-2.6 95%CI -4.3- -0.96). The edentulousness percentage was positively associated with smoking (B = 0.2 (with 95%CI: 0.07–0.37) and negatively with mean years of education (B =-1.08 (with 95%CI: -2.04- -0.12). DT was associated with expected years of schooling (B =-0.6 (with 95%CI: -1.07- -0.17), negatively. Mt was negatively associated with life expectancy (B =-0.5 (with 95%CI: -1.1- -0.007), fluoride concentration (B =-3.4 (with 95%CI: -4.5- -1.5) and number of dentists per 10,000 people (B =-0.4 (with 95%CI: -0.8- -0.01). Mean Years of Schooling (B = 0.5 (with 95%CI: 0.2–0.8) and number of dentists per 10,000 people (B =-0.62 (with 95%CI: 0.51 − 0.48) were positively in associated with FT. Conclusions The present findings indicate that there were differences in the oral health measures and their social determinants among the provinces of Iran. Regarding the limitations of the study especially the limitation of the number of independent variables, it seems, this discrepancy could be better explained by social variables of the provinces such as income than by environmental factors. Introduction Oral conditions have been considered as a major public health problem that affected approximately 3.5 billion cases worldwide in 2017.According to the Global Burden of Disease estimates, the highest prevalence of dental disease is mainly in low and middle-income countries [1].The DMFT of 35-44 years old adults in Iran, as a middleincome country in the Eastern Mediterranean region, is about 13.2 ± 0.16 and the prevalence of edentulousness is about 4% [2].However, obvious disparity in the oral health status of people has been shown among different provinces in Iran.The findings of a recent study among 128,813 adults aged 35 suggested that DMFT was mainly concentrated among the socioeconomically disadvantaged ones [3].Inequality in the number of nonreplaced extracted teeth also has been reported among adult populations in Iran (greater prevalence among participants who had less than 12 years of schooling and those in the poorest quintile regarding wealth index) [4].It was claimed that educational background, affects nonmaterial characteristics such as oral health literacy, oral health behaviors (like dietary and tooth brushing habits) and health service utilization frequency and patterns [4,5]. On the other hand, the role of structural determinants including economic, social and welfare policies in establishing social hierarchies and influencing the socioeconomic status of individuals within societies has been highlighted in the WHO conceptual framework for action on the social determinants of health [6].It is underlined that structural factors can impact health through intermediate determinants such as housing and working conditions, social capital, psychosocial factors, social support, and access to health care [7].It has been reported that there are substantial social disparities in the distribution of dental services across the country and generally dentists are more likely to be located in the provinces with better social ranks [8]. Over time, behavioral sciences have expanded our understanding of oral health beyond "disease" to a broader biopsychosocial concept of oral health and to the role of lifestyle or behavioral determinants, known as proximal risk factors [9,10].Diet high in free sugars and behaviors such as tobacco smoking, oral hygiene, and use of dental services have been identified as key behaviors that are critical for oral health [11].Good oral hygiene is key to preventing and maintaining periodontal health while its role in preventing dental caries is less conclusive [12].Regular dental attendance could be important in identifying oral health problems at early stages, managing them conservatively, and improving the adherence to preventive care [13].Sugar is increasingly recognized as a global public health issue that is related to both social and commercial determinants of health.Based on the WHO updated guidance on the sugar intake for adults and children, the intake of free sugars throughout the life course is currently high, and it should be reduced to less than 10% of total energy intake.However, WHO suggests a further reduction of the intake of free sugars to below 5% of total energy intake as a conditional recommendation [14].Smoking affects oral disease locally and systemically through vasoconstriction caused by nicotine leading to the enhancement of subgingival anaerobic bacteria colonization.Recent research also reported that tobacco smoke alter the bacterial surface and promote biofilm formation [15]. Recognition of the social determinants of oral health inequalities has crucial implications for strategy development at the local, regional and national levels [16].Based on the current evidence, it is emphasized that future action on tackling oral health inequalities requires a reorientation of oral health policy away from a mere focus on changing oral health behaviors to further actions on the common social determinants [17].However, as oral health surveys do not usually provide information on the role of socioeconomic factors, other study designs such as ecological studies are usually needed as complementary analyses.Therefore, this ecological study aimed to assess the association between different relevant factors and caries experience in 35-to 44-year-old adults in Iran. Materials and methods The framework developed by Peres et al. [18], based on the Watt and Sheiham's framework of social determinants of oral health [9], was used as the guiding map in order to decide which determining factors be considered in the analysis (Fig. 1).The unit of analysis was the provinces.No sampling method was used because the data of all 31 provinces were retrieved.All of the recruited data were published at the province-level. Oral health status Mean DMFT and percentage of edentulous population were used as outcome variable.National statistics on dental caries experience for 35-to 44-year-old adults were obtained from the latest published National Oral Health Survey conducted during the 2010-2011 period, which was based on the WHO Oral Health Survey basic method [19].This survey was undertaken by the Oral Health Bureau in the Ministry of Health and Medical Education. Behavioral factors To examine the association of the behavioral determinants with outcome measures, the diet and smoking status were chosen; the provincial level of percentage of smokers among the 35-44 year old population was obtained from the Non-Communicable Diseases Risk Factors Surveillance Provincial report 2009 [20].The provincial per capita consumption of free sugars was also found in the 2010 provincial report of the free sugar and other foods consumption in Iran [21]. Environmental factor The concentration of fluoride in drinking water was considered as one of the (environmental) determinants with the data obtained from the report of the provincial concentration of 15,039 water samples taken from rural and urban water resources in 31 provinces of Iran in 2010, including wells, springs, rivers, water reservoirs, water distribution, and aqueducts-a main source of drinking water in Iran [22]. Health service availability factor To estimate the role of health service availability, the provincial-level data on the number of dentists per 10,000 people practicing in public and private sectors in each province based on the 2010 report of Iran Medical Council was used [23]. Socio-economic factors To consider the socio-economic status at provincial level, the indices of Subnational Human Development Index (SHDI) [24] were used.SHDI is an average of the subnational values for three main dimensions of education, health and standard of living which are measured through the following indicators: 'Mean years of schooling of adults aged 25+' and 'expected years of schooling of children aged 6' for measuring the education level; 'life expectancy at birth' , as the major indicator of health, and 'Gross National Income per capita' (PPP, 2011 US$) for measuring the standard of living. The provincial data set of the year 2011 for Iran was elicited from the website of Global Data Lab (GDL) [25]. Statistical analysis Since both the outcome and the independent variables were measured on a continuous scale, data was analyzed using simple and multiple linear regression to estimate the association of independent (explanatory) factors with the DMFT index and its components (DT, MT and FT), as well as the edentulousness percentage, using SPSS (IBM SPSS Statistics 22).The level of significance was considered as 0.05. Results The proposed data set was obtained for all 31 provinces in Iran, except sugar consumption, which was available only for 20 provinces at the time of the study. Table 1 presents descriptive statistics for the outcome and independent variables used in this study.Regarding the dental caries, the means of DT, FT and MT were 4.4 ± 1.2 (1.8-6.9),2.1 ± 1.1 (0.3-5.1) and 7.3 ± 1.8 (2.8-10.8),respectively.In other words, MT defined about 52.3% of the total DMFT.The proportions of the FT and DT components in defining the total DMFT were 15.5% and 32.2%, respectively. The distribution of DMFT and edentulousness were normal (testing by Shapiro-Wilk normality test) therefore we used linear regression.The Shapiro-Wilk statistics for DMFT and mean percentage of edentulousness were 0.94 (p = 0.09) and 0.93 (p = 0.06), respectively.The results of the simple linear regression for DMFT and edentulousness are shown in Table 2.There was a positive correlation between the mean DMFT and the percentage of Similarly, regarding edentulousness, the only factor with a positive association was smoker percentages (p < 0.001, r = 0.5, B = 0.24 with 95%CI: 0.1-0.38). Multiple linear regression analysis was used to identify the best indicators of mean DMFT and edentulousness using a stepwise technique (Table 4).The fluoride concentration (B= -2.6 with 95%CI: -4.3 --0.94) and the percentage of smokers (B = 0.08 with 95%CI: 0.01-0.14)Regarding the edentulousness percentage as outcome measure, percentage of smokers and mean years of schooling were remained as the main indicators.The tolerance and VIF scores were 0.98 and 1.01, respectively.These two indicators explained 44% of the variation in percentage of the edentulousness.The indicator factor for DT was expected years of schooling, which explained 34% of the variation of DT.The factors with strongest association with MT were fluoride concentration, life expectancy and dentist per 10,000 people that had the tolerance of 0.95 to 0.89 and VIF of 1.04 to 1.1 and explained 67% of the MT variation.Regarding FT, mean years of schooling and dentist per 10,000 people remained in the model that explained 78% of the variation of the FT.The tolerance and the VIF scores were 0.52 and 1.9, respectively. Discussion In our ecological study, the association of the determinants of oral health at provincial level was assessed. Ecologic studies are valuable study designs that may be promising in casting light on etiologic relationships.However, they could not demonstrate the existence of true associations conclusively [26].The studies on aggregated data, as our ecological study, could benefit from some critical advantages over the individual-based studies [27], such as explaining the contextual effects on the prevalence of disease, lower cost, analytical simplicity and ethical appropriateness, plus the possibility of measuring the population-level exposures, e.g., the Human Development Index of a region.Our study also benefited from the availability of the national surveys data with the similar measurement scales of all provinces of Iran.The data made it possible to compare variables vigorously across the provinces.However, it is critical to avoid the ecological fallacy as the main limitation of inference from the ecological studies.It refers to the impossibility of inferring the results obtained at the population level to the individual level [26].Furthermore, in the current ecological study, the number of independent variables included in the analysis was limited and we were not able to consider all the proposed determinants based on the guiding frameworks.Specially, variables such as gender, urban/rural distribution, age groups and cumulative life-time effects of exposure to risks, ethnic variation within the provincial data, alcohol consumption, and oral health behavioral factors were not available.Besides, data for sugar consumption was only available from 20 provinces.Furthermore, the data might be old.However, as the last national survey among adults was held in 2011, we had to collect the other data for the same time period.Another limitation was that periodontal diseases were not assessed in the national survey and accordingly was not analyzed in our study.Availability of such data, might be precious for evaluating the proposed paths of determinants such as smoking and outcomes such as edentulousness or MT. Based on the results of simple regression analysis, the main two outcomes, i.e.DMFT and edentulousness, were associated with the percentage of smokers (positively) and the fluoride concentration (negatively).This finding was reconfirmed in the multiple regression model.Factors such as GNI, years of schooling and expected years of schooling significantly explained the variation of DT and FT components across provinces.In particular, based on the results of multiple regression models, the FT mean was highly and positively associated with the education level and the proportion of dentists to population. It was notable that while the lower mean years of schooling was not associated with the higher aggregate disease experience (i.e., DMFT), it was associated with the type of the received dental treatment, either filling or extraction.The same results were also reported in the study conducted by Mejia et al. on Australian adults [28], where social gradients in caries were evident but particularly notable in Missing and untreated Decay.Their findings, thus, indicated that social gradients for dental caries could have a greater effect on how the disease was treated, as compared to lifetime disease experience.The number of dentists per 10,000 people was another factor with a strong and direct impact just on FT.Since the per capita number of dentists was higher in the more affluent provinces with higher GNI, this finding indicated that dentists tended to be located in more advantaged parts of the country, while residents of disadvantaged provinces who faced more difficulty in meeting their basic dental needs had less access to dental services.It could serve as a good example of the inverse care law and might enforce inequities in the prevalence of untreated dental caries [29]. According to the results of the national survey, about 5% of the population in Iran could be considered edentulous and about 52.3% of the total DMFT was defined by the missing teeth (MT).Tooth loss is a complex outcome considered as an effective marker of a population's oral health, reflecting both the accumulated individuals' history of dental disease and the characteristics of the oral health care system [30].In a systematic review conducted to assess the main reasons for extractions of permanent teeth in adults, it was suggested that dental caries and periodontitis were the main indications for dentists to perform dental extractions [31]. The association of tooth loss with lower income and schooling, as reported in different other studies [32], could be explained, at least partly, by the fact that poorer and less educated individuals have less access to dental services and oral hygiene products [33].Also, they usually consume more sugar [34] and brush their teeth less frequently.On the other hand, people with higher schooling have more dental appointments and higher self-perception about the state of their oral health condition and the need for dental treatment [35].The reasons for more extractions among people with lower schooling, therefore, could be the result of extensive untreated disease or the tendency to choose the lowest cost services [36]. Although the prevalence of complete tooth loss in our study population was not considerable (about 5%), the impact of tobacco consumption and low exposure to systemic fluoride on edentulousness was visible.The association of tobacco consumption with MT and the eventual complete tooth loss might be mediated by the correlation of tobacco and periodontal disease as the main risk factor for tooth loss in adults [37].However, since the focus of our study was mainly on dental caries, a deeper analysis of the mediation by periodontal conditions would be helpful in the future studies. Low exposure to systemic fluoride has been recognized as one of the factors contributing to caries progression.In a study undertaken on Australian younger adults, the lifetime fluoridation exposure led to the significantly and substantially lower DMFT and number of filled teeth [38].Griffin et al., in their systematic review, also concluded that water fluoridation reduced caries by 27% in adults [39].Another study compared the state of teeth in young adults who had consumed fluoridated water from birth to 5-8 year of age with the subjects who had nonfluoridated water, revealing that systemic fluoride could lead to less missing teeth and lower progressed dental caries [40].In another ecological study conducted by Ekstrand et al., fluoride level of the drinking water was a significant variable explaining the variations in mean DMFS among municipalities in Denmark [41].The strong association of lower missing teeth and DMFT with a higher concentration of drinking water fluoride in our study might be explained by the same effect as the progressed dental caries could usually result in extraction. Findings of this study, in line with those of the ecological study of Antunes et al. [42], which was carried out at district level, showed that the more distant and impoverished areas had a higher level of dental caries prevalence.In Addition, the ecological study by Pattussi et al. [43] concluded that the social inequalities were associated in a significant way with the inequalities found in the distribution of cavities. To conclude, there was an apparent discrepancy among the provinces of Iran in the oral health measures and their social determinants.The social gradient was evident, particularly in the distribution of "decayed" and "filling" teeth.In light of the study's limitations, particularly the constraint on the number of independent variables, it appears that the observed disparity can be more effectively elucidated by social determinants such as the Human Development Index and provincial income levels rather than environmental factors.Nevertheless, in order to arrive at a more definitive conclusion and ascertain the precise extent to which each factor contributes to the explained variance, it is imperative to conduct more comprehensive investigations encompassing a broader range of variables.Average years of schooling could explain mean number of decayed, missing, and filling teeth, separately, but not the aggregate mean DMFT of the population-indicating the limitation of DMFT as the most commonly used dental indicators. Fig. 1 Fig. 1 Guiding framework for social and commercial determinants of oral diseases Table 1 descriptive indices of the outcome and independent variables at the province level Note-, Edentulousness has been reported as mean of prevalence in each province Table 2 Simple regression analysis of predictor factors on DMFT mean and edentulousness percentage in all 31 provinces Note-= P-value < 0.05, **= P-value < 0.001 were the strongest indicators for DMFT and remained in the model.This model explained 47% of the variation (R 2 ) in DMFT mean across provinces.The collinearity of indicators was checked by tolerance and Variance Inflation Factor (VIF) that were 0.87 and 1.1, respectively, for both variables indicating low level of collinearity. Table 4 Stepwise multiple linear regression analysis of predictor factors on DMFT and its components mean and edentulousness percentage in all 31 provinces	2023-11-23T14:50:46.844Z	2023-11-22T00:00:00.000	{ "year": 2023, "sha1": "799e05b7a4147ca29be6a54b77af091aeca73684", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Springer", "pdf_hash": "799e05b7a4147ca29be6a54b77af091aeca73684", "s2fieldsofstudy": [ "Medicine", "Sociology" ], "extfieldsofstudy": [ "Medicine" ] }
9022594	pes2o/s2orc	v3-fos-license	FRS2α Regulates Erk Levels to Control a Self-Renewal Target Hes1 and Proliferation of FGF-Responsive Neural Stem/Progenitor Cells Fibroblast growth factor (FGF) is among the most common growth factors used in cultures to maintain self-renewal and proliferative capabilities of a variety of stem cells, including neural stem cells (NSCs). However, the molecular mechanisms underlying the control by FGF have remained elusive. Studies on mutant mice of FGF receptor substrate 2α (FRS2α), a central mediator for FGF signaling, combined with FRS2α knockdown or gain-of-function experiments, allowed us to dissect the role of FGF signaling for the self-renewal and proliferation of NSCs and to provide novel molecular mechanisms for them. We identified Hes1 as a novel self-renewal target of FGF-signaling. Quantitatively different levels of Erk activation mediated by FRS2α may regulate self-renewal of NSCs and proliferation of neural stem/progenitor cells (NSPCs); low levels of Erk activation are sufficient for the former, however, higher levels are required for maximum activity of the latter. Thus, FRS2α fine-tunes the FGF-signaling to control qualitatively different biological activities, self-renewal at least partly through Hes1 versus proliferation of NSPCs. Stem Cells 2010; 28:1661–1673. INTRODUCTION Fibroblast growth factor (FGF) signaling mediates diverse cellular responses during embryonic development and plays critical roles in the physiological and pathological processes of the adult organism [1]. Particularly, FGF signaling plays a crucial role in the self-renewal and proliferation of various stem cells such as human embryonic stem cells, humaninduced pluripotent stem cells, trophoblast stem cells, and neural stem cells (NSCs) [2,3]. Moreover, it has been recently reported that cancer stem cells derived from various types of human tissues, such as brain and breast tissues, can be cultured in vitro in the presence of FGF [3]. Many studies indicate that FGF signaling has critical functions in both self-renewal and proliferation of neural stem/ progenitor cells (NSPCs) in vitro and in vivo [4][5][6][7]. The molecular mechanisms by which FGF controls the two distinct processes, that is, the self-renewal and proliferation of stem cells, are still unclear. The lack of such knowledge prevents the development of appropriate methods to culti-vate stem cells with suitable qualities, which are critical to regenerative medicine such as cell replacement therapy for disease-damaged tissues or organs. The membrane-linked docking/scaffolding adaptor FGF receptor substrate 2 (FRS2)-a acts as a central mediator for FGF signaling [1,[8][9][10][11]. Once stimulated by FGF, FRS2a becomes tyrosine-phosphorylated and creates two specific binding sites for Shp2, an SH2 domain-containing tyrosine phosphatase and four binding sites for the adaptor protein, Grb2, leading to activation of Ras-Erk pathway [12,13]. We previously examined contribution of each site for FGFinduced activation of Erk by exogenously expressing wildtype FRS2a or its mutants in Frs2a À/À mouse embryonic fibroblasts [13]. Strong activation of Erk is observed in cells expressing the wild-type FRS2a or FRS2a-4F, in which tyrosine residues of Grb2-binding sites are replaced with phenylalanine, though the cells expressing FRS2a-4F shows slightly lower levels of Erk activity than wild-type. FRS2a-2F, in which tyrosine residues of Shp2-binding sites are replaced with phenylalanine, shows more reduced levels of activation of Erk and FRS2a-6F, a combined mutant of both Grb2-and Shp2-binding sites, shows least levels of Erk activity. Thus, the Shp2-binding sites in FRS2a have a primary role in the activation of Erk, whereas the Grb2-binding sites have a secondary role in the activation of Erk [13][14][15]. Frs2a is ubiquitously expressed during development [16]. To analyze the functions of FRS2a, we previously constructed a Frs2a-knockout mouse and two knockin mice that express a mutant form of FRS2a whose tyrosine residues of Grb2 (Frs2a 4F mutant)-or Shp2 (Frs2a 2F mutant)-binding sites are replaced with phenylalanine and demonstrated its critical functions in multiple developmental processes that are dependent on FGF-signaling. Embryos of the Frs2a-knockout mouse have defects in the FGF4-dependent maintenance of trophoblast stem cells and shows developmental retardation, resulting in embryonic lethality by E8 [12]. Frs2a 4F/4F mice can survive as adults and show no gross morphological defects except for eyelid developmental defects that arise with low penetrance [15]. Frs2a 2F/2F mice display multiple developmental defects along with perinatal death. Embryos of Frs2a 2F/2F mice lack carotid bodies [17], have defective eye development and show anophthalmia or microphthalmia [15]. The expression of Frs2a is particularly strong in the ventricular zone (VZ) of the developing cortex, where radial glial cells (embryonic NSPCs) are localized [16]. In addition, Fgfs, together with the genes for their receptors (Fgfrs), are also expressed in the VZ [18][19][20][21]. By analyzing the Frs2a 2F mutant mice, we previously demonstrated that these sites are required for the proliferation of neural progenitor cells but are dispensable for the self-renewal of NSCs both in vivo and in vitro [22]. However, previous studies indicate that FGF signaling is required for the self-renewal of NSCs [23,24]. Given that FRS2a is a central mediator of FGF signaling, we decided to further explore the function of FRS2a in NSCs. In this study, we performed gain-of-and loss of-function analysis of FRS2a both in vitro and in vivo. These analyses showed that the quantitative regulation of Erk activation levels by FRS2a is important not only for the proliferation of NSPCs but also for the self-renewal of NSCs. Moreover, we identified Hes1 as a novel target of FGF-signaling for the self-renewal of NSCs. Our results suggest that FRS2a stands as a central player in the regulation of the delicate balance between the numbers of NSCs and progenitor cells to maintain proper homeostatic number of neural cells in cultural systems and cortical development. Statistical Analysis Data in the graphs are represented as mean 6 SD. The statistical significance was assessed with Student's t-test or Welch's t-test according to the variance between the two samples. Statistically significant differences are indicated with an asterisk (p < .05) or double asterisks (p < .01) in the figures. Overexpression of FRS2a Promotes FGF2-Induced Proliferation and Self-Renewal of NSPCs In Vitro It has been shown that NSPCs located in the telencephalon can be stimulated to proliferate in response to FGF2 or EGF stimulation and form spherical cell clusters called neurospheres in vitro [25]. The neurosphere cells are composed of heterogenous cell populations, including stem cells and multipotent progenitor cells [26]. Because self-renewing NSCs are capable of reforming new neurospheres and each neurosphere is derived from a single NSC, the frequency of neurosphere formation corresponds to the self-renewing capacity of the NSCs in the original neurospheres [22,27,28]. We first performed a gain-of-function analysis of FRS2a in vitro. We used wild-type FRS2a and two mutants: FRS2a-6F and FRS2a-8V. FRS2a-6F has all six tyrosine phosphorylation sites replaced by phenylalanine residues. FRS2a-6F does not interact with Shp2 or Grb2, and the introduction of an FRS2a-6F-expressing vector into FRS2a-deficient mouse embryonic fibroblasts could not rescue the activation of Erk in these cells in response to FGF [13,14]. The other FRS2a mutant FRS2a-8V has eight threonine phosphorylation sites replaced by valine residues, and it is thought to act as a dominant active form of FRS2a, which is sensitized to FGF stimulation due to the lack of negative regulatory phosphorylation sites [29]. We obtained NSPCs from the telencephalons of embryonic day (E) 14.5 mouse embryos and cultured them in the presence of FGF2 or EGF to obtain primary neurospheres. The primary neurospheres were dissociated to single cells and transduced with retroviral expression vectors encoding wildtype FRS2a or its mutant forms, FRS2a-6F or FRS2a-8V, together with GFP and the cells were cultured in the presence of FGF2 or EGF to form secondary neurospheres. The secondary neurospheres were dissociated, and cells expressing GFP were sorted by FACS; these sorted cells were cultured again in the presence of FGF2 or EGF to form tertiary neurospheres. When NSPCs were cultured in the presence of FGF2, cells expressing FRS2a or FRS2a-8V, but not FRS2a-6F, formed significantly larger tertiary neurospheres than did control neurospheres transduced with an empty vector (Fig. 1A). In contrast, when NSPCs were cultured in the presence of EGF, the expression of FRS2a or its mutant forms had no effect on the size of neurospheres (Fig. 1B). Interestingly, the expression of wild-type FRS2a or FRS2a-8V, but not FRS2a-6F, also significantly increased the frequency of FGF2induced tertiary neurosphere formation ( Fig. 1C) but not that of EGF-induced neurosphere formation (Fig. 1D). We obtained similar results using NSPCs from E12.5 telencephalons ( Fig. 1E and Supporting Information Fig. 1). These results suggest that the FGF2-induced tyrosine phosphorylation of FRS2a is important not only for the proliferation of NSPCs but also for the self-renewal of NSCs in vitro. To confirm this further, we passaged FGF2-induced secondary neurospheres and then cultured the NSPCs in the presence of EGF. The frequency of EGF-induced tertiary neurosphere formation was increased when using FRS2a or FRS2a-8V, but not FRS2a-6F, was expressed (Fig. 1F). Expression of FRS2a or FRS2a-8V had no effect on the formation of EGF-induced tertiary neurospheres when EGF-induced secondary neurospheres were used (Fig. 1D). This result indicates that the expression of FRS2a or FRS2a-8V enriched the number of self-renewing NSCs in each neurosphere induced by FGF2 but not by EGF. To confirm the expression of FRS2a protein in neurosphere cells, we performed western blotting. Strong expression of FRS2a was observed in cells expressing the wild-type or mutant forms (Fig. 1G). Both wild-type FRS2a and FRS2a-6F showed a strong shift in electrophoretic mobility in response to FGF2 (Fig. 1G). Consistent with the notion that the shift is largely caused by the phosphorylation of threonines in FRS2a by activated Erk, FRS2a-8V showed only a slight shift [29]. Western blotting was used to examine the activation of components of the FGF signaling pathways downstream of FRS2a in NSPCs expressing wild-type or mutant forms of FRS2a in response to FGF2. Stronger activation of Erk or Akt was observed in cells overexpressing FRS2a or FRS2a-8V after stimulation with FGF2 compared with FRS2a-6F-expressing cells or control cells (Fig. 1H, 1I). Enhanced Akt activation is consistent with a previous report that Grb2-binding to Gab1 contributes to activation of Akt [13]. We also evaluated the activation of Shp2 and Gab1 by immunoblotting with anti-phospho-Shp2 and anti-phospho-Gab1 antibodies, respectively. Activation of Shp2 and Gab1 was enhanced in cells expressing wild-type FRS2a or FRS2a-8V compared with control cells or cells expressing 6F (Fig. 1G). Overexpression of FRS2a Promotes FGF2-Induced Proliferation and Self-Renewal of NSPCs In Vivo To examine the function of FRS2a in the self-renewal of NSCs in vivo, we overexpressed wild-type FRS2a or its mutant forms in the developing mouse cortex using a retroviral expression system. We injected retrovirus into the telencephalic lateral ventricles of E12.5 mouse embryos in utero and then sacrificed them at E15.5. At these stages, NSPCs are normally localized at the VZ/subventricular zone (SVZ) and generate neurons, and those neurons migrate out of the VZ/SVZ through the intermediate zone (IZ) to settle at the cortical plate (CP) [30]. Immunohistochemistry of the viral GFP marker in cortical sections showed that a large number of control or FRS2a-6F-expressing cells migrate out of the VZ/ SVZ into the IZ/CP ( Fig. 2A, 2B, 2E). In contrast, many FRS2a-or FRS2a-8V-expressing cells remained in the VZ/ SVZ, and the number of cells in the IZ/CP was decreased compared with controls ( Fig. 2C-2E). These results imply that the expression of FRS2a or FRS2a-8V, but not FRS2a-6F, represses the differentiation of NSPCs. To examine the identity of virus-infected cells, we performed double-immunostaining against GFP and TuJ1 (a neuronal marker) or Musashi-1 (a NSPC marker). This analysis showed that the overexpression of FRS2a or FRS2a-8V, but not FRS2a-6F, increased the ratio of NSPCs to neurons ( Fig. 2F-2N). To confirm that cells expressing FRS2a or FRS2a-8V are ''bona fide'' NSCs, we examined whether they express nestin, another marker of NSCs, and undergo interkinetic nuclear migration (INM), a cell cycle-dependent somal translocation process unique to radial glial cells [31]. During the S-phase, the soma of radial glial cells is located at the basal (abventricular) side of VZ, but descends toward the apical (ventricular) surface during the G2 phase. The cell body is present at the ventricular surface during the M phase, and ascends basally during the G1 phase. We coinjected vectors expressing FRS2a or FRS2a-8V together with GFP into E13.5 cortex and performed in utero electroporation, and then sacrificed the embryos at E15.5. BrdU was administered 30 minutes or 12 hours before sacrifice to label cells in the S-phase or mainly the G1 phase, respectively [32]. Then, cortical sections of the embryos were subjected to double-immunostaining with antibodies against GFP and nestin or BrdU. This analysis showed that most cells in the VZ expressing FRS2a or FRS2a-8V were nestin-positive (95.7% 6 5.4% of cells expressing FRS2a among a total of 691 cells counted in 22 randomly selected areas; 97.9% 6 5.5% of cells expressing FRS2a-8V among a total of 717 cells counted in 26 randomly selected areas; Fig. 3A). Although cell bodies expressing FRS2a or FRS2a-8V were present at the basal side of VZ when BrdU was administered 30 minutes before sacrifice, many of them were located at the apical side when BrdU was administered 12 hours before sacrifice ( Fig. 3B and 3C). These results strongly suggest that cells expressing FRS2a or FRS2a-8V underwent INM and remained bona fide NSCs. Together, our results raise the possibility that FGF signaling mediated by tyrosine phosphorylation of FRS2a represses NSPC differentiation and thereby promotes the self-renewal of NSPCs in vivo. Grb2-binding sites of FRS2a Are Required for the Maximum Proliferation of NSPCs, But Are Dispensable for the Self-Renewal of NSCs We have previously demonstrated that the Shp2-binding sites of FRS2a are dispensable for the self-renewal of NSCs [22]; therefore, we next analyzed the function of the Grb2-binding sites of FRS2a using an Frs2a 4F mutant mouse in which phenylalanine replaces four tyrosine residues in the Grb2-binding sites. Although FGF2-induced secondary neurospheres from the telencephalons of E14.5 Frs2a 4F mutant embryos were smaller than those of wild-type embryos (Fig. 4A), the frequency of secondary neurosphere formation was not significantly different (Fig. 4B). Thus, the self-renewal ability of NSCs from Frs2a 4F/4F mutant mice appears to be normal, although the proliferation of mutant NSPCs was slightly decreased in vitro. Immunohistochemical analysis of the telencephalons of E14.5 embryos using anti-phospho-histone H3 (pH3) antibody, an M-phase marker, showed that the mitotic activity of the mutant telencephalons was normal in the VZ, where radial glial cells divide. Cell division in the E14.5 SVZ of the cortex but not of the ganglionic eminence, where intermediate progenitor cells divide [22,33,34], was slightly reduced (Fig. 4C-4G). Intermediate progenitor cells are derived from radial glial cells and are committed to neuronal cells. This result confirms that the self-renewal ability of NSCs from Frs2a 4F/4F mutant mice appears to be normal, although the proliferation of mutant NSPCs was slightly decreased. In response to FGF2 stimulation, mutant NSPCs showed moderately decreased Erk activation compared with wild-type cells (Fig. 4H). However, the activation of Akt was not affected in the mutant NSPCs. In addition, western blotting using lysates of E11.5 brain tissue showed that Erk activation, but not the Akt activation level, was moderately decreased in mutant brains (Fig. 4I). Tyrosine phosphorylation of one of the Grb2-binding sites of FRS2a, tyrosine-196, was reduced in mutant brains, confirming that the mutant lacks these sites. Thus, although the proliferation and Erk activation of Frs2a 4F NSPCs are moderately impaired, the self-renewal ability of the mutant NSCs appears to be normal both in vivo and in vitro. Erk Activation via FRS2a Is Required for the Full Activity of FGF-Induced Self-Renewal of NSCs It seems paradoxical that the data from the gain-of-function experiments suggest that tyrosine phosphorylation of FRS2a enhances the self-renewal of NSCs; however, the loss of the Shp2-binding sites [22] or the Grb2-binding sites (Fig. 4B, 4G) of FRS2a does not affect the self-renewal ability of NSCs. Although Erk activation, but not Akt activation, was moderately reduced in both the Frs2a 4F (Fig. 4H, 4I) and Frs2a 2F mutants [22], Erk activation via either the Shp2-or Grb2-binding sites of FRS2a appears to be sufficient for the self-renewal of NSCs. If both of these sites are lost, however, Erk activation may be severely diminished, and the self-renewal ability of NSCs may be compromised. To test this possibility, we blocked Frs2a translation in NSPCs with short hairpin (sh) RNA-expressing retroviral vectors. shRNAs for Frs2a (shFrs2a-1 or shFrs2a-2) effectively downregulated the expression of FRS2a (Fig. 5A). Then, we transduced the retroviral vectors into NSPCs from E14.5 telencephalons and cultured them in the presence of FGF2. We found that cells expressing shFrs2a-1 or shFrs2a-2 formed smaller and fewer secondary neurospheres than cells expressing control shRNA (Fig. 5B, 5C). Furthermore, neurospheres with decreased Frs2a expression showed strongly reduced activation of Erk in response to FGF2 stimulation, but the activation of Akt was not affected (Fig. 5D, 5E). These results suggest that the FRS2a-Erk axis, but not the FRS2a-Akt axis, contributes to the self-renewal of NSCs and to the efficient proliferation of NSPCs in response to FGF2. To examine the relationship between the Erk activation level and self-renewal as well as the proliferation of NSPCs, we treated NSPCs from E14.5 mouse telencephalons with different doses of a MEK inhibitor U0126. We found that Erk activation was decreased in a dose-dependent manner (Fig. 5F). Next, we cultured them in the presence of FGF2. The resulting primary neurospheres were dissociated and cell numbers were counted; then, the same number of NSPCs were cultured again to form secondary neurospheres. Our results demonstrated that the number of cells and the number of neurospheres both were decreased with treatment with U0126 in a dose-dependent manner (Fig. 5G). It thus appears that there is a close correlation between the activation levels of Erk and the self-renewal of NSCs as well as the proliferation of NSPCs. Moreover, we noticed that the number of cells was www.StemCells.com more severely suppressed than that of neurospheres at the same dose of U0126. When we used U0126 at lower doses, less than 0.7 lM, the number of cells was moderately suppressed, while the number of neurospheres was not significantly changed (Fig. 5H). To further examine the correlation between Erk activation, self-renewal, and proliferation, we used PD98058, another MEK inhibitor. PD98059 moderately inhibited Erk activation in a dose-dependent manner, but it was less effective than U0126 (Supporting Information Fig. 3A and Fig. 5F). Next, we treated NSPCs from E14.5 mouse telencephalons with different doses of PD98059 and cultured them in the presence of FGF2. The resulting primary neurospheres were dissociated and cell numbers were counted; then, the same numbers of NSPCs were cultured again to form secondary neurospheres. As a result, although the numbers of cells were decreased in a dose-dependent manner, there was little change in the number of neurospheres at any dose (Supporting Information Fig. 3B). It appears that cell proliferation was inhibited in correlation with the moderately reduced levels of Erk activity; however, the inhibition levels of Erk activation by U0126 at low doses or by PD98059 were not sufficient for the inhibition of self-renewing activity in this assay condition. Therefore, a low level of Erk activation appears to be sufficient for the self-renewal of NSCs, whereas a high level of Erk activation may be required for the full proliferative activity of NSPCs. Hes1 Is a Target of FGF2-FRS2a Signaling to Maintain NSPCs in an Undifferentiated State In Vitro The identities of the targets of the FGF-FRS2a signaling pathway for the self-renewal of NSCs remain unknown. It is known that the transcription factor Bmi-1 [35] and Notch signaling [36] play an important role in the self-renewal of NSCs. Hes family proteins are transcription factors downstream of Notch signaling [36]. We next examined whether certain components of the Notch signaling pathway or Bmi-1 serve as targets of FGF signaling for the self-renewal of NSCs. Quantitative real-time reverse transcription polymerase chain reaction showed that the expression of Bmi-1, Notch1, Notch2, Hes3, or Hes5 mRNA was not upregulated in NSPCs in response to FGF2 stimulation (Supporting Information Fig. 2). Interestingly, we found that the expression of Hes1 was elevated and sustained for 24 hours in response to FGF2 in NSPCs (control; Fig. 5I). To examine whether FRS2a is involved in FGF2-induced Hes1 expression, we knocked down the expression of Frs2a in NSPCs. We found that expression of Hes1 mRNA was rapidly decreased to basal levels after 6 hours (Fig. 5I). Expression of Hes1 protein was not induced at significant levels by FGF2-stimulation (Fig. 5J). We next examined whether activation of Notch1 and Notch2 contributes to the FGF2-induced Hes1 expression. We used c-secretase inhibitor DAPT, which blocks cleavage of the cytoplasmic domain of Notch and thereby inhibits Notch signaling. We found that treatment with U0126, but not DAPT, inhibited the FGF2-induced expression of Hes1 (Fig. 5K). FGF2 stimulation did not induce activation of Notch as shown by expression of the intracellular domain of Notch1 and Notch2, nor induce expression of Delta-like one (Dll1), a Notch ligand. Treatment with U0126 did not affect Notch activity (Fig. 5K). These results suggest that FGF2-induced expression of Hes1 is mediated by FRS2a, and is independent of Notch signaling but dependent on the activation of Erk. To further examine whether the FGF2-FRS2a-Erk-Hes1 axis contributes to the self-renewal of NSCs, we expressed Hes1 in cultured NSPCs, in which Frs2a was knocked down or not, and examined the resulting number and size of the FGF2-induced neurospheres. Expression of Hes1 increased the number of FGF2-induced neurospheres in which FRS2a was intact (Fig. 5L). Moreover, although the knocking down of Frs2a reduced the number of FGF-induced neurospheres, the exogenous expression of Hes1 rescued this phenotype to some extent (Fig. 5L). In contrast, the exogenous expression of Hes1 neither significantly increase the size of neurospheres in which FRS2a was intact (data not shown) nor rescue the decreased size of the neurospheres caused by knocking down of Frs2a (Fig. 5B and data not shown). These results suggest that the FGF2-FRS2a-Hes1 axis promotes the self-renewal of NSCs. Hes1 Is a Target of FGF2-FRS2a Signaling to Maintain NSPCs in an Undifferentiated State In Vivo To examine whether Hes1 is a target of signaling via FRS2a for the self-renewal of NSCs in vivo, we inhibited the expression of FRS2a in the developing cortex by shRNA for Frs2a. We coinjected expression vectors for GFP and an shRNA for Frs2a or control into the telencephalic lateral ventricles of E12.5 cortex and performed in utero electroporation. Then, embryos were sacrificed at E13.5 and immunostained with antibodies against Hes1 and GFP. We found that knocking down of Frs2a caused a decrease in expression levels of Hes1 in the cortex (Fig. 6A, 6B). We next coinjected those expression vectors with or without vectors expressing Hes1. The knocking down of Frs2a reduced the number of cells in VZ/ SVZ (Fig. 6C, 6D). When exogenous Hes1 was coexpressed, many control or Frs2a knocked-down cells were localized in the VZ/SVZ region (Fig. 6C, 6D). Hence, the exogenous expression of Hes1 increased the number of NSPCs and rescued the reduced NSPCs caused by the knocking down of Frs2a in vivo. Double-immunostaining with antibodies against GFP and TuJ1 or Musashi-1 showed that knocking down of Frs2a increased the ratio of neurons at the expense of NSPCs ( Fig. 6E and 6F). On the other hand, when exogenous Hes1 was coexpressed, many control or Frs2a knocked-down cells were Musashi-1-positive, suggesting that they remained undifferentiated ( Fig. 6E and 6F). However, exogenous Hes1 expression did not increase the number of mitotic cells that were positive for pH3 in the VZ/SVZ region (Supporting Information Fig. 4). These results suggest that the FRS2a-Hes1 axis maintains undifferentiated NSPCs in vivo; however, exogenous Hes1 expression does not significantly promote cell division. To confirm that the rescued cells are bona fide NSCs, we examined whether they express nestin and undergo INM in a similar way analyzed for Figure 3. Most cells in VZ expressing Hes1 also expressed nestin (97.3% 6 3.5%, total 518 cells counted in 18 randomly selected areas; Fig. 7A). Although the GFP-positive cell bodies in the S-phase were present at the basal side of VZ, many of them were located at the apical side in the G1 phase ( Fig. 7B and 7C). Thus, these cells appear to be bona fide NSCs. It thus appears that Hes1 is a downstream target of FGF-FRS2a signaling for the selfrenewal of NSCs in vivo. As NSCs derived from the Frs2a 2F mutant appear to have intact self-renewing activity [22], the expression of Hes1 should be intact in NSPCs in these mutant mice. We found that full levels of Hes1 expression were induced by FGF2 stimulation in Frs2a 2F NSPCs in vitro (Supporting Information Fig. 5A). We also found that there was little difference in the expression levels of Hes1 in the cortex between the wild-type/heterozygote and the mutant (Supporting 1666 FRS2a-Erk Axis Regulates Neural Stem Cells Information Fig. 5B). In addition, we examined expression levels of Hes1 in Frs2a 4F mutant cortex. As expected, we found little difference in expression levels of Hes1 between wild-type/heterozygote and the Frs2a 4F mutant (data not shown). Moreover, though treatment with PD98059 moderately reduced FGF2 induced-Erk activation, it did not affect Hes1 expression (Supporting Information Fig. 5C). These results further support the notion that Hes1 is a low level target of Erk activated by FGF-FRS2a signaling. To examine the molecular mechanisms of Hes1 induction by FGF-FRS2a signaling, we performed a luciferase assay with a reporter vector containing the promoter region of Hes1 (-2570 to þ277) in cultured NSPCs. FGF2 stimulation increased promoter activity, and this was inhibited by treatment with U0126 ( Supporting Information Fig. 6A). Serial de-letion analysis of the promoter (À750, À487, and À12) showed that FGF2 stimulation increased the promoter activity up to À750, but this was inhibited in À487 or À12 construct (Supporting Information Fig. 6B). In the promoter region between À750 to À487, we identified a potential binding sequence of AP-1 (À498 to À492, TGACTCC) [37], an important transcriptional activator downstream of Erk, composed of members of Jun and Fos families [38]. Point mutations in this sequence (TGACTCC to CGTCTAC) abolished the activation of the reporter by FGF2 stimulation (Supporting Information Fig. 6B). The expression of c-Fos, a member of the Fos family, was rapidly induced after FGF2 stimulation in NSPCs (Supporting Information Fig. 6C), and chromatin immunoprecipitation assay showed that c-Fos interacted with the promoter region containing the putative AP-1-binding site in response to FGF2 stimulation (Supporting Information Fig. 6D). FGF2 stimulation transiently induced expression of c-Fos, reaching peak levels at $80 minutes, and formation of AP-1 complex, and then expression of c-Fos gradually declined to basal levels. On the other hand, expression levels of c-Jun were stable (Supporting Information Fig. 6E). Moreover, knocking down of c-Fos decreased FGF2-induced Hes1 expression (Supporting Information Fig. 6F). These results suggest that Erk activation by FGF2 stimulation induces the expression of c-Fos, and then, AP-1 containing c-Fos binds to the Hes1 promoter and activates transcription. It has been reported that expression of Hes1 oscillates in neural progenitor cells, though precise molecular mechanisms are still unclear. To examine whether FGF signaling is involved in this process, we incubated cultured NSPCs in the starvation media without growth factors for overnight, stimulated them with FGF2, and monitored the expression levels of Hes1, c-Fos, and c-Jun at every 20 minutes (Supporting Information Fig. 6G). After starvation, expression of Hes1 was at very low levels. Upon stimulation with FGF2, expression of Hes1 was strongly induced, reaching a peak level after $100 minutes and declined to a basal level after $180 minutes. Then Hes1 expression was increased again and reached the second peak level after $240 minutes, though the expression levels at the second peak were lower than those of the first. Expression of c-Fos was strongly induced by FGF2 stimulation, then declined gradually to a basal level and did not show the second peak (Supporting Information Fig. 6E and data not shown). When c-Fos was knocked down, expression levels of Hes1 was decreased without showing the second peak (data not shown). We also monitored phosphorylation levels of Erk in the same time course and found that levels of phosphorylation of Erk was transiently induced as those of c-Fos without showing the second peak, while expression levels of Erk were stable (Supporting Information Fig. 6E and data not shown). These results suggest that the FGF-AP-1 signaling is important for the induction of Hes1 expression to initiate oscillation in cultured NSPCs. DISCUSSION In this report, we dissected the role of FGF signaling for the self-renewal and proliferation of NSPCs. First, FRS2a, a central mediator of FGF signaling, appears to selectively activate Erk and induces little activation of Akt. Then, it is likely that low levels of Erk activation is sufficient for induction of Hes1, which allows NSCs to self-renew. Further, high levels of Erk activation may be required for full levels of proliferation of NSPCs ( Fig. 7D and 7E). FRS2a Controls the FGF-Erk Axis by the Degree of Tyrosine Phosphorylation on Grb2-and Shp2-Binding Sites This notion is well-supported by the phenotype of Frs2a mutants and NSPCs expressing reduced levels of FRS2a by the expression of shRNA for Frs2a. The Frs2a knocked-down NSPCs showed impaired self-renewing activity with reduced Hes1 expression and impaired proliferation. The Shp2-binding site mutant Frs2a 2F -derived NSPCs showed intact self-renewing activity with intact Hes1 expression but impaired proliferation. The Grb2-binding site mutant Frs2a 4F -derived NSPCs showed intact self-renewing activity but slightly impaired proliferation. On the other hand, FGF2-induced activation of Erk was most impaired in the Frs2a knocked-down NSPCs among the wild-type, Frs2a 2F , and Frs2a 4F NSPCs. We previously reported that the Frs2a 4F mutant is able to activate Erk at slightly higher levels than the Frs2a 2F mutant, although both mutants show reduced levels of Erk activation than wild-type FRS2a [13]. Therefore, it is likely that the differences in the activities of self-renewal and proliferation of NSPCs in each FRS2a mutant is at least partly due to different levels of Erk activity. In other words, we demonstrated that FRS2a controls the FGF-Erk axis by the degree of tyrosine phosphorylation on Grb2-and Shp2-binding sites (Fig. 7D and 7E). Low Erk activation levels via at least one of two tyrosine phosphorylation sites of FRS2a are sufficient for FGF-dependent enhancement of Hes1 expression and self-renewal of NSCs. In contrast, strong Erk activation via both the tyrosine phosphorylation sites are required for the full proliferation of NSPCs. Consistent with our results, there is a convincing evidence that Erk2 is required for the full activity of self-renewal of NSCs and proliferation of NSPCs in response to FGF2, based on the studies using conditional Erk2 knockout mice [39]. It was also reported that adhesion signals by b1 integrin partly contributes to Erk activation for NSC maintenance [40]. Coordinated signaling of both growth factors such as FGF and adhesion such as b1 integrin would be essential for NSC maintenance. FGF-FRS2a-Erk Signaling Regulates Hes1 Expression and Self-Renewal of NSCs It is known that Hes1 plays important roles for self-renewal of NSCs. Hes1 knockout mice show defects in self-renewal of NSCs [28]. In this article, the authors also showed that proliferation of NSPCs is not significantly affected in the Hes1 knockout mice. These findings are consistent with our results. Because it has been reported that the expression level of Hes5, but not Hes1, is reduced in Notch1 or CBF1 mutant mice, the expression of Hes1 has been predicted to also be regulated by signaling cascades other than Notch signaling [36]. Our results suggest that FGF-FRS2a-Erk signaling is indeed a distinct pathway from Notch signaling for the selfrenewal of NSCs. This notion is supported by the result that knockdown of Frs2a led to reduced self-renewing activity of NSCs, and the coexpression of Hes1 rescued the phenotype in vitro and in vivo. Consistent with this, it was reported that overexpression of Hes1 or activated forms of either Notch1 or FGF receptor two increases the self-renewing activity of NSCs in the cortex [23,41,42]. We showed that FGF activates Hes1 promoter dependent on activation of Erk and our results suggest that the transcription factor complex AP-1 binds to the AP-1 consensus binding site of Hes1 promoter. Therefore, it appears that FGF-Erk axis induces expression of Hes1 at the transcriptional levels. It is known that Hes1 expression oscillates in many cell types, including presomitic mesoderm and many cultured cells [43]. During mouse somitogenesis, oscillation of Hes7, another Hes family member, is initiated by FGF signaling and propagated or maintained by Notch signaling [44]. It was reported that the oscillation of Notch-induced Hes1 expression regulates the maintenance of NSCs [45]. Accumulating evidence indicates that negative feedback mechanisms lead to oscillatory responses [46]. Hes1 serves as a transcriptional repressor, acting in an autoregulatory loop in the negative feedback mechanisms. Hes1 represses its own promoter, resulting in disappearance of Hes1 mRNA and Hes1 protein. Then the disappearance of Hes1 protein leads to relief from the repression and allows the next round of expression. It was recently reported that FGF induces Hes1 expression in an oscillatory manner in a mesenchymal C3H 10T1/2 cell line [47]. In these cells, Erk activation also oscillates due to negative feedback mechanisms through Sos protein, a Ras activator. As we did not observe oscillation of FGF2-induced c-Fos expression or Erk activation, the autoregulatory loop involving Hes1 and Notch signaling might be sufficient for the Hes1 oscillation in NSPCs. Together with our results it appears that FGF-FRS2a-Erk-AP-1 signaling is important for the induction of Hes1 expression to the levels at which oscillation takes place in NSPCs. It is possible that FGF signaling keeps basal levels of Hes1 transcription. Nevertheless, it is possible that FGF signaling contributes to regulation of oscillation at another level. We found that the second peak levels of Hes1 expression in FGF2stimulated NSPCs were greatly reduced compared with the first peak levels. This may be because cells were desynchronized in the second round of Hes1 expression as previously described in other cells [43]. Therefore, to examine the precise role of FGF signaling for Hes1 oscillation in NSPCs, it would be important to monitor cycling activity of Hes1 promoter in individual cells by real-time imaging as previously reported [42,43]. CONCLUSION In response to FGF2, FRS2a signals increase Erk phosphorylation levels, controlled by the degree of its tyrosine phosphorylation. This leads to qualitatively different biological outputs: self-renewal at least partly mediated by Hes1, the master regulator of stemness, versus cell proliferation. The molecular mechanisms by which FRS2a controls NSCs could be generalized to a variety of other stem cells. Certain methods for modifying the functions of FRS2a may be useful for obtaining in vitro cultures of stem cells of desired quality.	2014-10-01T00:00:00.000Z	2010-07-22T00:00:00.000	{ "year": 2010, "sha1": "17e2ff0512e797173b485969c974c9780d713e4c", "oa_license": "CCBY", "oa_url": "https://stemcellsjournals.onlinelibrary.wiley.com/doi/pdfdirect/10.1002/stem.488", "oa_status": "BRONZE", "pdf_src": "PubMedCentral", "pdf_hash": "17e2ff0512e797173b485969c974c9780d713e4c", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
18855052	pes2o/s2orc	v3-fos-license	Sex-specific exposure prevalence of established risk factors for oesophageal adenocarcinoma Background: There is an unexplained male predominance in the incidence of oesophageal adenocarcinoma, and the sex-specific distribution of its risk factors in the general population is not known. Methods: A random sample of Swedish citizens aged 40–79 years completed a questionnaire for assessment of the prevalence of five risk factors for oesophageal adenocarcinoma: reflux symptoms, body mass index, tobacco smoking habits, socioeconomic status, and use of non-steroidal anti-inflammatory drugs (NSAIDs). Logistic regression was used to calculate odds ratios (ORs) with 95% confidence intervals (CIs) to evaluate the association of these risk factors, separately and combined, with male sex, with women as reference. Results: Among 6969 invited people, 4906 (70.4%) completed the questionnaire. Adjusted prevalence estimates showed a negative association with male sex with regard to reflux disease (OR=0.70, 95% CI=0.58–0.84), whereas overweight (OR=1.98, 95% CI=1.72–2.27) and obesity (OR=1.22, 95% CI=1.01–1.47), previous smoking (OR=1.50, 95% CI=1.30–1.72), and no NSAID use (OR=1.35, 95% CI=1.15–1.49) were positively associated. Conclusions: Exposure to some but not all established risk factors for oesophageal adenocarcinoma seems to be more common in men than in women, but the differences are small and unlikely to explain the male predominance of this tumour. The striking male predominance of oesophageal adenocarcinoma, with a male-to-female ratio ranging from 7 : 1 to 10 : 1 (Vizcaino et al, 2002), remains unexplained. The rapid rise in the incidence of oesophageal adenocarcinoma in Western societies during recent decades (Brown et al, 2008) has been especially pronounced in men (Vizcaino et al, 2002). Epidemiological studies evaluating the hypothesis that sex hormones have a role, including hormonal replacement therapy (Lindblad et al, 2006) and reproductive factors , have not provided support for oestrogen as an aetiological factor, whereas reports on antiandrogen therapy have shown mixed results (Lagergren and Nyren, 1998;Cooper et al, 2009). Furthermore, there seem to be no clear sex differences in the strength of the associations between known risk factors and risk of oesophageal adenocarcinoma (Hampel et al, 2005;Lindblad et al, 2005;Kubo and Corley, 2006). Thus, the explanation underlying the strong and age-specific male predominance in oesophageal adenocarcinoma remains unknown (Rutegard et al, 2010). The principal aetiological factors are gastrooesophageal reflux (reflux) (Lagergren et al, 1999a;Shaheen and Ransohoff, 2002) and a high body mass index (BMI; Chow et al, 1998;Lagergren et al, 1999b;Kubo and Corley, 2006;Abnet et al, 2008), whereas tobacco smoking (Gammon et al, 1997;Lagergren et al, 2000;Freedman et al, 2007) and low socioeconomic status are weaker factors; regular use of non-steroidal anti-inflammatory drugs (NSAIDs) seems to be protective (Abnet et al, 2009). The sex-specific distribution of the exposure to these five factors in an unselected general population has not hitherto been estimated. We hypothesised that these risk factors for oesophageal adenocarcinoma, individually or in different combinations, are unevenly distributed between men and women. Furthermore, such sex differences might relate to preand post-menopausal age. To test these hypotheses, we conducted a population-based prevalence study in Sweden. MATERIALS AND METHODS This was a population-based, cross-sectional study, with data collection during the period April -June 2008. The exposure prevalence rates of the five known aetiological factors, that is, reflux, BMI, tobacco smoking, socioeconomic status, and NSAID use, were compared between men and women in a random Swedish population sample of age 40 -79 years. Random sampling and data collection were carried out by Statistics Sweden, a Swedish authority that holds the highly complete and updated nation-wide Swedish Total Population Register, which was used for this study. The sampling was performed to mimic the age and sex distribution of oesophageal and gastric adenocarcinoma according to the new cases reported to the Swedish Cancer Register in the year 2006. This provided a sample with a higher proportion of women and a slightly younger female population than had the sample been matched to oesophageal adenocarcinoma only. This age discrepancy was adjusted for in all analyses, but influenced the unadjusted prevalence, whereas all results were stratified by sex. A validated questionnaire (Lane et al, 2002) was sent to the selected individuals. Up to two reminding letters were sent to non-responders. The questionnaire contained questions about the five study exposures, together with some general characteristics, including sex, age, and physical activity. Reflux was defined as heartburn or regurgitation occurring at least once a week during the last 3 months, or at least weekly use of anti-reflux medication during the same time period, a definition commonly used in epidemiological research. In addition, a reflux variable based on the Montreal definition (Vakil et al, 2006), including both frequency and severity of symptoms, was evaluated. Current BMI value was calculated as body weight in kilograms divided by the square of body height in metres. Cutoffs for BMI were predetermined and based on the World Health Organization classification of overweight and obesity (WHO, 2008). A subject with a BMI value below 18.5 kg m À2 was considered to be underweight, a value of 18.5 -24.9 kg m À2 was regarded as normal, 25 -29.9 kg m À2 was defined as overweight, and 30 kg m À2 and above as obese. Tobacco smoking status was defined as current, former, or never smoker. If the participants had ever smoked 'one or more cigarettes a day for a year or more' and 'smoked within the last 3 months', they were classified as current smokers. Previous smokers were those who had ever smoked 'one or more cigarettes a day for a year or more', but had not smoked during the past 3 months. Never smokers had never smoked 'one or more cigarettes a day for a year or more'. Formal education was used as a proxy for socioeconomic status, as supported by previous findings (Robert and House, 1996;Fuchs, 2004). Length of education was categorised into less than or equal to 9 years, 10 -12 years, or more than 12 years. The use of NSAID was defined as the use of predefined and well-known brands of NSAIDs within the last 3 months. This was categorised into four groups, namely, no use of NSAIDs (or less than once a month), monthly use, weekly use, and daily use, in accordance with previous research (Abnet et al, 2009). Aspirin was included in the NSAID variable, as it is considered equivalent to NSAIDs with regard to cancer preventive effects (Abnet et al, 2009). Statistical analysis The male and female prevalence rates of reflux, high BMI, tobacco smoking, socioeconomic status, and use of NSAIDs were compared, using exposure frequencies and relative risk estimations. To allow adjustment for potential confounding factors, unconditional multivariable logistic regression was used to calculate odds ratios (ORs) with 95% confidence intervals (CIs). In these analyses, aetiological factors were the exposures and male sex was the outcome, using female sex as reference. Two predefined multivariable models were applied, a basic model adjusted only for age (categorised into three groups: o60, 60 -70, or 470 years) and the full model further adjusted for physical activity (several times a week, once a week, or less than once a week), reflux (no or yes), BMI (o25, 25 -29.9, or X30 kg m À2 ), tobacco smoking status (never, previous, or current smoker), education (p9, 10 -12, or 412 years), and NSAID use (ever or never). Physical activity was included as a potential confounder because of reported differences between the sexes and a putative association with the evaluated risk factors (Young et al, 2009). Goodness-of-fit (Hosmer and Lemeshow, 1980) was found adequate for both models (data not shown). Furthermore, predefined exploratory analyses were conducted by combining study variables, in which individuals with non-exposure were compared with exposed individuals with regard to given combinations of the included variables. For example, individuals with BMI o25 kg m À2 , without reflux, who had never smoked were compared with individuals with BMI X25 kg m À2 , with reflux, who were ever smokers. Intermediate groups of exposure are not presented, but were included in the model, thus using all observations. Owing to the expected small numbers in each category, these analyses were only age-adjusted. Finally, the cutoff of 50 years was used to delineate the presumed effects of menopause, but power was inadequate. Instead, the sample median (65 years) was used to allow age-stratified analyses. All analyses were conducted using STATA 10.1 (StataCorp, College Station, TX, USA). The Regional Ethics Committee in Stockholm approved the study. RESULTS Among 6969 invited people, the 4906 (70.4%) who responded to the questionnaire were included in this study. Of them, 3220 (65.6%) were men and 1686 (34.4%) were women, with participation rates of 69.5 and 72.6%, respectively. Non-participation was more common in younger age groups; 53.0% of those invited between the ages of 40 and 44 years responded, whereas 75.2% of the ones aged 75 -79 years replied. The mean ages of the male and female participants were 65.2 (s.d. ¼ 9.4) and 63.9 (s.d. ¼ 10.7) years, respectively. The physical activity level was similar in men and women (data not shown). Results from the logistic regression analyses follow, but as the results were similar in the two adjusted models, only the full model is presented. Reflux was observed in 10.2 and 13.5% of men and women, respectively (Table 1). The adjusted logistic regression analysis identified a statistically significantly lower prevalence of reflux in men than in women (OR ¼ 0.70, 95% CI ¼ 0.58 -0.84; Table 1). Use of the Montreal definition of reflux did not notably alter the sex difference in prevalence (6.2% for men and 8.5% for women). Among men, 46.8% were overweight and 13.5% were obese, whereas the corresponding prevalence rates for women were 31.9 and 15.1%, respectively (Table 1). After adjustment for other risk factors and other potential confounders in the full regression model, there was an almost two-fold increase in the odds of being overweight in men, as compared with women (OR ¼ 1.98, 95% CI ¼ 1.72 -2.27). The prevalence of obesity was also higher in men, but this sex-associated difference was less marked (OR ¼ 1.22, 95% CI ¼ 1.01 -1.47; Table 1). The proportion of never smokers was higher among women than among men (52.8 and 45.3%, respectively). Former smoking was more prevalent in men (37.6 vs 28.5%), whereas prevalence of current smoking was similar in the two sexes (Table 1). Compared with never smokers, results from the logistic regression analysis suggested that previous smoking, adjusted for all other factors, was 50% more common among men than among women (OR ¼ 1.50, 95% CI ¼ 1.30 -1.72), whereas current smoking was slightly, and non-statistically significantly overrepresented among men (OR ¼ 1.18, 95% CI ¼ 0.98 -1.42; Table 1). A higher proportion of women than men had more than 12 years of formal education (28.2 vs 23.9%). The intermediate educational level (9 -12 years) was more common in men than in women (12.3 and 7.1%, respectively). The prevalence of less than 9 years of education was similar between the sexes (Table 1). The adjusted analyses revealed that, compared with the highest education level, the intermediate level was twice as common in men than in women, (OR ¼ 2.10, 95% CI ¼ 1.65 -2.68), whereas the lowest education level was equally distributed between the sexes (OR ¼ 1.07, 95% CI ¼ 0.92 -1.24; Table 1). Use of NSAIDs was more common among women than among men in all subcategories, and 18.9% of men and 21.1% of women were daily users ( Table 1). The adjusted estimates revealed that with daily use as reference, weekly (OR ¼ 0.83, 95% CI ¼ 0.64 -1.06) and monthly (OR ¼ 0.89, 95% CI ¼ 0.68 -1.15) use was non-significantly less common in men than in women. Furthermore, non-use of NSAIDs was more prevalent in men (OR ¼ 1.35, 95% CI ¼ 1.14 -1.59; Table 1). Simultaneous exposures to combinations of some or all study variables are shown in Table 2. A marked male predominance was observed in combined exposure to reflux, high BMI, and NSAID use (OR ¼ 1.62, 95% CI ¼ 1.09 -2.42), the combination of reflux, high BMI, tobacco smoking, and NSAID use (OR ¼ 2.59, 95% CI ¼ 1.42 -4.72), and the combined exposure to all five studied factors (OR ¼ 2.76, 95% CI ¼ 1.21 -6.32). Evaluated associations of male sex with other combinations of risk factors were not statistically significant (Table 2). DISCUSSION This study of a random sample of the general population indicates that exposure to risk factors for oesophageal adenocarcinoma is more common among men than among women. There was no male predominance regarding reflux alone, but each of the risk factors, namely, high BMI, tobacco smoking, and low socioeconomic status, was more common among men and use of NSAIDs was less prevalent among men. Combinations of these risk factors were more prevalent in men only when use of NSAID was included. Age-stratified analyses indicated that high BMI was more common in men at a younger age. The advantages of this study include a population-based design with a high participation rate, which reduces the risk of selection bias and facilitates generalisation. Moreover, the large sample size allowed robust estimations, combining of study variables and stratification. The availability of data on all known risk factors allowed adjustment for potential confounding. There are, however, several limitations. The use of questionnaires to evaluate variables, such as height and weight, could introduce misclassification, and women might underestimate weight and overestimate height more than men (Flood et al, 2000). This effect, however, is mostly mediated by socioeconomic differences (Bostrom and Diderichsen, Abbreviations: CI ¼ confidence interval; N ¼ number; NSAID ¼ non-steroidal anti-inflammatory drug; OR ¼ odds ratio; SES ¼ socioeconomic status. Sex-specific prevalence rates and results of logistic regression analyses with OR and 95% CIs values in a randomly selected sample of 4906 Swedish citizens, using risk factors as exposures and male sex as outcome. a Adjusted for age, physical activity, reflux, education, body mass index, smoking status, NSAID use. b Defined as at least weekly symptoms of acid regurgitation and/or heartburn and/or weekly use of gastro-oesophageal reflux disease treatment, such as proton pump inhibitors, antacids, or H2-blockers. c No use or less than once a month. Sex differences in risk factors for oesophageal adenocarcinoma M Rutegård et al 1997), for which adjustment was made in this study. Missing values could introduce biased results, but the extent of missing data was limited and any such effect should be non-differential and therefore not explain the positive associations. Risk factors for cancer development are commonly considered to have an impact over a number of years. It might therefore be argued that a younger population sample should have been chosen to reflect the risk factors when cancer development was initiated. However, the induction times for the mechanisms that cause oesophageal adenocarcinoma are not known, and habits already established in adulthood may not readily be prone to change (Prattala et al, 1994;Mulder et al, 1998;Benzies et al, 2008). Residual confounding from known risk factors and confounding from unknown variables are threats to all observational studies. However, adjustments were made for all known risk factors and the categorisation was comparatively detailed. Some data suggest that infection with Helicobacter pylori prevents the development of oesophageal adenocarcinoma (Rokkas et al, 2007), but this possible negative association remains to be established. Moreover, data regarding a possible sex difference in H. pylori prevalence are conflicting, at most showing a weak male predominance of infection in adults (de Martel and Parsonnet, 2006). Multiple testing is another issue to be considered in this study, as several analyses were conducted and we combined various risk factors; however, this concern should be mitigated by the fact that the hypotheses were predefined and the exploratory evaluation of the combination of variables was planned before the initiation of any analysis. In previous studies, the sex-specific prevalence rates of the five known risk factors have been evaluated separately. The reflux prevalence has not been observed to be higher in males (Locke et al, 1997;Nilsson et al, 2004), an observation confirmed by this study. Findings of the national surveys of BMI in the United States (Ogden et al, 2006) and Europe (Andreyeva et al, 2007) are consistent with the prevalence pattern observed in this study, that is, a higher BMI in men. Our finding of a lower frequency of nonsmoking in females is in line with most previous reports (CDC, 2007), although the prevalence rates in Sweden, especially at younger ages, have more recently been observed to be higher in women (Ali et al, 2009). In our study, women had, on an average, a longer education, whereas previous research indicated a more similar sex distribution regarding the number of years of formal education (Molarius et al, 2007). The use of NSAIDs and aspirin was more common in women than in men, which is supported by some previous data with regard to aspirin (Larsson et al, 2006). Thus, the external validity of our study seems to be adequate. Indeed, the result that reflux seems to be less common in men might be a product of the comparatively high average age of participants in this study, as reflux prevalence in older men as compared with older women has previously been shown to be Abbreviations: BMI ¼ body mass index; CI ¼ confidence interval; N ¼ number; NSAID ¼ non-steroidal anti-inflammatory drug; OR ¼ odds ratio; SES ¼ socioeconomic status. Sex-specific prevalence rates and results of logistic regression analyses with OR and 95% CIs values, in a randomly selected sample of 4906 Swedish citizens, using predefined combinations of risk factors as exposures and male sex as outcome. a Using male sex as outcome and adjusted for age only. b Gastro-oesophageal reflux disease, defined as at least weekly symptoms of acid regurgitation and/or heartburn and/or weekly use of reflux treatment such as proton pump inhibitors, antacids, H2-blockers, etc. lower (Locke et al, 1997;Nilsson et al, 2004). Disregarding reflux, the multivariable analysis revealed small but significant differences in separate risk factor exposure, favouring an increased exposure in men. Putting these results into perspective, population attributable risks were calculated (Levin, 1953;Taylor, 1977) for the main exposures, namely, reflux, high BMI, and tobacco smoking, using another Swedish database, incorporating oesophageal adenocarcinoma data (Lofdahl et al, 2008). For the presence of reflux disease, a BMI value 425 kg m À2 , and ever smoking, these attributable risks were 20, 40, and 43% for men and 33, 45, and 49% for women, respectively. The finding that a combination of the two main risk factors, reflux disease and high BMI, even when smoking status was included, did not result in significant associations with male sex, warrants a comment. The absence of clustering of these risk factors in men might be explained by women reporting more reflux, although overweight and ever smoking were more frequent in men. The present study was unable to take into account different types of overweight and reflux; for example, the predominantly abdominal type of obesity (Corley et al, 2008) and erosive reflux disease (Cook et al, 2005) are more common in males than in females, and both these exposures have been shown to be more harmful with regard to carcinogenesis (Cook et al, 2005). When combining all risk factors to evaluate clustering, the data in this study indicate predominance in men, which seems to be driven mainly by differential NSAID use in men and women, a comparatively weak and uncertain aetiological factor for this cancer, without which no significant difference could be discerned. There is mounting evidence that the male predominance in oesophageal adenocarcinoma is age specific, wherein the highest incidence rate ratios are observed at younger ages (Cook et al, 2009;Derakhshan et al, 2009;Rutegard et al, 2010). This may reflect changes during and after menopause, and therefore the cutoff of 50 years was attempted for stratification. However, the sample size only allowed the use of the sample median of 65 years, which may not be entirely appropriate from a biological perspective. Nevertheless, it seems that a high BMI value is even more prevalent in men at younger age. This is intriguing, particularly as some evidence indicates that this high BMI value in men confers higher risks of oesophageal adenocarcinoma compared with women (Ryan et al, 2006). This first and large population-based study with an unselected sampling of participants indicates that exposure to some, but not all, established risk factors for oesophageal adenocarcinoma is overrepresented in males compared with females. The male preponderance to simultaneous exposure to all risk factors is mainly due to differential NSAID use in men and women. Our findings seem unlikely to explain the male predominance. Abbreviations: CI ¼ confidence interval; N ¼ number; NSAID ¼ non-steroidal anti-inflammatory drug; OR ¼ odds ratio; SES ¼ socioeconomic status. Age-stratified sex-specific prevalence rates and results of logistic regression analyses OR and 95% CIs values, in a randomly selected sample of 4906 Swedish citizens, using predefined combinations of risk factors as exposures and male sex as outcome. a Adjusted for physical activity, reflux, education, body mass index, smoking status, NSAID use. b Defined as at least weekly symptoms of acid regurgitation and/or heartburn and/or weekly use of gastro-oesophageal reflux disease treatment such as proton pump inhibitors, antacids, or H2-blockers. c No use or less than once a month.	2014-10-01T00:00:00.000Z	2010-08-10T00:00:00.000	{ "year": 2010, "sha1": "6649640cf4799ed1f21a0be0e5a5c6b5ccb69fb4", "oa_license": "CCBY", "oa_url": "https://europepmc.org/articles/pmc2938252?pdf=render", "oa_status": "GREEN", "pdf_src": "PubMedCentral", "pdf_hash": "6649640cf4799ed1f21a0be0e5a5c6b5ccb69fb4", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
195346542	pes2o/s2orc	v3-fos-license	Round- and Message-Optimal Distributed Part-Wise Aggregation Distributed graph algorithms that separately optimize for either the number of rounds used or the total number of messages sent have been studied extensively. However, algorithms simultaneously efficient with respect to both measures have been elusive for a long time. For example, only very recently was it shown that for Minimum Spanning Tree (MST), an optimal message and round complexity is achievable (up to polylog terms) by a single algorithm in the CONGEST model of communication. In this paper we provide algorithms that are simultaneously round-optimal with near-linear message complexities for a number of well-studied distributed optimization problems. Our algorithmic centerpiece is such a distributed algorithm that solves what we dub Part-Wise Aggregation: computing simple functions over each part of a graph partition. From this algorithm we derive round-optimal algorithms for MST, Approximate Min-Cut and Approximate Single Source Shortest Paths (SSSP), all with $\tilde{O}(m)$ message complexities. On general graphs all of our algorithms achieve a worst-case optimal $\tilde{O}(D+\sqrt n)$ round complexities and $\tilde{O}(m)$ message complexities. Furthermore, our algorithms require even fewer rounds on many widely-studied classes of graphs, namely planar, genus-bounded, treewidth-bounded and pathwidth-bounded graphs. For these graphs our algorithms require an optimal $\tilde{O}(D)$ rounds and $\tilde{O}(m)$ messages. Our results are the first instance of distributed algorithms with $\tilde{O}(m)$ message complexities for Approximate Min-Cut and Approximate SSSP. Moreover, our algorithms are the first algorithms for any of these problems that beat the general graph round lower bound of $\tilde{\Omega}(D + \sqrt{n})$ on graph families of interest and simultaneously achieve an $\tilde{O}(m)$ message complexity. Introduction A great deal of research has focused on the achievable runtime of distributed optimization algorithms in the CONGEST model of communication. Fundamental problems studied include Shortest Paths [10,22,23,27,28,31], MST [13,24,25,36], Min-Cut [16,32], and Max Flow [17]. Runtime is measured by the number of synchronous rounds of communication, and for these problems Θ(D + √ n) round upper and lower bounds are known [5,7,16,36]. 1 Another common performance metric optimized for in the CONGEST model is the total number of messages sent. For MST, anΩ(m) lower bound is known [2]. 2 However, for several decades the only MST algorithms known to match these message lower bounds had sub-optimal round complexities [1,2,3,9,11,12]. The question of whether algorithms attaining both optimal round and message complexity has been a long-standing problem. For instance, Peleg and Rubinovich [36] asked whether it might be achievable for MST. In a recent breakthrough work Pandurangan et al. [34] answered this question in the affirmative, providing a randomized MST algorithm with simulataneously optimal round and message complexities (up to polylog terms). Shortly thereafter Elkin [8] provided the same result without randomization. However, algorithms which are simultaneously round-optimal with only linear message complexities for other fundamental problems have remained elusive. Our Main Result In this paper we advance the study of simultaneously message-efficient and round-optimal distributed algorithms. Our main result is an algorithm for solving a fundamental distributed problem -which we dub Part-Wise Aggregation (PA) -with optimal round and message complexities. From this algorithm we derive round-optimal andÕ(m)-message algorithms for MST, Approximate Min-Cut and SSSP. Informally, the PA problem asks to compute the result of a function applied to each part of a graph partition. Formally, the problem is as follows. The value of β determines a tradeoff between the quality of the SSSP approximation and the round and message complexity of our algorithm. Taking β = log −Θ(1/ ) n, Corollary 1.5 yields an O(L )-approximation algorithm usingÕ(bD + c) rounds andÕ(m) messages. Discussion of Our Results There are a number of salient points worth noting regarding our results. Beyond Worst-Case Optimality. As stated above, every graph admits tree-restricted shortcuts with block parameter b = 1 and congestion c = √ n. It follows that our PA algorithm, as well as our MST, Approximate Min-Cut and SSSP algorithms (for constant ) requireΘ(D + √ n) rounds on general graphs. However, as observed in prior work, a number of graph families of interestplanar, genus-bounded, bounded-treewidth and bounded-pathwidth graphs -admit shortcuts with better parameters [15,19,20]. As a result, our algorithms require a round complexity that is at mostÕ(D) times the relevant parameter of interest (e.g., genus, treewidth or pathwidth). Provided these parameters are constant or even polylogarithmic, our algorithms run inÕ(D) rounds. We elaborate on our results for these graphs in Appendix B. We also note here that our algorithms need not know the optimal values of the block parameter b and congestion c. In particular, they perform as well as the parameters of the best shortcut that the input graph admits. Message and Round Optimality of our PA Algorithm By our proof of Corollary 1.3, which relies on solving PA O(log n) times to solve MST, and by the known lower bounds on MST, it is apparent that ourÕ(D + √ n)-roundÕ(m)-message algorithm for PA is both round-and messageoptimal (up to polylog terms). 5 Relation to Previous Work. To our knowledge, our Approximate Min-Cut and Approximate SSSP algorithms are the first round-optimal andÕ(m)-message algorithms for these problems. Our MST algorithms match the round and message complexities of that of Elkin [8] on general graphs but require even fewer rounds on a number of graph families of interest. For all such graph families our algorithms are the first withΩ(m) message complexities and better than worst-case (and in particular, near-diameter) round complexities. Future Applications of Work Non-trivial shortcuts likely exist for graph families beyond those mentioned above. For instance, work in progress [21] suggests that efficient shortcuts exist for all minor-free families of graphs. As such, demonstrating even better runtimes for our algorithms on many networks may be achieved in the future by proving the existence of efficient shortcuts on said networks. Lastly, we emphasize that the three applications of our PA algorithm are likely non-exhaustive; we are hopeful that our PA algorithm will find applications in deriving round-and message-efficient (or even optimal) bounds for more problems. Preliminaries Before moving onto our formal results, we explicitly state the model of communication we consider and review relevant concepts from previous work. CONGEST Model of Communication Throughout this paper we work in the classic CONGEST model of communication [35]. In this model, the network is modeled as a graph G = (V, E) of diameter D with n = \|V \| nodes and m = \|E\| edges. Communication is conducted over discrete, synchronous rounds. During each round each node can send an O(log n)-bit message along each of its incident edges. Every node has an arbitrary and unique ID of O(log n) bits, first only known to itself (this is the KT 0 model of Awerbuch et al. [2]). Shortcuts and Tree-Restricted Shortcuts Intuitively, low congestion shortcuts allow high-diameter parts to communicate efficiently, by using edges outside of parts; this effectively decreases the diameter of the parts. They were originally introduced by Ghaffari and Haeupler [15] to solve PA. This work showed how, given a high quality shortcut, PA can be solved in an optimal number of rounds (e.g.Õ(D + √ n) in a general graph) w.h.p. Formally, a low congestion shortcut is as follows. However, it was not until the work of Haeupler, Izumi, and Zuzic [19] that it was demonstrated that shortcuts could be efficiently computed in general. This work showed that high quality instances of a certain type of shortcut -tree-restricted shortcuts -can be efficiently approximated. These types of shortcuts are defined as follows. [19]). Let G = (V, E) be a graph and Definition 2.2 (Tree-Restricted Shortcuts Since a rooted BFS tree has minimal depth, and theÕ(D)-roundÕ(m)-message deterministic leader election algorithm of Kutten et al. [26] allows us to compute a BFS tree in the same bounds, throughout this paper T will be a rooted BFS tree. The same work that introduced tree-restricted shortcuts also introduced a convenient alternative to dilation, block parameter. [19]). Let H = (H i ) n i=1 be a T -restricted shortcut on the graph G = (V, E) with respect to the parts (P i ) n i=1 . For any part P i , we call the non-empty connected components (V, H i ) the blocks of P i , and the number of blocks of P i its block parameter. The block parameter of H, b, is the maximum block parameter of any part P i . Definition 2.3 (Block Parameter Haeupler, Izumi, and Zuzic [19] demonstrate that if T is a depth-D tree, the dilation of a Trestricted shortcut with block parameter b is at most O(bD). As such, block parameter is a convenient alternative to diameter. Techniques In this section we outline our general algorithmic approach. We begin by demonstrating the message sub-optimality of previous shortcut algorithms for PA on a particular example. We then give a workaround for this example and sketch notable aspects of our approach, allowing us to extend this workaround into a full-fledged algorithm. Bad Example for Previous Shortcut-Based Algorithms Several prior round-optimal randomized algorithms for PA used tree-restricted shortcuts [19,20]. To this end, these algorithms repeatedly aggregate within blocks. To aggregate within a block, every node in the block transmits its value up the block (along the tree's edges); when values from the same part arrive at a node in the block, they are aggregated by applying f and then forwarded up the block as a single value. By the end of this process, the root of the block has computed f of the block and can broadcast the result back down. This approach can be implemented using an optimalÕ(D + √ n) rounds. Does it also require an optimalÕ(m) messages? Unfortunately, there exist PA instances for which the above approach requires ω(m) messages. For example, consider the D × (n − 1)/D grid graph with an additional node, r, neighboring all of the top row's nodes. Suppose each row is its own part, and all the column edges are shortcut edges, forming a single block rooted at r. See Figure 1a. Aggregating within this block requires Ω(nD) messages: a message cannot be combined with other messages in its part until it has at least reached r and so each node is responsible for sending a unique message to r along a path of length D/2 on average. Thus, aggregating in blocks in this way to solve PA requires Ω(nD) messages, which is sub-optimal for any D = ω(1), since m = O(n) for this network. A Workaround. We can improve the poor message complexity of aggregating within blocks on this particular network as follows. Partition each of the D parts into sub-parts, each with O(D) connected nodes; we have O(n/D) sub-parts in total. See Figure 1b. First, sub-parts aggregate: the right-most node in the sub-part broadcasts its value left and every other node broadcasts left the aggregation of its own value and what it receives from its neighbor to the right. The leftmost node of a sub-part then uses the block's edges to transmit the sub-part's aggregate value to r, which then computes the aggregate value for each part. Symmetrically to the above procedure, r then broadcasts to every node the aggregate value for its part. Aggregating within each sub-part requires O(n) messages, as it requires each node to broadcast at most once. Moreover, there are O(n/D) sub-parts, each responsible for broadcasting up and down the block once and so using the shortcut requires O(n/D)·O(D) = O(n) messages. Therefore, for this network at least, our workaround requires an optimal O(m) = O(n) messages. Overview of Our Approach The workaround of the previous subsection is heavily tailored to the particular example of Figure 1a. Moreover, it requires that nodes know significantly more about the network topology than our PA algorithm is privy to. However, the above example and workaround motivate and highlight some of the notable strategies of our algorithm for Part-Wise Aggregation. Known-Leader PA. First, the above example illustrates why we begin by considering an easier variant of PA, the known leader PA problem. In the preceding example, r served as a single node towards which all nodes can aggregate. However, in general, a part might be incident to many blocks so there might be many such r. Thus, we consider the known leader PA problem where every node in a part knows some leader for its part. This provides a single node for each part towards which every node in the part can aggregate. Sub-Part Divisions. Even if nodes know a leader towards which they can aggregate, they must be able to do so efficiently. As illustrated in the example, having all nodes use a shortcut in order to send their private information to their part leader rapidly exhausts ourÕ(m) message budget. To solve this issue, we refine the partition of our network into what we call a sub-part division. In a sub-part division each part P i containing more than D nodes is partitioned intoÕ(\|P i \|/D) sub-parts each with a spanning tree rooted at a designated node termed the representative of the sub-part. In the preceding example the representatives are the left-most nodes of each sub-part. Each subpart uses its spanning tree to aggregate towards its representative, who then alone is allowed to use shortcut edges to forward the result toward the part leader. This decreases the number of nodes that use the shortcut from O(n) toÕ(n/D), thereby reducing the message complexity of aggregating within a block from O(nD) toÕ(n). Message-Efficient (and Deterministic) Shortcut Construction. If our algorithms are to use shortcuts as we did in the preceding example, they must construct them message efficiently; i.e., withÕ(m) messages. No previous shortcut construction algorithm achieves low message complexity. We show that not only do sub-part divisions allow us to use shortcuts message efficiently, but they also allow us to construct shortcuts message efficiently. In this vein, we give both randomized and deterministic message-efficient shortcut construction algorithms (the latter is the first roundoptimal deterministic shortcut construction algorithm known to date). Star Joinings to Compute Sub-part Divisions and Solve PA. The remaining issues to address are message-efficient construction of sub-part divisions, and how to solve PA using our known-leader PA algorithm, both of which we solve in similar fashion. For brevity's sake we only sketch here how we use algorithm for known-leader PA to solve PA. We begin with a singleton partition in which every node is its own leader. We repeatedly coarsen this partition, using our known-leader PA algorithm to elect a new leader for each new, coarser part. We continue this coarsening until our partition matches that of our PA instance. At this point every part has an elected leader and so we can simply apply our known-leader PA algorithm to solve the original PA problem. However, it is not clear how, if many parts try to merge together at once, a leader for the new merged part can be efficiently elected, as the new part can have arbitrarily large diameter (even accounting for the shortcuts of the finer partition) -rendering communication within the part infeasible. We overcome this issue by always forcing parts to merge in a star-like fashionwhich we term star joinings -where each part adopts the leader of the center of the star. As we show, enforcing this behavior is easily accomplished with random coin flips. Accomplishing the same behavior deterministically is significantly more involved. To this end, we introduce a novel deterministic symmetry-breaking algorithm that enforces this behavior based on the coloring algorithm of Cole and Vishkin [4]. Known Leader PA Before we can detail our randomized and deterministic algorithms, we provide a description of technical details common to both techniques, starting with a relaxation of PA that is particularly convenient for our approach -the Known Leader PA problem (KLPA). As we mentioned and show formally later, solutions to KLPA can be used to solve PA with only logarithmic overhead. In order to solve KLPA we will use a T -restricted shortcut and a refinement of the input partition into sub-parts, defined as follows. Definition 4.2 (Sub-part division). Given partition Each sub-part F j also has a spanning tree of diameter O(D) rooted at a node r ∈ F j , termed the sub-part's representative. It is worth noting that sub-parts need not coincide with blocks in a meaningful way; a single sub-part might span multiple blocks and there might be blocks without any sub-part representatives in them. See Figure 2 for an illustration of how sub-parts and blocks might interact. Using Sub-Part Divisions and Shortcuts to Solve KLPA In this section, we describe how to solve KLPA using sub-part divisions and T -restricted shortcuts. Moreover, because our KLPA algorithm is essentially the same algorithm we later use to verify that our shortcuts have good block parameter, we detail how we verify that our shortcuts have good block parameter in this section as well. Aggregating on Families of Sub-trees We must first restate an algorithm of Haeupler, Izumi, and Zuzic [19], Algorithm 1, used to aggregate and spread information within shortcut blocks. Consider a rooted tree T of depth D where every node knows its parent and children. Also suppose we are given a family of rooted subtrees of . Every b j is associated with an R i with which it has non-empty intersection and no vertex is incident to more than one b j associated with the same . Algorithm 1 accomplishes this by greedily aggregating and forwarding outstanding messages, defined in Algorithm 1. The complexity of Algorithm 1 is given by the following lemma. Algorithm 1 Algorithm to aggregate within blocks We overload "v" with v's input value here 2: for each round do 3: For every i and for each (o, i) received from a child in the last round add o to S i 4: Let mî = (f (Sî),î) be the outstanding message such that the root of Bî(v) is of minimum depth in T ; send mî to the parent of v in T ; markm as no longer outstanding 6: end for Lemma 2]) If no node ever has more than c outstanding messages, Algorithm 1 Proof. Although correctness and round complexity are proven in Lemma 2 of [19], that work did not consider message complexity. A message complexity of O(D) per each node in i R i follows since each node is responsible for at most one message being sent up and down T . Solving KLPA and Verifying the Block Parameter We now show how given a sub-part division and a T -restricted shortcut, we can round-and messageefficiently solve KLPA using Algorithm 1 with and without randomization. Our method is given by Algorithm 2. Roughly, Algorithm 2 works as follows. First, each leader l i of part P i , broadcasts an arbitrary message m i to all nodes in P i . Then, symmetrically to how m i was broadcast, each l i can compute f (P i ) and broadcast f (P i ) to P i . The most technically involved aspect of our algorithm is how l i broadcasts m i to all nodes in P i . If \|P i \| is smaller than D -easily verifiable within our round and message complexity -then broadcast can be trivially performed along the spanning tree of the single sub-part of P i in O(D) rounds with O(\|P i \|) messages. However, if P i is larger than D, we use shortcuts; in particular, we repeatedly spread messages within sub-parts and then across sub-parts. We use our sub-part divisions to limit our message complexity by only allowing sub-part representatives to use shortcuts. For our randomized algorithm, each part leader independently delays itself at the start of the algorithm by a delay chosen uniformly in the range [c]. Moreover, Algorithm 1 is now executed as before but O(log n) rounds are taken between each round of Algorithm 1 for each node to forward up to O(log n) outstanding messages. We illustrate the broadcast of m i in Figure 3. Algorithm 2 KLPA Algorithm, given a shortcut and a sub-part division. Broadcast m i from l i to all of P i along P i 's spanning tree 3: else 4: If randomization: delay each part independently by ∼ U (c); O(log n) Algorithm 1 blowup 5: Route m i from l i to r(l i ) 6: Initialize the set of "active" and "inactive" representatives. 7: for iteration ∈ [b] do 8: Run Algorithm 1 on A to send m i to all nodes in r∈A B i (r) ∩ R j 9: For r ∈ A broadcast m i from r to S(r) along S(r)'s representing tree 11: Broadcast m i over edges in E that exit sub-parts in S(A) 12: Every vertex v not in S(A) or S(I) that received a message in line 11 routes m i to r(v) 13: I ← I ∪ A ; A ← representatives that received a message in line 12 14: end for 15: end if 16: Symmetrically to lines 1-13 compute f (P i ) at l i 17: Broadcast result of f (P i ) from l i as in lines 1-13 The following lemma states the performance of our algorithm. Active: Inactive: Active: Inactive: Active: Inactive: Lastly, computing f (P i ) and broadcasting f (P i ) symmetrically requireÕ(b(D + C)) rounds. We now prove a message complexity ofÕ(m). We start by proving this message complexity for broadcasting m i . Message complexity is trivial if the part is of fewer than D nodes. Next consider parts of more than D nodes. Notice that nodes in a given sub-part only send messages in those rounds where the sub-part is active. Moreover, once a sub-part becomes inactive, it never again becomes active. Consequently, routing m i to l i requires O(n) messages across all sub-parts in all parts. Moreover, each of theÕ \|P i \| Correctness of broadcasting m i is trivial if \|P i \| < D. Moreover, if \|P i \| > D, a simple argument by induction over blocks shows that b iterations suffices for parts of more than D nodes. Correctness of computing f (P i ) and broadcasting f (P i ) symmetrically follow. We now show how we verify the block parameter of parts in a similar fashion. Our algorithm is given by Algorithm 3 and Lemma 4.5 gives the properties of the algorithm. Algorithm 3 Algorithm to determine which parts have good block parameter. where v ∈ P i knows leader l i ; sub-part division; T -restricted shortcut; desired block parameter b Output: for every P i , v ∈ P i learns if P i has block parameter b in the input shortcut 1: Run Algorithm 2 to broadcast arbitrary message m i from l i for every P i 2: if ∃v ∈ P i that did not receive m i then 3: Every such v broadcasts that it did not receive m i to neighbors in P i 4: Run Algorithm 2 to broadcast fact that ∃v ∈ P i that did not receive m i 5: end if 6: if a node in P i did not receive m i or received a message saying ∃v ∈ P i that did not receive m i then it decides that the block parameter of P i exceeds b 7: else every node runs Algorithm 2 to compute the block number of P i and every node decides if its parts' block number exceeds b based on the result 8: end if then Lemma 4.5. Given parts (P i ) N i=1 , a sub-part division, a c-congestion T -restricted shortcut, H, and desired block parameter b, Algorithm 3 deterministically (resp., w.h.p.) informs every node if its part's block parameter in H exceeds b inÕ(b(D +c)) (resp.Õ(bD +c)) rounds withÕ(m) messages. Proof. Round and message complexities follow trivially from Lemma 4.4 and Lemma 4.3. We now argue correctness. If a node does not receive m i when Algorithm 2 is first run then the block parameter of P i is certainly larger than b. When this occurs, all nodes will either be told by l i that the block parameter is larger than b or they will not receive m i , implicitly informing them that the block parameter of P i is larger than b. If all nodes receive m i , then l i clearly distributes to all nodes in P i the number of blocks incident to P i and so the block number of P i is correctly determined to be above or below b as desired. Having shown how sub-part divisions coupled with shortcuts can solve KLPA with or without randomization, we now detail how these primitives can be constructed and how a solution for KLPA can be used to solve PA. We do so first with the use of randomization and then deterministically. Round-and Message-Optimal Randomized PA In this section we show that with randomization, it is possible to solve PA simultaneously roundand message-optimally. To do so, we show first how to construct sub-part divisions and shortcuts with randomization to solve KLPA. Next, we use our randomized KLPA algorithm to solve PA. Computing Sub-Part Divisions Randomly We begin by showing how a sub-part division can be easily calculated with randomization by Algorithm 4 by randomly sampling sub-part representatives. We prove the complexity and correctness of Algorithm 4 in Lemma 5.1. Algorithm 4 Randomized sub-part division algorithm. Input: partition of V given by (P i where v ∈ P i knows leader l i Output: sub-part division 1: if \|P i \| ≤ D then 2: Let P i have one sub-part with representative l i 3: Compute sub-part spanning tree by an O(D) round BFS restricted to P i starting at l i 4: else 5: v ∈ P i decides to be a representative with probability min(1, log n D ) 6: for O(D) rounds do 7: Once v ∈ P i hears a representative ID it broadcasts it to neighbors in P i once 8: v ∈ P i remembers the neighbor from which it first heard a representative ID as its parent Proof. Runtime and message complexity are trivial. Correctness is trivial for parts of fewer than D nodes, so consider parts of more than D nodes. By construction, each claimed sub-part has diameter O(D). It remains to show that every node has a representative and there areÕ \|P i \| D sub-parts in P i . Fix node v and consider the ball of radius D around v. Since P i has at least D nodes, this ball is of size at least D and so a Chernoff bound shows that w.h.p Θ(log n) nodes in this ball will elect themselves a representative, meaning v will have a representative. A union bound over all v shows this to hold for every node. Moreover, the expected number of representatives in part P i is \|P i \| log n D and so a Chernoff bound shows that w.h.p there areÕ \|P i \| D sub-parts in P i . A union bound shows this holds for all parts w.h.p. Computing Shortcuts with Randomization We now show in Algorithm 5 how we message-efficiently construct a T -restricted shortcut with randomization. We rely on the CoreFast shortcut construction algorithm of Haeupler et al. [19], limiting message complexity by only allowing messages to originate from sub-part representatives. The correctness and runtime of Algorithm 5 is given by Lemma 5.2. Algorithm 5 Randomized shortcut construction algorithm. where v ∈ P i knows leader l i ; BFS-tree T ; sub-part division Output: T -restricted shortcut with congestionÕ(c) and block parameter < 3b 1: Set all P i active 2: for O(log n) iterations do 3: Run CoreFast [19] shortcut construction algorithm on representatives in active parts 4: Run Algorithm 3; set every P i with block parameter < 3b on result of CoreFast to be inactive and let every such P i use the shortcut edges assigned to it by CoreFast 5: end for Proof. We first argue runtime and message complexity. Haeupler, Izumi, and Zuzic [19,Lemma 4] show that CoreFast takes O(D log n+c) rounds. However, in this algorithm every node potentially sends a message up T once leading to super-linear message complexity. By amending CoreSlow so only theÕ n D sub-part representatives send a message up T once as we do, it is easy to see that the algorithm uses onlyÕ(n) messages total. Lastly, Algorithm 3 shows that Algorithm 3 with randomization uses onlyÕ(bD + c)) andÕ(m) messages. We now argue correctness. Haeupler, Izumi, and Zuzic [19,Lemma 4] show that each time CoreSlow is run, it computes a T -restricted shortcut with block parameter at most 3b for at least half of the nodes and congestion at most 8c. It is easy to see that only having sub-part representatives participate in CoreSlow does not affect correctness and so we conclude that after O(log n) iterations every P i has been rendered inactive. By construction every P i has block parameter < 3b and since the congestion of any edge increases by at most 8c in any iteration of Algorithm 5, the total congestion of our returned shortcut isÕ(c). Solving KLPA and PA with Randomization Having shown how to construct sub-part divisions and shortcuts round-and message-efficiently with randomization, we now use these constructions in Algorithm 6 to solve KLPA. Algorithm 6 Randomized KLPA algorithm. Input: a KLPA instance Output: a solution to the KLPA instance 1: Compute BFS tree T from an arbitrary root 2: Run Algorithm 4 to compute a sub-part division 3: Run Algorithm 5 to compute a T -restricted shortcut 4: Run Algorithm 2 with randomization Lemma 5.3. Algorithm 6 solves KLPA w.h.p inÕ(bD + c) rounds withÕ(m) messages. Proof. By Kutten et al. [26] we can elect a leader and then compute a BFS tree within our bounds. The remaining complexities and correctness follow trivially from Lemmas 5.1, 5.2 and 4.4. We now use our randomized KLPA algorithm to solve PA in Algorithm 7. We do so by starting with the singleton partition. We repeatedly coarsen this partition with KLPA by randomly sampling parts whose neighboring parts then merge with it. At each step in the coarsening we maintain the invariant that every part knows a leader and so in the end we need only solve KLPA. Input: a PA instance given by parts (P Output: a solution to the input PA instanec 1: Set P i ← {v} and l i = v for i ∈ \|V \| Each P i will maintain a leader, initially v 2: for O(log n) iterations do 3: Every P i agrees on a random coin flip and an edge (u, v) s.t. u ∈ P i , v ∈ P j for P i = P j and u, v ∈ P k if such an edge exists by running Algorithm 6. 4: if P i flipped a tails and P j flipped a heads then 5: Merge P j and P i and inform v ∈ P i that l i ← l j by running Algorithm 6 Proof. We begin by bounding round and message complexities. Each of the O(log n) iterations requiresÕ(bD + c) rounds andÕ(m) messages: P j can agree on some edge, share randomness and broadcast the ID of P k inÕ(bD + c) rounds andÕ(m) messages by Lemma 5.3. We now argue correctness. Each iteration 1/4 of all P j s participating in the algorithm get to merge in expectation and so by a Chernoff bound and union bound over P i s, O(log n) repetitions are sufficient to coarsen every P j to a P i w.h.p. Moreover, P 1 , . . . , P N is a valid input to a KLPA instance since we maintain the invariant that every node in a P j knows its leader. At the end of this coarsening our PA instance is now a KLPA instance with the same partition but a leader for each part: by Lemma 5.3 this KLPA instance is solvable within our round and message complexities. Round-and Message-Optimal Deterministic PA In this section we show how to deterministically solve PA round-and message-optimally. To do so, we show how to construct sub-part divisions and shortcuts deterministically. By our results in Section 4, this allows us to solve KLPA round-and message-optimally. Finally, we use our deterministic solution to KLPA to solve PA deterministically. Deterministic Star Joinings Because we do not have access to the symmetry breaking that randomization enables we must first begin by providing a novel deterministic symmetry breaking primitive -star joinings. We say a star joining is computed over parts (P i ) N i=1 if the following holds: a constant fraction of the parts P i are designated as receivers, and the other parts P i are designated as joiners. For every joiner part P i , all v ∈ P i knows some (common) edge with one endpoint in P i and another end-point in some receiver part P j . Implicitly, what Algorithm 7 did was compute a star joining in expectation. However, it used random coin flips to do so. In the remainder of this section we show how a star joining can be computed deterministically, given a deterministic solution to KLPA. We use as a sub-routine the 3-coloring algorithm of Cole and Vishkin [4]. Roughly, the algorithm works as follows. Every node begins with its ID as its color, meaning there are initially n colors. Next, every node updates its color based on its neighbors' colors, logarithmically reducing the number of possible colors. This is then repeated log * n times. For more, see Cole and Vishkin [4]. . v ∈ P i knows edge e i exiting P i and leader l i ; KLPA algorithm A Output: a star joining 1: Let G be the super-graph whose nodes are P i and directed edges are e i 2: Use A to mark all P i with more than one incoming e i ; designate all marked P i receivers and all P i with an e i into a marked part joiners; remove designated parts from G 3: Run the 3-coloring algorithm of Cole and Vishkin [4] on G 4: Use A to do the following: for color k = 1, 2, 3: every P i colored k is designated a receiver and removed from G ; also its in-neighbors are designated joiners and removed from G We give our algorithm for deterministically computing star joinings in Algorithm 8 and prove its correctness and complexity in Lemma 6.3. Then, Algorithm 8 computes a star joining over Proof. We begin by proving correctness. Line 2 yields stars of joiners centered around receivers. Moreover, the union of all nodes designated in Line 2 from a forest with trees of internal degree at least 2. Therefore, the number of internal (marked) super-nodes (and therefore the number of stars) in Line 2 is at most one half of the super-nodes of the tree. Now consider the result of Line 4. As no super-node in G has in-degree at least two at this point in the algorithm, the super-graph considered in Line 4 consists of directed cycles and paths. Thus, each time we remove a P i from the super-graph we remove at most three super-nodes from the graph and P i gets to merge with its neighbor. It follows that at least 1 3 of these super-nodes are merged. Combining the first and second stage, we find that the super-nodes are combined into stars, where the number of obtained nodes is less than 2/3 of the original nodes. Therefore the above algorithm computes a star joining. We now argue that our algorithm requires O(log * n) runs of A. This clearly holds for all subroutines of our algorithm except for Line 3. In particular, we must argue how the Cole-Vishkin algorithm can be efficiently simulated on our super-graph using O(log * n) runs of A. We repeat the following O(log * n) times. Let l i be the known leader of P i . Each P i begins with the color of l i 's ID. Next, the node in P i incident to the edge chosen by P i routes the color it received to l i using A. Then, l i performs the Cole-Vishkin computation and then broadcasts V i 's new color to all nodes in P i using A. Armed with a deterministic star joining construction, we now show how to compute sub-part divisions deterministically. We will also later use star joinings to solve PA given a KLPA algorithm. Computing Sub-Part Divisions Deterministically We now show how to deterministically compute sub-part divisions with Algorithm 9. We do so by iteratively computing star joinings of sub-parts and then turning each resulting star of sub-parts into a single sub-part. Algorithm 9 Deterministic sub-part division algorithm. Output: a sub-part division 1: Divide part P i into \|P i \| incomplete singleton sub-parts each of which is a single vertex 2: for O(log n) iterations do 3: Run Algorithm 8 on incomplete sub-parts with agreed on edges 5: For joiner F j from line 4 with agreed on edge (u, v) have u remember (u, v) as its parent edge in the sub-part spanning tree and run KLPA to have every u ∈ F j orient its parent edge in its sub-part spanning tree as toward u 6: Run KLPA on incomplete sub-parts to mark sub-parts with more than D nodes complete 7: end for Lemma 6.4. Given partition Proof. Round and message complexities are trivial apart from the fact that we must show that KLPA can be solved within our bounds on incomplete sub-parts. However, notice that an incomplete sub-part has fewer than D nodes by definition along with a spanning tree in which every node knows its parent; as such aggregating within each incomplete sub-part is trivially achievable with O(D) rounds and O(m) messages. We now argue correctness. Correctness for parts of fewer than D nodes is trivial. Consider parts of more than D nodes. Sub-parts continue to merge until they are complete and have at least D nodes and so our division clearly hasÕ P i D sub-parts. It remains to show that every complete sub-part's spanning tree has diameterÕ(D). When a complete sub-part results from two incomplete sub-parts joining, its spanning tree has diameter at most 2D. Call these nodes the core of the complete sub-part. When an incomplete sub-part F j -which has spanning tree with diameter at most D since it has fewer than D nodes by definition -joins a complete sub-part, it necessarily joins by way of nodes in the core. Thus, any node in F j is within 3D of any node in the core by way of the resulting sub-part's spanning tree. Similarly, any other subsequent incomplete sub-part that joins the complete sub-part will be within 4D of any nodes in F j by way of the associated spanning tree. Thus, every complete sub-part has spanning tree with diameter at most 4D. Computing Shortcuts Deterministically Having shown how sub-part divisions can be computed in a deterministic fashion, we now turn to our deterministic shortcut construction. We rely on heavy path decompositions [37]. Definition 6.5 (Heavy Path Decomposition [37]). Given a directed tree T , an edge (u, v) of T is heavy if the number of v's descendants is more than half the number of u's descendants; otherwise, the edge is light. A heavy path decomposition of T consists of all the heavy edges in T . It is immediate from the definition that each leaf-to-root path on an n-node tree T intersects at most log 2 n different paths of T 's heavy path decomposition. Given a rooted tree T of depth D, a heavy path decomposition of T can be easily computed in O(D) rounds using O(n) messages. Our deterministic shortcut construction algorithm, Algorithm 10, works by computing a heavy path decomposition and then computing efficient shortcuts on the obtained paths in a bottom-up order. Thus, we first provide a sub-routine, Algorithm 10, that computes shortcuts of congestion O(c log D) on path P . This algorithm assumes every node v begins with a set S(v) of part IDs that would like to use v's parent edge in the path. For simplicity, we assume vertices of P are numbered by their height, v = 1, 2, . . . (i.e., the source of the path is number 1, its parent is numbered 2, etc'). Algorithm 10 iteratively extends paths used for shortcuts, repeatedly doubling them in length, unless too much congestion results. See Figure 4. This algorithm's properties are as follows. Algorithm 10 Deterministic shortcut construction algorithm for paths. from v to u = v + 2 r along P provided there are no broken edges between v and u and set S i+1 (u) to S i (u) ∪ S i (v) 6: end if 7: end for 8: end for 9: return S f = S log 2 D−1 Lemma 6.6. Given directed path P of length D, desired congestion c and S ι : which denotes which parts want to use which vertices' parent edges in P , Algorithm 10 returns Proof. To bound the running time, observe that iteration i of the algorithm can be implemented in c + 2 i rounds. Summing over all iterations i = 0, 1, . . . , log 2 D − 1, the bound on the number of rounds follows. To bound the congestion of the output shortcuts, we prove by induction that before the i-th iteration the congestion on any edge is at most 2ci. This clearly holds before round i = 0. Now, before round i, all edges are used by at most 2ci parts. After round i all edges (v, v + 1) for a node v with v mod 2 r+1 ∈ [0, 2 i − 1] have their congestion unchanged, while all edges (v, v + 1) for a node v with v mod 2 i+1 ∈ [2 i , 2 i+1 − 1] have their congestion increase by at most 2c, implying the claimed bound on the congestion. We now turn to describing the overall shortcut construction algorithm, Algorithm 11, and analyze the resulting block parameter there. Again, Algorithm 10, works by computing a heavy path decomposition and then computing efficient shortcuts on the obtained paths in a bottom-up order. We limit message complexity by only allowing sub-part representatives to send a message requesting that an edge be used in their part's shortcut. As we show, each bottom-up computation yields good shortcuts for a constant fraction of parts. Thus, after each bottom-up computation, we can use Algorithm 3 to identify the parts for which our shortcut construction succeeded and freeze the shortcut edges of said parts. The correctness and runtime of Algorithm 11 is given by Lemma 6.7. Algorithm 11 Deterministic shortcut construction algorithm. Input: BFS-tree T ; sub-part division; partition of V , (P i ) N i=1 with leader l i known by v ∈ P i Output: T -restricted shortcut with congestionÕ(c) and block parameter < 3b 1: Initially all P i are active 2: Compute heavy path decomposition of T 3: for iteration ι ∈ [O(log n)] do 4: For representative r in active part P i , set S ι (r) = {l i }; S ι (v) = ∅ for all other v 5: Set each heavy path with no incoming light edges active 6: for log n repetitions do 7: Let S f be the return of Algorithm 10 run in parallel on all active heavy paths 8: For active path sink node v and light edge (v, u) Set all active paths inactive and all heavy paths with source u as in Line 8 active 10: end for 11: Run Algorithm 3 deterministically to set parts with block parameter < 3b inactive 12: end for 13: return ∪ ι S i (v) as v's shortcut edges Lemma 6.7. Given: partition Proof. We first prove the runtime and message complexity. As mentioned above, a heavy path decomposition of T can be computed in O(D) rounds using O(n) messages. We now bound the message and round complexity of each iteration. First note that we can inform every path if it has a light edge in O(D) rounds with O(n) messages. Next, running Algorithm 10 O(log n) times -once on each heavy path -requires O(c log D log n + D log n) rounds and O(n log n) messages. Lastly, notice that informing every node in a path that the path is now active requires O(D) rounds using O(n) messages. Lastly, by Lemma 4.5, running Algorithm 10 is within our stated bounds.. Summing over iterations, we conclude that the overall round and message complexities areÕ(b(c + D)) andÕ(m) respectively. We now prove correctness. Notice that by Lemma 6.6 the number of parts assigned to an edge in any particular iteration is at most O(c log D) and so the overall congestion on any edge is at most O(c log D log n) =Õ(c). We now analyze the block parameter. In particular, we argue that the number of active parts is at least halved in each iteration. Let A ι be the set of active parts in iteration ι. Let U ι be the set of heavy edges used by H but broken in iteration ι and therefore not assigned to any parts by S ι . Each edge in U ι received at least 2c − c = c more requests by parts to use it than in H. Thus each edge in U ι receives at least 2c − c = c requests from parts in A ι . However, each part in A ι can contribute at most b such additional requests to a broken edge, as each block can only send one additional request towards the tree's root. Consequently, we have \|U ι \| ≤ A ι b 2c . Next, we say an active part is bad in iteration ι if more than 2b of its edges of H are broken in iteration ι, and good in iteration ι otherwise. Note that for a good part the number of blocks in the output shortcut is at most 3b = O(b). On the other hand, every broken heavy edge used in H is used at most c times in H. We conclude that the number of bad active parts is at most \|U ι \| c 2b . Combining both upper and lower bounds on \|U ι \|, the number of bad parts active parts is at most A ι /2 in iteration ι. Thus, after O(log n) iterations all parts will be marked inactive, meaning the block parameter in the returned shortcut is at most 3b. Solving KLPA and PA Deterministically We now use our deterministic sub-part division and shortcut construction to solve KLPA deterministically. Algorithm 12 Deterministic KLPA algorithm. Input: a KLPA instance Output: a solution to the KLPA instance 1: Compute BFS tree T from an arbitrary root 2: Run Algorithm 9 to compute a sub-part division 3: Run Algorithm 11 to compute a T -restricted shortcut 4: Run Algorithm 2 without randomization Lemma 6.8. Algorithm 12 solves KLPA deterministically inÕ(b(D + c)) rounds withÕ(m) messages. Proof. By Kutten et al. [26] we can elect a leader and then compute a BFS tree within our bounds. The remaining complexities and correctness follow trivially from Lemmas 6.4, 6.7 and 4.4. Lastly, we use our deterministic solution to KLPA and star joinings to solve PA deterministically. We do so by starting with the singleton partition. We repeatedly coarsen this partition until it matches our input PA partition by applying KLPA to merge the stars given by a star joining. At Algorithm 13 Deterministic PA algorithm. Input: a PA instance given by parts (P i Output: a solution to the input PA problem 1: Set P i ← {v} and l i = v for i ∈ \|V \| Each P i will maintain a leader, initially v 2: for O(log n) rounds do 3: Every P i agrees on an edge (u, v) s.t. u ∈ P i , v ∈ P j for P i = P j and u, v ∈ P k if such an edge exists by running Algorithm 12. 4: Run Algorithm 8 to compute a star joining over the P i s with the agreed on edges 5: If P i is joined to P j in the star joining then merge P i and P j and inform v ∈ P i that l i is now l j by running Algorithm 12 6: end for 7: Run Algorithm 12 on the KLPA consisting of P i s each with leader l i each step in the coarsening we maintain the invariant that every part knows a leader and so in the end we need only solve KLPA. Lemma 6.9. Algorithm 13 solves PA deterministically inÕ(b(D +c)) rounds withÕ(m) messages. Proof. We first prove round and message complexities. Our algorithm only runs Algorithm 12 and Algorithm 8 logarithmically many times the latter of which consists of O(log * n) calls to Algorithm 12. Thus, the stated round and message complexities follow trivially from Lemma 6.8. We now argue correctness. Each round a constant fraction of the P j s participating in the algorithm get to merge by definition of a star joining and so O(log n) repetitions are sufficient to coarsen every P j to a P i . Moreover, P 1 , . . . , P N is valid input to a KLPA instance since we maintain the invariant that every node in a P j has an elected leader. At the end of this coarsening our PA instance is now a KLPA instance with the same partition as our input PA but a leader for each part is known. By Lemma 5.3, Algorithm 12 solves this KLPA instance within our round and message complexities. Combining Lemma 6.9 and Lemma 5.4 we obtain our main result -Theorem 1.2. During this weighted BFS, nodes claim as part of their ball any nodes they reach that have not yet been claimed or started their weighted BFS. The weights used for the weighted BFS change in each round: any weight strictly inside a claimed ball is updated to have weight 0 and any edge incident to two claimed balls is additively increased. Moreover, the increments used by the weighted BFS geometrically increase in each iteration so that despite increasing edge weights, the weighted BFS can still efficiently proceed. Lastly, the union of all BFS trees returned by every weighted BFS is returned as a tree, T * , that approximates distance in the graph; to inform nodes of their approximate distance from the source, the source can simply broadcast on T * . What makes this algorithm difficult to implement is that running a weighted BFS with edge weights set to 0 requires that a weighted BFS traverse components connected by weight-zero edges of potentially large diameter "in a single round". To overcome this issue, Haeupler and Li [18] use PA to efficiently traverse these connected components. For more see Haeupler and Li [18]. Relying on our algorithm for PA and observing that these dominate the round and message complexity of the weighted BFS calls, our claimed round and message complexities follow. Proof. Haeupler and Li [18] provide a distributed algorithm with the stated round complexity and approximation factor based on a solution to PA. Roughly the algorithm works as follows. We compute O( log n β ) low diameter decomposition; in particular, as in Miller et al. [30] every node starts a weighted BFS at a randomly-chosen time and runs this weighted BFS for O( log n β ) rounds. During this weighted BFS, nodes claim as part of their ball any nodes they reach that have not yet been claimed or started their weighted BFS. The weights used for the weighted BFS change in each round: any weight strictly inside a claimed ball is updated to have weight 0 and any edge incident to two claimed balls is additively increased. Moreover, the increments used by the weighted BFS geometrically increase in each iteration so that despite increasing edge weights, the weighted BFS can still efficiently proceed. Lastly, the union of all BFS trees returned by every weighted BFS is returned as a tree, T * , that approximates distance in the graph; to inform nodes of their approximate distance from the source, the source can simply broadcast on T * . What makes this algorithm difficult to implement is that running a weighted BFS with edge weights set to 0 requires that a weighted BFS traverse components connected by weight-zero edges of potentially large diameter "in a single round". To overcome this issue, Haeupler and Li [18] use PA to efficiently traverse these connected components. For more see Haeupler and Li [18]. Relying on our algorithm for PA and observing that these dominate the round and message complexity of the weighted BFS calls, our claimed round and message complexities follow. B Our Results In Tabular Form Throughout the paper we state our results in utmost generality by giving our algorithms' running time in terms of the optimal block parameter b and congestion c. As stated in Theorem 1.2 and its Corollaries 1.3, 1.4 and 1.5, for PA and MST, our deterministic algorithms requireÕ(b(D + c)) rounds and for Partwise Aggregation, MST, L -approximate SSSP and (1 + )-approximate Min-Cut (for fixed ) our randomized algorithms requireÕ(bD + c) rounds. To make these bounds more concrete, we review some known bounds on the parameters b and c in Table 1, and then state the implied running times of our algorithms for the above problems in Table 2. General Planar Genus g Treewidth t Pathwidth p DeterministicÕ(D + Reviewing Table 2, we note that for all problems considered, a matching worst case round lower bound ofΩ(D + √ n) is given by Das Sarma et al. [5], while a trivial lower bound of Ω(D) holds for these problems for all graphs. Our algorithms match the worst case bounds and the Ω(D) lower bound (up to polylog terms) for any constant genus, treewidth and pathwidth, all while requiring onlyÕ(m) messages. What the exact optimal dependence on the parameters g, t and p remains an open question.	2018-01-16T06:22:32.000Z	2018-01-16T00:00:00.000	{ "year": 2018, "sha1": "a4fe76b157cec14ecb04dcc8a8cd6700d2ab0b0d", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "a4fe76b157cec14ecb04dcc8a8cd6700d2ab0b0d", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Computer Science" ] }
54061439	pes2o/s2orc	v3-fos-license	The Impact of Hepatitis B Virus and Epstein-Barr Virus in Pathogenesis of Rheumatoid Arthritis The study reviews the current impression that previous infections with Hepatitis B Virus (HBV) or Epstein-Barr virus (EBV) are possible etiology for rheumatoid arthritis (RA). Patients with rheumatoid arthritis (n=100) and a control group (healthy persons, n=50, and osteoarthritis patients (n=50) were tested by Enzyme-Linked Immunosorbent Assay (ELISA) for HBs Ag, IgG anti-HBs antibodies, total anti-HBc antibodies, and IgG antibodies specific to EBV. RA patients were also tested for rheumatoid factor and C-reactive protein by latex agglutination test, total WBCs count, and ESR. The study extended from July 2016February 2017 in Baghdad Teaching Hospital, Baghdad/ Iraq. The results revealed that differences in antibody titers of HBV markers (HBs Ag, IgG anti-HBs antibodies, total anti-HBc antibodies) in serum of RA group and control group were statistically not significant while the mean antibody titers of IgG antibodies for EBV in RA patients was higher than that in control group and the difference was statistically significant (P < 0.05). EBV infection might be a possible factor in pathogenesis of RA, while the role of HBV infection is not evident. INTRODUCTION Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease that primarily affects the synovial membranes of multiple joints in a characteristic fashion 1 .Despite intensive research over many decades, the cause of RA remains unknown.Three sectors of interconnected research are currently most encouraging: genetic elements, autoimmunity and immunoregulatory abnormalities, and possible microbial infection 2 . Microbial infections are regarded as the major environmental factors that may cause inflammatory arthritides in genetically susceptible persons 3 .Numerous studies have demonstrated that some microorganisms might be related with the pathogenesis of RA through their previous or their chronic infection 4 . Many clinical and laboratory methods used to find the role of microorganisms in the pathogenesis of RA.However, direct isolation of the microorganisms from the synovial tissue or synovial membrane almost always was negative or lacks the reproducibility 5 .Many investigators demonstrated the presence of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) of some microorganisms in the synovial tissues including the synovial membrane or synovial fluid using polymerase chain reaction (PCR), southern blot hybridization technique, and some other advanced nucleic acid detection methods 6,7 .Studies on the synovial fluid clarified that antibodies against some microorganisms could be detected in RA patients and their titers were higher than their corresponding in the control group 8 .Some studies showed that some microorganisms could be detected in the blood of patients with RA, while some investigators showed that the antibody titers for some microorganisms in the serum of patients with RA are higher than those in the control group 9,10 . Many viruses are implicated in the etiology of RA; the most proposed to play a role in RA, include: Hepatitis B virus, Hepatitis C virus, Epstein -Barr virus, Retroviruses, Cytomegalovirus, and Human parvovirus B19 11,12 . METHOD This study was conducted to estimate the Hepatitis B virus markers (HBs antigen, IgG anti-HBs antibodies, and IgG anti-HBc antibodies) ,This test was performed using commercially available kit (DRG.ELISA).and for IgG antibodies for Viral capsid antigen (VCA) of EBV using commercially available kit (DRG.ELISA). in the patients with rheumatoid arthritis and control groups in a period between July 2016 and January 2017, using ELISA technique .One hundred Iraqi patients with rheumatoid arthritis who were attending the Rheumatology Consultation Clinic or admitted to Baghdad Teaching Hospital.All of whom met the revised criteria for RA as defined by the American College of Rheumatology 1987.Control group was age and sex matched with the patients group (RA group).The control group was sub grouped into: -Osteoarthritis (O.A.) control subgroup included (50) patients with osteoarthritis of the knee and (50) healthy individuals chosen from blood bank donors, who have no clinical evidence of any obvious disease. The study protocol also included: serological tests for detection of rheumatoid factor (RF) and C-Reactive Protein (CRP) by latex test, hematological tests to enumerate the total WBCs count (cell count/ mm3 ) using hemocytometer manual cell counting, and measurement the erythrocyte sedimentation rate (ESR mm/hr) by westergren method. Data have been analyzed statistically using SPSS program ver.10.Results were expressed using simple statistical parameters such as mean ±SD.Analysis of qualitative data was assessed using ×² Chi square test.Analysis of quantitative data was estimated using t-test and ANOVA (analysis of variance).The strength of linear relationship between X and Y variables was determined by sample correlation coefficient (r). RESULTS The RA group involves 100 patients with rheumatoid arthritis; their diagnosis based on the American Rheumatism Association 1987 revised criteria for the classification of RA.The frequency distribution of these criteria in the RA group is demonstrated in table (1). Other clinical features revealed that only 31 RA patients (31%) have extra-articular manifestations, only 3 RA patients (3%) have other autoimmune diseases (one patient with systemic lupus erythematosis, while the other two with insulin dependent diabetes and alopecia areata; depending on history taking), while of the O.A. control subgroup, only one patient (2%) have autoimmune disease (vitiligo).Of the RA group, 15 patients have joint effusion (15%), while the O.A. control subgroup only 2 patients (4%) have joint effusion. The RA group involved 100 patients, their age ranged between (16-76 years); with mean age (44.8 ±10.2 years).The age of highest incidence is between 35-40 years.There were 82 females and 18 males with female to male ratio was (4.5:1.0) as shown in figure (1). In the RA group the rheumatoid factor was positive in 88 patients (88%) as mentioned in table (1), while it was positive in only one O. A. patient subgroup and none in the healthy control subgroup (P<0.0001). Males Females The results revealed that CRP was positive in 89 RA patients (89%), and negative in 11 RA patients (11%).The CRP was positive in 2 patients with osteoarthritis (4%) and negative in 48 (96%) patients with osteoarthritis, while all the healthy control subgroup was negative.There was statistically significant differences between the RA group and the control group (P<0.0001),table (2). The mean of total white blood cells count in the patients group (RA) was 6.548× 10/L (±2.207), while those for the healthy control subgroup and O. A. patient's subgroup were 7.354× 10/L (±1.877) and 7.204×10/L (±3.361) respectively.The results showed no significant differences (P=0.408). Erythrocyte sedimentation rate (ESR) was found to be elevated in RA group in comparison to the O.A. control subgroup and healthy control subgroup.There was statistical significant differences between the RA group and control group (P=0.042),table (3). The seroprevalence of anti-HBs Abs in patients group showed that the mean O.D. measured at 450 nm was 1.0900 (±0.3822); while those for the healthy control subgroup and OA control subgroup were 1.0512 (±0.3357) and 0.9612 (±0.3359) respectively.The difference between RA and control groups was statistically not significant (p=0.121);figure (3). The seroprevalence of anti-HBc Abs in RA group and control group showed that the mean O.D. for RA patients' sera measured at 450 nm was 0.6244 (±0.1990); while those for the healthy control subgroup and OA control subgroup were 0.6448 (±0.1882) and 0.6752 (±0.2052) respectively.The results were statistically not significant (p=0.334);figure (4). The Seroprevalence of IgG Antibodies specific to EBV in RA group and control group clarify that the mean absorbance value for the RA group sera measured at 450 nm was 0.7569 (±0.1892); while those for the healthy control subgroup and O.A. control subgroup were 0.6723 (±0.1329) and 0.6962 (±0.1059).This difference between the RA and control groups was statistically significant (p=0.005),figure (5). 3: The seroprevalence of anti-hepatitis B surface antibodies in study groups The results showed non-significant positive correlation between EBV antibodies and other parameters as shown in table (4). DISCUSSION Rheumatoid arthritis is not an infrequent disease in Iraq; it was observed in about 1% of Iraqi people 13 .In our study the age of highest incidence for RA patients is at the fourth decade (35-40years), while abroad studies had diverse data for the predominant age of disease [14][15][16] . Female to male ratio in this study was 4.5:1 which indicates female predominance.Many other studies revealed almost similar results 17,18 .Over presentation of women of RA is mostly linked to sex and hormonal predisposing factors 19 . Past hepatitis B infection and vaccination against it have been implicated in the potential triggering or flare of some autoimmune diseases, including rheumatoid arthritis (RA) 20 . T h i s s t u d y d e m o n s t r a t e d t h a t seroprevalence of HBs Ag, anti-HBs antibodies, and anti-HBc antibodies were of low antibody titers in RA and control groups.There were no significant differences between RA and control groups.Kurbanov SA, et al., did not find specific markers of HBV infection, which is in accordance to our results in that HBV is not a probable causative agent of RA.However, vaccination against HBV may be associated with the development of RA, which might represent the activation of RA in genetically susceptible individuals 21 . Maillefert et al., detected the development of RA in six patients, 2 months following hepatitis B vaccination 22 .On the contrary to these result, Elkayam et al. did not notice the development of RA in 44 subjects receiving hepatitis B vaccine 23 . There might be no link between HBV, weather past infection or vaccination with the development of RA.An alternative hypothesis supposed the presence of a link, thus a single or more peptides of HBV might bind to MHC class II molecules in a genetically susceptible patients leading to triggering RA 24 . Our study revealed that the mean of IgG antibody titers specific to VCA of EBV in RA group was higher than in control group.The results were statistically significant (p=0.005).It was the first study in Iraq that looks for the role of EBV in RA as far as I know.The seroprevalence of EBV is generally high among healthy adult population which indicates a previous infection with EBV may occur in early childhood 25 . A similar ELISA technique was used by Billings et al. to detect the same antibodies (anti-VCA antibodies) in serum of RA patients.They detected a twofold increase in mean of values for IgG antibodies compared with the control group 26 .The rise in antibodies specific to EBV antigens in existing RA more than in healthy control has been reported in other studies [27][28][29][30][31] . Possible mechanisms were suggested for the contribution of EBV in pathogenesis of RA.The sequence similarity of an EBV capsid antigen to HLA -DR β 1 RA susceptibility sequence QKRAA, could lead to antibody cross reaction and the production of autoimmune response 32 . T cells proliferating against gp110 might represent a chronic exposure to EBV antigens, and thus leading to a chronic inflammatory response in patients with RA 33 .L o n g i t u d i n a l d a t a o n R A p a t i e n t s demonstrated a non-significant positive correlation between CRP and EBV antibody titers (r =0.231).All other inflammatory markers done in the study showed almost similar results; like ESR, and WBCs count in blood.Other clinical features and parameters used in the study like number of inflamed joints, presence of extra-articular manifestations and joint effusion, age, sex, smoking, and allergy; did not show any significance in relation to anti-EBV antibody titers.So EBV might trigger the disease but has no effect on the next steps in pathogenesis of RA, or on the activity of the disease. In conclusion this study gave rise to that the rheumatoid factor were higher in RA patients than in control group.There was a significant difference of IgG anti-EBV (anti-VCA) in RA patients compared to control group which indicated that previous infection with EBV may trigger RA. The antibodies to Hepatitis B virus, were very low in RA patients as well as in control group, thus this virus might have no role in RA.There was no correlation between IgG antibody titers against EBV in serum with some parameters like RF, CRP,ESR, WBCs count in blood or synovial fluid , which indicated that this virus if play a role in RA, have no effect on the next steps in pathogenesis of RA. CONCLUSION So further studies including large sample size of cases are needed to reveal specific link between various infectious agents and RA.As well as molecular techniques including PCR and DNA hybridization test should be introduce for detection of viral infections.In addition to that searching for other autoantibodies rather than RF, like antinuclear antibodies and anti-keratin antibodies and study their association with features of infection in RA patients. Fig. 1 : Fig. 1: Age and sex distribution of patients with rheumatoid arthritis enrolled in the study Table 3 :Fig. 2 : Fig. 2: The seroprevalence of Hepatitis B surface antigen in study groups Fig. 4 :Fig. 5 : Fig. 4: The seroprevalence of anti-Hepatitis B core antibodies in study groups	2018-12-01T08:13:37.280Z	2017-09-25T00:00:00.000	{ "year": 2017, "sha1": "a90db9928bd764138c982c73d22dd629cc590517", "oa_license": "CCBY", "oa_url": "https://doi.org/10.13005/bpj/1258", "oa_status": "HYBRID", "pdf_src": "ScienceParseMerged", "pdf_hash": "a90db9928bd764138c982c73d22dd629cc590517", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
213478879	pes2o/s2orc	v3-fos-license	Analysis of the Influence of Coagulant on Phosphorus in Landscape Water Environment This study selected three kinds of inorganic polymer coagulant: polyferric sulfate, polyaluminium chloride, polyaluminium ferric chloride, two inorganic low molecular coagulants: aluminum chloride and aluminum sulfate. The coagulation experiment of lake water without “water bloom” was carried out. The results were analyzed by multivariate linear regression and principal component regression analysis. It showed that the effects of the five coagulants on the phosphorus were the same when the landscape water was treated with different dosages of coagulants. The sequence was: total nitrogen (TN) > dosage > ammonia nitrogen > CODcr > turbidity > PH. Among them: the highest effect of total nitrogen on phosphorus was aluminum chloride (71.73%). The most obvious effect of dosage on phosphorus was polyaluminium chloride (34.68%). The most significant effect of ammonia nitrogen on phosphorus was polyaluminum chloride (16.98%). Introduction The shortage of water resources is a major problem in China. Recycled water is widely used in landscape water environment, which solves the need of water shortage for urban water environment. It also was the best way to restore the natural restoration of water ecological cycle [1]. Phosphorus is a restrictive nutrient for algae growth in landscape water environment, playing an important role in the outbreak of "water bloom" and the growth process of phytoplankton, and is one of the main reasons for eutrophication in landscape water environment [2]. Controlling the concentration of phosphorus in landscape water environment, breaking the ratio of nitrogen and phosphorus between 10 and 20, preventing water bloom production, coagulant is the most direct, rapid and effective method. Taking the artificial lake of Beijing vocational college of agriculture and the lake of xiaoyue lake of yongding river --the lake without water bloom as test water, the author analyzed the treatment effect of phosphorus in landscape water environment with different coagulants, studied the influence of various pollutants and different amounts of coagulant on phosphorus in water environment, and provided reference for landscape water environment management. Test Method. The experiment was carried out with a beaker of a programmable six-step mixing and coagulation experiment device. The water was stirred and separated into six of the 1000 ml beaker, open the mixer switch, lowered stirrer, and graduated pipette will make good coagulant added to upper part of the dosing tube instrument in four stages: the first phase of speed (G) 300 RPM for 1 minute, the second stage is 132.2 RPM, working for 1 minute, the third stage G value is 28.8 RPM, working for 10 minutes, and stage 4 G value is RPM for 20 minutes. We took supernatant through sampling mouth, suveied and evaluated the water quality indicators. The Research Methods The method of multivariate linear regression and principal component regression analysis in DPS data processing system is adopted [3]. Its basic principle is to deal with collinear relations of various influencing factors by the situation of high correlation (complex collinear) between independent variables. The effects of main test items and different coagulants on phosphorus in reclaimed water environment were studied. The above experimental results show that the amount of phosphorus in water gradually decreases with the increase of dosage of polyaluminium chloride and polyaluminium ferric chloride. Polymeric ferric sulfate, aluminum sulfate and aluminum chloride decrease the amount of phosphorus in water with the increase of dosage. Analysis of Phosphorus Removal Effect of Coagulant In order to further study the experimental results of the five reagents, the phosphorus removal percentage was analyzed, as shown in figure 2. The above figure shows that aluminum chloride has the best effect of removing phosphorus with the same dosage among the five reagents, and the removal rate of phosphorus in water reaches 100 % when the dosage is 32mg/L. The second best effect is polyferric sulfate. When the dosage is 15 mg/L, the removal rate of phosphorus is 95.58%, while when the dosage is 30 mg/L, the removal rate of phosphorus reaches 97.35%. Thirdly, polyaluminium chloride, when the dosage is 15 mg/L, the removal rate of phosphorus is 81.40%, while when the dosage is 30 mg/L, the removal rate of phosphorus is 95.35%. Moreover, the polyaluminum ferric chloride, when the dosage is 15 mg/L, the removal rate of phosphorus is 46.67%, while when the dosage is 30 mg/L, the removal rate of phosphorus is 75.56%. The worst effect was aluminum sulfate, the removal rate of phosphorus was 12.0 % when the dosage was 15 mg/L, and 64.00 % when the dosage was 30 mg/L. At the same time, with the addition of aluminum ferric chloride, the phosphorus removal effect of the other four coagulants fluctuated with the increase of dosage. When the dosage of aluminum sulfate was 10 mg/L, the phosphorus removal rate was -14.0 %. Analysis of Factors Affecting Phosphorus Removal by Coagulant Because of different coagulation reagents and their input amount, the effect of phosphorus treatment in water environment is different. So we used linear regression and principal component regression analysis with the results of three kinds of polymeric coagulants and two kinds of molecular coagulant test results, to seek different coagulants, the PH, ammonia nitrogen, CODCr, total nitrogen and the amount of pharmaceutical inputs with the change of phosphorus and analysis of its remarkable. Five kind of coagulant with different coagulant dosage, PH, ammonia nitrogen, CODcr, total nitrogen and phosphorus by principal component regression linear regression analysis, the significance test is made to the regression model, the model factor regression coefficient significance test results: the regression model of F values were greater than F 0.05 , correlation coefficient R > R 0.05 , model fitting is good. The six main influencing factors of drug dosage, PH, turbidity, ammonia nitrogen, CODcr and total nitrogen were calculated respectively. The characteristic values and percentages of the factors affecting total phosphorus in the five coagulants are shown in Table 1. According to the above table, the order of influence factors on total phosphorus in each coagulant is the same, and the order is: total nitrogen (TN) > dosage > ammonia nitrogen > CODcr > turbidity > PH. The above table shows that the total nitrogen (TN) affects phosphorus in the range of 71.73 ~ 43.59 (%), the dosage of 34.68 ~ 14.96 (%), ammonia nitrogen accounted for 3.55 ~ 16.98 (%), CODcr accounted for 1.08 ~ 8.62 (%), turbid The degree is 0.12 to 5.17 (%), and the PH value is 0.00 to 1.11 (%). It can be seen that the influence value of each impact factor on phosphorus is different under the different percentage. Among them, aluminum chloride has the highest effect on phosphorus, accounting for 71.73%, followed by polyferric sulfate, accounting for 71.09%. The weakest effect is polyaluminium chloride, accounting for 43.59%. The most obvious effect of the dosage on phosphorus is Polyaluminium chloride, accounting for 34.68%, followed by polyaluminum ferric chloride, accounting for 29.22%, the most important is aluminum sulfate, accounting for 14.96%; the most significant effect of ammonia nitrogen on phosphorus is polyaluminium chloride, accounting for 16.98%, after that is a polyferric sulfate, accounting for 14.96%. The weakest effect is polyaluminium ferric chloride, accounting for 3.55%. The results showed that the turbidity and pH had the weakest effect on phosphorus. In order to serve the production better.The turbidity and pH of the coagulant were less than 10% were eliminated, and the principal component regression was combined. Linear regression simplified analysis, significant regression test of regression coefficient of each model factor: F in regression model was greater than Conclusion It can be seen that the removal effect of these five coagulants on phosphorus is: aluminum chloride > polyferric sulfate > polyferric chloride > polyferric aluminum chloride > aluminum sulfate. The amount of phosphorus in water delinces with the increase of dosage of polyaluminum chloride and ferric chloride. The amount of phosphorus in water decreases with the increase of dosage of polyferric sulfate, aluminum sulfate and aluminum chloride, but when the dosage of aluminum sulfate is 10mg/L, the amount of phosphorus in water decreases. Compared with raw water, the amount of phosphorus increased by 14.0%. After five kinds of coagulant use different dosage to treat the water quality of landscape environment, The main pollution indexes in the water environment have the same order of total phosphorus, and the sequence is: Total nitrogen (TN) and dosage of ammonia nitrogen >codcr> turbidity >PH. The highest impact of total nitrogen on phosphorus was aluminum chloride, accounting for 71.73 %, followed by polyferric sulfate, accounting for 71.09 %, and the weakest impact was polyaluminium chloride, accounting for 43.59 %. The most obvious effect of the dosage on phosphorus is Polyaluminium chloride, accounting for 34.68%, followed by polyaluminum ferric chloride, accounting for 29.22%, the most important is aluminum sulfate, accounting for 14.96%; the most significant effect of ammonia nitrogen on phosphorus is polyaluminium chloride, accounting for 16.98%, followed by it is a polyferric sulfate, accounting for 14.96%. The weakest effect is polyaluminium ferric chloride, accounting for 3.55%.	2020-02-13T09:12:33.800Z	2020-02-11T00:00:00.000	{ "year": 2020, "sha1": "1d5ad3546d7c7c8a867f4a24943487184e6d8f26", "oa_license": null, "oa_url": "https://doi.org/10.1088/1757-899x/730/1/012045", "oa_status": "GOLD", "pdf_src": "IOP", "pdf_hash": "30a8d06881cf7d5d6b4d1d8d1722966b2d5477dc", "s2fieldsofstudy": [], "extfieldsofstudy": [ "Environmental Science" ] }
17482340	pes2o/s2orc	v3-fos-license	Correlation functions of twist operators applied to single self-avoiding loops The O(n) spin model in two dimensions may equivalently be formulated as a loop model, and then mapped to a height model which is conjectured to flow under the renormalization group to a conformal field theory (CFT). At the critical point, the order n terms in the partition function and correlation functions describe single self-avoiding loops. We investigate the ensemble of these self-avoiding loops using twist operators, which count loops which wind non-trivially around them with a factor -1. These turn out to have level two null states and hence their correlators satisfy a set of partial differential equations. We show that partly-connected parts of the four point function count the expected number of loops which separate one pair of points from the other pair, and find an explicit expression for this. We argue that the differential equation satisfied by these expectation values should have an interpretation in terms of a stochastic(Schramm)-Loewner evolution (SLE_kappa) process with kappa=6. The two point function in a simply connected domain satisfies a closely related set of equations. We solve these and hence calculate the expected number of single loops which separate both points from the boundary. Introduction In a recent paper, Werner [1] has shown there exists a measure on simple loops on any Riemann surface which has the property of conformal restriction. This is to say that if D ′ ⊂ D are any two subdomains of the manifold, the measures on loops in D ′ obtained by (a) restriction of the measure on loops in D to those in D ′ , and (b) conformally mapping D → D ′ , are the same. Moreover this measure is unique up to multiplication by a constant. We refer to these loops throughout the paper as self-avoiding loops and to the mass of any subset under Werner's measure as the µ-mass. One may also consider the set of self-avoiding polygons on some regular lattice embedded in the manifold. The total number of such polygons of length l is known to grow as µ l where µ is lattice dependent. The measure which weights each polygon with a factor x l c (where x c = µ −1 ) has the restriction property, and is commonly conjectured also to be conformally invariant in the limit of vanishing lattice spacing, which means that it should give a particular example of Werner's measure. In this paper we assume this to be true, and hence conjecture values of the µ-mass of certain loop subsets, using (non-rigorous) Coulomb gas and CFT methods applied to a generalised model, the O(n) model. The O(n) model encompasses a large group of physically interesting models, including the Ising spin model, the percolation problem and self-avoiding loops. The theory may be written as a spin model with nearest neighbour interactions, or alternatively in the loop gas picture, in which the states of the model are non-intersecting loops constructed on the edges of the spin model. Each loop is weighted with a factor nx l , where x is related to the reduced coupling in the model and l is the length of the loop. In the Ising model, these loops are the cluster boundaries of a spin model defined on the dual lattice. At the critical point of the model, x = x c (n), it is conjectured to flow under the renormalization group to a Gaussian free field [2,3], which is a well known example of a conformal field theory (CFT). One of us [4] showed the existence of operators in this theory whose two point correlation functions count loops around one of two points with a weight n ′ rather than n, hence giving information about the distribution of loops. The choice n ′ = −n is of particular interest; these operators are then called twist operators and are at (r, s) = (1,2) in the Kac classification [5]. Hence, they have null states at level two. The two point correlation function is then equivalent to the expectation value of (−1) N in the loop ensemble, where N is the number of intersections of loops with a defect line, a continuous curve connecting the two points. The twist operators may therefore be thought of as a source and a sink for this defect line. Self-avoiding loops are described by the loop gas picture of the O(n) model with n → 0. The partition function and correlation functions are determined (to order n 1 ) by graphs with just a single loop. The two point correlation function of twist operators therefore leads to an analytic expression for the number of single loops which separate the locations of the operators, weighted by x l c as in figure 1. This number is logarithmically divergent in the continuum limit, however, due to the contribution from vanishingly small loops around each of the points. In this paper, we consider the four point correlation function of these twist operators on the Riemann sphere. In the loop gas picture, we may think of each operator as again being a source (or sink) of a defect line. Hence there is a defect line running between each pair of points. We argue that the choice of paths for the defect lines is unimportant and that they may run between any two distinct pairs of points. The four point correlation function can then be shown to be the expectation value of (−1) N where N is now the total number of crossings of loops with defect lines. Particular semi-connected parts of this four point function yield the expected numbers of weighted loops (the µ-mass of loops) which wind around two of the four locations of the operators, as in figure 2. CFT may be used to derive the form of the four point function, since the null states of the operators imply that the correlation functions obey a set of Belavin, Polyakov and Zamalodchikov (BPZ) type partial differential equations [6]. We solve these equations and hence find analytic expressions for the µ-mass of loops which separate one pair of points from the other. For loops Im Re Figure 1: Two examples of single loops winding around one of two operators for the theory on the Riemann sphere. The stars mark the locations of the operators and the dashed line indicates that the curve wraps around the back of the sphere. Two point function is the sum of such loops, weighted by x c to the power of their length. Notice that the sum contains loops with vanishingly small perimeter in the continuum limit. which separate (z 1 , z 2 ) from (z 3 , z 4 ), for example, we obtain the expression These expected numbers of weighted loops are finite in the continuum limit because there is no contribution from vanishingly small loops, and are invariant under conformal transformations. We show that the non-leading behaviour of these expressions as η → 0 reveals the derivative of the central charge with respect to n at n = 0. This is consistent with the interpretation [7] of the stress tensor as the spin-two component of the relative probability that a loop passes between two points in the limit z 12 → 0. We use similar arguments for the O(n) model in a simply connected domain. Such a domain may always be mapped via a conformal transformation to the upper half plane with the real axis as the boundary. The two point function in this domain satisfies the same partial differential equations as the four point function in the bulk, with the locations of the two operators and the reflections of these points in the real axis being the locations of the four operators in the bulk theory. We show that the connected two point function of twist operators in the n → 0 limit counts the mass of loops around the locations of both operators, and find the explicit expression where η is now the cross ratio of the two points together with their conformal images in the boundary. Im Re Figure 2: Two examples of single loops winding around the locations of two of four operators for the theory on the Riemann sphere. A particular semiconnected four point correlation function counts the number of such loops, weighted by x c to the power of their length. Note that, in contrast to the two point correlation function above, there are no vanishingly small loops contributing to the sum. The layout of this paper is as follows. In the next section, we recall the arguments leading to the Coulomb gas picture of the O(n) model. In section 3 we discuss twist operators in the O(n) model and derive the form of the two point correlation function of these operators. The small n limit of the theory is explored and the correlation function in this limit is shown to depend (to order n 1 ) only on the configurations of a single loop. In section 4 we discuss the four point correlation function of twist operators. Because the twist operators have null states at level two, this correlation function satisfies a set of partial differential equations, which we solve analytically. The interpretation of the four point function in terms of a pair of defect lines is explained, followed by the example of the four point function of twist operators as the four point function of spin operators in the Ising model. Section 4.1 is devoted to the small n limit of the four point function and contains a derivation of the mass of loops winding around two of the four points. We then explain how these numbers, as functions of the positions of the four points, hold a key to measuring the effective central charge of the O(n) model as n → 0. In section 6 we apply the theory of twist operators to the O(n) model in a simply connected domain and show that the mass of loops around two points in such a domain is invariant under conformal transformations and finite in the limit of vanishing lattice spacing. Finally, in section 7 we show that the mass of loops around two of four points on the Riemann sphere can be thought of in terms of an SLE 6 process. The Coulomb Gas Let us start with the following partition function for the O(n) model [2]: The s(r i ) are n-component spins on a lattice {r i } and the product is over pairs of nearest neighbours. The product in the partition function can be expanded into a sum of 2 N terms, where N is the number of nearest neighbours. Each term can be represented by a graph of open and closed loops in the following way: the neighbouring sites, i and j, are joined together by a line if the xs(r i ) · s(r j ) term was chosen in the expansion of equation (1), or left disconnected if the number 1 was chosen instead. The trace over spins may then be done for each graph individually. Since Tr s(r i ) odd = 0 because of the symmetry under the transformation s(r i ) → −s(r i ), all graphs containing an odd number of s(r i ) make no contribution to the partition function. These are the graphs with open loops, hence the partition function will only contain contributions from graphs with closed loops. Let us consider the honeycomb lattice for simplicity, then there may be 0,1,2 or 3 powers of a given s(r i ). The trace over spins of s(r i ) 0 will contribute a factor of 1, whereas, from Tr s a (r)s b (r) = δ ab , instances of s(r i ) 2 lead to a factor of n for each closed loop in the graph. The partition function is therefore equivalent to where l is the total length of all the loops in a given graph, v is the number of closed loops and the trace is now over all graphs of closed, non-intersecting loops. This form of the partition function may be used for general values of n, whereas equation (1) is applicable only to positive, integer n. There exists a critical point in the theory, at x = x c , where the mean loop length diverges and the model is supposed to become conformally invariant. In order to formulate a field theory, the non-local factors of n must be made local. This can be done by assigning orientations to the loops (either clockwise or anticlockwise) and inserting local factors of e iχ at each vertex where the curve turns to the right and e −iχ where the curve turns to the left. Summing over the two possible orientations for each loop leads to a contribution of e 6iχ + e −6iχ from each closed loop on the honeycomb lattice, where closed loops have a difference in the number of left and right turns of six with the sign depending on the orientation of the loop. In order to obtain the desired factor of n for each closed loop, χ must therefore be chosen such that e 6iχ + e −6iχ = 2 cos 6χ = n . This may now be transformed into a height model. The height variables are taken to be integer multiples of π and are assigned to sites on the dual lattice, such that the loops are contours of the landscape. Crossing a loop running from left to right leads to a decrease in the height of π, whilst crossing a loop running from right to left leads to an increase in the height of π. A given configuration of the heights corresponds to a unique graph of orientated loops. The assumption of the Coulomb gas is that this height model flows under the renormalisation group (RG) into a free field theory with action for some g(n). There is an additional subtlety regarding topology, which may be seen by considering the model defined on the cylinder. On a cylinder of circumference L, the loops wrapping around the circumference are counted incorrectly (for n = 2). Loops wrapping around have the same number of left turns as right turns. These loops are therefore not counted correctly. The situation is remedied by placing a vertex operator (also known as an electric charge) e i6χh/π at one end of the cylinder and an operator e −i6χh/π at the other end. Let these points be ±w/2. If there is a single loop wrapping around the cylinder, then there will be a height difference of π between the ends, the sign of which depends on the orientation of the loop. Summing over the two possibilities for the orientation then leads to the required contribution of e i6χ + e −i6χ . These charges at the ends of the cylinder lead to a modification of the partition function to where the abbreviation ffc signifies that the above expectation values and partition functions are for the free field theory on the cylinder and where we have used w ≫ L. The free energy per unit length on the cylinder for this Coulomb gas (CG) partition function may be calculated as This is the form of the free energy on the cylinder obtained via the Schwartzian derivative from a theory on the plane with central charge c [5] given by where the first term comes from the known behaviour of Z f f c [5]. All that remains is to fix the value of g, which may be obtained from the following argument: adding a term −λ cos(2h)d 2 r to the action should not affect the critical behaviour, since the height variables were defined to be integer multiples of π and cos(2mπ) = 1 for all integer m. Hence, this term should be marginal under renormalisation group flow and therefore must have scaling dimension 2. This determines [8] Twist operators In the loop (Coulomb) gas picture of the O(n) model, loops wrapping around the cylinder may be counted with a weight n ′ different from n by placing additional charges e ±6ih(χ ′ −χ)/π at the ends of the cylinder, with χ ′ chosen according to equation (3). The scaling dimension of these operators may be obtained from their two point correlation function in the loop gas ensemble. Placing the charges at ±s/2, π h(w/2) free-field, cyl. . These free field expectation values may be calculated explicitly since they are Gaussian moments. Then, in the limit s ≫ L, the two point function is seen to be This expression is of the general form of a two point function of operators on the cylinder with scaling dimension [9] x(n, n ′ ) = 6 2 (χ ′2 − χ 2 )/2π 2 g . A particular choice of interest is n ′ = −n, for which the scaling dimension is x(n, −n) = 3/2g − 1; this is the scaling dimension of φ 1,2 operators with level two null states in the Kac classification: These correspond, in string theory language, to operators which insert orbifold points corresponding to the global symmetry s → −s of the hamiltonian and are known as twist operators. They will be the focus of this paper. Their correlation functions may be considered in geometries other than the cylinder. For example, on the Riemann sphere, the two point function of such operators is fixed by scale invariance to be where we have inserted explicit factors of the lattice spacing, a, so as to make φ dimensionless. The two point correlation function in the loop gas may also be calculated on the sphere using the ensemble of equation (2). Each loop separating the points (z 1 , z 1 ) and (z 2 , z 2 ) will be counted with weight −n rather than n, hence graphs in G will be weighted with an additional factor of (−1) to the power of the number of loops separating the two points. Graphs in G with an odd number of loops separating point (z 1 , z 1 ) from point (z 2 , z 2 ) will be weighted by an additional factor of (−1) odd = −1, whilst those with an even number of loops will be unaffected, since (−1) even = 1. These two different types of graphs can be separated in the sum over graphs as follows where G odd (z 1 , z 2 ) is the set of graphs of closed, non-intersecting loops with an odd number of loops separating point (z 1 , z 1 ) and (z 2 , z 2 ) and the G even (z 1 , z 2 ) is the set with an even number of loops separating the two points. This two point correlation function has a natural interpretation in terms of a defect line joining points (z 1 , z 1 ) and (z 2 , z 2 ). If N 12 is the number of times loops cross the defect line in a given graph, then equation (8) may be rewritten as The defect line can take any path between the two points because, for a given loop configuration, (−1) N12 will be the same for all paths. This can be best understood with reference to figure 3. Combining equations (7) and (9), we find 3.1 The small n limit Self-avoiding loops correspond to g = 3/2, which is the dilute phase of the n → 0 limit of the O(n) model. The results of section 3 are summarized by equation (10), which for n ≪ 1 may be expanded in powers of n. The order n 1 term in the loop gas expansion (the left hand side of the equation) comes from graphs with a single loop. By equating this with the order n 1 term of the right hand side, we shall demonstrate a property of the sum over graphs with a single loop. The left hand side of equation (10) for n ≪ 1 becomes where G 0 is the set of all graphs with no loops and G 1 is the set of all graphs with one loop, hence their respective powers of n 0 and n 1 . G 0 contains a single graph with no edges, hence the first term in both brackets is equal to 1. The set G 1 contains graphs with only a single loop. This set is composed of two subsets with no overlap, the set G 1,z1\|z2 with a single loop separating the point (z 1 , z 1 ) from (z 2 , z 2 ) and the set G 1,z1z2\| which contains no loops separating the points. It should be noted that a single loop surrounding the point (z 1 , z 1 ) is classified in the same group as a loop around (z 2 , z 2 ) since on the Riemann sphere one can be continuously deformed into the other. Both have an odd number of intersections with a defect line between the two points, hence (−1) N12 = −1. For all graphs belonging to G 1,z1z2\| , it may be seen that (−1) N12 = 1. This is shown in figure 4. Im Re Figure 4: An example of a graph belonging to G 1,z1z2\| and one belonging to G 1,z1\|z2 for a choice of (z 1 , z 1 ) and (z 2 , z 2 ) on the Riemann sphere. Notice that a loop in G 1,z1\|z2 can be thought of as being either around (z 1 , z 1 ) or around (z 2 , z 2 ). Similarly, a loop in G 1,z1z2\| can be thought of as being around both points or neither. Equation (11) can therefore be rewritten as Equations (6), (3) and (5) may be combined and then expanded in powers of n to find the following form for the scaling dimension of the twist operators in the small n limit: x(n) = −1 + 3/2g(n) = n/3π + O(n 2 ) . Therefore, the expansion of the right hand side of equation (10) is Hence, from comparing the coefficients of n 1 in equations (12) and (13), we may conclude that In words, for a given pair of points (z 1 , z 1 ) and (z 2 , z 2 ), the number of weighted loops which separate the two points, weighted by x l c , diverges as (1/3π) ln \|1/a\| in the limit of the lattice spacing tending to zero. The cause of this is the diverging contribution from vanishingly small loops in the continuum limit. The factor x l c may be thought of as the measure, hence this weighted number of loops is equivalent to the expected number of loops around one point. It may be noted that the coefficient 1/3π differs from that seen on the annulus with shrinking internal radius. In the case of the annulus with vanishingly small modulus [10] a coefficient of 1/6π is seen, because there are only diverging contributions from small loops around one point, as opposed to the plane where small loops around both points contribute. 4 The four point correlation function of twist operators from conformal field theory Twist operators were introduced in section 3 as the operators responsible for counting loops with weight −n rather than n in the loop gas picture of the O(n) model. Their scaling dimension was calculated and it was seen that it corresponds to the scaling dimension of operators with null states at level two. Conformal field theory may be used to derive a set of partial differential equations satisfied by the correlation functions of such null state operators. In the case of the four point function, these differential equations may be solved analytically, as will be seen in this section. To make contact with the language of Schramm (stochastic)-Loewner Evolution (SLE), g is dropped in favour of the parameter κ used in SLE [11]. The two are related by the formula κ = 4/g. It was shown in section 3 that the twist operators have a null state at level two [5]. This null state is itself a highest weight state; it is annihilated by all raising operators, L n with n > 0. This is not the same as saying that the null state decouples from all other states in the theory, as it does in unitary theories. That said, it can be shown by a modular invariance argument that physical results are seen only if the null state decouples on the torus. This is because, barring unforeseen cancellations, the nondecoupling of the null states would lead to a density of states at high energies corresponding to c = 1 rather than c < 1 [12]. We shall therefore set the null state to zero in this theory and use the resulting partial differential equations for the correlation functions of the twist operators. In the complex plane, the four point function satisfies the following set of partial differential equations (for j = 1, 2, 3, 4) [6]: In terms of z ij ≡ z i − z j , the cross ratios are defined as η ≡ z 12 z 34 z 13 z 24 η ≡ z 12 z 34 z 13 z 24 . These cross ratios are invariant under global conformal transformations. The partial differential equations above have the solutions h 2 = 3κ/16 − 1/2 and h 3 = κ/2 − 1 is the value of the scaling dimension of an operator with a null state at level three. The form of B(κ) can be derived by requiring consistency under the permutation of the labels (z i , z i ) and is found to be [13,14] The form of A(κ) is dependent on the normalisation of the two point function since, as η → 0, the four point function becomes Interpretation of the four point correlation function Recall from section 3 that the correlation function of two φ operators has an interpretation in terms of a defect line in the loop gas ensemble. The operators are the source and sink of the defect line, which can take any path between the two points. Each graph in the sum is weighted by an additional factor (−1) N12 where N 12 is the number of intersections of loops in that graph with the defect line. There is a similar interpretation of the four point function in the loop gas ensemble, but now there are two defect lines. The four points are split into two pairs and defect lines run between the points in each pair. For the choice of pairing (z 1 , z 1 ) with (z 2 , z 2 ) and (z 3 , z 3 ) with (z 4 , z 4 ), the four point function can be seen to correspond to the following expectation value in the loop gas picture where N 12 is the number of crossings of loops across a defect line from (z 1 , z 1 ) to (z 2 , z 2 ) and N 34 is similarly defined. There are three ways in which the pairs may be chosen, but each choice leads to the same set of weights for the graphs in the partition function. The reason for this may be seen in figure 5. Thus, the four point function may also be considered to be the expectation value in the loop gas ensemble of (−1) N13 (−1) N24 or (−1) N14 (−1) N23 . Comparing equations (17) and (18) leads to the main result of this section (−1) N12 (−1) N34 loop gas = z 13 z 24 a 2 z 12 z 34 z 23 z 14 4h2 A(κ)ξ(η, η, κ) , where ξ(η, η, κ) is defined below equation (17) in section 4. Im Re Figure 5: (a) shows the choice of defect lines described in the text. As the defect lines may take any path between their endpoints, they may be re-routed to pass infinitesimally close to each other, as in (b). Since a loop crossing a defect line leads to an additional weight (−1) in the loop gas partition function, a pair of defect lines leads to a factor (−1) 2 = 1 and hence makes no contribution to the weight of any graph. Hence, the defect lines shown in (b) are equivalent to the choice in (c). A similar construction may be used to show the equivalence to a pair of defect lines drawn from (z 1 , z 1 ) to (z 2 , z 2 ) and from (z 3 , z 3 ) to (z 4 , z 4 ). Note that this feature is a result of the choice n ′ = −n in section 3. Example: the Ising model Boundaries between Ising spin clusters correspond to the loops of the O(n) model at n = 1, or κ = 3. The twist operators in this case are equivalent to magnetisation operators because the parity of the number of loops separating two spins depends on whether they are parallel or anti-parallel. That is to say that Similarly, the four point correlation function of the twist operators is the four point function of the magnetisation operators. The following function is the result of substituting κ = 3 into the results of section 4 z 13 z 24 a 2 z 12 z 34 z 23 z 14 Equation (20) is the well known four point correlation of spin operators in the Ising model at criticality [6]. The small n limit of the four point function In section 3, we examined the small n limit of the two point function of twist operators, equation (10). We found an exact expression for the µ-mass of loops surrounding one of the two points. This result is summarised by equation (14). A similar expansion to order n 1 of the four point correlation function (equation 19) yields information about the configurations of a single self-avoiding loop around four points. From the equations in section 4, the following small n expansions may be deduced The expansion of A(n) around n = 0 is A(n) = 1 + ̺n + O(n 2 ) where ̺ is a constant independent of n, since the correlation function should tend to 1 as n → 0. The Taylor series expansion of the right hand side of equation (19) is then The left hand side of equation (19) may also be expanded in powers of n. The graphs up to order n 1 in G may be split into the graph with no loops (n 0 ) and eight distinct sets of graphs with a single loop, as will be shown in the next section. The configurations of a single loop around four points In this section, we expand the left hand side of equation (19) for n ≪ 1 to order n 1 . The coefficient of n 1 may then be equated with the result for expansion of the right hand side, described in the previous section and leading to equation (21). Recall that the partition function for the loop gas expansion is equation (2). The sum may be decomposed into a term of order n 0 coming from the graph with no loops (hence the zeroth power of n) and graphs with a single loop weighted by n 1 . Other graphs will contribute terms of order n 2 or smaller. The graphs with a single loop on the Riemann sphere may be further split up into eight distinct subsets, which are shown in figure 6. Each configuration has a unique set of (−1) Nij where i = j and i, j ∈ {1, 2, 3, 4}. Let L be defined as Im Re The numerator of L can be calculated as The denominator takes the same form as the expression above, but without the factors of (1) and (−1). For ease of notation, define and similar expressions for the other graphs in figure 6. The elements of {W i } are sums over all loops of a single configuration type i, weighted by x c to the power of their length. They therefore represent the µ-masses of loops of configuration i. It may then be seen that The two point functions C {ij} ≡ φ(z i , z i )φ(z j , z j ) = (−1) Nij and products of pairs of two point functions can be expanded in terms of the {W i } also: Thus, it can be seen from equations (22),(23),(24) and (25) that a subset {W c } of the {W i } can be expressed in terms of partly-connected four point functions: These {W c } correspond to graphs with a loop winding around two of the four points and hence do not include contributions from vanishingly small loops in the continuum limit. By comparison with the small n expansions of L (section 5) and C {ij} (section 3.1 with the same choice for the normalisation of the twist operator as in the four point function) from conformal field theory, these weights are the following functions of η, η where q(η, η) = −1 24π Note that the W c above are finite in the continuum limit of vanishing lattice spacing. In fact, the expressions in equations (29),(30) and (31) are independent of a. They are finite and non-zero in the limit n → 0 and are invariant under conformal transformations, being functions only of the cross ratios. These expressions for the µ-masses {W c } constitute one of the main results of this paper. The central charge It is a standard result of conformal field theory that, given the explicit form of any four-point function, the central charge c may be determined. This is because the operator product expansion (OPE) of any scalar primary operator φ with itself takes the form φ(z 1 ,z 1 )φ(z 2 ,z 2 ) = \|z 12 \| −4h 1 + · · · + 2h c T (z 1 ) + · · · (32) where T is the holomorphic component of the stress tensor. The coefficient follows from consideration of the limit z 12 → 0 in the three point function T (z)φ(z 1 )φ(z 2 ) ∝ 2h and the two-point function T (z)T (z 1 ) = (c/2)(z − z 1 ) −4 . If this result is applied to the explicit expression (17), we find that the coefficient of η 2 is , and so obtain the known expression for the central charge in terms of κ This result is of interest in the limit n → 0 for the light it sheds on the interpretation of the stress tensor T in terms of an observable of a random curve given by Doyon, Riva and Cardy [7]. In that paper it was shown that, for any measure on simple random curves which satisfies conformal restriction, one may identify T (z) as being proportional to the spin-2 angular Fourier component of the probability that the curve intersects a small line segment of length ǫ centred at z. While in that paper the focus was on curves which connect two points on the boundary of a simply connected domain, described in the case when conformal restriction holds by SLE 8/3 , the theorem should equally well apply to Werner's measure on self-avoiding loops, if suitably re-interpreted in terms of µ-masses rather than probabilities. However our results in the present paper do not directly apply to the intersection with a line segment, but rather to the event that the loop passes (or does not pass) between pairs of points (which, however, may be taken to mark the ends of the line segment). Nevertheless, one might expect these to differ only by a constant of proportionality, and indeed our results in this paper support this, and suggest that the two-point function of the object T introduced in Ref. [7] is indeed given by the central charge for small n as expected. Indeed, let us consider the quantity W z1z4\|z2z3 as given in equation 29. This is the µ-mass of loops which separate (z 1 , z 4 ) from (z 2 , z 3 ). In the limit z 12 → 0, writing z 12 = ǫe iθ12 , we can define where the numerical prefactor is lim n→0 (c/2h 2 ). Our result equation 17 then implies that V has the same dependence on its arguments as does the O(n) term in the CFT correlation function This result generalises to other correlation functions, and implies that we may interpret the spin-2 component of the µ-mass of loops which pass between two nearby points as being, in some sense, the derivative T of the CFT stress tensor with respect to n at n = 0. With this definition we then find the result as expected, where the left hand side is It would, of course, be important to establish this interpretation directly from the restriction property. Twist operators in a simply connected domain The model may also be considered in a simply connected domain. All such domains may be conformally transformed to the upper half plane, with the real axis being the boundary. We may therefore derive the results for the upper half plane. The two point function of twist operators in the presence of this boundary satisfies the same set of partial differential equations as the four point function in the bulk, with (z 3 , z 3 ),(z 4 , z 4 ) assigned as the complex conjugates of the positions of the operators. This is a well known result in the theory of boundary conformal field theory [5] (BCFT) and follows from the condition of T = T on the boundary. The solution for the two point function is where now there is only one cross-ratio . B(κ) can be determined by looking at the boundary conditions as the two points approach the boundary, which is η → −∞. Equation (38) may be analytically continued to large η: . The two natural choices of B(κ) are those which pick out one or the other conformal block as the operators approach the boundary. In the limit n → 0, there are no loops in the loop gas picture so the correlation function should tend to unity for all η. This is only possible if the first term in the square brackets of equation (40) is not present. A (non-unique) choice of B(κ) which satisfies this requirement is that which picks out the second conformal block only: . It is important to note also that this boundary condition is not compatible with the vanishing of the four point function in the limit η → −∞. The form of A(κ) is dependent on the choice of normalisation of the twist operators. This can be seen from the η → 0 limit of equation (38): . A(κ) must be of the form A = 1 + σn+ O(n 2 ) due to the constraint that the four point function tends to unity as n → 0. For small n, the correlation function may be expanded as Im Re where . In analogy with the interpretation of the four point function in the bulk in the loop gas picture described in section 4.1, the following is the interpretation of the two point function in the presence of a boundary The left hand side is an expectation value in the ensemble of lattice loops, as before. N 1,1 * is the number of times loops cross a defect line from z 1 and z * 1 . The set of all graphs may be decomposed into the set with no loops and the set with one loop; the other graphs are of order n 2 . The possible configurations of a single loop with a boundary are shown in figure 7. The two and one point functions are then found from the loop gas to be M ≡ φ(z 1 )φ(z 2 ) = (−1) N 1,1 * (−1) N 2,2 * = 1 − 2n(W 1(2) + W (1)2 ) + O(n 2 ) C 1 ≡ φ(z 1 ) = (−1) N1,1 * = 1 − 2n(W (1)2 + W (12) ) + O(n 2 ) C 2 ≡ φ(z 2 ) = (−1) N2,2 * = 1 − 2n(W 1(2) + W (12) ) + O(n 2 ) . Just as for the case of the loop gas in the bulk, the µ-masses {W i } are the number of weighted loops belonging to the configuration i (see figure 7). They are defined as x l c . Of the four possible configurations, the only one finite in the limit a → 0 is W (12) : In terms of η, Note, as for the bulk case, that the term involving σ in M is cancelled by the subtraction of the product of one point functions. Interpretation as a stochastic process We have seen in section 4.1 that the µ-mass of loops which wind around two of four points is invariant under conformal transformations. In this section, we show that the differential equations they satisfy can be interpreted in terms of an SLE process. Recall that these functions were identified as semi-connected four point functions, for example equation (26) where C {ij} are the correlation functions of a pair of twist operators at the points (z i , z i ) and (z j , z j ). The two point function is fixed by scale invariance and the four point function was determined as a solution to the partial differential equations (15) and (16). Using these, we find that W z1z4\|z2z3 satisfies the PDE together with a similar equation in which all the z s are replaced byz. Note that in the BPZ equations z andz can be taken as independent complex numbers, and it is only in applying these equations to physical quantities that one needs to impose reality conditions. These equations have almost the second order linear form which would result from applying the Itô formula to a martingale of an SLE process started at z 1 . One difference is that they are complex. We can obtain a real equation by, for example, taking the real part of the sum of the holomorphic and antiholomorphic equations (the more general case will be discussed below), but note that when we do this we have the sum where x 1 = ℜe(z 1 ) and ∆ z1 is the Laplacian operator ∂ 2 x1 + ∂ 2 y1 . We also note that we can remove the inhomogenous part in (43) by defining Then we can rewrite the real part of (43) as Consider now a sequence of conformal maps g t (z) which satisfy the stochastic chordal Loewner equation to be resolved before one could actually derive our results from SLE. In particular, one should explain why it is necessary to consider SLE 6 rather than some other value of κ. This is presumably related to the fact that the hull of SLE 6 corresponds locally to SLE 8/3 , and that both correspond to CFTs with central charge c = 0. The choice of a chordal SLE reflected in ℑm(z) = ℑm(z 1 ) is clearly arbitrary; other choices correspond to taking different linear combinations of the holomorphic and anti-holomorphic equations. Perhaps using radial or whole-plane SLE would make the formula look more symmetrical. Conclusion In this paper we have studied scaling properties of loops in the loop gas picture of the O(n) model. In the picture, the partition function is a sum over all graphs of non-intersecting closed loops, weighted by nx l c where x is a function of the reduced coupling and l is the length of the loop. We introduced twist operators, whose correlation functions count the loops separating the locations of the operators with weight n ′ different to the usual weight n, or equivalently count the minimum number of crossings of defect lines running between these locations. For the particular choice n ′ = −n, the twist operators have level two null states and their correlation functions satisfy BPZ type partial differential equations on the Riemann sphere. Thus, conformal field theory may be used to determine the analytic form of the two and four point functions. In the loop gas picture, the choice n ′ = −n means that loops are counted are weighted by an additional factor of −1 to the power of the number of defect lines crossed, and the choice of path for the defect lines between the locations of the operators is unimportant. The limit n → 0 describes the theory of self-avoiding loops. In this limit, the partition function and correlation functions are dependent to first order in n only on the configurations of a single loop. Hence, by equating particular semi-connected parts of the four point function calculated using conformal field theory to the result from the Coulomb gas picture, we have deduced the expected number of weighted loops winding around two of the four points and shown that this number is invariant under conformal transformations. This is presumably the mass of such a subset under the measure on loops introduced by Werner. Other configurations of loops receive contributions from vanishingly small loops around a single point and are not finite in the limit of vanishing lattice spacing. The central charge of the O(n) model for small n may be found from the dependence of the µ-mass of loops around two of the four points as a function of the cross ratio of the positions of the four operators. The result c ∼ 5n/3π from the analytic results agrees with other methods of calculating the central charge of the O(n) model, and lends support to the interpretation of the stress tensor for curves satisfying conformal restriction given in Ref. [7]. A similar calculation was also carried out for the model defined in a simply connected domain. A general simply connected domain may be mapped via a conformal transformation to the upper half plane with the real axis as the boundary. The two point function of operators in the upper half plane satisfies the same partial differential equations as the four point function on the Riemann sphere, with two additional operators positioned at the complex conjugates of the original operators. By again equating the results from conformal field theory with those from the Coulomb gas picture, we have deduced the µ-mass of loops around the two points in the upper half plane. Finally, we have shown that the differential equations satisfied by the above quantities should have a stochastic interpretation in terms of a chordal SLE 6 process starting from one of the points z 1 , along with its reflection in a fixed line. [	2014-10-01T00:00:00.000Z	2006-06-28T00:00:00.000	{ "year": 2006, "sha1": "03d5dd3ecfb762cde53b69e14a0bd29395b38ecf", "oa_license": null, "oa_url": "http://arxiv.org/pdf/math-ph/0606065", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "03d5dd3ecfb762cde53b69e14a0bd29395b38ecf", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Physics", "Mathematics" ] }
249707798	pes2o/s2orc	v3-fos-license	Financial Markets, Financial Institutions and International Trade: Examining the causal links for Indian Economy This study investigates whether a uni-directional or bi-directional causal relationship exists between financial development and international trade for Indian economy, during the time period from 1980 to 2019. The empirical analysis utilizes three measures of financial development created by IMF, namely, financial institutional development index, financial market development index and a composite index of financial development, encompassing dimensions of financial access, depth and efficiency. Johansen cointegration, vector error correction model and vector auto regressive model are estimated to examine the long run relationship and short run dynamics among the variables of interest. The econometric results indicate that there is indeed a long run causal relationship between the composite index of financial development and trade openness. Cointegration is also found to exist between trade openness and index of financial market development. However, there is no evidence of cointegration between financial institutional development and trade openness. Granger causality test results indicate the presence of uni-directional causality running from composite index of financial development to trade openness. Financial market development is also found to Granger cause trade openness. In contrast, trade openness is found to promote financial institutional development in the short run. Empirical evidence thus underlines the importance of formulating policies which recognize the role of well-developed financial markets in accelerating international trade of Indian economy. Introduction Financial development and international trade are key drivers of the economic growth performance of a country. India has a strongly diversified financial sector comprising commercial banks, insurance companies, non-banking financial companies, cooperatives, pension funds and mutual funds. Financial sector reforms initiated in India in the 1990s, based on the report by Narasimhan Committee, mainly focused on banking system and capital market. The reforms aimed at putting an end to the then prevailing regime of financial repression, enable price discovery by the market determination of interest rates, and maintain financial stability even in the face of domestic or external shocks (RBI 2007). Financial development is increasingly becoming an important contributor to the economic growth of countries. Seminal work by (Goldsmith 1969), (McKinnon 1973) and (Shaw 1973) have analyzed the role of financial intermediaries in boosting long run growth of economies. Other theoretical studies also contributed to the debate in the successive years, which include (Levine 1997, 702, Levine andZervos 1998, 550); (Levine, Loayza, and Beck 2000, 40) and (Luintel and Musahid 1999, 399). Another work (Murinde and Eng 1994, 400) is examining the country specific study about Singapore for the period 1979 -1999, finds the causality from financial development to economic growth. Evidence obtained from the study in Turkey for the period 1989-2007, supports the bidirectional causality exists between financial development, trade openness and growth (Yucel 2009). arXiv PREPRINT 4 Studies which examined the role of financial development in economic growth of countries are numerous and well established in the literature, compared to studies which unravel the link between financial development and international trade. The present study aims to examine whether a unidirectional or bi-directional relationship exists between financial development and international trade in case of Indian economy, by using time series techniques. This study brings in a new dimension to existing studies on this subject, by resorting to the broad based measure of financial development created by IMF, to account for all possible variations in the financial system. Since financial development is a complex multidimensional variable, the typical proxies such as ratio of private credit to GDP or stock market capitalization can't fully account for the concept. Broad financial development index of IMF encompasses three dimensions of efficiency, access and depth, with respect to both financial institutions and financial markets (Svirydzenka 2016). Literature Review The Heckscher Ohlin and Vanek theory of international trade states that factor content of a commodity is most important for trade. It postulates that a country well-endowed with a particular factor will engage in the production and trade of that commodity which intensively use the abundant factor. (Kletzer and Bardhan 1987, 65) was the first study to link credit markets to international trade patterns. They postulated that countries with identical technology and endowments may still differ in terms of comparative cost advantages, because of credit market imperfections, arising from moral hazard considerations and asymmetric information. Path breaking work by (Beck 2002, 120) theoretically modeled the role of financial intermediaries in boosting large scale, high return projects, and proved that countries with a higher state of financial development have a comparative advantage in manufacturing. The model was validated using a panel dataset for 30 years for 65 countries. Trade in manufactured goods was specified as a function of private credit as a share of GDP and other control variables such as initial level of real per capita GDP, black market premium, real per capita capital, population and growth rate of terms of trade. The estimation was carried out using Ordinary Least squares and Instrumental Variable Technique. After controlling for country specific effects and possible reverse causality, the study found that financial development does exert a significant causal impact on two measures of international trade, namely the level of exports and the trade balance of manufactured goods. arXiv PREPRINT 5 Few studies (Beck 2002, 120) and (Vlachos and Svalryd 2005, 114) have analyzed the link between financial development and international trade, from the point of view of economies of scale. They found that financial sector can facilitate trade immensely. A well developed financial sector can source savings to private sector and assist entrepreneurs to engage in more business activities, thereby overcoming credit constraints. Since manufacturing sector exploits increasing economies of scale, it can reap higher profit coupled with the high level of financial development associated with manufacturing sector. In every country, the sector which faces demand shock has to protect from risk. This implies that international trade patterns are highly dependent on differences in financial development, with a highly developed financial system permitting the country to specialize in risky goods (Baldwin 1989, 145). Availability of trade credit is determined by the level of financial health and width of network of issuing banks. These factors can positively contribute to the flexibility and liquidity of funding and supply of trade credit (Jain, Gajbhiye, and Tewari 2019). (Do and Levchenko 2004) built a theoretical model wherein international trade boosts growth of financially dependent sectors and financial system in a wealthy country. The poor country begins to import the financially dependent good from the rich country rather than produce it domestically, implying a decline of the domestic financial system and demand for external finance. The model was empirically validated using a panel dataset of 77 countries, from 1965 to 1995. Financial development was specified as a function of trade openness and control variables such as initial level of private credit to GDP, initial per capita GDP, a measure of human capital (average years of secondary schooling in the population), as well as legal origin dummies. Financial development was measured using three alternative indicators, namely, the ratio of private credit to GDP, the ratio of liquid liabilities to GDP, and claims of deposit money banks on nonfinancial domestic sectors as share of GDP. This study thus provides theoretical and empirical basis for direction of causality running from trade openness to financial development. As trade finance is an important factor determining flow of imports and exports, financial development is always positively correlated with volume of trade (Liston and McNeil 2013, 13). Studies carried out using multi industry approach found that financial development and ratio of export to domestic sales is positively associated in capital intensive industries, whereas the trade share is much lower in labour intensive industries. But the net effect can be offset at the aggregate level (Leibovici 2018). Empirical studies indicate positive short run and negative long arXiv PREPRINT 6 run association between financial development and international trade, along with unidirectional causality running from financial development to international trade (Bilas, Bosnjak, and Novak 2017, 84). Economic growth is linked to international trade through the medium of financial development, whereby economic growth increases financial development and thus enhances trade participation of countries (Kar, Nazlioglu, and Agir. 2013, 139). Countries that trade manufactured commodities can elevate the financial system of that country because such economies demand more external finance, which leads to greater financial development of country (Samba and Yu 2009, 65). Export oriented firms demand more external finance to meet high fixed cost. Therefore, destabilizing financial conditions affect export oriented firms more than it affects domestic oriented ones (Feng and Lin2013, 44). Another interesting inter-relationship between financial development and international trade is found via the linkage between imports and debt financed consumption. Higher domestic demand is financed by inflow of foreign loans. Both the appreciation of domestic currency due to higher imports and inflow of foreign loans resulted in current account deficit in European transition countries (Aristovnik 2008;Zakharova 2008;Bakker and Gulde 2010, 120). When developing and developed countries were analyzed separately for the period between 1961 and 2020 to assess the linkage between financial development and international trade, financial development was found to be a key factor which promotes trade participation of countries. The direction of causality the presence of cointegration. Financial development was measured using the widely used indicator of private credit as a percentage of GDP. International trade performance was measured using three alternative indicators, namely, manufactured exports as a percentage of GDP, manufactured imports as a percentage of GDP and net manufactured exports as a percentage of GDP. International trade in primary goods and services were not covered in this study. They found the existence of a long run equilibrium relationship between international trade in manufactured goods and financial development and existence of unidirectional causality from financial development to net exports of manufactured goods. Empirical evidence indicated absence of reverse causality from international trade to financial development in case of Indian economy. Based on the above literature review, it is found that that there is no conclusive evidence with regard to the direction of causality between the state of financial development and international trade of a country. The present study is trying to contribute to this growing literature by analyzing the linkage between financial development and international trade specifically for Indian economy. There is a dearth of Indian studies which have examined the relationship between financial development and international trade in aggregate (inclusive of agriculture, manufacturing and services). This paper also adds a new dimension to the existing studies by measuring financial development using the broad based index created by IMF. This indicator is definitely superior to ratio of private credit to GDP, as a proxy for financial development. This study also throws light on the nature of relationship between trade openness and two of the subindices of IMF index of financial development, namely, financial market development and financial institutional development. Data and Methodology The empirical model to estimate the relationship between financial development and international trade in India is specified as given in equations (1), (2) and (3). The term TRADE in the equations (1), (2) and (3) (1988), which is based on maximum likelihood method, is most suitable for this study. After cointegration between the variables is established, the VEC Model is estimated to study the short run dynamics and direction of causality among the variables. In the absence of cointegration for any of the equations (1), (2) and (3), VAR (Vector Autoregression) Model is estimated. The causality test is employed as any cointegrated system establishes an error correction mechanism that restricts the variable to deviate from its long run equilibrium. In applied econometric literature, the direction of causal relationship can be examined by using the well known Granger causality test (1988). In every step, the dependent arXiv PREPRINT 9 variable is regressed on past values of itself and other independent variables. The optimum lag length for the model is chosen based on Akaike Information Criteria. VECM representation for the equations 1 and 3 are as follows: Here, ∆ indicates first difference operator, λ and ρ are representing speed of adjustment to attain long run equilibrium and εt and πt are error terms. Empirical Results Countries across the world are experiencing greater integration of their domestic economies with the world economy. Economic growth of a country is influenced to a great extent by the growth of its financial sector along with other factors such as international trading environment. Table A1, Appendix). Since the paper is considering macroeconomic variables, it is wise to use a stationarity test which allows for unknown structural breaks as it avoids the possibility of spurious regression. The sequential Bai- Perron (2003) test claims for the presence of structural breaks in the series (see Table A3, Appendix). In the current study, Perron test (1997) unit root which allows for endogenous structural breaks is used in order to identify whether the series is stationary or not. Table 1 Different lag selection criteria for the three models to be estimated, are reported in Tables 2, 3 and 4. This study uses Akaike Information Criteria for all three equations, which is in support of optimum lag length of 5 for equations (1) and (3) and lag length of 2 for equation (2). (1), (2) and (3) are presented in Table 5, Table 6 and Table 7 respectively. Values of both trace and maximum eigen statistics in Table 5 are showing statistical significance. This indicates the existence of two cointegrating equations relating trade openness, composite index of financial development and other control variables at the 5% level of significance. Table 6, there is no evidence for cointegration between trade openness, index of financial institutional development and other control variables. Trace and maximum eigen value statistics in Table 7 indicate the presence of two cointegrating equations relating trade openness, financial market development and other control variables. year's real exchange rate. Short run adjustment parameters of equation (4) and (5) based on VECM are estimated and associated results are reported in Tables 9 and 10 respectively. The error correction terms for both the estimated VEC models are found to be negative and statistically significant, further reinforcing the presence of cointegration among the variables. About 10% of the short run disequilibrium between financial market development, trade openness, GDP per capita and REER gets corrected in the long run. However, only 3.5% of the short run disequilibrium between the broad-based index of financial development, trade openness, GDP per capita and REER gets corrected in the long run. Table 11 indicates that all of the short run coefficients quantifying the impact of financial development on trade openness are statistically significant at the 5% level, except for the three year lag. Table 12 also exhibits a similar short run dynamics. arXiv PREPRINT 16 All the short run coefficients quantifying the impact of financial market development on trade openness are found to be statistically significant at the 5% level, except for the three year lag. Few of the lagged terms of trade openness, real exchange rate and log of GDP per capita are also found to have significant short run impact on trade openness, in case of Indian economy. it aggravates the chance for collinearity between explanatory variables. However, the significance of regression coefficients and lower variance inflation factor rule out the possibility of high multicollinearity in the model. Therefore, no serious remedial measures are required to arXiv PREPRINT 18 treat it. Other tests on residuals ensure that the model is adequately robust and does not suffer from serial autocorrelation, non-normality and heteroscedasticity. The Ramsey RESET (1969) test was conducted to check whether the functional form of model is correct or not. Result shows all three estimated models have correct functional form (see Table A2 in Appendix). The presence of structural breaks is identified by using sequential Bai-Perron (2003) test and F statistics spotted three structural breaks in the series, results of which are reported in Table A3 LGDP 0.20 0.14 I(0) 0.02 0.14 I(1) REER 0.19 0.14 I(0) 0.08 0.14 I(1) Source: Authors estimation. Note: Unit root tests are carried out at 5% significance level. indicates 1% level of significance and ** indicates 10% level of significance. The rest of unit root tests are given at 5% significance level. I(0) means integrated of order zero and I(1) means integrated of order one. The lag length is selected using Schwarz Info Criterion	2021-12-06T02:15:08.423Z	2021-12-03T00:00:00.000	{ "year": 2021, "sha1": "0ccecf01889431e84bff2dac1e1a1d0acaffaa48", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "0ccecf01889431e84bff2dac1e1a1d0acaffaa48", "s2fieldsofstudy": [ "Economics" ], "extfieldsofstudy": [ "Economics" ] }
58929170	pes2o/s2orc	v3-fos-license	DETECTION OF Toxoplasma gondii DNA IN SERA SAMPLES OF MICE EXPERIMENTALLY INFECTED Detection of Toxoplasma gondii (T. gondii) DNA in blood can help to diagnose the disease in its acute phase; however, it must be considered that hemoglobin, present in blood, can inhibit polymerase activity, making impracticable the detection of DNA in samples. Mice were experimentally infected via oral route with ME49 and BTU2 strains cysts and RH strain tachyzoites; polymerase chain reaction was used to detect T. gondii DNA in mice sera 18, 24, 48, 96, and 192 hours post infection (PI). Toxoplama gondii DNA was detected in only one animal infected with BTU2 strain, genotype III (isolated from a dog with neurological signs) 18 hours PI. The agent’s DNA was not detected in any sample of the other experimental groups. New studies must be carried out to verify the technique sensitivity in researches on this agent’s genetic material using sera samples of acute-phase toxoplasmosis patients, especially in cases of immunosuppression. INTRODUCTION Toxoplasma gondii, the agent of toxoplasmosis, is an obligatory intracellular Apicomplexa parasite that infects warm-blooded animals, being felids the definitive hosts.Toxoplasma gondii presents a heteroxenous life cycle and causes a high number of abortions in many species, mainly ovines.In relation to public health, this parasite is an important cause of abortion in humans too, causing a congenital disease and seriously infecting immunocompromised patients (18). It presents three genetic lineages -I, II, and III -and reproduces in asexual or sexual form (12). Its virulence varies with the host species.In mice, type I presents greater virulence than the other types, being frequently lethal.Types II and III cause asymptomatic infections, with formation of tissue cysts.In humans, type II infects immunodepressed patients, whereas type I predominantly occurs in cases of congenital infection (5,6,11).Apparently, immunity among them does not exist (2). There are many experimental models of laboratory animals to study this disease. Pathology of infection by T. gondii results from the interaction between parasitic factors and the host.It is difficult to compare results, which sometimes are disagreeing, since the experimental models may vary according to the parasite lineage, route of infection, agent's strain, maintenance conditions, and number of parasite passages (21).There are two ways of parasite dissemination by the oral route.In the first form, the parasite reaches the mesenteric circulation and then the liver, reaching the systemic circulation.In the second way, it passes to the lymphatic system and thoracic duct, reaching the systemic circulation; being the latter the most frequent form of dissemination (14). In relation to immune response in the initial weeks of infection, the organism starts an independent and non-specific response of T cell and major histocompatibility complex (MHC), using interferon-(IFN-), tumor necrosis factor (TNF), a population of cells from thymus (Thy-1), and natural killer cells (NK).This precocious nonspecific response limits the proliferation of tachyzoites until the specific response dependent on CD4 + and CD8 + cells is able to protect the animal in chronic infection (8).There are three groups related to T. gondii sensitivity grade among the rodents: the most sensitive are Chinese hamster, Syrian hamster, and mice; the moderately sensitive are gerbils and Mastomys; rats are very resistant (7). Congenital and acute infection has been detected by serological tests, like Sabin-Feldman test (SF), indirect immunofluorescence test (IFAT), modified agglutination test (MAT), hemagglutination test, and enzyme-linked immunosorbent assay (ELISA), which detect the presence of antibodies against T. gondii.Specificity and sensitivity of these tests are variable.Other limitation in diagnose is that the amount of antibodies varies with the time of infection and the age of the host (18). Immunocompromised patients, mainly the ones infected by human immunodeficiency virus (HIV), do not present significant titers of IgG and/or IgM anti-Toxoplasma, which makes the diagnosis using these serological tests more difficult (20). In experimentally infected animals, the presence of T. gondii can be investigated by polymerase chain reaction (PCR) too.The techniques used nowadays are aimed, in the majority of cases, at detecting sequences of SAG1 gene, which codifies P30 glycoprotein, the main protein of the membrane of T. gondii tachyzoites; sequences of B1 gene, which has a strange function; and parts of DNA-codifying ribosomal RNA (rDNA).In the genome of T. gondii, there is only one copy of SAG1 gene, 35 copies of B1 gene, and 110 copies of genes to rDNA, which in part explains the differences found in the sensitivity of many PCR protocols to detect Toxoplasma (5).Homan et al. (11) described a new repeated sequence, with 200-300 copies in the genome of T. gondii, which when used in PCR demonstrated sensitivity higher than that of B1 gene. Weiss et al. (20) demonstrated that DNA of T. gondii RH strain can be detected by PCR in tissues of acute-toxoplasmosis mice with high specificity and sensitivity inferior or equal to ten tachyzoites.Parasite was detected in tissues on the second day and in blood, 15 days PI.Using a protocol of nested PCR to detect this agent's B1 gene in blood samples of infected mice, Joss et al. (13) observed that with RH strain, 18 from 20 animals tested 16-66 hours PI were positive to PCR and the bioassay.Among 24 animals infected with Beverley strain, only two had the parasite reisolated in the bioassay, resulting in positive PCR for 15 animals from 1 to 38 days PI. In Swiss mice infected by oral route with cysts of C strain isolated from human placenta, Paugam et al. (14) tested two PCR protocols for the detection of Using sera samples to detect T. gondii DNA in the acute phase of infection can be more advantageous compared to serological methods due to sensitivity, as well as to the fact of being a direct demonstration of the parasite.On the other hand, the use of sera as an alternative to total blood samples gives as advantages the minor concentration of probable agents that interfere in PCR, facilitating the obtainment of higher-quality DNA.Therefore, the experiment protocols used until the moment have not been based on situations similar to T. gondii natural infection by using tachyzoites via intraperitoneal route, an effective artificial route.Thus, the objective of the present study was to verify the presence of T. gondii DNA in sera samples of mice infected with tissue cysts of three genetically different strains. We have to consider the presence of hemoglobin and to be careful in the extraction of samples contaminated by this blood component.Hemoglobin can inhibit PCR because the connection between the groups heme and/or perforin and Taq DNA polymerase inactivates the enzyme.Hemoglobin and lactoferrin were found to be major PCR inhibitors in erythrocytes and leukocytes, respectively.Both hemoglobin and lactoferrin contain iron.Therefore, the inhibitory effects of both proteins may be related, in part, because of their ability to release iron ions.Hemin, a hemoglobin derivative, and its breakdown products, bilirubin and bile salts, were also found to be PCR inhibitors.It has been suggested that heme regulates DNA polymerase activity and coordinates the synthesis of hemoglobin components in erythroid cells by feedback inhibition (1).To solve this problem, PCR inhibitors should be inactivated or removed from the sample. MATERIAL AND METHODS We used RH strain tachyzoites, genotype I isolated from a child from the United States by Sabin in 1939 ( 16) which were weekly inoculated by intraperitoneal route in albino Swiss mice.To obtain the tissue cysts and free bradyzoites for the inoculation, the strains ME49, genotype II (17), and BTU2, genotype III, isolated from a dog with neurological signs, were used.The cysts were obtained by orally inoculating 10 albino Swiss mice, female, 50-80g, with 20 tissue cysts of T. gondii ME49 and BTU2 strains.Animals were treated with sulfadiazine (400mg sulfadiazine + 10g sodium bicarbonate/liter of water) in drink water from the third to the twentieth day PI to induce chronic infection (19).Two months PI, they were killed in a chamber saturated of isoflurane vapor.Cysts purification was performed according to Dubey (4).All animals were gently supplied by Central Animal Facility, UNESP, Botucatu, São Paulo. Brains were obtained with thorough antisepsis and washed with 0.85% saline solution (5ml/brain) in order to remove red blood cells; they were kept in 0.85% saline solution (4ml/brain) at 4°C for 12 hours, macerated and mixed from seven to eight times using syringe and needle.The suspension was transferred to a 50ml-centrifuge tube containing 8ml of 25% Percoll (2ml Percoll + 6ml saline/brain) and centrifuged at 2000g for 30 minutes in "brake off" position.The cerebral material was carefully removed as well as the rest of the Percoll suspension, except for the final 0.5ml.In order to resuspend the sediment, 10ml of 0.85% saline solution was added, followed by centrifugation at 1200g for 10 minutes in "brake off" position.Supernatant was discarded, sediment was resuspended in 1ml of 0.85% saline solution, and cysts in samples of 8 l of the suspension were counted in an optic microscope.The cysts concentration was calculated using the formula: number of cysts counted / (16x1000).To obtain tissue cysts of RH strain, four Fischer rats were infected, because this species develops chronic infection by genotype I strains easier than the others.Rats are naturally more resistant to infection caused by T. gondii. Release of bradyzoites from the interior of the cysts was performed according to Dubey (8), completing the volume of the cysts suspension to 10ml with 0.85% saline solution; an equal volume of pepsin acid solution previously maintained at 37°C was added, followed by incubation for five minutes under constant agitation.The suspension was neutralized with 1.2% sodium bicarbonate and centrifuged at 1200g for 10 minutes.Supernatant was discarded and the sediment was resuspended with 0.85% saline solution to 1ml.Bradyzoites were counted in a Neubauer chamber.The concentration of bradyzoites was adjusted to 10 4 in 100 l of suspension. In experimental groups, we also used albino Swiss mice, female, weighting 50-80g. Four groups of animals were used: G1, with 15 mice infected by oral route with 10 4 tachyzoites of RH strain (genotype I); G2, with 15 mice infected by oral route with 10 4 bradyzoites of ME49 strain (genotype II); G3, with 15 mice infected by oral route with 10 4 bradyzoites of BTU2 strain (genotype III); and G4, with 5 mice injected by oral route with 100 l of sterile saline solution (control group).Sera samples were collected from each experimental group, and blood was collected by retro-orbital sinus puncture from three mice 18, 24, 48, 96, and 192 hours PI.Serum was obtained by sedimentation and kept in microtubes at -80°C until analysis. Polymerase chain reaction was carried out using the primers described by Homan et al. (11), which amplify a sequence of 529 base pairs (bp) of T. gondii nuclear genome.PCR protocol was performed according to Da Silva & Langoni (3), changing the concentration of MgCl 2 , deoxynucleotides and primers.Initially, various protocols of DNA extraction from the serum were compared.Toxoplasma gondii AS28 strain tachyzoites (10 3 , 10 2 , 10 1 , and 10 0 ) were added to the sera samples of not-infected mice, testing the following protocols of DNA extraction: Protocol 1 Serum sample (250 l) added of 250 phenol was vigorously homogenized in vortex and centrifuged at 13200g for three minutes.After this, 200 of the aqueous phase was carefully transferred to a new microtube of 1.5ml plus 100 of phenol:chloroform:isoamilic alcohol (25:24:1), homogenized and centrifuged as previously described.The aqueous phase (100 was transferred to a new microtube of 1.5ml, with the addition of 18 l of 2M sodium acetate and 236 l of frozen absolute ethanol; the microtubes were kept at -80°C for one hour.The samples were centrifuged at 13200g for ten minutes, ethanol was carefully spilled, and 236 l of 70% ethanol was added.After homogenization, the samples were centrifuged at 13200g for ten ethanol was removed, and microtubes were dried to environment temperature.They were resuspended in 100 l of ultrapure water, incubated at 56°C for 30 minutes, and kept at -20°C until PCR. Protocol 3 Initially, sera samples were incubated at 100°C for 15 minutes with 250 of the buffer solution described in protocol 2, without addition of proteinase K and SDS.Afterwards, the previously described conducts were carried out. Protocol 4 In the beginning, 100µl of serum was homogenized with sodium acetate and ethanol, and then protocol 1 conducts were performed. Protocol 5 First, 100µl of serum was homogenized with 1000µl of DNAzol extraction reagent (Invitrogen ) and centrifuged at 10000g for 10 minutes.Supernatant was transferred to a new microtube, and 500µl of absolute ethanol was added for DNA precipitation. The previously described conducts were maintained. RESULTS AND DISCUSSION Figure 1 presents the results of PCRs in sera samples contaminated with 10 3 , 10 2 , 10 1 , and 10 0 tachyzoites of T. gondii AS28 strain.Protocol 3 showed an analytical sensitivity of one parasite in 250µl sample, while protocols 1 and 2 showed 100 and 1000 parasites in 250µl sample, respectively.Protocol 5 revealed an analytical sensitivity of 40 parasites in 100µl sample.PCR was not carried out in samples extracted by protocol 4 because when the alcohol and the sodium acetate were homogenized to the sera sample, precipitation occurred, making the purification of DNA impracticable.Thus, we used protocol 3 for extracting DNA of the sera samples studied. As demonstrated in Figure 2, detection of T. gondii DNA in the samples, referring to the first moment of collection, that is 18 hours PI, was possible in G3 animals, which were infected with BTU2 strain, genotype III, isolated from a dog with neurological signs. Results were different from those of Hafid et al. (10), who after inoculating 10 3 tachyzoites of RH strain via intraperitoneal route, obtained 60% sensitivity 18 hours PI, and 100% from 21 hours to the seventh day PI.Detection of only one positive sample 18 hours PI in the group infected with BTU2 strain could be explained by the inoculation route, the infecting form, the virulence of the strain, the individual susceptibility, and the mice lineage.Intraperitoneal route shows a lower number of barriers for the parasite to reach the lymphatic and hematogenic route, compared to the oral route.Tachyzoites present active penetration into the organs, but bradyzoites need to break the cyst and transform themselves again into tachyzoites to cause the infection, which can influence the results because PCR disfavor the detection of T. gondii in the first hours PI, in other words, during the acute phase.We believe that the mice lineage was the predominant factor to detect T. gondii DNA in only one sample collected 18 hours PI.The lineage used is not considered isogenic, so no mouse could respond similarly in the same conditions of the experiment. Group 3 was, therefore, the one that most developed the disease, with death before 192 hours PI, which suggests major infectivity and virulence by BTU2 strain via oral route.The results obtained can also be explained by the large individual variability of the group, because the animals were not isogenic. Performing PCR from the serum of infected animals has the advantage of using material with minor quantity of agents that can interfere in the reaction.With the results obtained in the conditions of the present work, PCR did not show to be an efficient method in detecting the parasite DNA.Thus, new studies with other samples of T. gondii are needed to explain the validity of researches on this agent in serum of animals, as well as of human patients in the acute phase of infection, and mainly in cases of reactivation of toxoplasmosis, like in immunosuppression.An important fact would be to consider intraperitoneal route as the infection route of tachyzoites, comparing the results in the same studied moments, in albino Swiss mice as well as in those of isogenic lineage. We concluded that serum is a great tool for diagnosing T. gondii infection, since it is not so invasive like the others; however, it must be further investigated in experimental groups to detect infection in earlier periods. parasitemia.One directed to B1 gene and the other to TGR 1 E repetitive sequence.PCR-B1 detected parasitemia from 48 hours to the seventh day PI, whereas PCR-TGR 1 E detected it until the twenty-first day.Rodríguez et al. (15) collected serum and cerebro-spinal fluid of 70 immunocompetent and asymptomatic patients with previous toxoplasmosis; 70 immunocompetent patients without toxoplasmosis; 63 carriers of HIV with encephalitis but not toxoplasmic; 5 patients under anti-Toxoplasma therapy and with cerebral toxoplasmosis; and 11 patients not under therapy and without cerebral toxoplasmosis.They performed PCR-B1 followed by hybridization with a chemiluminescent probe; serology to detect IgG, IgM, and IgA levels; and detection of circulating antigen.The technique of PCR presented the best results: 62% sensitivity and 100% specificity with five tachyzoites per sample as detention limit.In a similar study, Hafid et al. (10) detected the agent's DNA in sera of mice infected with T. gondii RH strain.Five animals were killed 3, 6, 9, 12, 15, 18, and 24 hours PI and then daily until the seventh day.In 60% of the animals, the agent was detected 18 hours PI, and in 100%, from 24 hours until the seventh day PI.The detention limit of the agent was two tachyzoites in 200 l.Comparison between PCR-B1, and ELISA and immunoblotting tests carried out by Hafid et al. (9) in serum samples of experimentally infected mice showed that PCR was positive 18 hours PI, whereas the other methods were positive only 24 hours PI.Amplification of B1 gene by PCR has demonstrated to be more sensitive and faster than ELISA, immunoblotting and cell culture.The authors discussed that sensitivity of the results can depend on the strain, inoculation dose, and PCR protocol used.	2018-12-09T16:44:45.793Z	2006-04-01T00:00:00.000	{ "year": 2006, "sha1": "a4ecd7bb8145bc98183b605eea1fd274d70f99f3", "oa_license": "CCBY", "oa_url": "https://www.scielo.br/j/jvatitd/a/BKFGzXpRzWczWtYGhrJxWdd/?format=pdf&lang=en", "oa_status": "GOLD", "pdf_src": "ScienceParseMerged", "pdf_hash": "a4ecd7bb8145bc98183b605eea1fd274d70f99f3", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [ "Biology" ] }
121642561	pes2o/s2orc	v3-fos-license	Flux-gate magnetometer spin axis offset calibration using the electron drift instrument Spin-stabilization of spacecraft immensely supports the in-flight calibration of on-board flux-gate magnetometers (FGMs). From 12 calibration parameters in total, 8 can be easily obtained by spectral analysis. From the remaining 4, the spin axis offset is known to be particularly variable. It is usually determined by analysis of Alfvénic fluctuations that are embedded in the solar wind. In the absence of solar wind observations, the spin axis offset may be obtained by comparison of FGM and electron drift instrument (EDI) measurements. The aim of our study is to develop methods that are readily usable for routine FGM spin axis offset calibration with EDI. This paper represents a major step forward in this direction. We improve an existing method to determine FGM spin axis offsets from EDI time-of-flight measurements by providing it with a comprehensive error analysis. In addition, we introduce a new, complementary method that uses EDI beam direction data instead of time-of-flight data. Using Cluster data, we show that both methods yield similarly accurate results, which are comparable yet more stable than those from a commonly used solar wind-based method. Introduction The in-situ characterization of the magnetic field in space has been a major objective of numerous spacecraft missions. For that purpose, flux-gate magnetometers (FGMs) are commonly used due to their superior accuracy, stability, and robustness, paired with their relatively low mass and power demand, e.g. [1,4]. The primary output of FGMs are raw vectorial magnetic field measurements B raw in sensor coordinates. These need to be transformed into physically meaningful units and coordinate systems, e.g. with the following equation: Here, B represents the calibrated magnetic field measurements, M is a 3 × 3 matrix dependent on six angles that transforms from sensor coordinates to an orthogonal, spacecraft-fixed coordinate system, G is a diagonal matrix consisting of three gain parameters, and O is a 3D offset vector. Altogether, M, G, and O are dependent on 12 parameters (6 angles, 3 gains, and 3 offsets) that have to be determined by ground-based (pre-launch) or in-flight (post-launch) calibration procedures, e.g. [3,9,11,21]. The vector O may exhibit larger variations due to spacecraft contributions: the vector represents the FGM measurement in zero ambient field, i.e. to a major extend, the stray fields of the spacecraft on which the FGM is mounted. These fields result from remanently magnetized parts and/or electric currents, e.g. [14]. Changes thereof that directly affect O can only be accounted for by in-flight calibration. Fortunately, the in-flight calibration procedures are relatively easy for spacecraft that are spin-stabilized. In that case, 8 of the 12 calibration parameters influence the measurements' spectral power at spacecraft spin frequency and/or harmonics in any inertial (de-spun) coordinate system, e.g. [2], which facilitates their in-flight determination. Out of these eight parameters, two are the spin plane components of O, which we denote as O y and O z . However, determining the spin axis offset O x (spin axis in x direction) is much more difficult. Usually, O x is obtained by analyzing solar wind (SW) FGM measurements that contain Aflvénic disturbances, i.e. field rotations without changes in field strength, e.g. [10,13,18]. Alternatively, simultaneous observations from an electron drift instrument (EDI) may be used. EDI consists of two gun-detector units (GDUs) mounted on opposite sides of a spacecraft. Each GDU can emit modulated electron beams. The electrons perform one (or more) gyrations in the ambient magnetic field and are registered by the other GDU. The electron time-of-flight (TOF) T is in principle inversely proportional to the field strength: Here, m e is the relativistic electron mass, e is the elementary charge, and k = 2π m e /e. Differences in T as measured by both GDUs indicate the presence of an electric field or magnetic field gradient [17,19]. Since measurements of \| \| B by an EDI are independent from spacecraft stray fields, they can be used to determine O x : A correction to this offset is given by the difference between FGM and EDI measured \| \| B , if the field points in spin axis direction. In this paper, we denote such a method of determining or adjusting O x via EDI TOF measurements as time-of-flight method (TOF method). An early description of the TOF method can be found in [8]. Later [12] explored the possibility of determining some of the parameters of matrix G by a comparison of FGM and EDI measurements. Just recently [15] showed that the EDI TOF data are also subject to offsets, which systematically alter the O x results by the TOF method. These studies proved the feasibility of the TOF method, but fell short of presenting a mature method for FGM spin axis offset determination using EDI measurements, which takes into account TOF offsets and other inaccuracies in FGM and EDI observations. The importance of such a method and timeliness of its development is rooted in its relevance for NASA's upcoming Magnetospheric Multi-Scale (MMS) mission. The mission consists of four spacecraft (featuring FGM and EDI instruments) that will explore the Earth's magnetosphere. The primary objective of the mission is to investigate the physics of magnetic reconnection. During the first years of operation, the MMS spacecraft will rarely encounter the pristine SW, if at all, as their apogee distances from Earth will be too low. Hence, MMS will rely on the availability of a working, mature, EDIbased method for spin axis offset calibration. The purpose of this paper is to make a major step forward in the development of such a method. First, we provide the TOF method with a comprehensive error analysis that facilitates the determination of spin axis offsets with higher accuracy. Secondly, we introduce another, complementary method which makes use of EDI beam direction (BD) data instead of TOF data. Consequently, we denote this method as beam direction method (BD method). Finally, we compare the spin axis offset results of the TOF method, of the BD method, and of a typically used solar wind (SW) based method [13], which is henceforth denoted as SW method. The data set we base this comparison on consists of one year (2008) of Cluster 3 FGM and EDI measurements. Methods Apart from the spin axis offset O x , we assume the FGM measurements to be well-calibrated. Furthermore, we assume that for every EDI measurement there is a simultaneous FGM observation B available. We would like to point out that the methods described in this section provide spin axis offsets O x after equation (1) if the FGM observations B are initially computed from B raw using O x = 0. Otherwise, O x denotes a correction to the already applied spin axis offset rather than the entire offset itself. TOF method Step 1: In order to avoid systematic errors in O x , the EDI GDU and operation mode dependent TOF offsets have to be corrected first, as shown by Nakamura et al [15]. For this purpose, measurements need to be selected for which \| \| B is unaffected by O x (magnetic field close to the spin plane, i.e. y-z-plane): Here C sp is a threshold value; B x and \| \| B are FGM-measured quantities. From the selected FGM and simultaneous EDI data, differences δT between FGM-determined magnetic field strengths, converted to TOFs, and EDI measured TOFs are computed: Therewith, we obtain one TOF offset O T per GDU and operation mode: Note that O T is specific to each GDU and operation mode. Hence, for the computation of each offset O T only samples δT corresponding to the particular GDU and obtained when the instrument was in the respective mode may be used. Step 2: All TOF values T (belonging to a particular GDU and mode, not restricted by C sp ) are subsequently corrected with the corresponding TOF offsets O T : Therewith, estimates of the FGM spin axis offset (correction) O x can be computed: The uncertainty ΔO x of each O x estimate does not only stem from ΔT c , i.e. from the TOF offset computations, but also from the inherent FGM measurement and calibration uncertainty of B. Inaccuracies in G and M will result in errors of B that scale with the strength of the magnetic field. Furthermore, there is a FGM noise floor which defines a minimum uncertainty of B. Taking both uncertainties into account, we assume each component of B to be associated with an error of the following form: where Δg is a factor of unit 1 and ΔB n is the noise level in nT. From equations (9) and (10) we derive the error of O x (error propagation): As can be easily seen, ΔO x increases with the factor: Consequently, it is advantageous for spin axis offset determinations with the TOF method if the field is low in strength and directed along the spin axis. Step 3: The samples of O x (and corresponding ΔO x ) are the basis for the computation of a final offset (correction) value O xf for a specific time interval of length t int . Therefore, we select those O x within the time interval, for which ΔO x ⩽ C O holds (C O is a threshold value), and compute: x f (13) with upper/lower error bounds given by the 16th and 84th percentiles O xf − ΔO xf− and O xf + ΔO xf+ of the distribution of selected/contributing O x , if their number exceeds another threshold number C # . The percentiles yield a 2σ range around the mean, if the estimates O x are normally distributed, but also account for skewness of the distribution. The average error of O xf is given by: Clearly, the choice of t int , C O , and C # will influence the result O xf . Larger intervals increase the statistical basis at the expense of a larger spread of the O x estimates due to offset drift. Lowering C O and increasing C # will also increase the quality of O xf in general, but less intervals of length t int may fulfill the criteria and yield such an offset value. It should be noted that realistic uncertainties ΔO x facilitate the selection of the most accurate estimates O x for the computation of a final offset (correction) O xf . Hence, error analysis is not only necessary to assess the uncertainty of any result, but is also crucial, in this case, for maximizing the result's accuracy. BD method Only those electron beams, that are emitted by an EDI GDU perpendicular to the ambient magnetic field, are able to return to the spacecraft. Hence, EDI beam directions (BD), which we denote with D, should be perpendicular to B. Deviations in angles α (depicted in orange in figure 1) between FGM measured B and EDI measured D from 90° may be attributed to inaccuracies in spin axis offset O x . Step 1: BDs are not subject to TOF offsets. However, for any BD method to yield accurate spin axis offsets, the coordinate systems of the BDs and of the calibrated FGM data have to coincide. Any adjustment of the coordinate systems should be based on FGM and EDI measurements for which the angles α are least affected by the spin axis offset D ( close to the spin plane). Our selection criterion is: where \| \|= D 1. Therewith, GDU specific coordinate transformations for the EDI BDs are found by minimization of the mean of (α −90°) 2 . The standard deviation can be regarded as angular uncertainty of the BD vectors D. The target coordinate systems should ideally coincide with the coordinate system of the calibrated FGM measurements. All the EDI BDs D (not restricted by C sp ) are transformed into these systems. Step 2: Ideally, = B D · 0, as stated above. Deviations can be attributed to the FGM spin axis offset as follows: Here, the uncertainty ΔO x results from the uncertainties of the components of B (after equation (10)) and of the components of D : Note that Δβ and, hence, ΔD are GDU specific. Therewith, we obtain the error in O x in a similar manner to equation (11): Here ΔB and ΔD are computed in accordance to equations (10) and (18), and \| \| = D 1. As can be seen, ΔO x increases with decreasing D x 2 and also with increasing \| \| B 2 , as: The reason for this behavior stems from the sensitivity of α to changes in O x being given by the quotient of the two quantities D x 2 and \| \| B 2 : Consequently, it is advantageous for spin axis offset determinations with the BD method if the field is low in strength and pointing in perpendicular direction to the spin axis. Step 3: Following the description of step 3 of the TOF method, a final spin axis offset (correction) value O xf for a time interval of length t int can be computed from O x estimates pertaining to that interval, for which ΔO x < C O holds, via: if their number exceeds a minimum threshold of C # . Upper/ lower error bounds O xf − ΔO xf− and O xf + ΔO xf+ may again be given by the 16th and 84th percentiles of the distribution of selected/contributing O x , with equation (14) defining the average error 〈ΔO xf 〉 of the final spin axis offsets (or offset corrections) O xf . SW method Magnetic field fluctuations embedded in the solar wind (SW) are primarily Alfvénic in nature, i.e. the field changes rather in direction than in magnitude. Hence, the magnitude of the magnetic field tends to be more constant over time than any of its three components [16]. The spin axis offset of a FGM can be determined in the solar wind by making use of this property, as incorrect offsets lead to an artificial increase in fluctuation levels of the field magnitude [1,5,6,10,20]. The technique used herein introduced and described in [13] is an improved and automated version of the Davis-Smith method [6]. Henceforth, it is referred to as SW method. This method relies on a few hours of FGM solar wind data as input, and yields final spin axis offset (correction) values O xf (O 3 in [13]) as well as upper and lower error bounds O xf − ΔO xf− and O xf + ΔO xf+ (O 3min and O 3max in [13]), just as the TOF and BD methods. Application We apply the TOF, the BD, and the SW methods to one year (2008) of Cluster 3 EDI and FGM measurements. The FGM measurements were calibrated using the daily calibration files [7]. Roughly 25 million EDI data samples (i.e. combinations of D, T, as well as corresponding simultaneous FGM measurements B) are available for that year. It should be noted that the daily calibration files include a non-vanishing spin axis offset, which remains constant throughout the year 2008. Consequently, all values O x that are determined by the TOF, the BD, and the SW methods are corrections to that daily calibration file spin axis offset. TOF method Step 1 consists of the determination of GDU and EDI mode dependent TOF offsets. For this task, we selected EDI and FGM measurements in accordance to equation (3) with C sp = 0.1. Two oppositely directed GDUs are placed on each Cluster spacecraft. The mode of each of these units is characterized by three parameters: n, m, and the code flag f. These parameters control the pseudo noise sequence of electrons emitted by Table 1. TOF offsets and uncertainties O T ± ΔO T (in μs) dependent on GDU (1 or 2) and EDI mode (code length f and clock dividers n and m). As EDI is a system of 2 GDUs, each being able to emit modulated electron beams with 2 different code lengths, with chip lengths given by 6 × 2 combinations of m and n, in total 2 × 2 × 6 × 2 = 48 different TOF offsets O T need to be considered. However, for some parameter combinations, there are very few measurements left (less than 100), having taken into account equation (3). In these cases, we abstain from computing O T and disregard measurements of the respective modes for the following TOF method computations. For all other parameter combinations, TOF offsets O T and uncertainties ΔO T are listed in table 1. For step 2, assumptions with respect to Δg and ΔB n are required. Orthogonalization angles and gains (matrices G and M) are assumed to be accurate to the order Δg = 10 −4 . The noise level of the Cluster FGMs is on the order of ΔB n = 10 pT. Therewith, we obtain uncertainties ΔO x via equation (11). They are color coded in figure 2 and depicted as a function of \| \| B and the angle ξ B (blue in figure 1) between B and the spin axis x. As expected, the uncertainties ΔO x decrease with \| \| B and ξ B . Hence, low ΔO x are found in the lower left corner of that figure. The value of Δg = 10 −4 reflects the order of magnitude of long term variations of those calibration parameters, influencing G and M, that can be accurately determined in flight (e.g. the ratio of the spin plane component gains). However, some calibration parameters that influence \| \| B , as measured by the FGM, namely the absolute spin axis and spin plane gains, cannot be easily and/or accurately determined in flight. It has to be assumed, without possibility of verification, that their long term variations are of the same order of magnitude. Consequently, FGM measurements are not guaranteed to exhibit relative accuracies on the order of or better than Δg = 10 −4 . Nevertheless, for the purpose of spin axis offset determination, we necessarily have to assume that the only undetermined calibration parameter is, indeed, O x . In step 3, we compute O xf for sliding windows of t int = 15 min shifted by 5 min. The quality thresholds were set to C O = 0.2 nT and C # = 100, respectively. Therewith, we obtain 3523 final offset correction values O xf distributed over 68 different orbits. The distribution of O xf values is depicted by a red line in figure 3(c). The figure illustrates how a number of EDI measurements per orbit (panel (a)) taken at certain \| \| B conditions (panel (b)) is converted into a number of final offset correction values O xf (red line, panel (c)). Interestingly, O xf values were computed roughly for every second orbit. This can be easily explained: For O xf computations, we selected O x estimates that fulfill ΔO x ⩽ C O = 0.2 nT. As shown in figure 2, these estimates are obtained in significant numbers for magnetic field strengths below 60 nT. EDI measurements in this field range were taken roughly during every second orbit (see figure 3(b)). For those orbits, O xf values are computed. BD method Each GDU can emit electrons in a solid angle range larger than 2π around a central axis. On Cluster, this axis is perpendicular to the spin axis. For convenience, we assume it to point in z-direction. For angles ζ D between D and z (shown in green in figure 1) approaching or even surpassing 90°, the beam width is known to increase significantly. This effect would deteriorate the results of the BD method. Therefore, we exclude EDI measurements with ζ D > C D = 80°. For the coordinate system adjustment (step 1 of the BD method) we select EDI (and corresponding FGM) measurements for which D x < C sp = 0.1 holds (equation (15)). Minimal deviations on the order of 0.6° are found between the coordinate system transformations that are obtained from EDI/ FGM data comparison and the nominal transformations that are given by the Cluster spacecraft building plans. The angular uncertainties of the former transformations are Δβ = 0.25° for GDU 1 and Δβ = 0.28° for GDU 2. Again, we assume Δg = 10 −4 and ΔB n = 10 pT. The resulting uncertainties ΔO x are shown in figure 4, this time as a function of \| \| B and the angle ξ D between D and the spin axis x (depicted in red in figure 1). Figure 4 is somewhat similar to figure 2 as lower uncertainties ΔO x are also found in the lower left corner, corresponding to lower field values \| \| B and lower angles ξ D (i.e. D close to the spin axis). However, for equal field values \| \| B minimal uncertainties ΔO x are larger for the BD method than for the TOF method. The final offset correction values O xf are computed for t int = 15 min sliding windows shifted by 5 min, C O = 0.2 nT, and C # = 100. Therewith we obtain 2792 values O xf distributed over 46 orbits. The numbers of these values per orbit are depicted in figure 3(c) by green bars. SW method In 2008, the apogee distances of Cluster 3 to the Earth's center were larger than 20 Earth radii. Hence, dayside apogee positions were beyond the bow shock, in the pristine solar wind (SW). Due to the Earth's rotation around the Sun, the apogee positions rotated once around Earth over the course of the year. When they were at the night side, Cluster 3 stayed inside the magnetosphere and the SW was not observed. Consequently, SW measurements were performed by Cluster 3 not during the entire year, but are available just for the first 6 months and for December of 2008. We applied the SW method to the solar wind (spin averaged) FGM measurements of each orbit, if available. Specifically, we used the parameters and criteria given in the 'THEMIS' column of table 1 of [13]. As a result, we obtained 53 spin axis offset corrections O xf pertaining to 53 different orbits (49 for DOYs 2-150, and 4 for DOYs 357-364). The corresponding solar wind data intervals ranged between 32 and 57 h in length. Orbits for which a final spin axis offset correction O xf could be computed by the SW method are marked by black arrows in figure 3(c). Results and discussion The O xf results of the three methods are shown in figure 5(a) (red dots: TOF method, green dots: BD method, black crosses: SW method). In total, 6368 O xf values contribute to that figure: 3523 from the TOF method, 2792 from the BD method, and only 53 from SW method. The results show consistently that the spin axis offset (as part of the FGM calibration files for that year) is, in general, off by about −0.2 nT. This systematic deviation is expected as the spin axis offset value in the calibration files for 2008 was determined in 2003. Since then, the offset has been checked periodically, but has been kept constant, as its changes were found to lie within its margins of error (Fornaçon, The average errors 〈ΔO xf 〉 (after equation (14)) of all O xf values are depicted in figure 5(b). The minima and maxima of this quantity are 0.05 and 0.53 nT (TOF method), 0.06 and 0.59 nT (BD method), as well as 0.04 and 0.32 nT (SW method). The medians of 〈ΔO xf 〉, which are more representative of the typical uncertainties, are 0.14, 0.12 and 0.12 nT for the TOF, BD, and SW method offsets, respectively. Hence, they are practically equal. If the margins of error of the results from the different methods are associated with similar statistical confidence levels, then the accuracies of the results obtained with the three methods will be practically equal, on average, as well. The blue lines in figure 5(b) mark levels of uncertainty of 〈ΔO xf 〉 = 0.1 nT and 0.2 nT. These levels correspond with two MMS mission requirements: 0.1 nT and 0.2 nT are the desired and minimum accuracy goals for FGM magnetic field measurements (referring to \| − \| B B ) measaured t rue , to be achieved by the MMS spacecraft in science target (i.e. reconnection) regions e.g. [22]. The figure suggests that 〈ΔO xf 〉 ⩽ 0.1 nT is achievable by all three methods. It has to be pointed out that this conclusion is only valid if the calibration parameters other than the spin axis offset, in particular those parameters pertaining to the matrices G and M, are known with sufficient accuracy. Here, we assume relative uncertainties on the order of Δg = 10 −4 in equation (10), which result in small FGM measurement errors on the order of 10 pT for fields of 100 nT. Uncertainties of (much) higher order of magnitude in G and M, however, may noticeably alter the outcome of the spin axis offset correction estimates O x and, consequently, of O xf , without necessarily increasing 〈ΔO xf 〉. Numbers of O xf values from the TOF and BD methods that (nominally) fulfill the accuracy criteria with respect to 〈ΔO xf 〉 are given in table 2; the offsets are distributed over certain numbers of orbits that are given in brackets (maximum: 73 orbits, for which there are TOF and/or BD method offset corrections O xf ). O xf with 〈ΔO xf 〉 ⩽ 0.2 nT are obtained for 70 out of 73 orbits. If the stricter criterion of 〈ΔO xf 〉 ⩽ 0.1 nT is used, corresponding offsets are still obtained for 46 out of 73 orbits. Interestingly, only for 8 of these 46 orbits there are sufficiently accurate O xf values from both TOF and BD methods. For the other 38 orbits, either TOF or BD method offsets are obtained with sufficient accuracy. This finding is an indication of the complementarity of the methods. Obviously, the SW method is complementary to the TOF and BD methods, as it is dependent on measurements in the solar wind. EDI, instead, does not work in the solar wind, as typical magnetic field strengths there (of a few nT) are too low for operation. This can be seen in figures 2 and 4, which show only few measurements below \| \| = B 10 nT. It should be noted that the low magnetic field strengths in the solar wind relax the requirement for accurate knowledge of the matrices G and M prior to spin axis offset determination with the SW method, because the measurement uncertainties associated to G and M scale with \| \| B (the factor being given by Δg, see equation (10)). Although the TOF and BD methods are both based on EDI measurements, they are also complementary to each other. The reason is rooted in the perpendicularity of D and B. Most accurate O x estimates are obtained with the TOF method if B is close to the spin axis (small angle ξ B , see figure 2). For the BD method, it is more favorable if D is closer to the spin axis (small angle ξ D , see figure 4). As a matter of fact, for where ξ ∼ is an arbitrary angle between 0° and 90°, ξ ξ >°− ∼ 90 D necessarily follows (and vice versa; see figure 1). Consequently, EDI and simultaneous FGM measurements tend to be either favorable for the TOF or for the BD method, but cannot be perfect for both. Nevertheless, for observations in low \| \| B , there is an overlap in EDI/FGM measurements of acceptable quality for both methods (e.g. ΔO x < C O = 0.2 nT, see figures 2 and 4). These measurements result in simultaneous O xf values from both TOF and BD methods, which we can directly compare. Figure 6 shows the distribution of differences of the 1162 simultaneous BD and TOF O xf results. As can be seen, differences between the two methods are minor on average: the mean and median of the distribution is 0.06 nT. Hence, BD method-determined O xf tend to be slightly higher than those determined with the TOF method. The standard deviation of the distribution, however, is 0.11 nT, i.e. about twice as large as the mean and median values of the distribution. Hence, average, systematic differences between TOF and BD method O xf results are small in comparison to the width of the distribution. Furthermore, from the 1162 simultaneous offsets, 1130 BD method O xf are within the corresponding TOF method error margins, i.e. between O xf − ΔO xf− and O xf + ΔO xf+ . Conversely, 905 TOF method O xf values are within the error margins of the corresponding BD method offsets. Also in that respect, O xf results from both methods have to be regarded as quantitatively similar. As discussed above, a comparison of simultaneous O xf values resulting from the SW method and from the TOF or BD methods is impossible due to the complementarity of the methods. In the next best case, O xf values from either the TOF or the BD method are available before and after an O xf estimate by the SW method within a common orbit. This is the case for 13 orbits (see dates on the vertical axis of figure 7). Spin axis offset corrections were obtained by the TOF method during both outbound and inbound passes of Cluster 3 through the magnetosphere on those orbits. SW method O xf values are based on the solar wind intervals in between, around apogees. For each of those orbits, we linearly interpolate TOF method O xf values and error bounds (O xf + ΔO xf+ and O xf − ΔO xf− ) to the central times of the solar wind intervals that the SW method has been applied to. The interpolates are shown in figure 7 in red. The corresponding SW method results are depicted in black. In eight cases the SW method results are within the margins of error of the TOF method offset corrections (and vice versa). Furthermore, the margins of error overlap in 12 out of 13 cases (exception: DOY 81). The mean O xf values are −0.23 nT (SW method) and −0.18 nT (TOF method); they are quite similar to the results obtained for the entire year 2008. As can be seen, the TOF method-determined offsets are much more stable; this is reflected in the respective standard deviations of 0.16 nT (SW method) and 0.05 nT (TOF method). The relative stability of the TOF method results suggests that they may be more accurate than the SW method results. The relative stability of the TOF method results is surprising when taking into account that the TOF method results are based on data from relatively short periods of time (t int = 15 min intervals). The SW method O xf estimates, instead, are based on solar wind data intervals that are more than one day long. Consequently, we would expect the SW method-based offset corrections to be more time-averaged and, hence, stable than the TOF method results, contrary to our findings. Summary and conclusions In this paper, we improve the time-of-flight (TOF) method for FGM spin axis offset determination by using a comprehensive error analysis. It allows for the selection of the most accurate offset (correction) estimates O x for the computations of final spin axis offsets (or offset corrections) O xf . Furthermore, we introduce a new method for the same task, denoted as beam direction (BD) method, that is complementary to the TOF method. While both are based on the comparison of EDI and simultaneous FGM measurements, the TOF method benefits from low angles ξ B and the BD method from low angles ξ D . Both conditions are mutually exclusive. Under low magnetic field conditions, the two methods allow for computations of O xf independently of the ambient magnetic field direction with respect to the spin axis of the observing spacecraft. In addition, the error analysis enables us to sensibly choose among TOF and BD method results, when both are available. In fact, it facilitates the combination of both methods after their respective steps 2: O x estimates and the corresponding uncertainties ΔO x may be merged into a single data set. The increased sample size would improve the Furthermore, we compare the results of the TOF and BD methods with those from a typically used SW method. These are our findings: • In general, the offset correction results for the considered Cluster 3 FGM data from 2008, obtained with the three methods, tend to lie consistently around −0.2 nT. • Standard deviations of the results of either of the three methods are on the order of 0.1 nT. Systematic differences between results from different methods are equal or smaller. • Errors 〈ΔO xf 〉 are typically a little larger, yet very similar among the three methods: around 0.13 nT, on average. • O xf values from the TOF or BD methods are mostly included in the error margins of the corresponding O xf estimates from the respectively other method. This further indicates that the TOF and BD method results are quantitatively very similar. • The comparison of the TOF and SW method results shows that the latter seem to be less stable, in contrast to expectations. This finding suggests (without proof) that the TOF method results are more accurate than the SW method ones. Altogether, we find the TOF and BD methods to perform similarly well, while the SW method results may be equally or slightly less accurate. Furthermore, the TOF and BD methods are able to provide O xf estimates with much higher cadence (we used t int = 15 min). Instead, the SW method requires solar wind data intervals that are at least several hours long [13]. On the other hand, the TOF and BD methods rely more than the SW method on the accurate knowledge of calibration parameters other than the spin axis offset. Finally, we approach the question of how readily usable the TOF and BD methods are for routine FGM spin axis offset calibration, in reference to the upcoming MMS mission. We find that most accurate offset estimates are obtained from EDI and FGM measurements in low magnetic field regions. Magnetospheric field strengths per orbit observed by MMS are expected to be highest in the subsolar region. There, field strengths of \| \| ≈ B 50 nT are typical, just Earthward of the magnetopause. Even under these (worst case) conditions, accurate O x estimates can be provided at least by the TOF method, likely also by the BD method over a wide range of angles ξ B and ξ D (see figures 2 and 4). Furthermore, we find that both methods are able to yield O xf values with (the required) accuracies of 〈ΔO xf 〉 ⩽ 0.1 nT or 0.2 nT. Offsets (or, more precisely, offset corrections) fulfilling the latter condition are distributed over almost all orbits for which EDI measurements in low magnetic field regions are available. Hence, we can conclude that the TOF and BD methods, introduced herein, constitute working EDI-based methods that are suitable for routine FGM spin axis offset calibration. This applies in particular to the MMS flux-gate magnetometers, but is dependent on the precise knowledge of the other calibration parameters.	2019-04-19T13:06:15.309Z	2014-10-01T00:00:00.000	{ "year": 2014, "sha1": "e7036ea38494f9f61e480753b14e3e6c665fd5b3", "oa_license": "CCBY", "oa_url": "https://doi.org/10.1088/0957-0233/25/10/105008", "oa_status": "HYBRID", "pdf_src": "IOP", "pdf_hash": "fa22bcfaa69f3414820786ca9c030d7a7bda28b2", "s2fieldsofstudy": [ "Physics", "Engineering" ], "extfieldsofstudy": [ "Physics" ] }
92140236	pes2o/s2orc	v3-fos-license	Forest and land fires in Pelalawan District , Riau , Indonesia : Drivers , pressures , impacts and responses Tata HL, Narendra BH, Mawazin. 2018. Forest and land fires in Pelalawan District, Riau, Indonesia: Drivers, pressures, impacts and responses. Biodiversitas 19: 544-551. Pelalawan District of Riau Province, Indonesia was one of the districts most damaged by fire in 2015. Analysis of factors driving the fires, of pressures arising from the fires and of responses to the fires in Pelalawan District was conducted using two approaches: semi-structured interviews regarding social and policy aspects, and analysis of biophysical factors such as soil properties and spatial data. Results showed that forest functions (i.e. the functions served by different forest types) was positively related to hotspot density (R=0.9868), while distance to nearest road less affected hotspot distribution (R=0.1612). Multiple regression analysis of the relationship between hotspots density and four variables resulted in the following model: Y = 0.005384 + 0.000021 Soil Type + 0.000019 Distance to Road + 0.000038 Forest Functions + 0.000017 Land Use type. The pressures were expansion for agriculture, plantation and forest encroachment. Despite many negative impacts of fire, the burning practice on peatland could improve the pH and peat soil fertility (particularly ash and P contents). As a response to fire, a standard operational procedure for forestand land-fire prevention was launched by the Governor of Riau Province in late 2015. A comprehensive and integrated policy package for forest and land fire prevention and control should include a social dimension in order to effectively reduce fire risk in the district. INTRODUCTION Peatland is a unique ecosystem which stores large amount of carbon (Page et al. 2010;Warren et al. 2017) and has a large water holding capacity (Rezaneshad et al. 2016).However, the peatland ecosystem is fragile; when damaged its condition changes drastically.Channel development in peatland drains water and accelerates oxidation, which increases CO 2 emission into the atmosphere (Gaveau et al. 2014;Van Noordwijk et al. 2014;Marlier et al. 2015;Wilson et al. 2016).Once peatlands dry out, they are prone to fire (Taufik et al. 2015;Turetsky et al. 2015). For many years, Indonesia has been suffering from recurrent forest and land fires.A recent report showed total burnt area in Sumatra and Borneo in 2015 was 1.34 million km 2 (Miettinen et al. 2017).The year 2015 was the most catastrophic fire season on record in Indonesia (Field et al. 2016).The World Bank reported that economic loss due to forest and land fire in 2015 amounted to IDR 221 trillion (World Bank 2016).The fires also had significant negative impacts on human health (Gaveau et al. 2014;Reddington et al. 2014;Marlier et al. 2015). Geographical Information System (GIS) and remote sensing are a common tool used for forest and land fire susceptibility mapping (Dewi et al. 2015;Nurdiana and Risdianto 2015;Field et al. 2016;Harris et al. 2017).The map of fire susceptibility is important as part of the Early Warning System for fire prevention.In developing a map of fire susceptibility of an area, hotspot series data are recorded, collected and analyzed (Samsuri et al. 2012; Usman et al. 2015;Mukti and Rushayati 2016;Tata et al. 2017).One hotspot point represents a pixel size area on the land that has higher temperature compared to the surrounding based on certain temperature threshold detected by a satellite (Giglio et al. 2003;Amri and Sitanggang 2015;Harris et al. 2017). Riau Province was one of the five provinces in Indonesia that was most affected by fire in 2015 (Harris et al. 2017;Prayoto et al. 2017).Riau has a large peatland area, amounting to 4,062,420 ha, of which 66.75% has been converted into smallholder and industrial plantation area.Peatland conditions in these two land use types have changed dramatically owing to human intervention (Miettinen et al. 2016).The total burnt area in Riau Province indicated by hotspot analysis is reported to be about 90,709 km 2 , which is about 19.02% of the total burnt area in Sumatra Island (Mietttinen et al. 2017). Therefore, the objective of this study was to develop an understanding of the factors driving forest and land fire in Pelalawan District of Riau Province; of the impacts of these fires on the peat soils; and of the responses of local government to forest and land fire. Study site The study was conducted in Pelalawan District, Riau Province, Indonesia located between 0 48'32" N and 0 24'14" S and between 101 30'40" E and 103 23'22" E. It covers an area of 1,382,210 ha, consisting of 1,276,433.44ha land, 39,146 ha rivers and lakes, and 66,630 ha marine.Peatland covers 155,349.9ha.Of the total district area, 863,725 ha is forest area, which represents about 15.70% of the total forest area of Riau Province) (BPS Pelalawan, 2014). Procedures The primary field data consisted of vegetation conditions after fire in 2015 and soil samples.The burnt peatland use-types in Pelalawan District were selected purposively; namely forest, rubber plantation, oil palm plantation and agriculture.We also collected soil samples from unburnt secondary forest.The soil samples were collected by using an Eijkelkamp peat-soil auger from two plot samples; these were taken from the four land-use types, purposively.The soil from the unburnt secondary peat forest was taken as a control.The soils samples were analyzed for chemical and physical properties following regular procedures at the Indonesian Soil Research Institute in Bogor.Only peat depth of two layers, i.e. 0-50 cm and 50-100 cm, were sent to the laboratory.The chemical properties consisted of pH, water content, Nitrogen content (Kjeldhal), available Phosphorus (P 2 O 5 , Bray1), and pyrite.The ash content, organic matter, and C-organic were analyzed using the lost-on-ignition method (Maswar et al. 2011). Spatial data related to forest and peatland fires were collected, such as: hotspot NOAA18 data for the years 2013-2015 from the Mitigation Disaster Agency of Riau Province (BPBD, Badan Penanggulangan Bencana Daerah Riau); topography and village maps of Geospatial Information Agency (Badan Informasi Geospasial, BIG); land-use type for the year 2015; and forest functions for the year 2014 from the Directorate General of Planology (the Ministry of Environment and Forestry, MoEF): peatland hydrological data for the year 2015 from the Directorate General of Pollution Control and Environment Degradation (MoEF). Some key respondents were interviewed by using semistructured interview and questionnaires.The respondents were selected purposively at province, district, sub-district, and village levels.They consisted of representatives of the Forest Service of Riau Province; the Agency of Disaster Mitigation of Riau Province; the Forestry, and Estate Crops services of Pelalawan District; the Nature Conservation Unit of Riau; the Teso Nilo National Park; the head of subdistricts Kerumutan and Ukuy; the head of Kerumutan village and farmer groups of Teluk Meranti.Data collected concerned their perceptions of forest and land fires, and of fire prevention measures. Data analysis We analyzed factors (both biophysical and social factors) driving forest and land fire in Pelalawan District of Riau Province.The soil properties were analyzed using analysis of variance (ANOVA) of two factors, viz.peat depth and land-use types.A General Linear Model (GLM) for each parameter was estimated using software of IBM SPSS statistics ver.21. Spatial data were analyzed via the following four steps: (i) each variable of the model was classified; (ii) each factor of fire susceptibility was weighted; (iii) score and score estimation of each sub-factor was calculated, using formula of Samsuri et al. 2012 andTata et al. 2017;(iv) a rescaling score was calculated using the formula of Arianti et al. (2007) and Tata et al. (2017).Rescaling score for each factor was used to calculate multiple scores of several factors using Composite Mapping Analysis (CMA) method as explained by Samsuri et al. (2012) and Tata et al. (2017).Multiple regression analysis was used to determine the weight of each factor.The regression model shows the relation between estimation of hotspot density and composite score of each factor.The best regression model for each factor was chosen based on the coefficient determination (R 2 ).The weight of a factor was resulted from a comparison between regression coefficients of the factor to the total amount of regression coefficients.The fire susceptibility level of an area (polygon) was determined based on scores of rescaling and the weight of the factors.This was calculated using the field calculator in the ArcGIS software. The estimated value of hotspot density of each area represents the fire susceptibility level.This was classified into four classes (Tata et al. 2017) i.e.: (i) Less susceptible, when the estimated value of hotspot density per km 2 is less than 0.061; (ii) Rather susceptible, when the estimated value of hotspot density per km 2 is less than 0.121; (iii) Susceptible, when the estimated value of hotspot density per km 2 is less than 0.181; (iv) Very susceptible, when the estimated value of hotspot density per km 2 is more than or equal to 0.181. The map of fire susceptibility was then overlaid with the sub-district administrative map.The total area of each level of fire susceptibility was analyzed based on this map. The in-depth interview data with key stakeholders on policy and regulations related to fire prevention was analyzed and synthesized. Hotspot number and distribution According to NOAA18 satellite data, the number of hotspots in Pelalawan District decreased from 2911 to 2017 (30.71%) during the year 2013 to 2014.In 2015, the hotspot number again decreased to 1784 (11.55%).The majority of hotspots usually occurred in June to August. Hotspot density Hotspot density was calculated based on four factors, i.e. forest functions, land-use types, soil types, and distance to the nearest road.In the Indonesia Law no.41/1999, the state forest area can be distinguished based on its functions, which are classified into three main categories, namely conservation, preservation, and production forests.Regression analysis for the four factors is shown in Table 1.Table 1 showed that forest function (FF) has the strongest positive relationship with hotspot density compare to other factors (R 2 =0.9868).In contrast, distance to nearest road has the lowest relationship with hotspot density. Map of fire susceptibility Multiple regression analysis for the effect of the four variable determining hotspots density produced the following model: Y = 0.005384 + 0.000021 ST + 0.000019 DR + 0.000038 FF + 0.000017 LU, where ST is soil type, DR is distance to road, FF is forest functions, and LU is land use type. Each factor (independent variable) was weighted based on the regression model.The contribution of each of the four variables was estimated to be: soil types (22%), distance to road (20%), forest function (40%) and land use type (18%).The map of fire susceptibility was created, as shown in Figure 2. The level of fire susceptibility was calculated for each sub-district.Overall, 49.9% of the area of Pelalawan District is classified as least susceptible to fire, while 11.9% of the district is classified as highly prone to fire.Teluk Meranti sub-district, which is dominated by peatland, has the largest area classed as highly prone to fire (Table 2). Impact of fire on peat soils The soil properties on burnt peatland vary between four land use types (namely secondary forest, oil palm plantation, rubber plantation, and agricultural crops) as shown in Table 3.The land use types significantly affected several soils properties, namely: pH, water content, bulk density and content of P 2 O 5 .The water content was the only variable affected by the peat depth.The upper layer (0-50 cm) had lower water content than the lower peat layer (50-100 cm).Other properties, such as ash content, organic matter, C organic, N total , CEC, and pyrite, were not affected by land use type or by peat depth. Response to forest and land fire According to the records for the Strategic Forest Planning Office at Provincial level in the years 2014-2019, the government of Riau Province targeted a 20% reduction in total hotspots annually for the years 2014 to 2019 (Dinas Kehutanan Provinsi Riau 2014).The number of hotspot reduced from 2911 in year 2013 to 1784 in year 2015, which accorded with the target of the Strategic Forest Planning Office of Riau Province. The Governor of Riau Province released Regulation no.11 in the year 2014 regarding the Centre of Forest and Land Fire Control of Riau Province (Gubernur Riau 2014).This regulation aims to strengthen the unity of steps and actions in forest and land fire control.The organization and roles of the Centre are described in the regulation.The Riau Province aims for a policy of "non-burning"; all concessions operating in Riau Province have an obligation in fire prevention and control.In addition, a Community's Fire Care Unit (Kelompok Masyarakat Peduli Api) has been established at the village level. As a response to forest and land fire in 2015, the Governor of Riau Province also released Regulation No. 61/2015, on an Established Procedure for Forest and Land Fire Disaster Control.This regulation aims to provide guidance on forest and land fire disaster control in Riau Province (Gubernur Riau 2016).This is in accordance with the Government Regulation No. 21 of the year 2008, requiring disaster conditions to be officially announced and declared by the Governor at provincial level, or by Head of District at district level. Discussion The largest area of Pelalawan District that is very susceptible to fire is in the peatland, e.g.Teluk Meranti, where recurrent fires occurred in 2013-2015.Teluk Meranti is dominated by peatland.The community who live in the area use the peatland for agriculture such as oil palm, rubber and cash crops.Although a 'no-burning policy' was launched by the local government, nevertheless, up to mid-2015, fire was still being used in land preparation for agriculture.Some farmers selected as respondents for our interviews, stated that slash-and-burn has been used since long ago as part of their agricultural practices.The reason for their practice of prescribed burning was that it was a simple and fast technique for preparing land for agricultural use.The ash from the biomass burning is believed to improve soil pH and fertility.Saharjo (2007) reported pH of sapric peat soil was not affected by fire, while our study showed pH and ash content are affected by fire.Sulwinski et al. ( 2017) also reported soil pH and ash content of upper layer of fen-soil increased after it has burnt.It is shown in Table 3, that the upper layer of crop-land has the highest pH (4.70), ash content (11.38%) and P 2 O 5 (423.15%),but low CEC (58.76%), compared to other land use classes.The high recorded phosphorus content suggests that the farmers are also giving high inputs to those peatlands where they plant crops regularly., burned oil palm plantation, burned rubber farm, CRBburned crops, WC: water content, BD: bulk density, AC: Ash content, OM: organic matters, Corg: C organic, N: Nitrogen, P: Phosphor, CEC: cation exchange capacity.Numbers in parentheses are standard error of means.Means followed by the same letters showed in the same column are not significantly different at 95 % confidence level of Tukey's post hoc test. It is surprising that soil type has only a moderate relationship with hotspot density in Pelalawan District.This is connected to fact that frequent recurrent fires occurred in a national park which is located on mineral soils.It is evidence that fire incidence is not only affected by biophysical factors such as peat soil type.This contrasts with hotspot density in Kapuas district, Central Kalimantan Province (Thoha et al. 2014) and Musi Banyusin district, South Sumatra province (Tata et al. 2017), where hotspots mostly occur in peat soils. The hotspot and recurrent fires that occurred in Teso Nilo National Park in Pelalawan District were driven by human activities.People used fire as a weapon for land grabbing.The WWF-Riau programme has reported that land encroachment has occurred in the National Park.Many social problems, such as land claims, have been encountered as a result of recurrent fires, as has also been shown in other parts of Indonesia (Purnomo et al. 2017;Cattau et al. 2016;Gaveau et al. 2017). At the national and provincial level, the fire-prevention policy package has been comprehensive enough (Rosul 2015).However such regulations related to fire prevention appear not to be sufficient to stop fire hazard in the districts of Riau Province.Dewi et al. (2015) reported the existing law enforcement is inadequate to prevent fires in Kalimantan and Sumatra.We recommend the implementation of fire prevention on the ground needs also to include social participation approaches at the community level, such as: improving the capacity of the communities' fire care units; providing alternative and applicable nonburning technologies for preparation of agricultural lands; and creating incentive mechanism to reward the zeroburning practices, not only for the corporate sector, but also for the local communities. of each sub-factor Score R out = Score from rescaling calculation Score E input = Score of estimated input Score E min = Minimal value of estimated score Score E max = Maximal value of estimated score Score R max = Maximal score of rescaling calculation Score R min = Minimal score of rescaling calculation However, an anomaly was shown in 2014, when most hotspots occurred in February and March.Monthly hotspot data in the period 2013-2015 is shown in Figure 1.Hotspot distribution for Pelalawan District, Riau Province was developed based on the hotspot data from the years 2013 to 2015, as shown in Figure 1.Recurrent fires occurred in some places in Pelalawan District, e.g.Teso Nilo National Park, Teluk Meranti, and Ukuy sub-districts. Figure 2 . Figure 2. Map of fire susceptibility on forest and land in Pelalawan District, Riau Province, Indonesia Table 1 . Hotspot density and score resulting from re-scaling for values of the four variables: soil type, nearest distance to a road, forest function and land-use type Table 3 . The properties of peat soils from different land use types, namely unburnt secondary forest (SF), burnt secondary forest, burnt oil palm plantation, burnt rubber plantation, and burnt crop-lands, in Pelalawan District, Riau, Indonesia	2019-04-03T13:07:56.167Z	2018-03-01T00:00:00.000	{ "year": 2018, "sha1": "b9463b9fc524db74888ecde8dbc7a2f04dc9e437", "oa_license": null, "oa_url": "https://doi.org/10.13057/biodiv/d190224", "oa_status": "GOLD", "pdf_src": "ScienceParseMerged", "pdf_hash": "4a94d29a7eeeae30e510829fab618b2115e6ed9a", "s2fieldsofstudy": [ "Environmental Science", "Geography" ], "extfieldsofstudy": [ "Environmental Science" ] }
222843416	pes2o/s2orc	v3-fos-license	Advances in preclinical hematopoietic stem cell models and possible implications for improving therapeutic transplantation Abstract Hematopoietic stem cell transplantation (HSCT) is a treatment for many malignant, congenital, and acquired hematologic diseases. Some outstanding challenges in the HSCT field include the paucity of immunologically‐matched donors, our inability to effectively expand hematopoeitic stem cells (HSCs) ex vivo, and the high infection risk during engraftment. Scientists are striving to develop protocols to generate, expand, and maintain HSCs ex vivo, however these are not yet ready for clinical application. Given these problems, advancing our understanding of HSC specification, regulation, and differentiation in preclinical models is essential to improve the therapeutic utility of HSCT. In this review, we link biomedical researchers and transplantation clinicians by discussing the potential therapeutic implications of recent fundamental HSC research in model organisms. We consider deficiencies in current HSCT practice, such as problems achieving adequate cell dose for successful and rapid engraftment, immense inflammatory cascade activation after myeloablation, and graft‐vs‐host disease. Furthermore, we discuss recent advances in the field of HSC biology and transplantation made in preclinical models of zebrafish, mouse, and nonhuman primates that could inform emerging practice for clinical application. multipotent cells capable of both self-renewal and differentiation that give rise to transient amplifying progenitors of varying potencies, and ultimately to the diversity of mature lymphoid, myeloid, and erythroid blood lineages. The regenerative capacity of HSCs makes them valuable in treating patients with hematopoietic disorders via HSC transplantation (HSCT) following chemotherapy or radiotherapy. The gold-standard functional definition of an HSC is its ability to reestablish lifelong hematopoiesis upon transplantation into a lethally irradiated recipient. 1 This requires the graft HSCs to home to the niche, engraft and differentiate, while maintaining the "stem- can be used to explore HSC dynamics not only following transplantation but also by providing insight into native, unperturbed hematopoiesis. Herein, we describe HSCT limitations and some of the preclinical approaches being investigated to overcome these roadblocks. Furthermore, we discuss the impact of niche damage and inflammation on hematopoiesis and reconstitution, and the novel methods being employed to diminish toxicity to the niche. Finally, we explore the variety of transplantation and lineage tracing approaches utilized to further our fundamental understanding of HSC biology with an eye toward improving therapeutic HSCT in patients. \| HSCT IN THE CLINIC HSCT is used to treat many malignant, congenital, and acquired diseases, and depending on the indication for transplant, it provides replacement of the hematopoietic system, a graft-vs-leukemia (GVL) effect, or correction of a hematopoietic defect. 2 In high-risk hematologic malignancies such as acute myeloid leukemia, acute lymphoblastic leukemia, or aggressive lymphomas, HSCT is utilized after chemotherapy or radiotherapy to consolidate patient remission and provide a durable cure. 3 HSCT is curative for certain nonmalignant disorders, such as aplastic anemia, heritable hemoglobinopathies, and immunodeficiency syndromes. 4 There are two main types of human transplantation: autologous, in which the patient's own stem cells are harvested in advance and then reinfused post-treatment; and allogeneic, in which a separate individual donates HSCs for infusion. Determining the applicable HSCT type is based largely on the diagnosis. For example, an autologous graft is typically utilized after highdose chemotherapy for solid tumors, multiple myeloma, or lymphoma. 2 The uses for allogeneic HSCT, however, are more varied. Allogeneic HSCT are used in malignant settings for hematopoietic rescue after intensive chemotherapy, providing a GVL effect that can increase the likelihood of leukemia eradication. Healthy donors can also provide a genetic correction or phenotypic enzyme replacement for hereditary conditions like storage and metabolic disorders, immunodeficiencies, and hemoglobinopathies. 2 Donor HSCs are harvested via bone marrow aspiration or apheresis of growth factor-mobilized HSCs in the peripheral blood. Alternatively, umbilical cord blood is collected immediately postnatally. Although cord blood units are small in volume they are rich in HSCs and immunologically naïve T-cells, which allows greater latitude in Human Leukocyte Antigen (HLA) matching between donor and recipient. 5 The cell source chosen depends on multiple patient-and diagnosis-related factors, as well as the availability of appropriately-matched donors. A preconditioning regimen is given in preparation for transplantation to clear endogenous hematopoietic stem and progenitor cells (HSPCs) in the marrow niche thereby creating space for the incoming graft, to suppress the host immune system capability to reject the foreign graft cells, and to clear the disease that the transplant is intended to treat. Preconditioning processes are termed myeloablation and immunoablation, and each can be intensified or reduced based upon the needs of the patient and the underlying indication for transplant. For example, a nonmyeloablative, or reduced intensity conditioning (RIC) regimen may be used when the patient is too ill or frail to tolerate the intensity of standard myeloablative therapy. \| ADDRESSING CURRENT TRANSPLANT LIMITATIONS Challenges remain in the HSCT field despite immense advances in conditioning regimens, immune matching, graft manipulations, and supportive care. A critical issue post-transplant is low-level or delayed engraftment ( Figure 1A). This greatly increases the risk of infection, which remains one of the highest causes of post-HSCT mortality, particularly during the postconditioning phase of neutropenia. 6 Prophylactic antimicrobial agents are commonly administered to high-risk HSCT patients to mitigate the risk of viral, bacterial, or fungal infection; however, such treatments carry risk of their own adverse effects including drug-related complications, antimicrobial resistance and host microbiome disruption. 6 Significance statement This review considers deficiencies in current hematopoietic stem cell transplantation and the fundamental insights from preclinical studies in diverse model organisms including zebrafish, mouse, and nonhuman primates. The effect of cell dose on both engraftment speed and risk of graft rejection has long been known. 7 In preclinical studies, humanized ossicles have been engineered to facilitate targeted investigation of HSC interactions with the niche in healthy and malignant hematopoietic settings. 12 Humanized ossicles are bone marrow-like organoids generated using primary human mesenchymal stem cells (MSCs) that are embedded in scaffolds and implanted into mice. By recreating the HSC niche or microenvironment, researchers aim to not only interrogate niche-HSC interactions, but also to promote in vivo expansion of HSCs for transplantation. The use of stromal cells and/or animal-derived serum and growth factors in preclinical expansion models are inappropriate for clinical applications. To address this, protocols advancing cell purification and ex vivo graft manipulations that enhance HSC replicative potential are in development. For example, culture in defined, serum-free media with a polyvinyl alcohol (PVA) substrate has enabled massive expansion of both mouse and human umbilical cord HSPCs, while maintaining multilineage engraftment capacity upon serial transplantation in mice. 13 In addition, co-culture with endothelial cells derived from primary bone marrow or human umbilical vein endothelial cells (HUVECs) mimics the HSC microenvironment and has been shown to support ex vivo expansion of mouse, nonhuman primate and human HSPCs while maintaining multilineage engraftment and serial repopulation capacity. 10,[14][15][16][17][18] In vivo approaches are also being pursued to improve engraftment or repopulation capacity of available HSCs, for example Tumor Necrosis Factor (TNF)-α inhibition posttransplant, which has been shown in murine models to improve engraftment. 19 However, further optimization is required before any of these techniques can be utilized clinically. Overall, an array of methods to culture and/or support HSC expansion and repopulation capacity are under development in preclinical and clinical models, and translation of these findings could overcome the limitation of low HSC numbers in cord blood grafts. The toxicity of HSCT conditioning regimens also requires improvement. Preconditioning with myeloablative radiation to clear the bone marrow for HSCT significantly disrupts the bone marrow microenvironment, damaging the endothelium and triggering inflammatory signaling cascades ( Figure 1C). HSCs reside in the bone marrow within a perivascular niche composed of a complex milieu of cells, extracellular matrix, substrate tension, and soluble factors. 20,21 It is well-established that bone marrow vasculature functions not only as the circulation conduit, but also actively contributes to niche signaling to regulate HSC self-renewal, differentiation, and regeneration. 22 Sublethal myeloablative irradiation depletes vascular and perivascular cells in the bone marrow via p53 pathway activation. 23 In turn, the stress on bone marrow endothelial cells causes HSC depletion and egress from the bone marrow to the periphery. 23 Myelosuppressive drugs or lethal irradiation in mice induce release of inflammatory cytokines and significant bone loss due to increased osteoclast activity. 24 Consistent with these findings, elevated levels of bone turnover markers persist for 12 months after therapy in human patients following HSCT. 24 Combined, these studies demonstrate that the architecture and signaling of the HSC microenvironment are disrupted following myeloablation, which impairs HSC engraftment and function. Preclinical efforts to decrease the deleterious effects of myeloablative conditioning on the marrow niche include investigations into the potential utility of bone marrow stromal cells (BMSCs). In a murine model, freshly isolated primary CD73 + CD105 − SCA1 + BMSCs were co-transplanted with the stem cell graft and were shown not only to reconstitute stromal function but also to improve HSC reconstitution and abundance after HSCT. 25 As an alternative to the toxic myeloablative radiation, the explosion of RIC regimens and indications where they may be used with success has revolutionized the transplant field, opening up this therapy to patients who were previously considered too elderly, too sick, or with pre-existing organ damage too severe. Unfortunately, RIC is not currently applicable for all patients or conditions, and there is a risk balance when choosing RIC over conventional myeloablation. With improved understanding of the stem cell niche, and more targeted, less-toxic conditioning therapies, HSCT may become safer and more widely available. One unique inflammatory consequence encountered in allogeneic HSCT is GVHD, in which the immune system of the graft sees host tissues as immunologically foreign and mounts an immune attack ( Figure 1D). The inflammatory pathway activation in acute and chronic GVHD lead to poor outcomes for the recipient. 26 Acute GVHD pathogenesis involves the activation of antigen presenting cells and donor T cells by foreign host cell antigens, and results in host tissue injuries. 26,27 In humans, acute GVHD manifests within the first 100 days post-transplant, and varies in intensity. 28 In chronic GVHD, inflammation is mediated by alloreactive CD4 + T cells, B-cell activation, and generalized fibrosis/sclerosis perpetrated by macrophages. 26 Chronic GVHD may result as progression from acute GVHD or arise de novo. 28 Common pro-inflammatory cytokines (ie, TNF-α, interferons (IFNs), interleukin (IL)-6, and IL-1) that are produced in transplantation and contribute to GVHD are known to influence HSC engraftment and function. [29][30][31] Despite advances in understanding the pathophysiological under- 43 Four MPP subsets have been identified and extensively studied, revealing distinct immunophenotypes, lineage bias, and cell cycle status. 44,45 In addition, serial transplantation is useful for defining the self-renewal capacity of HSCs. Studies in animal models have shown progressively reduced output with repeated HSC isolation and transplantation, suggesting a state of HSC exhaustion. 46 This assay has facilitated identification of critical regulators of HSC self-renewal capacity such as reactive oxygen species (ROS) 47 and epigenetic regulators Ezh2 48 and DNMT3A. 47,49 The impact of post-transplant inflammation is not restricted to the niche. Inflammatory signaling also has important consequences on donor-derived HSC function, aging, and lineage output. In murine HSCT, inflammatory signaling increases apoptosis of differentiated cells and HSCs, and alters HSC differentiation. 31,50 Specific to the transplantation procedure, a recent study demonstrated that irradiation damage of murine bone marrow induced ROS accumulation and elevated TNFα levels in transplanted HSCs. 29 This finding is important because TNFα receptors negatively regulate HSC terminal differentiation and expansion. 51 Moreover, HSCT upregulates IFNγ and IL-1, which affects HSC subset differentiation, skewing toward myeloid lineages. 45,50,52 Overall, transplantation-related inflammation and altered niche impairs HSC function and engraftment, highlighting the need to understand the HSC-niche interplay within the healthy, intact setting in order to improve transplant outcome. It is critical to have a comprehensive understanding of normal HSC biology to optimally utilize them in a therapeutic HSCT setting. Building on insights about HSCs from HSCT studies, in vivo genetic lineage tracing systems have been employed in murine, zebrafish, and nonhuman primate models to genetically or fluorescently label HSC clones and their progeny allowing reconstruction of lineage fate maps. 53,54 The earliest clonal marking studies assessed HSC differentiation via the transplantation of retrovirally-transduced HSCs. 55,56 Through this work, they not only found that a small number of HSC clones could reconstitute and maintain hematopoiesis in recipients, but they also provided the foundation for modern gene therapy. Reports tracking the reconstitution of transplanted fluorescentlybarcoded cells support the finding that few HSC clones are needed to reconstitute irradiated organisms. 57,58 However, steady-state lineage tracing analysis demonstrates that many different HSC clones maintain homeostatic blood production, suggesting HSC contribution is polyclonal or heterogenous at baseline. 57,58 Additionally, lineage tracing has supplemented our understanding of lineage-biased HSC subsets which are primed to produce certain cell types over others. Lineage bias identified in HSCT is retained upon serial transplantation, 59,60 but megakaryocyte/platelet-biased HSC subsets in a steady-state context do not maintain their primed state and instead revert to multipotency upon HSCT. 61 Lastly, in the native, 71 From this study, it was computationally determined that HSC self-renewal occurs in the range of 2-20 months and approximately 50,000-200,000 total HSCs actively contribute to hematopoiesis in adult humans. Comparing HSCs by their somatic DNA changes revealed that human hematopoiesis is polyclonal and maintained by cells exhibiting multipotency. 72 As seen in murine, zebrafish, and nonhuman primates models, heterogenous HSC contribution to human hematopoiesis is supported by the lineage tracing studies with evidence of HSC lineage priming toward megakaryocytes 72 or other mature blood cell types. 71 Deep targeted sequencing for somatic mutations is both costly and highly error-prone, but recent studies detecting mtDNA mutations with bulk Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) or singlecell RNA-sequencing are establishing more cost-effective lineage tracing approaches that have similar utility to exogenous lentiviral barcoding. 73,74 Lineage tracing with mtDNA has provided additional evidence for the heterogeneity of the "preleukemic" HSC population that evolve into acute myeloid leukemia. 74 Genomic and mitochondrial mutations are thus providing new ways to investigate human HSC properties in situ and add to our existing knowledge of human hematopoiesis.	2020-10-17T13:06:25.764Z	2020-10-15T00:00:00.000	{ "year": 2020, "sha1": "46cb4b78f82cd292c6eddcedf1f81df5e9592f8e", "oa_license": "CCBY", "oa_url": "https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/sctm.20-0294", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "d2a015ea3f694f9622e7bb684789d819141e4a50", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
245827667	pes2o/s2orc	v3-fos-license	A sinusoidal signal reconstruction method for the inversion of the mel-spectrogram The synthesis of sound via deep learning methods has recently received much attention. Some problems for deep learning approaches to sound synthesis relate to the amount of data needed to specify an audio signal and the necessity of preserving both the long and short time coherence of the synthesised signal. Visual time-frequency representations such as the log-mel-spectrogram have gained in popularity. The log-mel-spectrogram is a perceptually informed representation of audio that greatly compresses the amount of information required for the description of the sound. However, because of this compression, this representation is not directly invertible. Both signal processing and machine learning techniques have previously been applied to the inversion of the log-mel-spectrogram but they both caused audible distortions in the synthesized sounds due to issues of temporal and spectral coherence. In this paper, we outline the application of a sinusoidal model to the inversion of the log-mel-spectrogram for pitched musical instrument sounds outperforming state-of-the-art deep learning methods. The approach could be later used as a general decoding step from spectral to time intervals in neural applications. I. INTRODUCTION Digitised audio signals can be represented in different forms depending on the application and the available resources. Raw audio is a representation of the time domain acoustic signal specified by the sampling rate and bit depth. This form is typically the end goal of sound synthesis algorithms. However, as we perceive sound in terms of time evolving spectral content, time-frequency representations are often appropriate. Such representations include the spectrogram based on short time Fourier transform (STFT), and the log-mel-spectrogram, which is a compressed form of spectrogram modelled after the frequency resolution of the human ear. During the last years, many different approaches have been applied to sound synthesis. Basic signal processing methods, e.g. concatenative synthesis [1] and statistical parametric synthesis [2] were very influential in the field, especially in the area of speech synthesis. More recently, the rise of computational power and deep learning architectures have led to the development of high quality results for sound synthesis. WaveNet [3] is a state-of-the-art vocoder 1 based on dilated convolutional neural networks. WaveNet's high quality depends on synthesis directly in the time domain; every new sample is based on previous ones. Therefore, the system is computationally expensive and it needs a large amount of data in order to be trained. Many deep learning algorithms have employed spectrogram like representations [4] [5] [6] [7] [8]. A major issue with this representation is that it is not directly invertible as it only describes the general spectral distribution over time. In this paper we use the log-mel-spectrogram to generate parameters for a signal processing synthesis model. We implemented a modification of the classic sinusoidal model generating amplitudes that approximate the spectral distribution of the log-mel-spectrogram; the phase calculation takes advantage of the continuous nature of quasi-sinusoidal oscillations over time. Thus, our system overcomes some of the issues of deep learning based inversion, such as that used in Tacotron 2 [9], being a less computationally expensive decoding step and shows the potential of producing higher quality results for the class of sounds under consideration. The paper is organized as follows. In Section 2 we review previous works on sound reconstruction methods, and in Section 3 we describe the sinusoidal model and our modification for the inversion of the log-mel-spectrogram. Results are presented in Section 4, and conclusions in Section 5. II. RELATED WORK In many visual representations of sound, the phase part of a time-frequency representation is discarded. This is generally appropriate for sustained sounds as the ear is not sensitive to absolute phase. To reconstruct a pitched waveform from its given "phaseless" spectrogram, many signal processing-based and machine learning-based methods have been proposed. Signal processing methods for sound generation focus directly on attempting to reconstruct the phase from the spectrogram. A much used solution for phase estimation is the Griffin-Lim algorithm [10] [11]. This method manages to estimate the parameters by minimizing a least square error criterion between the generated and the original spectrum. The algorithm is able to attain impressive results and it is used by many researchers even today [12]. Another significant vocoder for generating sound from a time-frequency spectrogram is STRAIGHT [13]. The key objective of this algorithm is to eliminate the periodicity of a speech signal and then use a sampling operation to generate the parameters. WORLD [14] is designed to reconstruct the sound by its fundamental frequency, the spectral envelope and an aperiodic parameter. Due to its quick processing and its low computational cost, WORLD is able to generate sound with adequate quality in real time. Most of the models lack of sound naturalness, producing a "robotic" effect. Consequently, machine learning has been adopted widely. WaveNet [3] is an autoregressive and probabilistic neural network-based architecture applied on raw audio. Although WaveNet was initially proposed as a stand-alone application, in Tacotron 2 [9], it was conditioned on log-melspectrogram and used as a vocoder. Another implementation of deep learning for the inversion of the log-mel-spectrogram is WaveGlow [4]. This method models multiple invertible transformations to estimate and sample from the distribution of the training data. The generated sound achieved demonstrates the same quality as WaveNet but with less computational cost. To take advantage of the strengths of both methodologies, some contemporary attempts include hybrid models. LPCNet [15] was proposed as an improvement to the WaveRNN [16]. A Linear Prediction Coefficient analysis on Bark-scale cepstral coefficients was applied to generate the training data of a neural network. This way, they managed to model the response of the vocal tract and improve the synthesised sound. Although our approach clearly falls into the signal processing category, our short term plan includes the investment to a more hybrid method, using the method as a general decoding step of a neural architecture; an approach conceptually similar to the recent DDSP [17]. In DDSP, a differentiable autoencoder has been applied for the parameter estimation in a supervised or unsupervised manner. The model manages to synthesise monophonic sounds using the fundamental frequency, the loudness and a latent representation which forms the timbre. The computed parameters are then fed to a harmonic oscillator and a noise filtering component. III. SINUSOIDAL MODEL Although deep learning models demonstrated some results, their demand for a large amount of data as well as their high computational cost lead researchers to prior signal processing or hybrid methods. The sinusoidal model [18] constitutes a signal processing method that provides a parametric approximation of a signal. In the following section, after introducing the mel-scale, we will explain the original sinusoidal model, and then we will apply the effective inversion of the log-melspectrogram. A. The mel-spectrogram In 1937, a group of psychologists conducted a psychoacoustical experiment concerning the perceived distance in pitch between sounds with varying frequency [19]. The outcome was that the human ear has greater resolution at lower frequencies. This observation led to the development of the mel-scale (mel from 'melody'). The conversion of the frequency in hertz (f) to the mel-scale is illustrated in Eq.1. The mel-spectrogram demonstrates a compressed form of sound in the time-frequency domain. This nonlinear transformation constitutes the outcome of the Short Time Fourier Transform (STFT) after the application of mel-filters (a bank of bandpass filters with bandwidths modelled after the melscale). B. Sinusoidal Model from Linear Spectrogram The sinusoidal model [18] models signals as a linear combination of sinusoids, each having independent time varying amplitude and phase trajectories. The modeled signal is given by where θ s (t) = t 0 2πf l (τ ) dτ + φ l is the time evolving phase of the s th sinusoid 2 . The basic steps of analysis include a peak detection of the spectral shape in each frame of the STFT, a parameter estimation using parabolic interpolation, and a peak matching across frames of the SFTF. The synthesis is then the addition of the sinusoids modelled from the time evolving trajectories. C. Sinusoidal Model from Mel-Spectrogram The following method uses the log-mel-spectrogram and an estimate of the fundamental frequency to synthesise the target sound. The log-mel-spectrogram constitutes a non-invertible representation since many frequencies can co-exist in the same mel-band. Furthermore, the mel-spectrogram encodes only the energy distribution over time-frequency of the given sound, therefore, it represents a phaseless representation. In this paper, we propose a sinusoidal model to model a harmonic source, using the log-mel-spectrogram to inform the spectral distribution. While not attempting to estimate the absolute phase of the original signal, this will preserve continuity in the oscillations of the signal. The approach outlined here can be seen as a source/filter approach -shaping a spectrally flat source with a time variant filter. 1) Frequency Estimation: For monophonic harmonic target signals, the sinusoid partials are described as integer multiples of the fundamental frequency [20]. Therefore, the harmonic frequencies can be calculated according to the Eq. 3 where f 1 n is the fundamental frequency of the nth frame, i=[1, f loor fs/2 where f i m is the frequency of the ith sinusoid in the mth frame and f s is the sampling rate. In addition, in order to guarantee no distortions of the synthesised signal based on fluctuations of the fundamental frequency, a 6% tolerance is adopted between adjacent frames. 2) Amplitude Computation: To estimate the amplitude of each sinusoid, the energy of every frame of the log-melspectrogram needs to be preserved. Conversely, having the mel-spectrogram and the information of the mel-filter banks, the amplitude of each sinusoid can be approximated using a source filter model. As this project demonstrates an initial attempt of this idea, a filter equivalent to the rectangular one is applied. Finally, because a single frequency can be present in more than one filter, the amplitude computed is the average of those estimated in overlapping filters. 3) Phase Reconstruction: The phase of each estimated frequency peak is calculated by the phase of the previous frame and the frequencies on both the previous and the current frame interpolated by the hope size as it is expressed in Eq. 4. where θ i n is the phase of the ith sinusoid in the nth frame, T is the hop size multiplied by f s . IV. EXPERIMENTS To evaluate the performance of the modified sinusoidal model, we examined the inversion of the mel-spectrogram for independent pitches of a variety of instruments. The results are compared to a pre-trained WaveNet conditioned on the logmel-spectrogram and to a pre-trained WaveNet autoencoder [21]. The sounds used in this experiment can be found here: https://anastasianat57.github.io/sinusoidalreconstruction/. A. Dataset For the conducted experiments, we used a subsample of the NSynth dataset [22], trying to be as diverse as possible. For each instrument, three different music notes were captured; a low, a medium and a high pitch, in a range of 80Hz to 2100Hz. The sounds could belong in different categories as per their velocity or acoustic quality. B. Description of the experiments The sinusoidal model approach as well as WaveNet are utilised in this paper in order to reconstruct the original music notes from their the log-mel-spectrogram. As we explained before, the models are based on two completely different technologies, therefore the comparison occurs between signal processing and deep learning approaches independently for the conversion of a frequency representation to time domain. The log-mel-spectrogram was created after the raw audio of the original sound with 80 bins, originating from an STFT of 1024 window length, 256 hop size and 1024 FFT size. More details about the parameters of both models are given in the following sections. 1) WaveNet: The sinusoidal model is compared with an already pre-trained WaveNet on speech [23]. These results can be considered as baseline because both the models are conditioned on the same log-mel-spectrogram. 2) WaveNet Autoencoder: The main comparison can be considered between our approach and a WaveNet autoencoder pre-trained on the NSynth dataset [24]. Although the network is not conditioned on the log-mel-spectrogram, the original tests [21] demonstrated that the representation generated from the autoencoder can outperform a baseline using spectrograms. However, we did not consider their baseline as an interesting comparison to our method. 3) Sinusoidal Model: For the sinusoidal model, a normalized blackman window is used to address the expectations of the amplitude computation and the yin algorithm searches for fundamental frequencies between 80Hz and 3000Hz. C. Evaluation After reconstructing the sample from its original representation, the models are evaluated using spectral convergence. For this purpose, the synthesised sounds are aligned with the original music note and the error is calculated by the Eq. 5, where S represents the power spectrogram of the original, S the power spectrogram of the synthesised and n the FFT size of the power spectrogram. Finally, the two aligned sounds are normalized by a total energy calculated using the energy of the original and the energy of the generated sound as it is demonstrated in the Eq. 6. D. Results The results, aggregating them by instrument, are illustrated in Fig. 1. Using the spectral convergence, one can identify that the modified sinusoidal model is more precise than the WaveNet autoencoder in all the cases. We encourage the reader to listen to the samples provided. The musical note synthesised by the sinusoidal model sounds more natural, presenting phase coherence. Another advantage of the modified sinusoidal model over deep learning approaches is the low computational cost. The synthesis of a four second musical note using a pre-trained WaveNet model needs significantly more time than the synthesis of the same sound using the sinusoidal model on an average laptop. In cases where the model needs to be trained again, the memory needed and the computational cost exceed the capabilities of an average laptop. V. CONCLUSION Deep learning architectures have been proven very effective in sound synthesis applications. WaveNet is a state-of-theart signal reconstruction model based on dilated convolutional neural networks and it has been used for the inversion of the log-mel-spectrogram, a non-invertible audio representation. While deep learning models do show promise, they can be difficult to interpret and are computationally inefficient at the final synthesis step. Comparing it with a signal processing method, WaveNet autoencoder presents lack of continuity and phase coherence while the requirements for computational power are severe. In the current paper, we proposed a signal processing method based on a modified sinusoidal model for the inversion of the log-mel-spectrogram. The results seem promising, achieving more natural synthesised musical notes than WaveNet with a significantly lower computational cost. Although the outcome of the sinusoidal model illustrated high quality, the current version of the model is not appropriate for the generation of non harmonic signals. With the suitable extraction of the parameters, this model could be used for any sound. This research work illustrates an early investigation of marrying signal processing with deep learning. An extension of the presented model could be used as the last step of a deep learning architecture for decoding spectrogram-based representations for a more general class of single source sounds. ACKNOWLEDGMENT This work was funded by Science Foundation Ireland through the SFI Centre for Research Training in Machine Learning (18/CRT/6183)	2022-01-10T02:15:26.673Z	2021-11-01T00:00:00.000	{ "year": 2022, "sha1": "6df067f5157ac6686373cbece0a010ba6f11644f", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "6df067f5157ac6686373cbece0a010ba6f11644f", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Computer Science", "Engineering" ] }
58591213	pes2o/s2orc	v3-fos-license	Positive Selection at Key Residues in the HIV Envelope Distinguishes Broad and Strain-Specific Plasma Neutralizing Antibodies Millions of people are still being infected with HIV decades after the first recognition of the virus. Currently, no vaccine is able to elicit bNAbs that will prevent infection by global HIV strains. Several studies have implicated HIV Env diversity in the development of breadth. However, Env evolution in individuals who fail to develop breadth despite mounting potent strain-specific neutralizing responses has not been well defined. Using longitudinal neutralization, epitope mapping, and sequence data from 14 participants, we found that overall measures of viral diversity were similar in all donors. However, the number of positively selected sites within Env epitopes was higher in bNAb participants than in strain-specific donors. We further identified common sites that were positively selected as bNAbs developed. These data indicate that while viral diversity is required for breadth, this should be highly targeted to specific residues to shape the elicitation of bNAbs by vaccination. non-bNAb donors (with Ͻ10% breadth) and 8 bNAb donors (with Ͼ40% breadth) over 3 years of infection (Table 1). Since viral diversity is closely linked to viral load, we selected, where possible, non-bNAb participants with similarly high viral loads to the bNAb donors. Two participants, CAP45 and CAP228, who had consistently low viral loads, were also included, as we have extensively characterized their neutralizing responses (Fig. 1A). We generated between 29 and 171 single-genome-derived env sequences per donor from multiple time points spanning 3 years (Table 1). Maximum likelihood analysis of transmitted/founder viruses from bNAb and non-bNAb donors confirmed that these were phylogenetically unlinked (data not shown). Since dual infection results in increased genetic diversity, we used BEAST to compare the estimated time of infection (tMRCA), based on the sequence diversity, to the actual time of infection for each donor (41,42). For dual infection, sequence analysis predicts a greater estimated duration of infection than the known time of infection; this is indicated by orange shading for CAP256 (a donor known to be superinfected), but not for CAP88, in Fig. 1B. Twelve out of 14 data sets had sufficiently strong temporal signals (as determined by the positive slope [rate] and correlation coefficient) for phylogenetic molecular clock analysis. The two donors that were excluded from this analysis were CAP225 and CAP229. BEAST analysis indicated single infections in 6 donors, CAP88, CAP206, CAP248, CAP255, CAP357, and CAP257 (Fig. 1C, bNAb participants are ranked by breadth), consistent with the fact that most HIV-1 infections are initiated by a single variant (44,45). In contrast, we saw evidence of dual infection (indicated by asterisks in Fig. 1C) for 5 donors, CAP200, CAP228, CAP8, CAP177, and CAP256. Dual infections were observed both in individuals with bNAbs (CAP8, CAP177, and CAP256) and without bNAbs (CAP200 and CAP228). We next compared the levels of overall Env glycoprotein diversity in participants with and without bNAbs at 3 years postinfection (Fig. 1D). We observed the highest overall diversity in CAP228, CAP8, CAP256, and CAP257, with a mean overall diversity Ͼ4% amino acid differences per site at 3 years postinfection (Fig. 1D). With the exception of CAP257, this is likely a result of their infection with multiple viruses or variants (Fig. 1C). Among these donors, CAP228, who failed to develop breadth, had relatively high diversity despite low viral loads (Fig. 1A). However, overall, there was no statistically significant difference in Env diversity between participants with or without bNAbs (P ϭ 0.7758, two-tailed Mann-Whitney test). Similarly, evolutionary rates across the envelope glycoprotein, based on nucleotide substitution rates estimated in BEAST, showed no significant differences between these groups (data not shown). Comparison of Env evolution within antibody-targeted regions between bNAb and non-bNAb donors. We have previously mapped the targets of both strain-specific (Table 1). Although CAP177 developed bNAbs to the C3V4 region, this participant also had a strain-specific V1V2 response (38) and is included as a strain-specific donor for this epitope. The two participants with V1V2-directed bNAbs (CAP256 and CAP257) had higher levels of V1V2 amino acid diversity (20.7% and 14.5%, respectively) than did non-bNAb participants (ranging from 6.9 to 11.9% amino acid differences per site, though this difference failed to reach significance; Fig. 2B). Similarly, the two bNAb participants showed slightly higher V1V2 evolutionary rates than those of non-bNAb donors; however, these differences were also not significant, with estimates within the same high posterior density intervals (Fig. 2C). We similarly compared two donors who developed N332-specific bNAbs (CAP177 and CAP255; dark green in Fig. 2A) with three donors shown to have strain-specific C3V4 responses (CAP88, CAP206, and CAP228; light green in Fig. 2A). CAP206 developed bNAbs that targeted the MPER (4), but in this analysis, we focused on the strain-specific C3V4 response. bNAb donors showed no difference in overall C3V4 diversity and nucleotide substitution rates from those of the strain-specific C3V4 responders ( Fig. 2D and E). Overall, analysis of viral evolution within antibody-targeted regions did not show evidence of unique viral evolutionary pathways for donors with N332-directed bNAbs. Participants with bNAbs show high levels of positive selection within targeted epitopes. We compared levels of positive selection within epitope regions in bNAb and non-bNAb donors by estimating the ratio of nonsynonymous to synonymous substitutions per site (47). We first compared two approaches for assessing selection, cumulative and a snapshot selection analyses. In the cumulative analysis, all sequences at preceding time points were combined, whereas in the snapshot analysis, we analyzed sequence data only from two consecutive time points. The cumulative analysis iden- responses are shown in red, and those with C3V4 responses are shown in green. The dark shading and light shading represent participants with and without bNAbs, respectively. The asterisk (*) denotes participants who have bNAb responses to other regions but also developed strain-specific antibodies to V1V2 or C3V4. Epitope diversity (average number of amino acid differences per site) and nucleotide substitution rates within V1V2 (B and C) and C3V4 (D and E) in participants with/without bNAbs. Broad participants arranged in order of increasing neutralization breadth. subs, substitutions. tified the same positively selected sites as the snapshot analysis, as well as additional sites, and unlike the snapshot analysis, it retained all sequences for analysis (data not shown). All subsequent analyses were therefore done using the cumulative analysis. As expected, the number of positively selected sites increased over time in all participants regardless of whether they developed breadth or not ( Fig. 3A and D). However, participants with V1V2-directed bNAbs exhibited a rapid increase in the number of positively selected sites (peaking at 26 to 27 amino acid residues) in V1V2 within the first year, compared to those without bNAbs (6 to 11 amino acid residues), and this increased level of positive selection persisted over 3 years of infection ( Fig. 3A to C). Similarly, participants with C3V4-directed bNAb responses had an increased number of positively selected sites (peaking at 11 to 12 amino acid residues FIG 3 bNAb participants have more sites under positive selection than non-bNAb individuals. Positive selection was higher in the V1V2 region of participants who developed bNAbs (dark red) than in those with strain-specific neutralizing antibodies (dull red) (A to C). In participants that had C3V4 responses, positive selection was higher in the C3V4 region of bNAb participants (dark green) than in those with strain-specific neutralizing antibodies (dull green) only in the first year of infection (D to F). at 1 year) in C3V4 during the first year of infection compared to that of strain-specific donors (3 to 4 amino acid residues at 1 year) ( Fig. 3D to F), though this difference was less pronounced than that observed in V1V2 and was not sustained in later years. These differences between bNAb and non-bNAb donors were driven largely by the C3 region ( Fig. 3D to F), consistent with the fact that bNAbs to this region are known to be focused on the N332 glycan in C3, whereas strain-specific antibodies also target the more sequence-variable V4 region (39,48). Common sites in the V2, C3, and V3 regions are targeted by bNAbs. We next compared the specific sites under positive selection between participants with and without bNAbs. We focused on the V2 and C3 regions only, as the V1 and V4 regions were highly variable and prone to indels, making accurate identification of specific sites difficult. Using FUBAR, we identified positively selected sites in the V2 and C3 regions in each of the donors and compared their positions between bNAb and non-bNAb donors. In the V2 region, we identified 28 positively selected sites across all donors with V1V2 responses. bNAb donors CAP256 and CAP257 had 13 and 11 positively selected sites in V2, respectively (Fig. 4A). Of these, six sites (160 and 162, which together form the highly conserved N160 glycosylation sequon, 166, 169, 181, 185, and 187) were common in both V2 bNAb participants (CAP256 and CAP257) (highlighted in red in Fig. 4A and B). Four of these sites were also targeted by at least one of the non-bNAb donors, indicating that strain-specific V1V2 antibodies overlapped somewhat with bNAbs. Many of these sites (160/162, 166, 169, and 181) have previously been reported to mediate escape from V1V2-directed bNAbs (17,(49)(50)(51), and all are located proximal to one another at the apex of the envelope trimer (Fig. 4B). However, overall positive selection at these sites, particularly at sites 166 and 181, was less common in donors who developed strain-specific V1V2 responses (Fig. 4C). Similarly, 22 positively selected sites were identified within the C3 region of the 5 participants with C3V4 responses (Fig. 5A). Of these, seven sites (332/334, which form part of mutually exclusive glycosylation sequons, 337, 339, 340, 341, 343, and 344) were common in both participants who developed bNAbs targeting the N332 supersite (highlighted in red in Fig. 5A and B). Unlike the two bNAb participants, donors with strain-specific responses to the C3V4 region did not show evidence of positive selection at residue 332 or 334. In addition to the glycan, residues 341 and 344 were not under positive selection in any of the three strain-specific donors (CAP88, CAP206, and CAP228). Sites 337, 339, 340, and 343, under positive selection in both bNAb donors, were also sometimes under positive selection in participants who failed to develop bNAbs (Fig. 5C). Of note, we identified other common sites (351, 354, 355, 356, and 357) under selection in both CAP177 and CAP206 that clustered together within the C3 region on the envelope trimer (highlighted in yellow in Fig. 5B), suggesting another distinct epitope within this region. bNAbs to the N332 supersite have also been shown to depend on the 324 GDIRQA H 330 motif at the base of the V3 loop (52). We therefore analyzed selection at these sites. Although none reached statistical significance for positive selection, we observed toggling at sites 325 and 330 in both bNAb donors (CAP177 and CAP255) but also in strain-specific donor CAP228. No such toggling was observed in CAP206 and CAP88 (Fig. 5D). Sites 325 and 330 have been reported to impact neutralization sensitivity when these mutations are accompanied by changes at other sites, such as 301, 324, 326, and 327 (52). The similarity of toggling of V3 residues in CAP177, CAP255, and CAP228 might suggest a similar epitope for both broad antibodies and some strainspecific antibodies, raising the question of how they differ in their mode of recognition. Increased positive selection is associated with neutralization breadth. We next assessed whether positive selection at key sites increased with the acquisition of breadth. For this analysis, we used previously published data defining the kinetics of bNAbs (gray shading, Fig. 6) and strain-specific responses to the same epitope (dotted line, Fig. 6) (4,16,17,23,24,33,38). Selected residues are shown compared to the T/F virus, except for CAP256, where the comparison is with the superinfecting virus previously shown to elicit bNAbs in this donor (16). For V1V2 bNAb donors CAP256 and CAP257, we observed positive selection at sites 169/185 and 166/169/181, respectively, prior to the development of breadth, likely driven by the earlier strain-specific responses (Fig. 6A). In CAP256, the positively charged K169 in the superinfecting virus was replaced with a 169Q, which is rare among subtype C viruses but present in the primary infecting virus and confers partial escape from V2 directed antibodies (16). Similarly, in CAP257, a charge-changing minority K169E mutation emerged prior to the development of breadth. In both donors, positive selection expanded to additional sites, including the N160 glycan, with the onset of neutralization breadth. In CAP257, additional mutually exclusive glycans at positions at 185 and 187 were selected for after bNAb development. In C3V4 donors CAP177 and CAP255, strain-specific responses drove positive selection at two to three sites (N332 and 344 for CAP177 and sites N339 and 340 for CAP255) (Fig. 6B). However, the emergence of bNAbs was associated with a significant increase in levels of positive selection. For CAP177, we have previously shown that the T/F does not contain a N332 glycan, and that early strain-specific neutralizing antibodies (nAbs) drive selection of this glycan, which is subsequently lost as bNAbs develop (23). For CAP255, the N332 glycan is under positive selection but is nonetheless largely maintained in most late viral variants. Overall, we observed a significant increase in the number of key sites exhibiting selection pressure with the onset of breadth (Fig. 6), both within the V1V2 and C3V4 bNAb donors. DISCUSSION The study of virus-antibody coevolution in infected donors who develop bNAbs has provided crucial insights into how viral variation drives antibody lineages toward breadth. However, most studies have focused on bNAb donors, making the assumption that specific Env evolutionary patterns are unique to this group. The existence of infected donors who develop strain-specific responses raises the question of whether viral evolutionary pathways in these donors differ from those of the well-described bNAb donors. The purpose of this study was therefore to compare the evolutionary pathways of HIV envelope glycoproteins in bNAb and non-bNAb donors to identify whether there are distinct viral features that are associated with breadth. Moreover, we used previously generated data describing the targets of both strain-specific antibodies and bNAbs to compare viral evolution specifically within epitope regions. We showed that neither overall viral diversity nor local diversity in targeted regions is sufficient for breadth. However, we observed higher numbers of positively selected sites in broad neutralizers and identified several positively selected sites in the V2 and C3 regions that were common to bNAb donors and limited or absent in participants without neutral- ization breadth. Selection pressure at these sites also increased with onset of neutralization breadth, highlighting the role of targeted viral selection in the development of breadth. One of the consistent factors associated with the development of bNAbs in cohort studies is the presence of high levels of viral diversity (5,6,9,14,15). However, in this study, Env diversity at 3 years of infection showed no significant differences between bNAb and non-bNAb donors. While this may appear to contradict findings from previous cohort studies, it is important to note that most non-bNAb donors in this study were selected to have a viral load similar to that of the bNAb donors. As viral load and diversity are linked, this analysis indicates that in donors with similar viral loads, overall diversity does not distinguish bNAb donors from non-bNAb donors, and it suggests that additional factors influence breadth. The lack of difference in overall Env diversity between the two groups is consistent with several studies showing that bNAbs often represent only a small proportion of the overall neutralizing responses in an infected donor (53; see also D. Kitchin and P. L. Moore, unpublished data). Although most of the strain-specific neutralizing antibodies that comprise the overall neutralizing response fail to acquire breadth (as is evident from the low proportion of bNAb donors in infected cohorts), they nonetheless drive substantial diversity through viral escape. Indeed, there appears to be no clear association between overall autologous neutralization titers and breadth (4,54,55). Superinfection has been reported to be important in the development of breadth, although the effect is small (16,56,57). Our study included two donors where the tMRCA and actual time of infection differed significantly, suggesting (CAP8) or confirming (CAP256) superinfection. Both donors developed bNAbs, and CAP256 has been intensively studied over many years (16,17,46,58). However, in CAP256, we have recently demonstrated that superinfection played no significant role in driving breadth (55). Furthermore, within the CAPRISA cohorts, we see no association between superinfection and breadth (55). Many studies defining viral escape from bNAbs have confirmed that despite the conserved nature of these epitopes, HIV can utilize multiple pathways to escape (23,24,59). For this reason, bNAbs fail to confer any clinical benefit to those donors in whom they develop (4). In donors who develop breadth, extensive toggling with epitopes has been shown to precede the development of breadth (17,18). Our observation of higher numbers of positively selected sites in bNAb donors, particularly within the first year of infection, supports the notion that this early but targeted viral toggling is required for the development of breadth, and it differentiates bNAb donors from those who remain strain specific. The underlying reason for a higher number of positively selected sites in bNAb donors is of interest for HIV vaccine design. This is unlikely to be a consequence of higher titers, as many of the strain-specific responses we have previously mapped have equivalently high titers to bNAb responses (38,60). The targeting of intrinsically more conserved epitopes may result in fitness costs for some escape mutations and, therefore, higher levels of compensatory mutations at proximal sites (61). Alternatively, it is possible that antibodies with the potential to mature toward breadth bind the virus using a wider "footprint," i.e., they have more contact sites and therefore drive early escape mutations at more sites. The finding that mutations at many of these positively selected sites, when introduced into heterologous viruses, confer neutralization escape from bNAbs may support the latter possibility (17,18,49,52,(62)(63)(64). The identification of sites common to bNAb donors and their clustered location on the trimeric Env may emphasize the importance of accurate early targeting by bNAbs. Studies of the ontogeny of V1V2-directed bNAb donors have confirmed the need for precursors encoding long third complementarity-determining regions to penetrate the glycans shielding the V2 C-strand (where many of the positively selected sites we identify here are located). The tight clustering of positively selected sites for C3V4 bNAb donors in this study may suggest that initial targeting is important here, too, though mature members of V3-directed bNAb lineages show much more promiscuity in their angles of approach as they mature to accommodate sequence variation and glycosylation (65)(66)(67)(68)(69)(70)(71). We selected 14 participants for the in-depth epitope study because of the availability of both sequence and mapping data. Furthermore, both V1V2-and N332-directed bNAbs are among the most common bNAb specificities elicited during infection (4,6,54,72,73) and may require relatively less somatic hypermutation to acquire breadth (16,20,74). This makes these bNAb specificities attractive vaccine targets. However, this study could be improved by performing similar analyses on additional participants with/without bNAbs, including other less commonly elicited bNAb specificities. Since the number of participants in this study was relatively small, larger cohorts and inclusion of donors infected by other viral subtypes would be valuable in the future. We also utilized single-genome Sanger sequencing data with limited depth, whereas sequences generated through next-generation sequencing would give a more accurate representation of viral diversity. Nonetheless, this study has highlighted key differences between the evolutionary pathways of HIV env glycoproteins in participants with and without bNAbs. Furthermore, it has clarified that amino acid diversity at key positions within the V1V2 and C3V4 epitopes is more important than overall env diversity. The association of shared positively selected sites with the onset of breadth highlights the importance of diversity at these positions in bNAb development. Designing immunogens against HIV that include targeted diversity within these common sites may thus be critical in the maturation of V1V2-and C3V4-directed bNAbs. Evidence of HIV infection in participants was determined using two rapid HIV-1 antibody tests (Determine, Abbott 89 Laboratories, Tokyo, Japan; and Capillus, Trinity Biotech, Jamestown, NY). False positives and negatives were eliminated by using PCR for the negative samples (Ampliscreen version 1.5; Roche Diagnostics, Rotkreuz, Switzerland) and an HIV enzyme immunoassay (EIA; BEP2000; Dade Behring, Marburg, Germany) for positive samples. Viral loads were estimated using the Cobas Amplicor HIV-1 Monitor test version 1.5 (Roche Diagnostics). The time of infection was estimated as either the midpoint between the last antibody-negative visit and first antibody-positive visit, or 14 days prior to an RNApositive antibody-negative sample. Written informed consent to conduct research on stored specimens was obtained from all women at enrollment, and ethics clearance for the use of plasma samples was obtained from the Human Research Ethics Committee (Medical) from the University of Witwatersrand (MM040202), the University of Cape Town (025/2004), and CAPRISA at the University of KwaZulu-Natal (E013/04). Single-genome envelope amplification and sequencing. HIV-1 RNA was isolated from plasma samples, and full-length envelope genes were amplified from single genomes at multiple time points by nested PCR and sequenced using Sanger sequencing, as described previously (77). Sequence alignments and data partitioning. Sequence alignments were made in MUSCLE (78) and edited manually in BioEdit (79). The longitudinal envelope (env) glycoprotein sequences were analyzed as a whole and subsequently partitioned into variable and constant regions for subsequent analyses using the Se-Al software (80). The partitions were defined according to the HXB2 reference sequence as follows: V1, amino acids 131 to 156; V2, amino acids 157 to 196; V3, amino acids 296 to 330; C3, amino acids 329 to 383; and V4, amino acids 385 to 418. Estimating diversity, nucleotide substitution rates, and time of infection. Overall Env, C3V4, and V1V2 diversity was calculated using the Poisson correction distance model implemented in MEGA6 (81,82). The model assumes equality of substitution rates among sites and equal amino acid frequencies, while correcting for multiple substitutions at the same site (81,82). The presence of temporal signal was estimated in TempEst using maximum likelihood trees constructed with PhyML (83,84). Phylogenetic analyses were performed using BEAST version 1.8.4 (85). Best-fit nucleotide substitution models were selected based on the Bayesian information criteria (BIC) and Akaike information criterion, corrected (AICc) values estimated using the MEGA 6 software (81). The best-fit demographic and clock models were selected with marginal likelihood estimations using the generalized path sampling and stepping stone methods with 50 path steps (86,87). The Monte Carlo Markov chains were run until convergence (effective sample sizes, above 200), and the chain lengths were between 50 and 500 million steps. The log files generated were analyzed in Tracer version 1.5, and the maximum clade credibility (MCC) tree annotated in TreeAnnotator and viewed in FigTree (88).	2019-01-22T22:20:09.046Z	2018-12-19T00:00:00.000	{ "year": 2018, "sha1": "f24a8eb3b9a954b3e861deae0a50c3488efe3593", "oa_license": "CCBY", "oa_url": "https://jvi.asm.org/content/jvi/93/6/e01685-18.full.pdf", "oa_status": "HYBRID", "pdf_src": "PubMedCentral", "pdf_hash": "f24a8eb3b9a954b3e861deae0a50c3488efe3593", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Medicine", "Biology" ] }
87033528	pes2o/s2orc	v3-fos-license	Effect of Different Processing Methods on Anti-Nutrients Content and Protein Quality of Improved Lupin ( Lupinus Albus L.) Cultivar Seeds Lupin seeds of genetically improved cultivar (Golo) were subjected to different processing methods and investigated according to anti-nutritional factors content and protein quality. Results showed that tannin content of raw seeds was significantly increased in sprouted and debittered seeds before and after boiling but in fermented seeds it declined significantly. Phytate content was significantly decreased in all processed seeds with a significant reduction observed in germinated seeds. The reduction in Phytate as a result of processing was accompanied by a significant improvement in protein digestibility. The protein content of lupin seeds decreased in sprouted seeds and increased in fermented and debittered ones. Boiling of the seeds even the sprouted ones significantly increased the protein content compared to raw lupin seeds. In raw lupin seeds, globulins comprised the major fraction followed by glutelin. Debittered seeds characterized by high glutelin, fermented are characterized by high globulin while germinated characterized by both fractions. Most of the amino acids level was increased after processing of the seeds. Introduction Interest in lupin production is increasing due to its potential as a source of protein, pharmaceutical purposes, green manure and as a natural component of plant pesticides due to the high alkaloid content (James et al., 2004;Lampart-Szczapa et al., 2003). Lupin is a legume rich in protein, essential amino acids, dietary lipids, fiber, minerals and vitamins (KohaJdoVá et al., 2011). Lupin is considered as an excellent source of protein for both human and animal nutrition (Martınez-Villaluenga et al., 2007) that could be used instead of soybean in countries that do not produce soybean. Lupin seed protein is an excellent source of lysine but poor in the sulfurcontaining amino acids (El-Adawy et al., 2001). According to Osborne fractionation, lupin proteins can be divided into water-soluble albumins, salt-soluble globulins, alcohol-soluble prolamins and acid/alkalisoluble glutelins (Mandal and Mandal, 2000). Globulins are the major storage proteins of lupin seeds while prolamines and glutelins are detected in small amounts (Mandal and Mandal, 2000). The production of flatulence from the α-galactoside oligosaccharides, and the presence of antinutrients, such as tannins are the major reasons that preclude a greater inclusion of lupin and cowpea in human or animal diets (Ibrahim et al., 2002). Processing methods, such as soaking, germination, fermentation, boiling and addition of enzymes has been reported by many researchers to reduce the amount of antinutritional factors and to improve the nutritional value of cereals and legume seeds (Frías et al., 2000;Idris et al. 2007;Mohiedeen et al., 2010, Mohamed Nour et al., 2010Osman et al., 2010;Sokrab et al., 2012). Germination of seed legumes is practiced in many countries and reported to be associated with improvements in the nutritive value of seeds and their quality (Mohamed Nour et al., 2010;Sokrab et al., 2012). It has also been reported that germination and fermentation are effective methods of reducing polyphenols, phytic acid and thereby increasing the protein digestibility and improving sensory properties. On the other hand, fermentation is a common practice in African and Southeast Asian countries, is believed to enhance the nutritional quality of foods in addition to increasing shelf life. Breakdown of food components by microbial enzymes may explain the enrichment for such properties during fermentation (Kim et al., 2004;Wong and Mine, 2004). Fermentation, however, offers potential for extensive applications, particularly with respect to the preservation of legumes, cereals, and root crops and the provision of safe foods for developing countries (Deshpande et al., 2000). In Sudan, lupin seeds are processed in several ways including boiling in water in the presence or absence of table salt, germination followed by boiling or fermentation for a short period of time (2 days) followed by boiling. The main aim of this study was to evaluate the effect of different processing methods on antinutrients contents and protein quality of lupin seeds. Materials Lupin seeds (Lupinus albus L.) of cultivar Golo (white lupin) which was improved by conventional breeding were obtained from Crop Research Station, Shambat, Sudan. The seeds were carefully cleaned and freed from foreign materials and stored in polyethylene bags at 4 o C. Chemicals used in the present study were of reagent grade. Processing Methods Home debittering process: The lupin seeds were steeped overnight in water containing 0.5% NaCl. Then, soaking water was decanted, and the seeds were boiled in fresh water until the color changed from white to light yellow (3-5 h). Boiling of the seeds was repeated, and the seeds were steeped twice as above mentioned. Then the boiled and untreated (control) seeds were freeze-dried (Viritis Unitop 600SL, New York), milled (KENWOOD Model CG510, China) to pass a 0.25 mm sieve and kept in polyethylene bags at 4 °C for further analysis. Germination process: Germination was carried out in an incubator at 20°C for 6 days according to Mohamed Nour et al. (2010). Samples were periodically taken at 2, 4 and 6 days intervals. Part of the sprouted seeds was boiled in water for 30 minutes. Both raw and boiled samples were freeze-dried, milled to pass a 0.25 mm sieve and kept in polyethylene bags at 4°C for further analysis. Fermentation process: About 100 grams of lupin seeds were washed and suspended in sterile distilled water (333 mL) using a 500 mL Erlenmeyer flask protected from the daylight with aluminum foil. The suspension was left to ferment naturally in an orbital shaker incubator (Unitrin, Infors AG, Switzerland) at 220 rpm for 2 days at 37 °C. Part of the fermented seeds was boiled in water for 30 minutes. Then both raw and boiled fermented seeds were freeze-dried and milled and then stored at 4°C for further analysis. Analytical Methods Tannin determination: The modified vanillin-HCl method was used for tannin determination as described by Price et al. (1978). A 200 mg sample was extracted with 10 mL of 1% HCl in methanol (v/v) for 20 min in capped rotating test tubes. Then, 5 mL of 0.5% Vanillin reagent was added to 1 mL of the extract. Thereafter, the mixture was incubated at 30°C for 20 min and then the absorbance was read at 500 nm. A standard curve was prepared to express the results as catechin equivalents, i.e., amount of catechin (mg per mL) that gives a color intensity equal to that given by tannins after correcting for blank. Phytate determination: The phytic acid content was determined as described by Wheeler and Ferrel (1971) using 2.0 g of dried sample. A standard curve of various concentrations of Fe(NO 3 ) 2 was plotted to calculate the ferric ion concentration. The phytate phosphorus was calculated from the standard curve assuming 4:6 iron to phosphorus molar ratio. In vitro protein digestibility determination: The in vitro digestibility of the protein was determined as described by Monjula and John (1991). Triplicate samples with a known weight containing 16 mg nitrogen were digested with pepsin (1 mg) in 15 mL of 0.1 M HCl at 37 o C for 3 h. The reaction was terminated by the addition of 15 mL of 10% trichloroacetic acid (TCA). Then, the mixture was filtered through Whatman No. 1 filter paper. The TCA soluble fraction was assayed for nitrogen using the micro-Kjeldahl's method. Digestibility was calculated by using the following equation: PD: Protein digestibility Determination of protein content and fractionation: The protein content was determined according to AOAC method (1990). For protein fractionation, the stepwise extraction of nitrogen using various solvents was applied according to the method of Landry and Moureaux (1970). Firstly, the salt-soluble globulin was obtained by adding 0.5 M NaCl to the sample, and the mixture was then stirred three times for 0.5, 1, and 30 min at 4°C. Secondly, the water-soluble albumin was obtained by twice extraction of the residue with an equal volume of distilled water for 15 min at 4°C. Thirdly, the alcoholsoluble prolamin was obtained by twice extraction of the residue with 60% ethanol for 30 min at 20°C and then at 60°C for 30 min, followed by extraction with 55% isopropanol (Pr-OH) at 20°C. Fourthly, G1-glutelin was extracted from the residue using 60% ethanol plus 0.6% of 2-mercaptoethanol (2-ME) and stirred twice for 30 min (20°C), then extracted with 55% Pr-OH containing 2-ME (0.6%) at 20°C (twice) for 30 min. Lastly, G2and G3glutelins were achieved following treatment of the remaining residue with 0.6% 2-ME and 0.5 M NaCl in 0.0125 M borate buffer (pH 10), and with 0.6% 2-ME and 0.5 M sodium dodecyl sulfate (SDS) in 0.0125 M borate buffer (pH 10), respectively. The nitrogen of each of these fractions was determined by the micro-Kjeldahl method. Amino acids composition: The amino acids composition was estimated according to the standard methods using high-performance liquid chromatography (HPLC) (Sykam system Model S7130, Sykam GmbH, Eresing, Germany). About 200 mg of the sample was taken in a hydrolysis tube, and 5 ml of 6 N HCl was added. The tube was tightly closed and incubated at 110 o C for 24 h. After incubation the solution was filtered, and 200 ml of the filtrate was evaporated at 140 o C for 1 h. The hydrolysate after drying was diluted with 1.0 ml of 0.12 N citrate buffer (pH 2.2). Aliquot (150 µl) of the sample hydrolysates was injected into an action separation column at 130 o C. Elution buffers (solvent A, pH 3.45 and solvent B, pH 10.85) and ninhydrin solution were simultaneously delivered to a high-temperature reactor coil (16 m) at 0.7 ml/min flow rate. The buffer/ninhydrin mixture was heated in the reactor at 130 o C for 2 min, and then the formed products were detected at wavelengths of 570 and 440 nm on a dual channel photometer. The amino acids composition was calculated from the area under the curve of the standards and expressed as mg/100gm. The sulphur-containing amino acids were oxidized using performic acid before the acid hydrolysis. Statistical Analysis Each determination was carried out on three separate samples and then triplicates samples were analyzed and figures were then averaged. Data were measured by the analysis of variance (ANOVA) (Snedecor and Cochran, 1987) while mean values were compared by Duncan's test (P≤0.05). Figures 1 and 2 show the effect of different processing methods on anti-nutritional factors and in vitro protein digestibility (IVPD) of lupin seeds, respectively. Tannin content of raw lupin seeds was 100.02 mg/100gm. Boiling of lupin seeds significantly (P≤0.05) increased tannin content. Germination of the seeds alone significantly (P≤0.05) increased tannin content, and further significant increment in tannin was observed when germination was accompanied by boiling process, with a maximum value (134.56 mg/100g) obtained for 6 days germinated and boiled seeds. This could be a result of solubilization of tannin when the seeds were soaked in water and migration of tannin to the outer layer as a result of germination, as indicated by the browning of the germinated seeds (Ahmed et al., 1996). However, fermentation alone and when accompanied by boiling significantly (P≤0.05) decreased the level of tannin of lupin seeds. The reduction in the degree of tannin during fermentation may be attributed to the action of the enzyme tannase released during fermentation. Moreover, boiling significantly (P≤0.05) increased tannin content of fermented seeds but still lower than that of lupin seeds tannin. Debittering significantly (P≤0.05) increased the level of tannin of lupin seeds. This could be due to the fact that boiling of the seeds in the presence of salt may solubilize the tannin of the seeds as well as the migration of the soluble tannin to the inner layers of the seeds. The phytate content of lupin seeds was 280.53 mg/100g. The seeds are rich in protein (38.52%). Therefore, they had high phytate levels. It has been reported that in legumes, phytates are associated with protein bodies (Sulieman et al., 2007) and therefore, phytate levels should increase with increasing protein content. All processing methods significantly reduced (P≤0.05) phytate content of lupin seeds and further reduction was observed when the process was accompanied by boiling. Ordinary boiling of lupin seeds brought about a significant decrease in phytic acid content when compared to raw. The loss of phytic acid during boiling could probably be explained on the basis that phytase activity at a temperature of 40-55 o C may degrade inositol hexaphosphate to the pentaphosphate or lower molecular weight forms (ElMaki et al., 2007). Further, they observed that phytic acid content decreased while boiling because insoluble complexes between phytate and other components were formed and, accordingly, the amount of free phytate was reduced (ElMaki et al., 2007). The loss in phytate during debittering might be due to discharge of phytate ions into the soaking water under the effect of a concentration gradient, which governs the rate of diffusion. Moreover, low level of phytate in germinated and fermented seeds is likely due to the action of the enzyme phytase on phytate. Yorgancilar and Bilgiçli (2014) reported that bulgur processing of bitter and sweet lupin seeds (Lupinus albus L.) caused a significant reduction (P≤0.05) in phytic acid content. The IVPD of lupin seeds was 68.26%. Boiling of lupin seeds significantly (P≤0.05) increased the IVPD of the seeds (Fig. 2). Each of sprouting, fermentation and debittering significantly (P≤0.05) improved the IVPD of the seeds and further improvement in IVPD was observed after boiling of sprouted, fermented and debittered seeds. Boiling of the seeds had been reported to reduce the IVPD but when preceded by germination or fermentation the effect of boiling was alleviated. Germination, among other technological processes, has been widely used for its ability to decrease the level of antinutritional factors existing in legume seeds in the meanwhile improving the concentration and bioavailability of their nutrients (Urbano et al., 2005). The comprehensive degradation of seed storage proteins that occurs during this process improves the protein digestibility and the essential amino acid content, consequently enhancing the nutritional value of legumes (Kuo et al., 2004). It has been reported that fermentation and boiling of Sicklepod-fermented flour significantly (P≤0.05) increased the protein digestibility compared to that of the raw sample (Osman et al., 2010). However, contradictory results showed that the protein digestibility of fermented Uji flour (Onyango et al., 2004) and maize (Yousif and El Tinay, 2000) was decreased after boiling. The decline in IVPD after boiling was possible because of the formation of protein-antinutrient complexes which are resistant to pepsin attack as explained by Onyango et al. (2004) or may be as a result of the formation of cross-linkages within and between protein chains such as lysinoalanine and lanthionine, and partly because of the loss of certain attacking sites of the digestive enzyme and thus affected the protein quality. Effect of Processing Methods on Antinutrients and Protein Digestibility of Lupin Seeds However, in the present study the improvement in IVPD of sprouted, fermented or debittered lupin seeds before and after boiling may be attributed not only to the removal/reduction of antinutrients Mohiedeen et al., 2010, Mohamed Nour et al., 2010Osman et al., 2010;Sokrab et al., 2012), but also to the structural disintegration of the native protein, including enzyme inhibitors and lectins, phytase activity to break down phytic acid (Sulieman et al., 2007) in the seeds. Moreover, the study revealed that the phytate had a stronger impact on IVPD than tannin as indicated by the direct relation between tannins and IVPD (as the tannins increases, the rate of digestion increases) and reverse relation between phytate and IVPD. Table 1 shows the effect of different processing methods on protein content and Osborne fractions of raw and processed lupin seeds. The protein content (38.52%) of lupin cultivar examined was the major nutrient exceeding the values obtained for carbohydrates (37.62%). The results obtained confirm that lupin seeds are protein-rich and agree with the data reported by many researchers for different lupin seeds (Fernandez and Batterham, 1995;Martınez-Villaluenga et al., 2007). Boiling of lupin seeds significantly (P≤0.05) increased the protein to 42.12%, but germination alone slightly decreased the protein content of the seeds. However, boiling of germinated seeds significantly (P≤0.05) increased the protein content. Fermentation before and after boiling and debittering process significantly (P≤0.05) increased the protein content of lupin seeds. The increment in protein content after boiling of germinated and fermented seeds could be attributed to solubilization and concentration of the protein. Moreover, the extensive breakdown of seed-storage proteins that takes place during this process enhanced the nutritional value of legumes (Kuo et al., 2004). The predominant fraction of lupin seed before and after boiling was globulins, which was 45% and 57.24%, respectively of the total protein followed by albumin, prolamin, glutelin and the insoluble protein fractions. Boiling of lupin seeds significantly (P≤0.05) increased all protein fractions due to an increase in protein content of the seeds. Most of the data in the literature coincide with high values for the globulin fraction in legume seeds (Duranti et al., 1990;Gulewicz et al., 2008). Each of germination and fermentation before and after boiling significantly (P≤0.05) reduced the nitrogen content of albumin fraction. The reduction in the nitrogen content of albumin, with significant amounts, continued during the germination period prolonged, reaching a minimum value of 11.14% at the end of germination period and further reduction was observed after boiling (9.35%). The reduction in albumin is likely to be due to the fact that albumin is a water soluble protein and may be extracted in soaking water prior and after germination and fermentation. A decrease in the content of albumin and globulin fractions after germination was reported by Gulewicz et al. (2008) in some lupin cultivars. Debittering of the seeds significantly (P≤0.05) decreased the level of all fractions except glutelin that was significant (P≤0.05) increased to 34.94% compared to that of raw lupin seeds (15.50%). Fermentation alone resulted in a slight rise in the level of prolamin (5.74%) but decreased significantly (P ≤0.05) to 2.88% after boiling. Glutelin content was significantly (P≤0.05) increased for all processing methods with higher value observed after boiling of raw seeds (51.90%). Insoluble protein fraction was significantly (P≤0.05) increased after germination and natural sweetening but significantly (P≤0.05) decreased after fermentation. However, boiling of fermented seeds significantly (P≤0.05) increased the level of insoluble protein. This variation in insoluble protein between processing methods is likely to be attributed to the fact that during fermentation the enzymes of the fermenting media may act on the protein fraction and lower its level. Mohiedeen et al. (2010) proved the effect of heat treatment on the solubility of the protein fractions. The observed variations in the globulin fractions after germination, fermentation, and boiling are the consequences of the changes in the molecular mass of different proteins. The dissociation, denaturation or hydrolysis of the proteins may modify their solubility, therefore; they are extracted in other conditions (Yagoub et al., 2004). Chilomer et al. (2010) reported an increase in the residual protein fraction after germination of lupin seeds. Table 2 shows the amino acids composition of raw and processed lupin seeds. Glutamic acid, arginine, leucine and aspartic acid are the major amino acids in lupin seed with values of 4.64, 2.99, 2.15 and 2.08 mg/100gm protein, respectively. Relative to the FAO reference protein pattern (FAO/WHO, 1991), methionine was the limiting amino acid. Lupin seed was found to have low sulfur-containing amino acids as reported by Martinez-Villaluenga et al. (2007) and Guewicz et al. (2008). Lysine content of the seed was 1.14 mg/100gm protein, which is significantly (P≤0.05) lower than that of the FAO reference protein. Lone et al. (2003) reported higher lysine content in lupin seed than the value of the present study. Other essential amino acids (Histidine, valine, isoleucine, leucine and phenylalanine plus tyrosine) are comparable to the reference protein (FAO/WHO, 1991). Non-essential amino acids such as serine, glycine, alanine, and proline are found to be 0.50, 0.76, 1.31 and 1.43 mg/100gm protein, respectively. The amino acids composition was changed with varied patterns during processing. Boiling of lupin seeds and debittering increased the level of most amino acids. Germinated and fermented seeds before and after boiling had a variable impact on amino acids composition but most of the amino acids content was increased especially after boiling of the treated seeds. Mubarak (2005) reported a decrease in some essential amino acids after germination of mung bean seeds for 3 days. Transamination and deamination reactions that may occur during fermentation could be responsible for the changes observed in the amino acids profile of the seeds. Boiling in water had been reported to increase the essential amino acids in some legume seeds (Khattab et al., 2009). Yorgancilar and Bilgiçli (2014) declared that bulgur processing of bitter and sweet lupin seeds (Lupinus albus L.) caused a significant increase (P≤0.05) in protein and amino acid content. The results obtained showed that processing of lupin seeds improved the level of some amino acids such as lysine. Conclusion In conclusion, the present study revealed that Golo cultivar had a considerable amount of protein and EAA. The seeds contained an appreciable amount of antinutritional factors (tannin and phytate). However, processing of the seeds alleviate the effect of such factors and improve the protein quality.	2019-03-31T13:41:40.560Z	2016-01-19T00:00:00.000	{ "year": 2016, "sha1": "52313e3c299aec9f3002173be276a3165ac1b02d", "oa_license": "CCBYNC", "oa_url": "http://www.agrifoodscience.com/index.php/TURJAF/article/download/404/244", "oa_status": "GOLD", "pdf_src": "Adhoc", "pdf_hash": "74f52b83673716de92796a20b3a1401f73861671", "s2fieldsofstudy": [ "Agricultural And Food Sciences" ], "extfieldsofstudy": [ "Chemistry" ] }
220405809	pes2o/s2orc	v3-fos-license	Reference interval and the role of soluble suppression of tumorigenicity 2 (sST2) in subclinical cardiac dysfunction at health checkups Abstract Background Soluble ST2 (sST2) is known to predict adverse outcomes and death in individuals with established heart failure. However, the role of sST2 testing in the general population has not been established. The aims of this study were to determine the reference interval (RI) and the clinical utility of sST2 in subclinical cardiac dysfunction in general population. Methods This cross‐sectional study consecutively selected 41,806 general subjects at health checkups who underwent echocardiography and sST2 testing at 16 health promotion centers in 13 Korean cities. The reference subjects were obtained among those with normal findings in echocardiography. Sex‐specific RIs were established according to the CLSI C28‐A3 guidelines. sST2 was measured using immunoassay with the Presage ST2 assay (Critical Diagnostics). Results In the general subjects, age, sex, BMI, systolic blood pressure, blood glucose, creatinine, liver function, and triglycerides were associated with the sST2 levels. The RI for sST2 was higher in males (≤49.6 ng/mL, 95% CI = 48.5‐51.5) than in females (≤44.5 ng/mL, 95% CI = 43.5‐45.6) and higher in subjects aged < 40 years than ≥ 40 years in both sexes. The sST2 levels were 29.1 ± 10.7 (mean ± SD) and 29.1 ± 14.4 ng/mL in the groups with normal cardiac function and subclinical cardiac dysfunction, respectively. The sST2 level was not associated with subclinical cardiac dysfunction (odd ratio = 1.002, P = .13). Conclusions RIs obtained from a large and echocardiography‐proven healthy community‐based sample are presented. Subclinical cardiac dysfunction was associated with older age, male sex, and metabolic factors but not with the sST2 level. \| INTRODUC TI ON The wide range of cardiovascular disorders that result in an impaired ability of the heart to fill or to pump blood may eventually lead to the clinical syndrome of heart failure (HF). The incidence of cardiovascular diseases is increasing (including in younger subjects) due to changes in lifestyles and dietary patterns, and there have also been increases in the rates of progression to HF. 1 Patients with HF often present with non-specific signs and symptoms. Moreover, systolic dysfunction is frequently present in community-dwelling individuals without recognized symptoms of HF. 2 Accordingly, biomarkers for identifying the presence of HF before it is fully developed are needed in community-dwelling individuals. Suppression of tumorigenicity 2 (ST2) is the receptor for interleukin-33 (IL-33), which is an IL-1-like cytokine that is secreted by cardiac cells in response to myocardial stress. ST2 has two main isoforms: (a) transmembrane or cellular (ST2L) and (b) soluble or circulating (sST2). Interactions between IL-33 and ST2L are cardioprotective since they reduce myocardial fibrosis, cardiomyocyte hypertrophy, and apoptosis. However, when the soluble receptor is shed in cases of cardiac distress, sST2 binds to IL-33 in competition with ST2L, blocking the IL-33/ST2L system and eliminating the cardioprotective effects. Therefore, sST2 is considered a decoy receptor. [3][4][5] Increased sST2 levels are clinically predictive of adverse outcomes in acute myocardial infarction, 6 acute decompensated HF, 7 and chronic HF. 8 The sST2 level has an impact in the prognosis and risk stratification of patients with established HF. However, the role of sST2 testing in the general population without apparent cardiac symptoms has not been established. Meanwhile, the sST2 level is also increased in several non-cardiac conditions such as asthma, pulmonary fibrosis, rheumatoid arthritis, collagen vascular disease, sepsis, trauma, malignancy, and helminthic infections. 9 The aims of this study were to determine (a) the factors associated with sST2 and the reference interval (RI) in echocardiography-proven healthy reference subjects and (b) the utility of sST2 in preventive strategies at a population level through screening the sST2 level at health checkups. \| MATERIAL S AND ME THODS This study was approved by the Institutional Review Board of the Korea Association of Health Promotion (approval no. 130750-202005-HR-008). \| Study subjects This cross-sectional retrospective study consecutively selected subjects at health checkups who underwent echocardiography and sST2 testing at 16 health promotion centers in Korea between January 2018 and September 2019. The self-reported personal medical history, subjective symptoms and signs, and lifestyle information were obtained from all participants at time of health checkups. Their medical records were also reviewed. Individuals with non-cardiac conditions such as asthma, pulmonary fibrosis, rheumatoid arthritis, collagen vascular disease, sepsis, trauma, and malignancy were excluded through evaluation of medical records and personal medical history. Subjects who had echocardiography-detected HF, atrial fibrillation, or acute myocardial infarct or who were younger than 19 years were not eligible for inclusion. The general subjects comprised 41,806 individuals. Echocarciography-normal individuals defined as preserved left ventricular systolic function (LVEF > 50%) and those who do not have any abnormal findings in echocardiography, such as valvular insufficiency, diastolic dysfunction, atrial fibrillation, heart failure, pulmonary hypertension, or atrial enlargement. The 7090 reference subjects had normal findings in echocardiography and did not have diabetes, hypertension, obesity (body mass index [BMI] > 25 kg/m 2 ), renal disease (eGFR < 60 mL/min/1.73 m 2 or creatinine > 1.4 mg/dL), or hepatic dysfunction. Subclinical cardiac dysfunction in the general subjects was defined as any abnormal findings in echocardiography, such as a mild-to-moderate degree of valvular insufficiency, diastolic dysfunction, or atrial enlargement ( Figure 1). \| Echocardiography The echocardiographic investigations were carried out using a Philips/Hewlett-Packard Sono 5500 ultrasound device (Philips Ultrasound). M-mode, two-dimensional, and hemodynamic Doppler images were acquired using a standardized protocol with a 3.5-MHz transducer. The left ventricular ejection fraction was calculated using the modified Simpson method. 10 \| Laboratory measurements Venous blood was drawn after an overnight fast for health checkups that included the complete blood count (CBC), biochemical measurements, and the sST2 level. The CBC and biochemical parameters were measured using the Sysmex XE-2100D analyzer (Sysmex) and the Hitachi 7600 analyzer (Hitach), respectively. Metabolic syndrome was defined in accordance with the National Cholesterol Education Program Adult Treatment Panel III. 11 The serum sST2 level was measured using a quantitative sandwich monoclonal ELISA in a 96-well plate format with the Presage ST2 assay (Critical Diagnostics). Presage ST2 ELISA was measured on GEMINI COMBO (Stratec Biomedical). Serum is loaded into appropriate wells in the anti-ST2 antibody-coated plate and incubated at room temperature (18-25°C) for 60 minutes. Following a series of steps where reagents are washed from plate, and additional reagents are added and subsequently washed out, the analyte is finally detected by addition of a colorimetric reagent, and the resulting signal is measured spectroscopically at 450 nm. The lower limit of detection of the assay is 1.8 ng/mL. The assay has a within-run CV of 6.5% and a total CV of 9.1% at a mean concentration of 16.9 ng/mL, within-run CV of 3.4% and a total CV of 5.5% at a mean concentration of 33.1 ng/mL, and within-run CV of 2.4% and a total CV of 4.8% at a mean concentration of 159.1 ng/mL. \| Statistical analysis and calculation of RIs Statistical analyses were performed using SAS version 9.4 (SAS Institute). Multivariate (adjusted) regression analysis was performed to determine the variables affecting an increased sST2 level. Q-Q plots were used to confirm normality of residuals, and Durbin-Watson D statistics was used to check non-autocorrelation. The variables considered in the analysis included age, sex, BMI, systolic blood pressure (SBP), diastolic blood pressure (DBP), fasting blood glucose (FBS), triglyceride (TG) level, liver function tests, and creatinine. The RI for sST2 was calculated for the 7,090 reference subjects. The sST2 levels were analyzed according to the CLSI C28-A3 guidelines. 12 Scatter and distribution plots were used to inspect the data. The data in each partition were transformed using the Box-Cox transformation method. RIs for all of the partitions were calculated using non-parametric methods. To analyze the variations in sST2 according to age and sex, the levels of sST2 for each sex were grouped into the following age groups: <30, 30-39, 40-49, 50-59, 60-69, and ≥70 years. The Wilcoxon rank-sum test was used to compare the levels of sST2 according to age groups. Multivariate (adjusted) logistic regression analysis was performed to evaluate the association between the sST2 level and subclinical cardiac dysfunction. A P value of < .05 was considered statistically significant. Table 1 presents the characteristics of the general and reference subjects. The male and female general subjects were aged 56.7 ± 10.8 and 59.3 ± 10.0 years, respectively, and their serum levels of sST2 were 30.8 ± 13.7 and 27.5 ± 12.1 ng/mL. TA B L E 2 Variables associated with sST2 in the general subjects a higher serum sST2 levels, as were younger age and lower BMI, alanine aminotransferase (ALT), and TG levels (P < .01). The levels of sST2 were higher in subjects aged 30-39 years than in those aged 40-49 years in both sexes (P < .01) (Figure 3). The sex-specific and age-specific (<40 and ≥40 years) RIs for sST2 are presented in Table 3. The one-side upper 95th percentiles of the sST2 level were 49.6 ng/mL (95% confidence interval [CI] = 48.5-51.5) and 44.5 ng/ mL (95% CI = 43.5-45.6) in males and females, respectively. The sST2 levels were generally higher in males than females. \| Logistic regression of the association between sST2 and cardiac dysfunction in the general subjects Subclinical cardiac dysfunction was detected using echocardiography in 24,628 (58.9%) of the general subjects. These subjects were older and had higher BMI, blood pressure, FBS, HbA1c, and TG levels and a lower high-density lipoprotein cholesterol (HDL-C) levels. However, the sST2 level did not differ significantly between subjects with and without subclinical cardiac dysfunction. While older age, female sex, and higher BMI, HbA1c, and TG levels were associated with subclinical cardiac dysfunction (P < .001), this was not associated with the sST2 level in multiple logistic regression analysis (P = .130) ( Table 4). An association of sST2 with FBS was found in our study. Although the underlying mechanisms are not known, animal studies have shown that sST2 signaling may be important in modulating the autoimmune effects on the pancreas associated with diabetes. 16 Miller et al 17 also reported that the sST2 level was strongly associated with markers of diabetes, including triglyceride, liver function, and blood glucose. We also found that the sST2 level was associated with AST and GGT. It was found previously 18 that the sST2 level was significantly higher in patients with chronic hepatitis C than in healthy controls. Roth et al 19 reported that the serum sST2 level was higher in patients with acute liver failure than in patients with chronic liver failure and healthy controls, which suggests that sST2 is an early biomarker of liver injury. \| D ISCUSS I ON We determined the RIs of sST2 using a well-characterized ref- 20 The association between the sST2 level and age seems to be controversial among studies. Some previous studies 21,22 showed that links between the sST2 level, age, and male sex, whereas Lu et al 23 did not find any association of sST2 with age. In our study, the RIs of sST2 were higher in the subjects aged <40 years than in those aged ≥40 years in both sexes. We attempted to evaluate the utility of sST2 in screening subclinical cardiac dysfunction in terms of preventive strategies at a population level. The subjects with subclinical cardiac dysfunction were older and had a higher BMI, blood pressure, FBS, and triglyceride levels, and a lower HDL-C level. However, the sST2 level did not differ significantly between subjects with and without subclinical cardiac dysfunction. To investigate this relationship further, we created a model that included covariates that are associated with cardiac function. We observed associations of subclinical cardiac dysfunction with cardiovascular risk factors such as higher BMI, DBP, HbA1c, triglycerides, and low-density lipoprotein cholesterol levels, but not with the sST2 level. The ST2 system is known to be induced when cardiac fibroblasts or cardiomyocytes are subjected to mechanical stresses 24 and appear to be intimately involved in cardiac remodeling and fibrosis in HF. However, ST2 was initially described in the context of cell proliferation, inflammatory states, and autoimmune disease. 25 Data are mean ± SD or N (%) values, except where indicated otherwise. TA B L E 4 Logistic regression of the association between sST2 and cardiac dysfunction in the general subject	2020-07-09T09:07:13.186Z	2020-07-07T00:00:00.000	{ "year": 2020, "sha1": "81292337d5a72794eb9c6b181df6ad1115af3c2e", "oa_license": "CCBYNCND", "oa_url": "https://doi.org/10.1002/jcla.23461", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "220492936076f703c70a6d06a330c6edd5d8b050", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
255713279	pes2o/s2orc	v3-fos-license	Metamaterial Based Ku-Band Antenna for Low Earth Orbit Nanosatellite Payload System The concept of nanosatellite technology becomes a viable platform for earth and space observation research to minimize cost and build time for the payload. The communication approach is the essential fundamental attribute of a satellite, of which the antenna is a crucial component for forming a communication link between the nanosatellite and the earth. The nanosatellite antenna must comply with some special requirements like compact size, lightweight, and high gain with a space-compatible structure. This paper proposes a compact metamaterial-based Ku-band antenna with circular polarization for the nanosatellite communication system. The designed antenna obtained an impedance bandwidth of 2.275 GHz with a realized gain of 6.74 dBi and 3 dB axial beamwidth of 165° at 12.10 GHz. The overall antenna size of the designed is 0.51λ × 0.51λ × 0.17λ, which is fabricated on Rogers 5880 substrate material. The antenna results performance has been examined with a 1 U nanosatellite structure and found suitable to integrate with metallic and nonmetallic surfaces of any miniature nanosatellite structure. Introduction With the advancement of technology, nanosatellite has drawn remarkable contemplation from space researchers due to the feasibility of payload missions within a few cubic centimeter's size satellite structure and minimal expense. Nowadays, this type of small satellite is widely used in various sectors, like astronomy, astrobiology, earth observation, atmospheric science, ecology, meteorology, telecommunications, disasters mitigation and management, education, and training [1][2][3][4]. Smooth data communication is very crucial for nanosatellite missions, where the antenna plays a key role in establishing communication between the satellite and the earth. The nanosatellite antenna design becomes complex to antenna researchers due to the inverse proportionality relation between antenna performance and size [5][6][7]. Most nanosatellite payloads demand high gain, compact and circular polarized antenna for a smooth communication system [2]. Various antennae have been designed in Ku-band applications, but most have linear polarization. A linear polarized dual-band patch dipole antenna was developed in [8] for Ku and Ka-band uses, with resonant frequencies at 16.5 and 32.5 GHz. In [9], a larger size patch antenna was presented for S, C and Ku band frequencies. The antenna shows the resonant frequency at 2.4, 5.0, and 15.5 GHz frequencies with a low gain value. Different designs of Ku band antenna are presented in [10][11][12][13][14], where all the design has linear polarization and lower gain. Several researchers also focused on designing compact circular polarized (CP) high-gain antenna for small satellite communication systems [7,15,16]. In [12], a circular patch antenna was designed, which has CP, but the antenna size is 40 × 48 mm 2 . In [17], a patch antenna has been developed for Ku-band satellite application with a size of 20 × 20 mm 2 . The antenna shows circular polarization from 12.30 to 12.46 GHz. However, the gain of this antenna at this band is 1.6 dB. Similarly, Vijayvergiya et al. proposed a patch antenna with the size of 22.13 × 21.9 mm 2 , which operates at 12.2-14.5 GHz and has a linear polarized gain of 4.8 dB at 12.2 GHz [18]. In [19], a frequency selective surface (FSS) integrated patch antenna has been presented for a wide X and Ku band communication system, where the antenna can operate from 5 GHz to 24.5 GHz with the highest gain of 5.9 dB. The major limitation of this antenna is the overall size of 52.8 mm × 52.8 mm × 21.2 mm, which is not compatible with the 1 U (One unit) nanosatellite system. In [20], a printed monofilar antenna is offered for the Ku-band CubeSat application, which provides a gain of 8.5 dBi at 12.2 GHz with an overall size of 18 mm × 18 mm × 14 mm. The square cavity structure of the antenna makes complexity to mount with a 1 U nanosatellite structure, which is a major concern for nanosatellite antenna researchers. Therefore, from the study, it is shown that there is a great demand for designing a Ku-band antenna for a 1 U nanosatellite communication system to overcome the limitations of lower gain, larger size, complex structure, and circular polarization, etc. The metamaterial structure is attractive in antenna design, improving the gain and reducing interference with the satellite structure [21][22][23]. This paper proposes a metamaterial-inspired Ku band antenna for a 1 U nanosatellite communication system. The proposed antenna is a non-deployable planar structure with sufficient mechanical robustness to realize the maximum design flexibility of the limited volume 1 U nanosatellite structure. Antenna Design Methodology The developed antenna is intended to provide effective uplink and downlink communication between small satellites and the earth. The antenna prototype consists of two layers, shown in Figure 1. One layer is a truncated square patch antenna (Figure 1a), and another layer is an interconnected split rectangular metamaterial array ( Figure 1b). Both layers are interconnected with conducting reflectors shown in Figure 1c. The space between the antenna and the metamaterial layer is 1.69 mm. Both layers are connected with a 0.25 mm thick conductive reflector wall, which also provides a strong mechanical strength of the structure. The antenna is fabricated on space-quality substrate material Rogers 5880 with a thickness of 1.575 mm, a dielectric constant of 2.2 and a dielectric loss tangent of 0.0009. The antenna is fed by a 50 Ω Sub-Miniature version A (SMA) connector. The optimized structural layout parameters are listed in Table 1. The overall size of the antenna will be 15 mm × 15 mm. Figure 1b shows the designed net-like hexagonal split ring base metamaterial structure, which has equal dimensions to the antenna and all other design parameters of the antenna listed in Table 1. The metamaterial structure has been characterized by considering perfect magnetic (PMC) and perfect electric (PEC) boundary conditions along the y and x-axis. The designed metamaterial-based antenna has been presented in Figure 1c. The designed metamaterial structure simulation setup is presented in Figure 1d [24]. The metamaterial property like relative permittivity (ε r ) and permeability (µ r ) has been calculated from scattering parameters by using Equations (1) and (2) [25][26][27]. Figure 2a,b shows the metamaterial property of the designed metamaterial reflector. The higher negative permeability has been achieved at the operating frequency of the antenna (10.18-13.05 GHz), which is µ r ≥ −600. On the other hand, higher positive permittivity appeared in the same frequency range. This single negative feature reflected back the incident electromagnetic wave. The phase information of the reflected wave can also be understood from the phase value of the reflection coefficient. Figure 2c shows the phase value of the proposed metamaterial reflection operating frequency relayed in the +90 • to −90 • range. This is characterized as an artificial magnetic conductor (AMC) because the operating bandwidth of the AMC is considered from +90 • to −90 • , and the resonant frequency is considered at 0-degree. The AMC-type metamaterial structure mimics the attributes of the perfect magnetic conductor. The AMC, which has PMC characteristics, can reflect the incident wave with a 0-degree phase, which made a constructive interference with the antenna-radiated wave in the forward direction shown in Figure 2d. Hence, the directivity and gain of the antenna improve significantly, and interference of the signal with the back side element of the antenna has been reduced. Results and Discussions The simulated antenna has been fabricated and measured to validate performances. The antenna's reflection coefficient is presented in Figure 3. The antenna achieves −10 dB impedance bandwidth of 1.87 GHz (11.18 GHz to 13.05 GHz) in simulation and 2.275 GHz (10.85 to 13.125 GHz) in measurement. Both results seem identical. However, a little mismatch is observed due to fabrication and measurement tolerances. Results and Discussions The simulated antenna has been fabricated and measured to validate performances. The antenna's reflection coefficient is presented in Figure 3. The antenna achieves −10 dB impedance bandwidth of 1.87 GHz (11.18 GHz to 13.05 GHz) in simulation and 2.275 GHz (10.85 to 13.125 GHz) in measurement. Both results seem identical. However, a little mismatch is observed due to fabrication and measurement tolerances. Results and Discussions The simulated antenna has been fabricated and measured to validate performances. The antenna's reflection coefficient is presented in Figure 3. The antenna achieves −10 dB impedance bandwidth of 1.87 GHz (11.18 GHz to 13.05 GHz) in simulation and 2.275 GHz (10.85 to 13.125 GHz) in measurement. Both results seem identical. However, a little mismatch is observed due to fabrication and measurement tolerances. The simulated 3D radiation pattern of the projected antenna at 12.1 GHz is demonstrated in Figure 4a. The antenna shows 6.53 dBi of realized gain with very low back radiation. The substantial decrease in back radiation occurred due to the AMC metamaterial layer. Additionally, the 3 dB simulated axial ratio is also presented in Figure 4b. The antenna shows approximately 165 • of 3 dB axial beamwidth with stable circular polarization. The simulated 3D radiation pattern of the projected antenna at 12.1 GHz is demonstrated in Figure 4a. The antenna shows 6.53 dBi of realized gain with very low back radiation. The substantial decrease in back radiation occurred due to the AMC metamaterial layer. Additionally, the 3 dB simulated axial ratio is also presented in Figure 4b. The antenna shows approximately 165° of 3 dB axial beamwidth with stable circular polarization. The radiation characteristics have been measured in Satimo nearfield measurement system, shown in Figure 5. The antenna exhibits realized gain of 6.61 dB and 6.7 dB without nanosatellite (free space) and with nanosatellite structure, respectively. Besides, axial beamwidth at 12.1 GHz has also been measured, shown in Figure 6. The antenna attained 3 dB axial beamwidth of 162° and 158° for phi 0° and phi 90°, correspondingly. The radiation characteristics have been measured in Satimo nearfield measurement system, shown in Figure 5. The antenna exhibits realized gain of 6.61 dB and 6.7 dB without nanosatellite (free space) and with nanosatellite structure, respectively. Besides, axial beamwidth at 12.1 GHz has also been measured, shown in Figure 6. The antenna attained 3 dB axial beamwidth of 162 • and 158 • for phi 0 • and phi 90 • , correspondingly. The developed antenna has been integrated into the standard 1 U nanosatellite architecture to investigate the performance in the real environment. In both simulation and measurement, the antenna reveals well agreement in both measurements. The radiation pattern with 1 U nanosatellite structure at 12.1 GHz is depicted in Figure 7. In Figure 7a, only antenna (without metamaterial layer) has been integrated, and radiation performances have been observed where the antenna reveals realized gain of 4.7 dB. On the other hand, the antenna with a metamaterial layer has been integrated to observe radiation performances, where the antenna reveals realized gain of 6.69 dB. Therefore, the metamaterial significantly improves antenna gain and reduces the coupling effect with satellite structure. The developed antenna has been integrated into the standard 1 U nanosatellite architecture to investigate the performance in the real environment. In both simulation and measurement, the antenna reveals well agreement in both measurements. The radiation pattern with 1 U nanosatellite structure at 12.1 GHz is depicted in Figure 7. In Figure 7a, only antenna (without metamaterial layer) has been integrated, and radiation performances have been observed where the antenna reveals realized gain of 4.7 dB. On the other hand, the antenna with a metamaterial layer has been integrated to observe radiation performances, where the antenna reveals realized gain of 6.69 dB. Therefore, the metamaterial significantly improves antenna gain and reduces the coupling effect with satellite structure. The developed antenna has been integrated into the standard 1 U nanosatellite architecture to investigate the performance in the real environment. In both simulation and measurement, the antenna reveals well agreement in both measurements. The radiation pattern with 1 U nanosatellite structure at 12.1 GHz is depicted in Figure 7. In Figure 7a, only antenna (without metamaterial layer) has been integrated, and radiation performances have been observed where the antenna reveals realized gain of 4.7 dB. On the other hand, the antenna with a metamaterial layer has been integrated to observe radiation performances, where the antenna reveals realized gain of 6.69 dB. Therefore, the metamaterial significantly improves antenna gain and reduces the coupling effect with satellite structure. This article presents a compact Ku-band CP antenna for the nanosatellite communication system. The antenna's gain significantly increases by placing an AMC in the back side of the antenna, which also reduces the antenna's signal interference with other nanosatellite components. The antenna's performance has been investigated with a 1 U nanosatellite structure and found suitable to integrate with metallic and nonmetallic surfaces of any miniature nanosatellite structure. A details comparison table of the projected antenna with the existing antenna has been presented in Table 2. Where most of the shows liner polarization except [12], but the antenna size is larger than the projected antenna. Additionally, antenna performance does not investigate with satellite structure. The antenna presented in [28] and [11] shows higher gain, but the antenna size is very large compared to the nanosatellite structure; polarization is the liner. Finally, the compact size, high gain, circular polarization, and investigation with 1 U nanosatellite structure makes proposed antenna a potential candidate for Ku-band 1 U nanosatellite communication systems. Conclusions This paper presents a non-deployable circular polarized antenna for the Ku-band 1 U nanosatellite communication system, which is highly mechanical robust with 1 U structure. The antenna prototype has been designed, fabricated, and measured. By adopting AMC metamaterial layer with reflector wall technique, the antenna achieves circular polarization with a realized gain of 6.69 dB with 1 U nanosatellite structure. Therefore, an extensive and comprehensive performance analysis of the proposed antenna shows its suitability with a nanosatellite environment.	2023-01-12T17:59:04.596Z	2023-01-01T00:00:00.000	{ "year": 2023, "sha1": "2f6df79f6ab62280bedb45658694884db7230d4e", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2079-4991/13/2/228/pdf?version=1672826623", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "67108cdfcdc7c208d1c8dc871032e836c726a2cd", "s2fieldsofstudy": [ "Engineering", "Physics" ], "extfieldsofstudy": [ "Medicine" ] }
170373697	pes2o/s2orc	v3-fos-license	God Beyond Theism? Bishop Spong, Paul Tillich and the Unicorn John Shelby Spong has recently advocated belief in a “God beyond theism”. While rejecting traditional theism, he also distinguishes his position from atheism. Spong suggests that there is a divine reality, which may be described as “being itself” and which reveals itself in our commitment to unconditional ideals. It is argued that this notion of God is vacuous, the product of a confused belief that “being” is a characteristic of individual beings which may be universalised. Belief in such a God is also unmotivated, since there exist naturalistic explanations of the phenomena to which Spong appeals. actually required occasional divine interventions to shore up the mechanism of the universe.I) But despite these signs of sloppy scholarship, many of Spong's central ideas have a respectable intellectual pedigree. Therefore what he is saying deserves closer scrutiny by students of religion. In what follows I want to illustrate this claim by reference to one of Spong's most recent themes: the idea that our understanding of God must progress beyond the "theism" of Christian history. In his espousal of belief in a "God beyond theism", Bishop Spong is clearly dependent on the work of the twentieth-century theologian Paul Tillich. 3 It was Tillich who first spoke of the need for Christianity to transcend "theism in all its forms", so as to begin speaking about "the God above God", who is the ultimate source of our "courage to be".4 S 0 if we are looking for a developed form of the views held by Bishop Spong -one which is worthy of intellectual engagement -we may find this in the work of Paul Tillich. In what follows I will use Tillich's work to interpret Spong's views, before posing a few questions. I will not spend much time on the critical side of Spong's work, with which (as it happens) I am fundamentally in agreement. I am sympathetic to the view that "the gods" are nothing other than what Tillich calls "images of human nature or subhuman powers raised to a superhuman realm".5 To this extent, I would accept the classic modern criticism of religion, dating from the time of Ludwig Feuerbach, which regards the gods as creations of human beings, projections of elements of human experience into an unseen world. I am happy to accept the "postmodern" claim that many of the entities posited by the modern sciences must be seen in a similar light. These, too, draw upon analogies in everyday experience to create models which are used to explain the underlying reality of the world," The difference -and it is a key one -is that the models created by the sciences are subject to a rigorous process of criticism. In other words, any such model will eventually be abandoned if it is not performing its explanatory role. I therefore regard the modern sciences as the most reliable means we have of arriving at a knowledge of reality, without (I think) falling into a kind of uncritical realism about their results. Religions, on the other hand, are notoriously resistant to criticism, being inclined to the view that their models are divinely revealed and therefore the subject of certain knowledge. I am also inclined to agree with Bishop Spong that since the emergence of the modern sciences in the seventeenth century -with their impersonal models of explanation and their tradition of critical rationality -there is little point in trying to explain the way things are by reference to a divine being." Indeed insofar as religions employ personal rather than impersonal models to describe the underlying reality of the world, their claims simply fall outside what Michel Foucault would call the" episteme" of modern knowledge.f It is this realisation that lies behind Spong's references -not entirely accurate, as we have seen -to Sir Isaac Newton, to suggest that the modern sciences have gradually made appeal to divine activity redundant. It lies behind the claim made by New Testament scholar turned atheist Michael Goulder and endorsed by Spong, that "the God of the past 'no longer [has] any real work to do'."? It also explains Spong's attitude to prayer, expressed in the tenth of his Twelve Theses (apparently modelled on Martin Luther's), where he writes that "prayer cannot be a request made to a theistic deity to act in human history in a particular way". 10 For if one can no longer appeal to a divine being to explain the course of events, then any attempt to persuade that deity to alter the course of events is obviously doomed to failure. While others may wish to debate these views, their defence would be the work of another day. For the moment, I need only note that I have no serious disagreement with Spong's more substantive conclusions. Where we differ is in the consequences we draw from these conclusions. If one accepts the critical arguments outlined above, the obvious consequence would seem to be atheism. One would not need to adopt a "strong" atheism, which would deny the existence of God outright. But these arguments do seem to entail at the least a "weak" atheism, or (if one prefers) a strong agnosticism, which denies that we have sufficient reason for affirming God's existence. For if belief in God can be accounted for in purely naturalistic terms and if appeals to the actions of God are no longer a plausible way of explaining the existence and shape of the world, it is hard to see what other grounds we could have for affirming his reality. Yet Bishop Spong claims not to be an atheist. He continues to use religious language and his words imply that this language has a distinctive referent, albeit one about which we can say very little. For instance, Spong speaks of "experiencing God" in 7. John Shelby Spong, "Can One Be a Christian Without Being a Theist?", The Voice (Diocese of Newark) http://www.dioceseofnewark.org/vox21096.htrnl ( terms which suggest that this is an experience of something, or someone, who cannot be simply identified with the other objects of our experience. He says that this God is the "ultimate reality" in his life, that he lives in "a constant and almost mystical awareness of the divine presence'i.'! He says that he is among those who "cannot cease believing", since God is "too real" to allow them to do soP But what is this reality? Where is it to be found? How can we know about it? It is at this point that Spong's language becomes both "elusive and allusive'i.P Often his position looks like yet another retreat to religious experience, a tactic characteristic of liberal theology since the time of Friedrich Schleiermacher. We can (it seems) no longer speak of God, in any traditional sense, but we can continue to speak of our "Godexperiences". Yet Spong's position is not identical with that of theological liberalism. He does speak of an experience of God, which is the basis of his faith, but it is a very particular kind of experience. God is the source of human love which (or whom) we know in the very act of loving wastefully; he is the Ground of Being which (or whom) we come to know when we ourselves have the courage to be.l 4 For the philosopher, of course, such expressions are infuriatingly vague. (Indeed one is tempted to say that their vagueness is their strength, since Spong's readers can find in them whatever meaning they want.) But they take on a more precise meaning in the work of Tillich. We may begin with Tillich's analysis of human rationality, which he understands in a very broad sense, as encompassing all of our cultural life. Tillich argues that there exists a depth dimension to human reason, which precedes the division into knowing subject and known object.l> This takes the form of a quest for an limitless and unconditioned reality, which is implicit in our all dealings with the limited and conditioned objects of experience. 16 In the field of cognition, this involves a striving for what Tillich calls "truth itself", a truth that is not relative and partial but absolute and complete. In the field of aesthetics, it takes the form of striving for "beauty itself", a striving which underlies every artistic work. In the field of law, this depth dimension has the form of a striving for "justice itself", while in the field of personal relations it takes the form of a striving for "love itself" .17 These are all examples of what Tillich famously calls our "ultimate concern". 18 The existence of this ultimate concern raises the central question of religion. Is there a way in which the conflicts which arise in the exercise of reason -conflicts between the conditioned and the unconditionedcan be overcomert? Revelation answers this question in symbolic language. It does so by way of insights received in what Tillich calls a state of "ecstasy". A state of ecstasy is a "state of mind in which reason is beyond itself, that is, beyond its subject-object structure" and thus capable of grasping the reality for which it is striving.s? The reality it grasps is that of the ground or power of being,21 which Tillich identifies with God. 22 As the ground or power of being, God is "being itself". 23 Incidentally, it is because God is "being itself" that He cannot be thought of as a being among other beings, whose existence could be a matter of dispute.is Even to talk about God as the "highest being" is to reduce Him to the level of other beings and to deny His true nature/" To speak of God as a "person" without due qualifications is to fall into the same trap. 26 What can we make of these ideas? Let me begin with some positive comments. I believe that, at least in the first part of this argument, Tillich (and by association Bishop Spong) have identified something of philosophical interest. If we assume the most plausible view of human origins we have, namely the Darwinian one, there is something remarkable about our commitment to certain ideals -let's call them the ideals of truth, beauty and goodness -in a world in which they seem impossible of realisation. At first sight this commitment is not readily explicable as the product of an evolutionary process which has no other "purpose" (loosely speaking) than the successful propagation of organisms. Indeed at least two contemporary philosophers have suggested that it simply cannot be accounted for on evolutionary grounds at all. 27 This is not a question I wish to adjudicate. All I wish to note is that there is a question here worthy of investigation. However, there is a theological tradition dating to the time of Immanuel Kant which goes further. It suggests that the existence of at least some of these ideals implies the existence of God. Loosely speaking, it is this tradition to which Tillich and Bishop Spong seem to belong.P More precisely, for Tillich the experience of the finitude -the limited and conditioned character -of human existence raises the question to which the Christian revelation of God is the symbolic answer. As "being itself", God is the implicit goal of our strivings for truth, beauty and goodness. He makes possible a life lived in hope in pursuit of these goals, a life which Tillich describes as the "New Being" of faith-filled existence. 29 Such claims seem to go far beyond what is warranted by the evidence. First of all, there are some philosophical objections to the way in which both Tillich and Bishop Spong describe the reality of God. As we have seen, Tillich's preferred designation of God is "being itself", a phrase which Spong also uses. 30 This is, of course, a very traditional designation of God. No less a figure than Thomas Aquinas refers to God as ipsum esse subsistens: "being itself existing".31 But at least as used by Tillich, this expression seems to be the product of a twofold confusion. The first mistake is that of regarding the word "being" as a descriptive word, capable of picking out some characteristic which all beings have in common.F The problem here, as Kant pointed out, is that "being" is not a descriptive term.P I take nothing away from the idea of a unicorn -I deprive it of none of its characteristics -if I judge that no unicorns exist. A second error lies in imagining that "being" can be meaningfully spoken of as a universal, as having some kind of quasiindependent existence, so that one can speak not just of the being of individual beings, but of "being itself". 34 It is true that Bishop Spong seems to prefer what is for Tillich an equivalent term,35 namely "ground of being".36 At first sight, this suggests a very traditional conception of God: an infinite, necessary being who sustains the world of contingent, finite beings. Yet such a God is nothing less than the God of "theism", which Spong and Tillich reject.F Secondly, on the very grounds that Spong has brought forward, it is not clear why we need to use this word "God" at all. For Spong's mysterious "ground of being" is apparently not responsible for the way the world is. As we have seen, Spong has already argued that the sciences have made such explanatory appeals to divine action redundant. If, with Tillich, Spong wishes to see mystical depths in our strivings for truth, beauty and goodness, then it is not at first sight clear why we need God in this context either. We can regard such ideals as simply projections to an ideal limit of qualities which we happen to value for all sorts of ultimately practical reasons. In this case, they are no more pointers to a divine "ground of being" than is the mathematician's parallel creation of the idea of infinity." Incidentally, to recognise that all these ideals are our creations -that they are to a certain extent fictions, to which no reality completely corresponds -is not necessarily to undermine their force. A world without God, contrary to much theological (and even "postmodern") polemics, is not necessarily a world without truth or value.'? In a word, what is most problematic about Spong's position is not his criticism of traditional religious language. It is the fact that he continues to use language about God, when that language seems to have been emptied of its content and stripped of its necessity. Spong will not only need to show his theological opponents that this "God beyond God" has religious power. He will need to show his philosophical opponents that we need to continue to speak of God, in a world in which entirely naturalistic explanations are on offer for the phenomena to which he appeals. He will also need to show that the term "God", which he continues to employ, is something more than an empty abstraction. For there are good reasons to believe that a God so stripped of all the characteristics of an individual being has, in fact, no reality at all. 38. See	2019-05-31T13:10:21.958Z	2002-02-01T00:00:00.000	{ "year": 2002, "sha1": "9a1f6cc619b562e5bb47f3b6bc8bd7c5917c35b4", "oa_license": "CCBYSA", "oa_url": "https://ourarchive.otago.ac.nz/bitstream/10523/566/1/DAWES-God%20beyond%20Theism.pdf", "oa_status": "GREEN", "pdf_src": "Sage", "pdf_hash": "35b8cde140619ab50f815c9cee33fdbb5f7f4c0e", "s2fieldsofstudy": [ "Philosophy" ], "extfieldsofstudy": [ "Philosophy" ] }
14920796	pes2o/s2orc	v3-fos-license	Enhancing effect of lysine combined with other compounds on cephamycin C production in Streptomyces clavuligerus Background Lysine plays an important role in Streptomyces clavuligerus metabolism; it takes part in its catabolism, via cadaverine, and in its secondary metabolism, in which lysine is converted via 1-piperideine-6-carboxylate to alpha-aminoadipic acid, a beta-lactam antibiotic precursor. The role of lysine as an enhancer of cephamycin C production, when added to production medium at concentrations above 50 mmol l-1, has already been reported in the literature, with some studies attributing a positive influence to multifunctional diamines, among other compounds. However, there is a lack of research on the combined effect of these compounds on antibiotic production. Results Results from experimental design-based tests were used to conduct response surface-based optimization studies in order to investigate the synergistic effect of combining lysine with cadaverine, putrescine, 1,3-diaminopropane, or alpha-aminoadipic acid on cephamycin C volumetric production. Lysine combined with cadaverine influenced production positively, but only at low lysine concentrations. On the whole, higher putrescine concentrations (0.4 g l-1) affected negatively cephamycin C volumetric production. In comparison to culture media containing only lysine as additive, combinations of this amino acid with alpha-aminoadipic acid or 1,3-diaminopropane increased cephamycin C production by more than 100%. Conclusion This study demonstrated that different combinations of lysine with diamines or lysine with alpha-aminoadipic acid engender significant differences with respect to antibiotic volumetric production, with emphasis on the benefits observed for lysine combined with alpha-aminoadipic acid or 1,3-diaminopropane. This increase is explained by mathematical models and demonstrated by means of bioreactor cultivations. Moreover, it is consistent with the positive influence of these compounds on lysine conversion to alpha-aminoadipic acid, a limiting step in cephamycin C production. Background The commercial importance of the actinomycete Streptomyces clavuligerus lies in its ability to produce several secondary metabolites of therapeutic interest [1]. Among these compounds are: cephamycin C, a beta-lactam antibiotic more resistant to beta-lactamases than the structurally similar antibiotic cephalosporin C produced by filamentous fungi, and for this reason used as raw material for production of semi-synthetic antibiotics (cefotetan, cefoxitin, cefmetazole, and temocillin) [2,3]; clavulanic acid, a beta-lactamases inhibitor whose use in conjunction with amoxicillin is the most important commercial example [4]; other clavams, which have antifungal properties [5]; and non-beta-lactam compounds such as holomycin and tunicamycin, which have antibiotic and antitumor properties [5][6][7]. The biosynthetic diversity inherent to S. clavuligerus results in extremely complex metabolic regulation [8][9][10][11][12][13][14], which has led to different studies aimed at increasing the biosynthesis of relevant biocompounds. Among these compounds, cephamycin C has been one of the most extensively investigated [15][16][17][18][19][20][21][22][23]. The basic structure of this biocompound and of all other beta-lactam antibiotics produced by prokaryotes or eukaryotes derives from L-cysteine, L-valine, and L-alpha-aminoadipic acid. In prokaryotes, alpha-aminoadipic acid is the product of lysine degradation via 1-piperideine-6-carboxylate [24][25][26]. The use of exogenous lysine to enhance cephamycin C biosynthesis in cultures of producer species has been known for over thirty years [16,20,23,27,28]. Studies have shown that high lysine concentrations (above 50 mmol l -1 ) promote higher cephamycin C production as compared to that of culture media containing little or no lysine. Researchers have obtained increases of up to 300% in cephamycin C production by S. clavuligerus in a culture medium containing about 100 mmol l -1 of lysine [14,20,21]. In spite of lysine degradation via 1-piperideine-6-carboxylate pathway producing the precursor alphaaminoadipic acid [25,26], complete lysine catabolism occurs via cadaverine [24,29,30]. Cadaverine and other diamines, such as diaminopropane and putrescine, promote beta-lactam antibiotic production in Nocardia lactamdurans or S. clavuligerus [31][32][33][34]. Nevertheless, it is difficult to determine the extent to which these compounds influence antibiotic biosynthesis, since diamines act as modulators of several cell functions [32,33,35]. Thus, there is scarce quantitative research on the use of lysine combined with other diamines or other compounds that can potentially enhance beta-lactam antibiotic production in S. clavuligerus [16,23,33]. This was explored in this study, which investigates increases in cephamycin C production by adding cadaverine, putrescine, 1,3-diaminopropane or alpha-aminoadipic acid in culture media containing lysine as compared to those obtained in culture media containing lysine alone. Cultivations were performed in accordance with a central composite-based, face-centered experimental design (CCF) whereas concentrations of lysine combined with every compound were optimized using Response Surface Methodology. Best conditions were validated by means of batch cultivations in a stirred and aerated benchscale bioreactor. Escherichia coli ESS 2235 supersensitive to beta-lactam antibiotics was employed as test organism. The strain was cultivated in nutrient agar medium (Difco™ Nutrient Agar) at 37°C for 24 hours. The cells were stored at −80°C in 2 ml cryotube vials. In order to investigate the influence of different compounds on cephamycin C biosynthesis, the production medium was supplemented with lysine, alpha-aminoadipic acid, and diamines 1,3-diaminopropane, putrescine, and cadaverine. These compound concentrations were established according to the purpose of each experiment. Experimental procedure Spore germination and inoculum preparation consisted of two pre-cultures with 24-hour cultivation each in shake flasks. Inoculum volume comprised 10% of suspension cell volume per culture medium volume throughout this study. Submerged cultures for cephamycin C production were performed in 500 ml Erlenmeyer shake flasks at 28°C and 260 rpm (5 cm eccentricity). To prevent problems of oxygen limitation during the shake-flask procedure, the broth volume was kept under 10% of the Erlenmeyer flask nominal volume. Samples were collected at 24-hour intervals. Experiments in the bench-scale bioreactor (New Brunswick Bioflo 2000; 5 l working volume) were performed at 1.0 vvm aeration rate, 6.8 ± 0.1 pH, 28°C temperature, and 50% dissolved oxygen saturation level automatically controlled by varying the agitation speed. Analytical methods The supernatant was obtained after centrifugation of the culture medium at 15,550 x g for 10 min, 4°C, for further analyses. The cell density was quantified as grams of dry weight per liter of sample (gDWC l -1 ). Cephamycin C was determined by means of the agar-diffusion assay method using cephalosporin C zinc salt (Sigma) as standard. Penase® (BD Difco) was employed at 20 μL per ml of sample, reacting at 25°C for 20 min to degrade penicillin N. In this method, the measure of cephamycin C represents the total amount of cephalosporins in the sample (in mg l -1 ) [36]. A calibration curve was performed using ten cephalosporin C concentration values from 5 to 120 mg l -1 and 24 replicates for each concentration. Antibiotic analyses were also carried out via high-performance liquid chromatography as described in Baptista Neto et al. [37]. Lysine and alpha-aminoadipic acid analyses were conducted by means of the post-column derivatization method with orthophtalaldehyde and quantified in a fluorescence detector [38]. The starch concentration was determined after acid hydrolysis, by quantifying the total reducing sugars by the dinitrosalicylic acid method [39]. Experimental design CCF experimental designs, including four replicates of an experiment under the same conditions, were employed to investigate individual and combined effects of lysine and compounds, one at a time, putrescine, 1,3-diaminopropane, cadaverine, and alpha-aminoadipic acid, on cephamycin C production. The response surface methodology was used to investigate the relationship between cephamycin C production (response variable) and the compounds that enhance beta-lactam antibiotic production (independent variables) [40,41]. The chosen experimental design and the established concentration limits of the compounds under investigation (independent variables) were appropriate for adjusting models represented by second-order polynomials according to the following equation: whereŷ represents the dependent variable, cephamycin C production (mg l -1 ), a 0 is the interception coefficient (average value of cephamycin C, in mg l -1 ), x represents the independent variables in coded unities: x Lys = lysine, and x i represents the other compounds studied (i = alpha- Table 2 Range and levels of independent variables lysine (Lys), 1,3-diaminopropane (1,3D), cadaverine (Cad), and putrescine (Put), in coded and original units, according to two-factor, three-level central-composite-based, face-centered, experimental designs (CCF); the response variable is cephamycin C concentration (CephC) obtained at 72-hour cultivation Independent variables Response Coded units Original units (g l -1 ) CephC (mg l -1 ) aminoadipic, 1,3-diaminopropane, cadaverine or putrescine); a 1 and a i are the linear terms, a 11 and a ii are the quadratic terms, a 1i is the interaction term. Statistica software (7.0 version) was used for regression and graphical analyses of experimental data. The conditions of independent variables and cephamycin C production results (observed and predicted) are shown in Tables 1 and 2. Results and discussion Individual effect of diamines and precursors on cephamycin C production For this study, two concentrations for each diamine were defined based on literature data obtained for other betalactam antibiotic producing microorganisms [32,33,35,42]. Cephamycin C biosynthesis precursors lysine and alphaaminoadipic acid were tested at several concentrations in order to define ranges of adequate values for the experimental designs. Cephamycin C production and cell growth obtained at 48 h and 72 h cultivations in basal medium without additives and supplemented with putrescine, 1,3-diaminopropane, and cadaverine are shown in Figure 1. Leitão et al. [32] found that all three diamines promoted cephamycin C production by N. lactamdurans, albeit at different levels. The largest increase was observed in culture media containing 2.5 or 5.0 g l -1 of 1,3-diaminopropane. In this study, this diamine also produced a similar effect: a 100% increase in volumetric production was observed after the addition of 5.0 g l -1 of the compound as compared to that of the culture medium with no additive. Also, the addition of 1,3-diaminopropane alone promoted higher specific production than that obtained at the control condition ( Figure 1C). Similarly, Martín et al. [42] observed that adding 5.0 mM (0.37 g l -1 ) or 10 mM (0.74 g l -1 ) of 1,3diaminopropane enhanced Penicillium chrysogenum betalactam antibiotic production by approximately 100%. It is likely that one of the effects of 1,3-diaminopropane is to maintain high mRNA transcript levels during the production phase [43]. In the present work, putrescine did not affect antibiotic production by S. clavuligerus ( Figure 1B), as Martín et al. [42,43] observed with P. chrysogenum. However, Leitão et al. [32] observed positive effects on cephamycin C production with N. lactamdurans when 0.20 g l -1 of putrescine was added. With regard to cadaverine, volumetric production almost doubled by adding 7.0 g l -1 of this diamine ( Figure 1B). However, specific production was not higher than that obtained in media without additives ( Figure 1C). For cultivations with N. lactamdurans, a threefold increase was obtained using 5.0 g l -1 of this diamine [32]. In general, the increase in biomass observed at the end of cultivations ( Figure 1A) suggests that these diamines acted as sources of carbon and energy (C) and/or nitrogen (N), thereby supplementing the basal medium sources (starch and PROFLO®). Cephamycin C production was evaluated at several lysine and alpha-aminoadipic acid concentrations (Figures 2 and 3). Consistent with the literature, high concentrations of exogenous lysine strongly affected cephamycin C production [20,28]. After adding 14.6 g l -1 of this amino acid, biomass almost doubled ( Figure 2A) and cephamycin C production increased about six fold ( Figure 2B) as compared to data from the basal medium. However, residual concentration values of this amino acid at 14.6 g l -1 and 18.3 g l -1 of lysine were approximately 25% and 35%, respectively. This surplus was not observed at concentrations lower than 11 g l -1 . Moreover, a fivefold global increase in antibiotic volumetric production was obtained between 0 and 11 g l -1 of lysine, whereas biomass increased only 1.5 times. Adding up to 1.6 g l -1 of alpha-aminoadipic acid did not influence biomass formation, which was in the same order of magnitude as that in the basal medium with no additives. Adding 0.64 g l -1 of alpha-aminoadipic acid to the basal medium resulted in the largest increase in cephamycin C production, four times larger than that obtained with the basal medium. Alpha-aminoadipic acid concentrations higher than 0.64 g l -1 did not promote higher antibiotic volumetric production, in spite of the amino acid having been completely consumed. Henriksen et al. [44] reported that alpha-aminoadipic acid can be metabolized into 6-oxo-piperideine-2-carboxylic acid (OPC), which is secreted into the culture medium during penicillin production by P. chrysogenum. The authors suggested that OPC formation would divert alpha-aminoadipic acid from antibiotic synthesis and lead to lower levels of penicillin production. A similar phenomenon may have occurred in S. clavuligerus. Effect of lysine in conjunction with diamines or alphaaminoadipic acid on cephamycin C production The concentration of independent variables enabled the investigation of ranges in which, according to the literature and data from this study, it is possible to maximize cephamycin C production by S. clavuligerus or N. lactamdurans [16,20,21,[31][32][33][34]42,43]. Based on results of cultivations using only lysine as additive (Figure 2), concentrations of amino acid ranging from 0 to 7.4 g l -1 were selected in order to minimize its effect on biomass production. With respect to alpha-aminoadipic acid, concentrations ranging from 0 to 0.64 g l -1 were selected due to superior cephamycin C volumetric production results obtained in this range ( Figure 3). As to lysine, the highest volumetric production of cephamycin C was observed at 48 hours, which varied little at 72 hours ( Figure 2B). The highest volumetric production values for the basal medium with 1,3-diaminopropane or alpha-aminoadipic acid were observed at 72 hours. With respect to cadaverine and putrescine, the highest volumetric production values observed at 48 and 72 hours were almost the same. For this reason, cultivation time was standardized to 72 hours for the experimental designs and bioreactor processes. The chosen experimental design (CCF) and the concentration range employed for the compounds under investigation (independent variables), together with the use of response surface methodology for statistical treatment of the data obtained at 72 h cultivation, allowed for the adjustment of quadratic models to predict cephamycin C production at 90% confidence level. The generated response surfaces and their corresponding second-order polynomials are shown in Figure 4. Table 3 shows the analyses of variance (ANOVA) of the fitted models, including the F-test to verify the overall significance of each model, its associated probabilities p(F), and determination coefficient R 2 . Cephamycin C production was affected differently for lysine combined with the remaining four compounds. The resulting response surfaces of experimental designs using lysine and alpha-aminoadipic acid ( Figure 4A) and lysine and 1,3-diaminopropane ( Figure 4B) showed curves and parameters of the same order of magnitude, thereby providing comparable production values. The adjusted mathematical models provide the highest cephamycin C concentrations of approximately 126 and 140 mg l -1 when 0.6 g l -1 of alpha-aminoadipic acid and 5.3 g l -1 of lysine and 5.2 g l -1 of 1,3-diaminopropane and 7.0 g l -1 of lysine were added, respectively. In culture media containing just lysine, a production of about 120 mg l -1 was obtained, but only at high amino acid concentrations (14.6 g l -1 ) (Figure 2). It should be remarked that alpha-aminoadipic acid has a strong impact on cephamycin C production even when added at concentrations nine times lower than those of 1,3-diaminopropane. This is probably due to its being a direct precursor of the beta-lactam antibiotic molecule [20,21,33]. On the other hand, 1,3-diaminopropane acts indirectly on beta-lactam antibiotic biosynthesis at the genetic and transcriptional levels [32,43]. Leitão et al. [32] showed that this diamine increases the concentration of lysine-6-aminotransferase and P6C dehydrogenase, which are enzymes responsible for alpha-aminoadipic acid formation. This complex mechanism may support the need for adding larger amounts of 1,3-diaminopropane to produce the same effect as that obtained with alphaaminoadipic acid at lower concentrations, which is in line with the results obtained in this study. These data and those found in the literature clearly demonstrate, albeit through different methods, that lysine conversion to alpha-aminoadipic acid is a limiting step to cephamycin C biosynthesis. For this reason, adding alpha-aminoadipic acid or 1,3-diaminopropane, though at different concentration levels, was equally effective to overcoming this bottleneck. Fitted response surfaces for cultivations in culture media containing lysine combined with cadaverine indicate that this diamine only exerts influence on antibiotic production when lysine is added at low concentrations. When the amino acid concentration was increased, the effect of adding diamine gradually waned. It has been suggested that intracellular accumulation of cadaverine may regulate the lysine catabolic pathway through a feedback control mechanism. In this manner, the lysine that would be decarboxylated to form cadaverine is spared, thus increasing lysine supply for cephamycin biosynthesis via the alpha-aminoadipate pathway. The fitted model shows that this behavior only happens at low lysine concentrations. At higher concentrations, lysine would supply both cadaverine and alpha-aminoadipic acid pathways, thereby decreasing the influence of cadaverine. For this reason, as predicted by the model, there is little antibiotic variation (73-77 mg l -1 of cephamycin C) at the highest lysine concentration (7.4 g l -1 ) within the entire cadaverine concentration range under investigation. This is due to the fact that the linear effect of lysine is about thrice stronger than that of this diamine. With respect to lysine combined with putrescine, adding 0.20 g l -1 of this diamine to media containing 3.7 g l -1 of amino acid increased production by approximately 40% as compared to that obtained with medium containing just lysine at the same concentration (Table 2). On the other hand, adding this diamine to media with higher lysine concentrations (7.4 g l -1 ) adversely affected production due to the negative effect stemming from the interaction between the compounds ( Figure 4D). Thus, the highest production value predicted for 7.7 g l -1 of lysine combined with 0.13 g l -1 of putrescine is just 76 mg l -1 . Similar volumetric production values were obtained with basal culture media containing 7.4 g l -1 of lysine as additive ( Figure 2). Martín et al. [43] observed that supplementation with putrescine provided much lower mRNA levels than those obtained with 1,3-diaminopropane in P. chrysogenum cultures. Despite structural similarity between 1,3-diaminopropane and putrescine, these authors suggest that the positive effect obtained with diamines is probably attributable to the three-carbon structure of diamines. On the other hand, Leitão et al. [32] observed an approximately threefold increase when 0.2 g l -1 of putrescine was added to N. lactamdurans cultures. Figures 5 and 6 show the results of two cultivations in bioreactor using 7.0 g l -1 of lysine combined with 5.2 g l -1 of 1,3-diaminopropane and 5.3 g l -1 of lysine combined with 0.64 g l -1 of alpha-aminoadipic acid. These concentrations, predicted by the models as optimal production conditions, resulted in 190 mg l -1 and 160 mg l -1 of cephamycin C for lysine combined with 1,3-diaminopropane and lysine combined with alpha-aminoadipic acid, respectively. When compared to top values predicted by the mathematical models, these results represent increases of approximately 35% for lysine combined with 1,3diaminopropane and approximately 27% for lysine combined with alpha-aminoadipic acid. While diamine supplementation favored cell growth, because it can act as an additional source of C and N, alpha-aminoadipic acid did not affect biomass production. Thus, the specific production at the end of cultivation with lysine combined with alpha-aminoadipic acid was approximately 30% higher as compared to that of lysine combined with 1,3-diaminopropane, reaching values of up to 40 mg l -1 and 30 mg l -1 , respectively. Results obtained in bioreactor employing medium without additives (control condition) are also shown in Figures 5 and 6. Conclusions It has been known for a long time that adding lysine enhances cephamycin C production. However, its use as the sole enhancer does not take full advantage of the antibiotic productivity of S. clavuligerus. In this study, an experimental design method (CCF) and Response Surface Methodology are successfully employed to adjust mathematical models to describe the effects of lysine combined with cadaverine, putrescine, 1,3-diaminopropane or alpha-aminoadipic acid on cephamycin C production ) control Figure 6 Batch cultivation in agitated and aerated bench-bioreactor for lysine combined with alpha-aminoadipic acid. Cephamycin C concentration (CephC), specific production, and biomass; basal medium containing cephamycin C production-enhancing compounds at their optimal values (in parentheses), lysine (5.3 g.l -1 ) and alpha-aminoadipic acid (0.6 g.l -1 ) (open symbols); control condition: basal medium without additives (solid symbols). ) control Figure 5 Batch cultivation in agitated and aerated bench-bioreactor for lysine combined with 1,3-diaminopropane. Cephamycin C concentration (CephC), specific production, and biomass; basal medium containing cephamycin C production-enhancing compounds at their optimal values (in parentheses), lysine (7.0 g l -1 ) and 1,3-diaminopropane (5.2 g l -1 ) (open symbols); control condition: basal medium without additives (solid symbols). by S. clavuligerus. Moreover, the interactions observed and validated by the fitted models are shown to be compatible to biochemical data already established in the literature about the pathway of beta-lactam antibiotics in S. clavuligerus. This study demonstrates that different combinations of lysine with other compounds promote significant variations in antibiotic production, with emphasis on the benefits obtained from using lysine combined with alpha-aminoadipic acid or 1,3-diaminopropane. These combinations increased cephamycin C production by more than 100% as compared to that with culture media containing just lysine as additive at the same concentrations. This positive effect may be attributed to alpha-aminoadipic acid or 1,3-diaminopropane in conjunction with lysine acting to overcome the bottleneck caused by lysine conversion to alpha-aminoadipic acid, albeit via different mechanisms. In the case of lysine combined with cadaverine, there was a positive effect on cephamycin C production by the diamine, especially when lysine was added at low concentrations. Cadaverine acted by decreasing lysine catabolism. However, as the amino acid concentration increased, the diamine effect waned, as the model clearly indicates. On the other hand, the highest volumetric production obtained with lysine combined with putrescine was approximately twice lower than that obtained with lysine combined with alpha-aminoadipic acid or 1,3diaminopropane.	2016-05-04T20:20:58.661Z	2013-12-20T00:00:00.000	{ "year": 2013, "sha1": "6828237ec7a822b69e4447a24bfd2df82c9a8508", "oa_license": "CCBY", "oa_url": "https://bmcmicrobiol.biomedcentral.com/track/pdf/10.1186/1471-2180-13-296", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "7fb39a8f2078f1018bbec2b1e33d3d94d9e463da", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
259252529	pes2o/s2orc	v3-fos-license	JSEEGraph: Joint Structured Event Extraction as Graph Parsing We propose a graph-based event extraction framework JSEEGraph that approaches the task of event extraction as general graph parsing in the tradition of Meaning Representation Parsing. It explicitly encodes entities and events in a single semantic graph, and further has the flexibility to encode a wider range of additional IE relations and jointly infer individual tasks. JSEEGraph performs in an end-to-end manner via general graph parsing: (1) instead of flat sequence labelling, nested structures between entities/triggers are efficiently encoded as separate nodes in the graph, allowing for nested and overlapping entities and triggers; (2) both entities, relations, and events can be encoded in the same graph, where entities and event triggers are represented as nodes and entity relations and event arguments are constructed via edges; (3) joint inference avoids error propagation and enhances the interpolation of different IE tasks. We experiment on two benchmark datasets of varying structural complexities; ACE05 and Rich ERE, covering three languages: English, Chinese, and Spanish. Experimental results show that JSEEGraph can handle nested event structures, that it is beneficial to solve different IE tasks jointly, and that event argument extraction in particular benefits from entity extraction. Our code and models are released as open-source. Introduction Event extraction (EE) deals with the extraction of complex, structured representations of events from text, including overlapping and nested structures (Sheng et al., 2021;Cao et al., 2022). While there are existing datasets annotated with such rich representations (Doddington et al., 2004;Song et al., 2015), a majority of current approaches model this task using simplified versions of these datasets or sequence-labeling-based encodings which are 1 https://github.com/huiling-y/ JSEEGraph sentence "I, purposely buy things made in Canada or USA.", taken from Rich ERE (Song et al., 2015). not capable of capturing the full complexity of the events. Figure 1 shows an example from the Rich ERE dataset (Song et al., 2015) of a sentence containing both nested and overlapping events: "buy" serves as trigger for two overlapping events, transfermoney and transferownership with their respective argument roles, and similarly "made" for two artifact events triggered by the coordination of two GPE entities Canada and USA; at the same time, the event trigger "made" is nested inside the entity span "things made in Canada or USA". For this example, models based on token tagging (such as the commonly used BIO-encoding) would fail completely when a token contributes to multiple information extraction elements. In this case, the version of the ACE05 dataset widely employed for EE would not fully capture the doubletagged event triggers, by simply disregarding one of the two events, and the nested entity "things made in Canada or USA" would be "things". Event extraction is a subtask of a wider set of Information Extraction (IE) tasks, jointly dealing with extracting various types of structured information from unstructured texts, from named entities, relations, to events. There have been continued efforts in creating benchmark datasets that can be used for evaluating a wide range of IE tasks. Both ACE05 (Doddington et al., 2004) (Song et al., 2015). ERE (Song et al., 2015) 3 provide consistent annotations of entities, relations, and events. While there are clear inter-relations between these different elements, and despite the availability of rich annotations, existing works often deal with individual tasks, such as named entity recognition (NER) (Chiu and Nichols, 2016;Bekoulis et al., 2018) or event extraction (EE) (Yang and Mitchell, 2016;Du and Cardie, 2020;. Recently there have been some efforts in jointly modelling multiple IE tasks (Wadden et al., 2019;Lin et al., 2020;Nguyen et al., 2022), but these methods explicitly avoid nested instances. We here propose to represent events, along with entities and relations, as general graphs and approach the task of event extraction as Meaning Representation Parsing (Oepen et al., 2020;Samuel and Straka, 2020). As shown in Figure 2, in such an information graph, event triggers and entities are represented as nodes; event types, argument roles, and relations are constrained edges; and nested/overlapped structures are straightforwardly represented, since a surface string can be abstracted into an unlimited number of nodes, as illustrated by the two separate nodes for the event triggers for "cost". Our approach does not rely on ontology-or language-specific features or any external syntactic/semantic parsers, but directly parses raw text into an information graph. We experiment on the benchmark datasets ACE05 (Doddington et al., 2004) and Rich ERE (Song et al., 2015), zooming in on nested structures. Our results show JSEE-Graph to be versatile in solving entity, relation, and event extraction jointly, even for heavily nested instances and across three different languages. Abla-3 https://catalog.ldc.upenn.edu/ LDC2020T18 tion studies consistently show that event extraction especially benefits from entity extraction. The paper is structured as follows: section 2 provides the relevant background for our work, and section 3 further describes the tasks addressed and the datasets we employ, focusing in particular on their complexity, as measured by level of nesting. Section 4 presents the JSEE graph parsing framework and section 5 the experimental setup for evaluating the JSEE parser. Section 6 presents the results of our evaluations and provides a study of the performance for nested structures, as well as an ablation study assessing the effect of joint IE modeling and an error analysis. Finally we provide conclusions (Section 7) and discuss limitations of our work. Related work Event extraction is commonly approached as supervised classification, even though other approaches relying e.g. on generation (Paolini et al., 2021;Lu et al., 2021;Hsu et al., 2022) or prompt tuning inspired by natural language understanding tasks (Shin et al., 2020;Gao et al., 2021;Li and Liang, 2021;Liu et al., 2022) also are gaining ground. Classification-based methods break event extraction into several subtasks (trigger detection/classification, argument detection/classification), and either solve them separately in a pipeline-based manner (Ji and Grishman, 2008;Li et al., 2013;Liu et al., 2020;Du and Cardie, 2020; or jointly infer them as multiple subtasks (Yang and Mitchell, 2016;Nguyen et al., 2016;Liu et al., 2018;Wadden et al., 2019;Lin et al., 2020). Classification-based joint methods typically apply sequence-labeling-based encoding and extract all event components in one pass, whereas pipeline methods break the problem into separate stages which are performed sequentially. Whereas sequence-labeling approaches cannot distinguish overlapping events/arguments by the nature of the BIO-encoding, pipeline methods may in principle detect these. However, they typically suffer from error propagation and are not equipped to model the interactions between the different event elements (triggers, arguments). Nested events Some previous work addresses the problem of overlapping or nested arguments in EE. Xu et al. (2020) address overlapping arguments in the Chinese part of the ACE05 dataset and jointly perform predictions for event triggers and argu-ments based on common feature representations derived from a pre-trained language model. Sheng et al. (2021) propose a joint framework with cascaded decoding to tackle overlapping events, and sequentially perform type detection, event and argument extraction in a Chinese financial event dataset. They deal with cases of both "overlapping events" and "overlapping arguments", however, their approach may suffer from error propagation due to the cascading approach. Cao et al. (2022) distinguish between overlapped and nested events and propose the OneEE tagging scheme which formulates EE as a word-to-word relation recognition, distinguishing separate span and role relations. OneEE is evaluated on the FewFC Chinese financial event dataset and the biomedical event datasets Genia11 and Genia13. While specifically focusing on nested events, these previous works are limited by focusing only on one language or on specialized (financial/biomedical) domains. In this work we aim to provide a more comprehensive evaluation over two datasets in several versions with increasing levels of structural complexity (see below) and across three different languages. Wadden et al. (2019) propose the DyGIE++ model which approaches joint modeling of IE entities and relations via span-based prediction of entities and event triggers, and subsequent dynamic graph propagation based on relations. They evaluate on ACE05 and Genia datasets and limit their experiments to English only. Their approach is restricted to a certain span width, limiting the length of possible entities. OneIE (Lin et al., 2020) is a joint system for IE using global features to model cross-subtask or cross-instance interactions between the subtasks and predict an information graph. They propose the E+ extension of ACE05 which includes multi-token events (E + ) as we do. As in our work, they also present results on Spanish and Chinese as well and develop a multilingual model, but their experiments avoid nested structures, by using only the head of entity mentions and specifically removing overlapped entities. Nguyen et al. (2022) model joint IE in a two-stage procedure which first identifies entities and event triggers and subsequently classify relations between these starting from a fully connected dependency graph; a GCN is employed to encode the resulting dependency graphs for computation of the joint distribution. While the approach is shown to be effective, it is still a pipeline approach which can suffer from error propagation. Since it relies on sequence labeling for entity/event detection, it cannot identify overlapping entities/event triggers. Furthermore, the approach relies on syntactic information from an external parser and focuses only on English and Spanish in the Light ERE dataset (Song et al., 2015). Joint IE approaches Meaning Representation Parsing Meaning Representation Parsing (MRP) (Oepen et al., 2014(Oepen et al., , 2015(Oepen et al., , 2020) is a framework covering several types of dependency-based semantic graph frameworks. Unlike syntactic dependency representations, these semantic representations are not trees, but rather general graphs, characterised by potentially having multiple top nodes (roots) and not necessarily being connected, since not every token is necessarily a node in the graph. The semantic frameworks include representations with varying levels of "anchoring" to the input string (Oepen et al., 2020), ranging from the so-called "bi-lexical" representations where every node in the graph corresponds to a token in the input string to a framework like AMR (Banarescu et al., 2013) which constitutes the most abstract and unanchored type of framework, such that the correspondence between the nodes in a graph and tokens in the string is completely flexible. This allows for straightforward representation of nesting and overlapping structures, where multiple nodes may be anchored to overlapping sub-strings. There have been considerable progress in developing variants of both transitionbased and graph-based dependency parsers capable of producing such semantic graphs (Hershcovich et al., 2017;Dozat and Manning, 2018;Samuel and Straka, 2020). Previous research has further made use of AMR-based input representations to constrain the tasks of event extraction ) and more recently joint information extraction (Zhang and Ji, 2021), where an off-the-shelf AMR parser is used to derive candidate enitity and event trigger nodes before classifying pairwise relations guided by the AMR hierarchical structure. While there are clear parallels between the MRP semantic frameworks and the tasks proposed in IE, little work has focused on the direct application of MRP parsing techniques to these tasks. You et al. (2022) is a notable exception in this respect, who presents an adaptation of the PERIN semantic parser (Samuel and Straka, 2020) to the event extraction task. While their work is promising it is limited to only one dataset (ACE05), which does not contain a lot of nested structures and is further limited to English event extraction only. In this work we extend their approach to the task of joint information extraction, covering both entities, events and relations taken from two different datasets in several versions and for three languages, and further demonstrates the effectiveness of approaching general information extraction from text via graph-parsing and the interpolation of different IE tasks. Task and Data While the main focus of this work is on event extraction, we hypothesize that our graph-based approach lends itself to dealing with two challenging aspects of current research on this task: the processing of nested and overlapping event structures, and the joint modeling of inter-related IE structures. In the following we quantify the level of nesting in two widely used datasets which contain rich annotations for both entities, events, and relations. We further propose two versions of each dataset with varying potential for nesting, which allows us to focus on this aspect during evaluation. Event Extraction is the task of extracting events into structured forms, namely event triggers and their arguments. An event trigger is the word(s) that most clearly describes an event, such as "buy", which evokes a transferownership and an transfermoney event in Figure 1. Event arguments are the participants and attributes of an event, and can be tagged as entities at the same time, as demonstrated in Figure 2. We use the benchmark datasets ACE05 (Doddington et al., 2004) 2015), both containing consistent annotations for entities, relations, and events, for joint evaluation of multiple IE tasks and in multiple languages (ACE05 in English and Chinese, and ERE in English, Chinese, and Spanish). Table 1 summarizes the relevant statistics of the datasets. The inventory of event types, argument roles, entity types and relation types are listed in Table 2. Despite targeting the same IE tasks, from ACE05 to Rich ERE, the annotation guidelines have shifted towards more sophisticated representations, resulting in more complex structures in Rich ERE (Song et al., 2015). Prominent differences between ACE05 and Rich ERE are: • Entities, and hence event arguments, are more fine-grained in Rich ERE, with 15 entity types, as compared to 7 types in ACE05. In terms of entity spans, ACE05 explicitly marks the head of the entity versus the entire mention, providing the possibility of solving a simpler task for entity extraction and recognizing only the head token as opposed to the full span of the entity in question. This is commonly done for this task in previous work of EE. However, in Rich ERE, the entire string of text is annotated for entity mentions, and heads are only marked explicitly for nominal mentions that are not named entities or pronominal entities. • Event triggers can be double-tagged in Rich ERE, namely one trigger can serve multiple event mentions, giving rise to overlapping events, as shown in Figure 1, while in ACE05, an event trigger only evokes one event. This means that Rich ERE presents a more complex task of event extraction. We measure the nested instances in ACE05 and Rich ERE as a way to showcase different levels of complexity for extracting entities, relations, and events. More specifically, we quantify nested instances in two versions of each dataset, one using only the head of an entity mention (when it is annotated), and the other with the entire mention text. Following Lin et al. (2020) Rich ERE-E + , and introduce two additional versions of the datasets, dubbed, ACE-E ++ and Rich ERE-E ++ which retain the full annotated mention text span. Nesting is measured between any pair of triggers and entities. Note that our notion of nesting subsumes both overlapping and nested target/entities (Cao et al., 2022), i.e. both full and partial overlap of text spans. As shown in Table 3, Rich ERE features many cases of nested triggers, while these are not found in ACE05, due to the aforementioned double-tagging in Rich ERE (see Figure 1); when only considering the head of an entity, ACE05 exhibits very little nesting, but Rich ERE exhibits a considerable amount of nesting within entities, as well as between entity-trigger. The reason for this is that in Rich ERE, only certain nominal mentions are marked with explicit heads; when the full entity mentions are considered, both datasets are heavily nested. As mentioned above, this work deals with three IE tasks, as exemplified by Figure 2: entities, relations, and events. Given a sentence, our JSEE-Graph framework extracts its entity mentions, relations, and event mentions. In addition to event extraction, we thus target two additional IE tasks in our graph-based model: Entity Extraction is to identify entity mentions from text and classify them into types according to a pre-defined ontology. For example, in Figure 2, "district" is an organization (ORG) entity. Relation Extraction aims to assign a relation type to an ordered pair of entity mentions, based on a pre-defined relation ontology. For example, in Figure 2, the relation between PER "officials" and ORG "district" is orgaffiliation. Graph parsing framework Our JSEEGraph framework is a text-to-graph parser tailored for EE tasks, additionally with different IE components explicitly encoded in a single graph, as shown in Figure 2. Our framework builds on Samuel and Straka (2020) who developed the PERIN parser in the context of Meaning Representation Parsing (Oepen et al., 2020), as well as (You et al., 2022) who applied PERIN to the task of event extraction. We here extend this parser to the IE graphs shown in Figure 2 in a multilingual setting. Given a sentence, as the example shown in Figure 3, JSEEGraph encodes the input tokens with the pre-trained language model XLM-R (Conneau et al., 2020) to obtain the contextualized embeddings and further maps the embeddings onto queries; nodes (triggers and entities) are predicted by classifying the queries and anchored to surface tokens via a deep biaffine classifier (Dozat and Manning, 2017); edges are constructed between nodes with two biaffine classifiers, assigning arguments to predicted events and relations to entity pairs. We describe each module in detail in what follows. Sentence encoding We use XLM-R (Conneau et al., 2020) to obtain the contextualized embeddings of the input sequence. To be specific, a trainable weight w l is used to get a weighted sum of representations of different layers, so the final contextual embedding e = L l=1 softmax(w l )e l with e l as the intermediate output from the l th layer. If an input token consists of multiple subwords, the final contextual embedding will be the weighted sum over all subword embeddings with a learned subword attention. Each contextual embedding is mapped into q = {q 1 , · · · , q n } queries via a linear layer, and further transformed into hidden features h = {h 1 , · · · , h n } with a stack of transformer encoder layers, which models inter-query dependency with multi-head self-attention. Node prediction The node prediction module consists of a node label classifier and an anchor biaffine attention classifier. Node anchoring, as shown in Equation (1), is performed by biaffine attention (Dozat and Manning, 2018) between the contextual embeddings e and hidden feature of queries h, to map each query (a candidate node) to surface tokens, as shown in Equation (3). For each query, every input token is binary classified into anchor or non-anchor. Node prediction is complete with queries that are classified into nodes and anchored to corresponding surface tokens. Predicted nodes are either event triggers or entities, labeled as "trigger" or entity type. A dummy node is randomly generated to add to predicted nodes to play the role of <root> node, and always holds the first position. Edge prediction Edge prediction between nodes is performed with two deep biaffine classifiers, as in Equation (6), one to predict edge presence between a pair of nodes and the other to predict the corresponding edge label. To construct edges between nodes, only queries from which nodes have been constructed will be used, and the new hidden features is h ′ , which are further split into two parts with a singlelayer FNN, as show in Equation (4) and (5). The edge presence biaffine classifier performs binary classification, deciding whether or not an edge should be constructed between a pair of nodes. The edge label biaffine classifier performs multiclass classification, and the edge label set is the union of argument roles and relation types. Constrained decoding During inference, we apply a set of constraints specifically developed for the correct treatment of event arguments and entity relations based on the graph encoding we define for the information graph ( Figure 2): 1) directed edges from the <root> node can only connect to a trigger node, and the corresponding edge label is an event type; 2) directed edges from a trigger node to an entity indicates an event argument, with the argument role placed as edge label; 3) directed edges between a pair of entities indicate an entity relation, and the corresponding relation type is assigned to the edge label. 120 5 Experimental setup Data As mentioned above, we evaluate our system on the benchmark datasets ACE05 4 (LDC2006T06) and Rich ERE 5 (LDC2020T18). As mentioned above, Table 1 summarizes the statistics of the preprocessed datasets. Following Lin et al. (2020), we keep 33 event types, 22 argument roles, 7 entity types, and 6 relation types for both the English and Chinese parts of ACE05. We follow You et al. (2022) in employing the ACE-E ++ version of this data, which uses the full text span of entity mentions instead of only the head, as described in section 3 above. For Rich ERE, we keep 18 out of 38 event types defined in the Rich ERE event ontology 6 , 18 out of 21 argument roles 7 , 15 entity types, and 6 relation types for English, Chinese, and Spanish. Given no existing data splits, we randomly sample similar proportions of documents for train, development, and testing as the split proportions in ACE05. • Entity An entity mention is correctly extracted if its offsets and entity type match a reference entity. • Relation A relation is correctly extracted if its relation type, and offsets of both entity mentions match those of reference entities. • Event trigger An event trigger is correctly identified (Trg-I) if its offsets match a reference trigger, and correctly classified (Trg-C) if its event type also matches a reference trigger. • Event argument The evaluation of an argument is conditioned on correct event type prediction; if a predicted argument plays a role in an event that does not match any reference event types, the argument is automatically considered a wrong prediction. An argument is 4 https://catalog.ldc.upenn.edu/ LDC2006T06 5 https://catalog.ldc.upenn.edu/ LDC2020T18 6 The Rich ERE event ontology defines 38 event types, but for Chinese and Spanish data, only 18 event types are annotated. For consistency, we also use the same 18 event types for the English part. 7 3 argument roles for the reduced event types are thus excluded. correctly identified (Arg-I) if its offsets match a reference argument, and correctly classified (Arg-C) if its argument role also matches the reference argument. Implementation detail We adopt multi-lingual training for each dataset for the reported results. Results of monolingual models are listed in Appendix B. Detailed hyperparameter settings and runtimes are included in Appendix A. System comparison We compare our JSEEGraph to the following systems: 1) ONEIE (Lin et al., 2020); 2) GraphIE (Nguyen et al., 2022); 3) FourIE (Nguyen et al., 2021); 4) JMCEE (Xu et al., 2020); 5) EventGraph (You et al., 2022) on the ACE05 dataset. For Rich ERE there is little previous work to compare to; the only previously reported results for EE only solve the task of argument extraction, using gold entity and trigger information, hence their work is not included in our system comparison. Results and discussion We here present the results for our JSEEGraph model for the EE task, as well as its performance for the additional IE components: entities and relations, evaluated as described above. We further zoom in on the nested structures identified in Section 3 and assess the performance of our system on these rich structures which have largely been overlooked in previous work on event extraction. We go on to assess the influence of inter-related IE components in an ablation study. Finally we provide an error analysis of our model's predictions. Overall performance As shown in Table 4, on ACE-E + , our overall results align with other systems. Our JSEEGraph results are especially strong for event argument extraction, with an improvement of around 10 percentage points from the best results of the previous best performing systems in our comparison. On the newly introduced ACE-E ++ , despite having more complex structures, with a higher degree of nested structures, the results of JSEEGraph on trigger extraction remain stable. We further note that our results on argument, entity, and relation extraction suffers some loss from highly nested entities, which is not surprising. Model Trg-I Trg-C Arg-I Arg-C Entity Relation From Table 5, we find that the scores on Rich ERE are consistently lower compared to those of ACE05. The double-tagging of event triggers described in Section 3 clearly pose a certain level of difficulty for the model to disambiguate events with a shared trigger. Argument and entity extraction also suffers from more fined-grained entity types. Nesting In order to directly evaluate our model's performance on nested instances, we split each test set into nested and non-nested parts and report the corresponding scores, as shown in Table 6 8 . We observe that JSEEGraph is quite robust in tackling nested instances across different IE tasks and languages. On ACE05-E ++ , more than half of the test data are nested for both English and Chinese, and the results on the nested parts are lower, however consistently comparable with the non-nested parts of the datasets. On Rich ERE-E + , nested instances make up only a small part of 8 ACE05-E + is not included as it lacks sufficient nested instances. Lang Nested #sents Trg-I Trg-C Arg-I Arg-C Entity Relation To conclude, JSEEGraph does not suffer considerable performance loss from nesting among different IE elements, and in many cases actually gains in performance from more complex structures, notably for trigger, entity, and relation extraction. It is clear that the system can make use of inter-relations between the different IE elements of the information graph in order to resolve these structures. Ablation study In order to gauge the effect of the joint modeling of entities, events, and relations, we perform an ablation study where we remove the entity and relation information from our information graph, hence only performing the task of event extraction directly from text. In the reduced information graph, node labels for entity types are removed, and relation edges between entities are also removed. We find that event extraction clearly benefits from entity and relation extraction, especially for event argument extraction. As shown in Table 4 and Table 5, when we train our model only for event extraction, the performance on argument extraction drops consistently across different datasets and languages, but the performance on trigger extraction remains quite stable. Error analysis The experimental results show that JSEEGraph has an advantage when it comes to the task of argument extraction. In a manual error analysis we therefore focus on the errors of event trigger extraction. After a manual inspection of our model's predictions on the test data, we find that the errors fall into the following main categories. Over-predict non-event sentences. Our system tends to be more greedy in extracting event mentions, and wrongly classifies some tokens as event triggers even though the sentence does not contain event annotation. For instance, the sentence "Anne-Marie will get the couple's 19-room home in New York state" (from ACE05) does not have annotated events, but our system extracts "get" as trigger for a Transfer-Ownership event; in this case, however, one could argue that the Transfer-Ownership should be annotated. Under-predict multi-event sentences When a sentence contains multiple event mentions, JSEE-Graph sometimes fails to extract all of the event triggers. For example, this sentence "Kelly , the US assistant secretary for East Asia and Pacific Affairs , arrived in Seoul from Beijing Friday to brief Yoon, the foreign minister" from ACE05 contains a Transport event triggered by "arrived" and a Meet event triggered by "brief", but our system fails to extract the trigger for the Meet event; in this example, it requires a certain level of knowledge to be able to identify "brief" as an event trigger, which is beyond the capacity of our model. Wrong event types In some cases, even though our model successfully identifies an event trigger, it assigns a wrong event type. Some event types can easily be confused with each other. In this sentence from Rich ERE, "The University of Arkansas campus was buzzing Friday after a student hurt himself when a gun went off in his backpack in the KUAF building", an Injure event is evoked by "hurt", but our model assigns an event type of Attack. Clearly, Injure and Attack events are one typical case of event types that can be easily confused. Context beyond sentence This error applies specifically to Rich ERE: even though the annotation of events is on a sentence level, annotators were instructed to take into account the context of the whole article. Our model fails completely when a trigger requires context beyond the sentence. For instance, this sentence "If Mickey can do it , so can we!" is taken from an article describing an on-going demonstration in Disney Land, and "it" is the trigger for a demonstrate event; without the context, our model fails to identify the trigger. These are cases which would require information about event coreference. Conclusion In this paper, we have proposed JSEEG, a graphbased approach for joint structured event extraction, alongside entity, and relation extraction. We experiment on two benchmark datasets ACE05 and Rich ERE, covering the three languages English, Chinese, and Spanish. We find that our proposed JSEEGraph is robust in solving nested event structures, and is especially strong in event argument extraction. We further demonstrate that it is beneficial to jointly perform EE with other IE tasks, and event argument extraction especially gains from entity extraction. Limitations Our work has two main limitations. Firstly, we do not compare our system to previous works on the Rich ERE dataset. This is mainly due to the fact that most work use the light ERE (Song et al., 2015) dataset. We were unfortunately not able to got access to this version of the data 9 , which is why no experiments were carried out on it. Secondly, we only experiment with one language model, the multilingual model XLM-R. As our model is language agnostic, and we aimed to test its performance on datasets in different languages, the choice of a multilingual model was obvious. XLM-R has been chosen based on its good performance in other tasks, and to make our work comparable to previous work (You et al., 2022). However, another approach would be to test our model with a selection of language-specific language models. Media Technology and Innovation, through the centers for Research-based Innovation scheme, project number 309339. Lang Trg-I Trg-C Arg-I Arg-C Entity Relation B Monolingual training results Apart from multilingual training, we also train two monolingual models for each language, one for joint event extraction with entity and relation and the for event extraction only. Results of monolingual models are summerized in Table 9.	2023-06-27T01:01:30.023Z	2023-06-26T00:00:00.000	{ "year": 2023, "sha1": "817322a14e12d4bfa528d0ad5229e4778bf5eb96", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "ACL", "pdf_hash": "84c8f3d6310b9553b466c5538a7726acb18e5f74", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Computer Science" ] }
76662758	pes2o/s2orc	v3-fos-license	Integrated Analyses of Phenotype and Quantitative Proteome of CMTM4 Deficient Mice Reveal Its Association with Male Fertility* CMTM4 is aberrantly expressed in spermatozoa from infertility patients and testes from elderly adults and NOA patients. Cmtm4 KO mouse was generated to study its involvement in male fertility in vivo. Integrative phenotypic and quantitative proteomics analyses reveal an association of CMTM4 with histone-to-protamine exchange, sperm motility and induction of the acrosome reaction, and demonstrate that CMTM4 is associated with spermatogenesis and sperm quality. Graphical Abstract Highlights CMTM4 is associated with human spermatogenesis and sperm quality. Cmtm4 knockout mouse were generated by CRISPR/Cas9 technology for male fertility research. CMTM4 is required for male fertility but not female fertility. Phenotype and quantitative proteomics of Cmtm4 KO mice reveal an association of CMTM4 with histone-to-protamine exchange, sperm motility and induction of the acrosome reaction. The chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing family (CMTM) is a gene family that has been implicated in male reproduction. CMTM4 is an evolutionarily conserved member that is highly expressed in the testis. However, its function in male fertility remains unknown. Here, we demonstrate that CMTM4 is associated with spermatogenesis and sperm quality. Using Western blotting and immunohistochemical analyses, we found CMTM4 expression to be decreased in poor-quality human spermatozoa, old human testes, and testicular biopsies with nonobstructive azoospermia. Using CRISPR-Cas9 technology, we knocked out the Cmtm4 gene in mice. These Cmtm4 knockout (KO) mice showed reduced testicular daily sperm production, lower epididymal sperm motility and increased proportion of abnormally backward-curved sperm heads and bent sperm midpieces. These mice also had an evident sub-fertile phenotype, characterized by low pregnancy rates on prolonged breeding with wild type female mice, reduced in vitro fertilization efficiency and a reduced percentage of acrosome reactions. We then performed quantitative proteomic analysis of the testes, where we identified 139 proteins to be downregulated in Cmtm4-KO mice, 100 (71.9%) of which were related to sperm motility and acrosome reaction. The same proteomic analysis was performed on sperm, where we identified 3588 proteins with 409 being differentially regulated in Cmtm4-KO mice. Our enrichment analysis showed that upregulated proteins were enriched with nucleosomal DNA binding functions and the downregulated proteins were enriched with actin binding functions. These findings elucidate the roles of CMTM4 in male fertility and demonstrates its potential as a promising molecular candidate for sperm quality assessment and the diagnosis or treatment of male infertility. The human chemokine-like factor (CKLF) 1 -like MARVEL transmembrane domain-containing (CMTM) family is a gene family encoding proteins that link classical chemokines and the transmembrane 4 superfamily (TM4SF). In humans, the nine members include CKLF and CMTM1-8, which encode proteins that play important roles in the immune system, tumorigenesis, and the male reproductive system (1,2). Our previous studies have reported that CMTM3, CMTM4, CMTM5, and CMTM7 function as tumor suppressors in the development and progression of carcinomas (3)(4)(5)(6). CMTM3 and CMTM7 colocalize with RAB5 in early endosomes and facilitate epidermal growth factor receptor (EGFR) internalization and degradation by enhancing RAB5 activity and early endosome fusion (7,8). CMTM3 and CMTM4 mediate cell-cell adhesion by involvement in VE-cadherin turnover, and this process is involved in the regulation of angiogenesis (9,10). CMTM3 and CMTM7 also initiate B-cell linker (BLNK)-mediated signal transduction (11,12). CMTM6 and CMTM4 have been identified as programmed death-1 (PD-L1) regulators that inhibit immune function (13,14). These studies showed that the CMTM family has important regulatory effects on the trafficking, degradation, and signal transduction of membrane molecules. Interestingly, CMTM1, CMTM2, and CMTM4 are highly expressed in the human testis, implying biological roles in male reproduction (2). CMTM1 is predominantly expressed in the human testis, with at least 23 alternative splicing isoforms (15). However, Cmtm1 knockout (KO) has no significant influence on male fertility (16). CMTM2 is highly expressed in the testis and is closely correlated with spermatogenic defects (17,18). Its two homologs in the mouse, Cmtm2a and Cmtm2b, serve as androgen receptor corepressor and enhancer, respectively (19 -22). Coexpression of Cmtm2a and Cmtm2b is essential for male fertility in mice (16). These findings indicate that CMTM family members may play important roles in spermatogenesis or testicular development. CMTM4 is the most conserved member of the CMTM family, and forms a gene cluster with CKLF and CMTM1-3 on chromosome 16q22.1 (2). Our previous studies showed higher expression of CMTM4 in the testis than in other tissues (2,23), which warrants exploration of its significance in male reproduction. Given its sequence structure and expression characteristics, CMTM4 might also play crucial roles in male fertility as do CMTM2 (16,17). Previous studies have indicated that CMTM4 acts as a tumor suppressor through its involvement in cell growth and cell cycle regulation (4,23,24). However, its roles in male reproduction remain unknown. In the present study, we first assessed the expression of CMTM4 in the spermatozoa and testes of patients with male infertility to characterize its association with spermatogenesis and sperm quality. Because the amino acid sequences are highly homologous between human and mouse CMTM4, the functions of CMTM4 in male fertility were examined in a KO mouse model, and the underlying mechanism was investigated using isobaric tags for relative and absolute quantification (iTRAQ)-based proteomics. Consistent with the association of CMTM4 expression with sperm quality in patients, Cmtm4 KO mice showed male subfertility with phenotypes of decreased sperm motility and aberrant acrosome reaction. Gene ontology (GO) term analysis revealed that proteins downregulated in KO mice testis and spermatozoa compared with wild-type (WT) were mainly involved in spermatogenesis and sperm functions including motility, the acrosome reaction, and histone-to-protamine exchange. This study also provided in-depth proteomic mapping of the mouse testis and sperm that will facilitate to understand of spermatogenesis and sperm functions. Combining phenotypic characteristics and proteomic analyses of Cmtm4 KO mice, we have shown that CMTM4 plays key roles in regulating sperm function and male fertility by affecting sperm motility and the acrosome reaction. EXPERIMENTAL PROCEDURES Ethical Statement-The present study was approved by the Medical Ethical Committee of the YuHuangDing hospital and all participants provided written informed consent. All experiments were performed in accordance with the guidelines provided by YuHuangDing hospital. All animal experiments were carried out in according with the guidelines of the care and use of laboratory animals. All mice were kept under light/dark cycles of 12/12 h with free access to food and water. Sample Preparation-Samples of human semen were collected from the YuHuangDing hospital, and were classified into four groups without leukocytospermia as follows: the normozoospermia group (24 -37 years old, sperm count Ͼ39 ϫ 10 6 , progressive motile spermatozoa Ͼ40%), asthenozoospermia group (27-36 years old, progressive motile spermatozoa Ͻ32%), oligoasthenozoospermia group (27-39 years old, sperm count Ͻ39 ϫ 10 6 , progressive motile spermatozoa Ͻ32%), and teratozoospermia (27-40 years old, normal morphology Ͻ4%). Young testes were obtained from five young fathers (23-27 years old) who died in car accidents, had no history of pathology that could affect reproductive function, and had previously indicated a willingness to donate their bodies to medical research. Donations of their organs for medical research were approved by their immediate family members. Aged testes samples were obtained from five elderly fathers (70 -74 years old) who were prostate cancer patients with no anti-androgen treatment before orchiectomy and who had given written informed consent. All procedures were approved by the Ethics Committee of YuHuangDing Hospital. A total of 25 patients with azoospermia (26 -37 years old) who underwent surgical testicular sperm extraction were recruited and divided into the obstructive azoospermia (OA) group (n ϭ 5) and the nonobstructive azoospermia (NOA) group (n ϭ 20). In OA, spermatozoa were produced normally inside the testicle, whereas in NOA, spermatogenesis problems were observed with a very low level of sperm production or a total lack of production. Patients with abnormal karyotypes and those who had previously suffered from an injury to the genitals were excluded. Human testicular quality was evaluated by the modified Johnsen score according to our previous studies (25,26). Protein Extraction-Human sperm samples exhibiting normozoospermia, asthenozoospermia, oligoasthenozoospermia, and teratozoospermia were collected for protein extraction. As in our previous reports (26), after centrifugation at 800 ϫ g for 20 min at 4°C in a 50% step Percoll gradient in phosphate buffered saline (PBS) media, the seminal plasma and other contaminating cells in semen were removed. Sperm pellets were dissolved in lysis solution and sonicated for three times at 5 s intervals. Then they were kept at 4°C for 2 h before the solution was centrifuged at 12,000 ϫ g for 45 min, and the supernatant was collected. After determination of protein concentration, protein samples were stored at Ϫ80°C. Assessment of KO Mouse Fertility and Fecundity-To assess fertility and fecundity, one littermate male (6 weeks old) was placed in cages with two mature WT females for two months or more. Two littermate females were caged with a WT fertile male for the same period. The number of female mice achieving pregnancy and the number of offspring were recorded. Mouse sperm suspensions were prepared by mincing the cauda epididymidis of a mature male mouse (WT or KO) in 1 ml IVF medium for about 1 h. Part of this sperm suspension was divided into 30 l pellets using a micropipette in a plastic culture dish (35 mm ϫ 11 mm) covered with paraffin oil, and the rest was treated by 10 M calcium ionophore A23187 (Sigma-Aldrich, St. Louis, MO) for assessing the induction of the acrosome reaction. Mature female mice (WT or KO) were induced to super-ovulate by i.p. injection of 10 i.u. pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG) 48 h apart. The oocytes were recovered from the oviducts 14 h after injection of hCG and were introduced into each sperm suspension. The sperm concentration was counted by using improved-Neubauer counting chamber, and at least 200 cells were counted. About 10 6 spermatozoa/ml was used for insemination. The development stage and morphology of embryos were recorded at 24 h interval after zygote collection. Evaluation of KO Mouse Sperm Quality-Epididymal spermatozoa was collected from the caudal part of the epididymis. The cauda epididymidis was placed in PBS supplemented with 10% (w/v) bovine serum albumin (BSA) and cut into small pieces followed by incubation on a warming metal plate at 35°C for 5 min. Sperm motility was measured with Computer-aided Semen Analysis system (CASA) System (Medealab™, Erlangen, Germany). For each sample, 12 fields were examined. To evaluate the acrosome reaction, spermatozoa were suspended in Biggers-Whitten-Whittingham (BWW) and incubated at 37°C, 5% CO 2 for 3 h to get capacitation. Capacitated sperm was exposed to vehicle alone (DMSO, dimethyl sulfoxide) or calcium ionophore A23187 (10 M) for 30 min, subjected to Coomassie brilliant blue staining (2% w/v G250) for 3 min, and then washed with PBS three times before mounting on slides for observation. Four hundred spermatozoa were evaluated under a light microscope (ϫ400). Spermatozoa were scored as acrosome-intact when a bright blue staining was observed in the dorsal region of the head or as acrosomereacted when no labeling was observed. Daily Sperm Production (DSP)-DSP was estimated according to a previous report (28). Briefly, after the testes were crushed in 600 l SMT solution containing 0.05% (v/v) Triton X-100, 0.9% NaCl and 0.01% Azide by ultrasonic pulverizer, the solution was homogenized again. Aliquots of 10 l were placed on a makler counting chamber and sperm heads were counted twice under microscopic visualization to determine the average number of spermatids per sample. Sperm heads in 80 small squares were assessed. These values were divided by testicular weight to give the number of spermatids per gram of testis. Because developing spermatids spend 4.84 days in steps 14 -16 of spermatogenesis, the number of spermatids per gram of testis was divided by 4.84 to provide the DSP. iTRAQ Labeling of Mouse Testicular Samples-The iTRAQ procedures were described in previous publication (29,30). Briefly, the testicular or spermatozoa protein samples from the wild and Cmtm4 KO mice were separately pooled. The pH was adjusted to 8.5 by adding 25 l triethylammonium bicarbonate (TEAB) buffer and samples were treated with 20 mM dithiothreitol (DTT) at 56°C for 60 min and 50 mM iodoacetamide in dark for 30 min. After digestion with 3 g trypsin (sequencing grade, Promega, Madison, WI) at 37°C overnight, the peptides were labeled with iTRAQ tags and dried. 2-D Reversed-phase (RP) Separation and Mass Spectrometer Data Acquisition-The first dimension RP separation by microLC was performed by using a Durashell RP column (5 m, 150 Å, 250 mm ϫ4.6 mm i.d., Agela, Tianjin, China). Mobile phases A (2% v/v acetonitrile, 20 mM ammonium formate, adjusted to pH 10.0 with NH 4 OH) and B (98% acetonitrile, 20 mM ammonium formate, adjusted to pH 10.0 using NH 4 OH) were used to develop a gradient. The peptides were eluted over 45 min by a gradient of 100% buffer A to 100% buffer B at a flow rate of 800 l/min. In total, 10 fractions were generated for further LC-MS/MS analysis, for which a nanoflow HPLC instrument (EASY-nLC 1000 system, Thermo Fisher Scientific, Waltham, MA) coupled to an on-line Q Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA) with a nanoelectrospray ion source (Thermo Fisher Scientific) was used. The chromatography columns were packed in-house with Ultimate XB-C18 3 m resin (Welch Materials, Shanghai, China). The peptide mixtures were loaded onto the C18 RP column (10-cm length, 75 m inner diameter) with buffer A (99.5% water and 0.5% formic acid, v/v) and separated with a 75-min linear gradient of 5-100% buffer B (99.5% acetonitrile and 0.5% formic acid, v/v) at a flow rate of 300 nl/min. Including the loading and washing steps, the total time for an LC-MS/MS run was 90 min. The electrospray voltage was 2.0 kV. Peptides were analyzed by data-dependent MS/MS acquisition with a dynamic exclusion duration of 18 s. In MS1, the resolution was 70,000, the automatic gain control (AGC) target was 3e6, and the maximum injection time was 20 ms. In MS2, the resolution was 17,500, the AGC target was 1e6, and the maximum injection time was 60 ms. The scan range was 300 -1400 m/z, and the top 75 intensive precursor ions were selected for MS/MS analysis. Identification and Quantification of Mouse Testicular or Spermatozoa Proteins-The raw data were processed by using the proteomic workflow of Proteome Discoverer 1.3. The fragmentation spectra were searched against the UniProt reviewed mouse database (2017_12, 16950 sequences) by the Mascot search engine (version 2.2.06) with the precursor and fragment mass tolerances set to 15 ppm and 20 milli-mass units (mmu), respectively. The algorithm was set to use trypsin as the enzyme, allowing for two missed cleavage sites. The fixed modification was carbamidomethylation (cysteine), and the variable modifications were oxidation (methionine), acetylation (protein N terminus), and iTRAQ labeling (tyrosine and lysine, N-terminal residues). Peptide ions were filtered from the cut-off scores of Percolator based on p Ͻ 0.01. The false discovery rate was set to 1% for peptide identifications. An additional filter was applied with the removal of spectrum matches with scores lower than 10. The iTRAQ quantization values were automatically calculated based on the intensity of the iTRAQ reporter ions in the dissociation scans with higher collision energy using Proteome Discoverer. All protein iTRAQ ratios were exported to an Excel file, the Gaussian distribution of ratios was recalculated manually, and all ratios were transformed to base 10 logarithm values. A confidence interval of 99% (testis proteome, 0.754 -1.24; sperm proteome, 0.84 -1. 19) was used to determine the cut-off values for statistically significant changes. Bioinformatics-Broad functional classification of molecular function and biological process were performed by using Protein Analysis Through Evolutionary Relationships (PANTHER) tools (http://www. pantherdb.org/). The over-representation analyses of GO (http:// www.geneontology.org/) for main biological processes were performed by using the Database for Annotation, Visualization and Integrated Discovery (DAVID) tools (https://david.ncifcrf.gov/). The GO level 2 and 3 categories and a p value cut-off of 0.01 were selected. Knockout and mutant phenotypes related to male reproduction and fertility were obtained by batch query from Mouse Genome Informatics (http://www.informatics.jax.org). Western Blotting-For protein separation, 50 g proteins from each sample were loaded on 12% gels for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS)-PAGE. This was transferred to polyvinylidene difluoride membranes at 100 v for 1 h, blocked with 5% (w/v) skimmed milk for 1 h, and incubated with primary antibodies (diluted 1:800 in blocking solution) (supplemental Table S1) at 4°C overnight with gentle agitation. The membranes were washed with 0.5% (v/v) Tween-20 in Tris-buffered saline (TBS) three times and incubated with horseradish peroxidase (HRP)-conjugated anti-IgG for 1 h at room temperature (RT). The immune-reactive complexes were detected by an enhanced chemiluminescence (ECL) kit (Amersham Biosciences Life Science, Cleveland, OH). The images were analyzed by commercial image analysis software (Gene Tools, version 4.02; Syngene, Cambridge, UK). The integrated optical density (IOD) of positive immune-staining was calculated, and the IOD ratio of target protein to housekeeping proteins was used to express the results of the Western blot analysis. Immunohistochemistry (IHC)-Testicular tissue blocks were fixed in Bouin's solution for 12 h, and then embedded in paraffin according to conventional methods. Sections of 4 m thickness were de-waxed. Antigen retrieval was performed in a microwave oven above 94°C for 20 min. Endogenous peroxidases of sections were inhibited by incubation with 3% (v/v) H 2 O 2 for 10 min. Then, 3% (v/v) BSA in TBS was used to block nonspecific binding with antibodies at RT for 1 h. Sections were then incubated with corresponding primary antibodies (diluted 1:100 in blocking solution) overnight at 4°C. After washing three times with TBS, the sections were incubated with horseradish peroxidase-conjugated IgG (Zhong-Shan Biotechnology, Beijing, China) at a final dilution of 1:400 for 1 h at 37°C. A 3,3Ј-diaminobenzidine (DAB) kit (Zhong-Shan Biotechnology, Beijing, China) was used to visualize peroxidase activity at the binding sites. Hematoxylin was used to counterstain the sections. After dehydration, the sections were mounted for bright-field microscopy (DM LB2, Leica, Nussloch, Germany). Preimmune IgG was used as a negative control. Image-Pro analysis software was used to analyze the images of immunostained sections. Immunofluorescence Analysis-The mouse spermatozoa were smeared on slides precoated with 1% (w/v) gelatin. The air-dried slides were fixed with cold methanol for 10 min, blocked for 1 h at room temperature with 3% (w/v) BSA in PBS and incubated at 4°C overnight with primary antibody against CMTM4, ODF2 (diluted 1:50 in 3% BSA). After three washes with PBS, the anti-rabbit IgG (Alexa Fluor® 488 Conjugate) (1:100 in 3% BSA) was applied and incubated at room temperature for 1 h; rabbit IgG was used as a negative control. Samples were subsequently washed with PBS. Propidiumiodide (0.01 mg/ml, Invitrogen, Carlsbad, CA) counterstaining visualized the nuclei and sections were mounted in 80% (v/v) glycerol. Quantitative assessment of protein expression in spermatozoa was achieved by a Zeiss LSM 510 laser confocal microscope (LSM, Carl Zeiss Microimaging, Thornwood, NY). Slides were systematically examined at 400ϫ magnification according to the World Health Organization manual 2010. Inspection was performed in sequence until a total of 200 spermatozoa had been assessed. By using Zeiss LSM510 Meta software (LSM5 version 3.2, Carl Zeiss Microimaging, Thornwood, NY), the fluorescence intensity value per stained sperm was calculated automatically by subtracting the background fluorescence intensity, which was determined by scanning a sperm-free area. Experimental Design and Statistical Rationale-The expression of CMTM4 in spermatozoa from normozoospermic, asthenozoospermic, oligoasthenozoospermic, and teratozoospermic men was evaluated by Western blotting. Its cellular localization and expression intensity in testis from young adult, elderly adult, and NOA patients were detected by IHC. Cmtm4 KO mice were constructed to investigate CMTM4 function in male infertility. The testis proteomes, epididymal sperm proteome from Cmtm4 WT and KO mice were compared by iTRAQ, each group was a pool from 10 mice and performed in 2 technical replicates. The MS data were processed by using Proteome Discoverer 1.3 and searched against the UniProt (2017_12, 16950 sequences) with the Mascot search engine (version 2.2.06). The false discovery rate was set to 1% for peptide identifications. Data are reported as meansϮS.D. Means of two groups were analyzed with Student's t test, and more than two group means were analyzed by one-way analysis of variance (ANOVA). A commercial software package (SPSS 18.0; SPSS, Chicago, IL) was used to perform the correlation analysis. A value of p Ͻ 0.05 was statistically significant. Association of CMTM4 with Human Spermatogenesis and Sperm Quality-Western blots were performed on protein extracts from the spermatozoa of fertile and infertile men. Spermatozoa from patients diagnosed with asthenozoospermia, oligoasthenozoospermia, or teratozoospermia exhibited obvious poor sperm quality with decreased sperm motility or sperm count (Fig. 1A) (supplemental Table S2). As shown in Fig. 1C-1D, the expression of CMTM4 in spermatozoa from asthenozoospermic, oligoasthenozoospermic, and teratozoospermic men was significantly lower than that in spermatozoa from normazoospermic men. Especially, significantly decreased expression of CMTM4 was observed in spermatozoa from teratozoospermic patients. An analysis of gene expression omnibus (GEO) data from GSE6968, which shows gene expression profiles of normazoospermic and teratozoospermic spermatozoa, demonstrated significant downregulation of CMTM4 in teratozoospermic spermatozoa (Fig. 1B). To further explore the association of CMTM4 with spermatogenesis, we compared the expression levels of CMTM4 in the testes of young adults with those of elderly men, who have poor spermatogenesis associated with poor sperm quality as described in our previous study (26,31), and with NOA patients, who have impaired spermatogenesis because of Sertoli cell-only pattern (Fig. 2F), maturation arrest (Fig. 2G), and hypospermatogenesis ( Fig. 2H) (supplemental Table S2). Testes from 10 young adults (including 5 biopsies from OA patients) (score ϭ 8), 5 elderly adults (score ϭ 6), and 20 NOA patients (score ϭ 0) were evaluated as shown in Fig. 2A. Morphologically, elderly testes showed hyperplasia of the interstitial layer and vacuolization of seminiferous tubules. Compared with young adults, the testes of elderly adults and NOA patients had significantly decreased CMTM4 expression by immunohistochemical analysis (Fig. 2B). FIG. 1. Expression of CMTM4 in human spermatozoa. Human spermatozoa were collected from normozoospermic, asthenozoospermic, oligoasthenozoospermic, teratozoospermic patients and showed significant differences in progressive sperm motility (A); comparison of CMTM4 expression in spermatozoa in normozoospermia and teratozoospermia was analyzed by retrieving GEO data from GSE6968 (B); Western blot analysis of CMTM4 in human spermatozoa in normozoospermia, asthenozoospermia, oligoasthenozoospermia and teratozoospermia. The quantification of CMTM4 expression was calculated by normalizing band intensities to the respective TUBB expressions by Image Pro software (C-E). Asth, asthenozoospermia, Olig, oligoasthenozoospermia, Tera, teratozoospermia; Lines (A) indicate mean values, whiskers indicate standard deviation; Data were compared by one-way analysis of variance (ANOVA); *, p Ͻ 0.05. Cmtm4 KO Mice Were Constructed by Cas9 Microinjection-To further investigate the role of CMTM4 in spermatogenesis and male fertility, we constructed Cmtm4 KO mice using CRISPR-Cas9. A pair of independent sgRNAs targeting exon 1 of the Cmtm4 locus was designed to establish the homozygous KO mouse model. Coinjection of the two independent sgRNAs with Cas9 mRNA caused a 179-bp deletion spanning the 5Ј untranslated region (UTR) and exon 1 in the open reading frame of the Cmtm4 locus, resulting in a frameshift mutation and KO of Cmtm4 (Fig. 3A). Deletion of the Cmtm4 gene was confirmed by PCR and sequencing analyses (Fig. 3B). Western blot analysis confirmed the absence of CMTM4 protein in KO mice (Fig. 3C). IHC analysis showed that CMTM4 was mainly localized in the cytoplasm of spermatocytes and round spermatids of WT mice, whereas no signal was detected in the testis of KO mice (Fig. 3D, 3E). CMTM4 Requirement for Male and Female Fertility-All germ cells (i.e. spermatogonia, primary spermatocytes, round spermatids, elongated spermatids, and Sertoli cells) were examined in the seminiferous tubules of Cmtm4 KO mouse testes, and no obvious morphological abnormalities were found. No significant differences were observed in the testis/body weight ratio, cauda epididymal sperm content, or serum testosterone concentration between WT and KO mice (Fig. 4A-4C). DSP and progressive motility were significantly reduced in adult KO males compared with adult WT males (Fig. 4D-4G). A higher percentage of abnormal epididymal spermatozoa with backward-curved heads and bent midpieces were observed in Cmtm4 KO mice (Fig. 4H, 4K, supplemental Table S3). KO males mating with WT females produced fewer pregnancies after 2 months of continuous cohabitation. The Cmtm4 ϩ/Ϫ littermates were all fertile, and produced the same number of offspring as WT littermates. Cmtm4 KO females gestated normally when mated with WT males (Table I). We then examined the in vitro fertilization (IVF) capacities of spermatozoa obtained from the epididymides of WT and KO male mice. IVF results showed that the spermatozoa of KO mice had low fertilization abilities (Table II, Fig. 4J, 4L), implying impaired sperm function. There was no difference in the per- centage of intact acrosomes between spermatozoa from WT and KO mice, whereas a significantly lower percentage of acrosome reactions was induced by the calcium ionophore A23187 in spermatozoa from KO mice (Fig. 4I, 4M). Differentially Expressed Proteins in KO Mouse Testes Identified Through Quantitative Proteomics-To identify testicular proteins and biological processes that were associated with CMTM4 in male fertility, we performed comprehensive differ-ential proteomics analysis of WT and Cmtm4 KO mouse testes. iTRAQ was applied to compare the proteomes of whole testes from 8-week-old WT and Cmtm4 KO mice, and each group was pooled from 10 mice and performed in duplicate (Fig. 5A). A total of 5394 testicular proteins were identified (supplemental Table S5). There was an overlap of 1702(31.6%) proteins with the reported mouse germ cell proteome (32) (Fig. 5B) and 2052 proteins with the reported mouse sperm cell proteome (33) (Fig. 5C). A broad functional category classification was performed (Fig. 5D, 5E). In total, 351 proteins were differentially expressed between WT and Cmtm4 KO testes, including 212 upregulated and 139 downregulated proteins in the KO testes (supplemental Table S6). A broad functional classification showed that majority of proteins related to signal transduction (16%), actin/tubulin binding (9%), and protease/protease inhibitor (8%) functions were downregulated in KO mice, whereas more proteins involved in transport (11%) and DNA binding (9%) functions were upregulated in KO mice (Figs. 6A, 6B). Enrichment analysis indicated that most of the downregulated proteins were involved in spermatogenesis and regulation of sperm quality, such as sperm motility and the acrosome reaction (Fig. 6C), whereas the upregulated proteins were mainly involved in metabolic processes (Fig. 6D). Several well-known proteins involved in the acrosome reaction (IZUMO1, CRISP1, AKAP1, AKAP3, AKAP4, PLB1, and TRIM36) and sperm motility (ODF1, ODF2, ATP1A4, PGK2, SORD, AKAP3, AKAP4, SMCP, and GAPDHS) were downregulated in the KO mice (Fig. 6E), whereas some upregulated proteins belonged to the chromatin components involved in the DNA packaging complex (e.g. HIST1H1B, H2AFX, HIST1H3A, H1FX, and H3F3C). Using retrieved GEO data set GDS3142 that includes mouse transcriptome of different tissues, differentially expressed gene products in the KO testis was grouped into specific, high, wide, and undetectable (low) expression patterns. Notably, 100 (71.9%) of the downregulated proteins were specifically expressed in the testis (Figs. 6F, 6G). Characterization of germ cell-type specific expression of the differentially expressed proteins showed that 57% of downregulated proteins were specifically expressed in round spermatids (Figs. 6H, 6I). The proteomics results indicated that CMTM4 deficiency mainly affected the expression of proteins involved in sperm motility and the acrosome reaction. The differential expression of selected proteins (PGK2, SORD, HSPA1L, and HSPA9) was confirmed by Western blotting and IHC analyses. The IHC results indicated that PGK2 and SORD were mainly expressed in round spermatids, whereas HAPA1L and HSPA9 were mainly expressed in spermatocytes (Fig. 7). All these proteins showed downregulated expression in the KO mouse testis. In agreement with the IHC results, lower levels of these proteins were observed in the KO mice in Western blot analysis (Fig. 7). Enrichment analysis indicated that the upregulated proteins were mainly related to functions of DNA binding including histones H2A, H2B, H4 and their variants in KO sperm (Fig. 8D). The abnormal upregulated levels of H2A, H2B, H4 were validated by Western blotting. Given that nucleosomal histones were involved histone-to-protamine exchange, the expression of PRM1, PRM2 were also validated. PRM1 and PRM2 showed downregulated expressions in KO sperm (Fig. 9A, 9B). Downregulated proteins in KO sperm were mainly related to actin binding functions, which are well-known related to sperm motility and acrosome reaction (Fig. 8E). 15 proteins are characterized with male reproductive phenotypes of abnormal sperm function (supplemental Table S9). ODF2, CFL1, MYH11, TPM1, ␣-tubulin, and ␤-tubulin were validated to be lower expressed in KO sperm. Immunofluorescence analysis demonstrated that Cmtm4 was localized in sperm acrosome and middle piece, and ODF2 showed low intensity in KO sperm (WT sperm:178 Ϯ 18.5; KO sperm:98.6 Ϯ 10.7, p Ͻ 0.05) (Fig. 9C). Sperm quality is the decisive factor in male fertility (35). Most infertile male patients are characterized by poor sperm quality (36,37,38). Because they are likely to affect spermatogenesis or sperm quality, genes and gene products that are highly expressed in the testis are promising targets for the diagnosis or treatment of male infertility (39 -42). Our previous studies have identified CMTM4 is highly expressed in the human testes (2,23); thus, we hypothesized that CMTM4 was correlated with spermatogenesis, and its altered testicular expression would consequently be related to sperm quality. In the present study, we investigated the association of CMTM4 with spermatogenesis and sperm quality and demonstrated a role of CMTM4 in male fertility by phenotypic and quantitative proteomic analyses of the Cmtm4 KO mouse model. This study provides novel information in the functional research of the CMTM family in male reproduction. In the present study, CMTM4 was observed to be aberrantly expressed in the spermatozoa of infertile individuals, indicating its association with sperm quality. As in our previous reports, the elderly testis showed decreased spermatogenesis, resulting in poor sperm quality (26,31). Here, IHC analysis showed downregulated expression of CMTM4 in elderly and NOA testes, with especially low expression in NOA testes. The combined results imply that CMTM4 is a potential key protein in spermatogenesis and sperm quality regulation. There is a high homology of the amino acid sequences (95.7%) between human and mouse CMTM4, and thus the Cmtm4 KO mouse is an ideal model for investigating the functions of CMTM4 in male reproduction in vivo. The Cmtm4 KO mouse revealed subfertility in males but no significant morphological or behavioral abnormalities. No significant difference in serum testosterone level in KO mice compared with WT mice was observed. This indicates that Cmtm4 is not actively involved in androgen regulation, whereas other members of the CMTM family, such as mouse Cmtm2a and Cmtm2b and human CMTM1-v17, CMTM2, and CMTM3, are reported to affect androgen receptor activity (19,22,43). However, Cmtm4 KO mice showed an obvious phenotype of reduced progressive sperm motility, abnormal spermatozoa with backward-curved heads or bent midpieces, leading to male subfertility. A previous study reported that CMTM4 knockdown in HeLa cell resulted in cytokinesis defects (44). Thus, Cmtm4 deficiency may affect male meiotic cytokinesis, thereby resulting in lower DSP and abnormal spermatozoa. These distinct phenotypes between WT and KO mice suggest that they may alter sperm functions in KO mouse. Observation of the acrosome reaction indicated that a significantly reduced percentage of acrosome reactions was induced in KO mice sperm by the calcium ionophore A23187. These findings imply that CMTM4 is actively involved in spermatogenesis and sperm quality regulation. Further in vivo fertility assessment showed that male KO mice were subfertile, whereas female KO mice had normal fertility. In vitro fertility experiments also confirmed that spermatozoa from KO mice was inefficient in producing a two-cell stage. Collectively, these results indicate that Cmtm4 is associated with spermatogenesis and sperm quality and is essential for male fertility. Further, quantitative proteomic analyses were performed to detect the molecules and biological processes in testes affected by Cmtm4 deficiency. Our study identified 5840 mouse testis proteins, which provides an inventory of mouse testic- ular proteins useful for the understanding of spermatogenesis. Of these, 351 proteins showed differential expression between WT and KO mice. Bioinformatic analysis revealed notable effects of Cmtm4 deficiency on male infertility. Downregulated proteins in KO mice included those that function in spermatogenesis and sperm functions. Of the downregulated proteins, 100 (71.9%) were testis-specifically expressed, reflecting an association with impaired spermatogenesis, and 57% of downregulated proteins were exclusively expressed in round spermatids, indicating the association with sperm quality might involve regulation of post-meiotic biological processes. These downregulated proteins are involved in fertilization, sperm motility, the acrosome reaction, and spermoocyte recognition. Several of the downregulated proteins are known to be involved in spermatogenesis; for example, PKG2 and GAPDHS are more abundant in round spermatids, and are testis-specific glycolytic enzymes positively correlated with sperm motility (45)(46)(47). Odf1 and Odf2 are major components of the outer dense fibers that modulate sperm motility (48). Spata20, Spata3, Spata6, Spata7, and Spert are well-known spermatogenesis-related proteins (49). Inactivation of Spata6 in mice leads to acephalic spermatozoa and male sterility (50). IZUMO1, CRISP1, AKAP3, AKAP4, PLB1, and TRIM36 are classified in the literatures by bioinformatic analyses as having functional involvement in the acrosome reaction (51)(52)(53)(54), and their downregulated expression may contribute to the deficient acrosome reaction of KO mouse spermatozoa. The downregulation of these spermatogenesisrelated proteins may contribute to lower sperm quality and reduced fertility. To further understand the effect of CMTM4 knockout on mouse sperm quality, a quantitative proteomics was performed to detect the affected sperm molecules by Cmtm4 deficiency. A total of 3588 proteins were identified and 1997 proteins were included in current testis proteome, which provides complementary information for sperm protein profiles (supplemental Fig. S1). Chromatin DNA binding function that was overrepresented in upregulated proteins of KO mouse sperm are known to participate in chromatin remodeling and meiosis during spermatogenesis (55). Particularly, Western blotting demonstrated the increased levels of H2A, H2B, and H4 in KO sperm, and conversely, reduced levels of PRM1 and PRM2 in KO sperm. The results suggest that impaired histone-to-protamine exchange exists in KO sperm, which may contribute to abnormal sperm morphology and function (56). Most of downregulated proteins in KO sperm perform actin binding functions, which are well-known to play crucial roles in maintenance of sperm morphology and motility (57). Cofilin in sperm is involved in acrosome reaction and its downregulation in sperm is correlated with poor sperm quality (58). ODF2 is related to sperm motility (59). Interestingly, ␣-tubulin and ␤-tubulin are downregulated in KO sperm, suggesting the impaired sperm motility and acrosome function (57). Collectively, proteomic alterations in KO sperm indicate its influence on sperm motility and acrosome reaction, which is consistent with its functions of localization in acrosome and midpiece, and implications of proteomic alterations in KO testes. This study revealed an important role of CMTM4 in male reproduction. CMTM4 expression level was associated with human spermatogenesis and sperm quality. Phenotypic and quantitative proteomic analyses of Cmtm4 KO mice revealed that CMTM4 mainly affects histone-to-protamine exchange during spermatogenesis process, and functions in sperm motility and acrosome reaction. Proteomic analysis also identified a group of essential proteins as being affected by Cmtm4 deficiency. Their interactions and functions in male fertility are warranted to be further study in the future. This study provides an impetus for investigating the roles of other members of the CMTM family in male reproduction, and shows that CMTM4 is a promising molecular candidate for the assessment of sperm quality and the diagnosis or treatment of male infertility.	2019-03-15T02:58:02.782Z	2019-03-13T00:00:00.000	{ "year": 2019, "sha1": "64f0e62675a2323539bbd5748b88c4b337df7fd8", "oa_license": "CCBY", "oa_url": "https://doi.org/10.1074/mcp.ra119.001416", "oa_status": "HYBRID", "pdf_src": "Highwire", "pdf_hash": "faf53a5bfbcc91d47ad92142aaf69b1958896da9", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Medicine", "Biology" ] }
55595896	pes2o/s2orc	v3-fos-license	Biodiesel in Brazil : Agricultural R & D at Petrobras Biocombustível The expansion of the use of biofuels is based on the implementation of public policies to support production, technological development and the market. In Brazil, the National Program for the Production and Use of Biodiesel (PNPB) seeks to articulate these incentives and attract private initiative in promoting sustainable production. Investigating the business actions for the development of new technologies in the face of public stimulation motivates this article that aims to analyze the Research and Development (R&D) strategies of Petrobras Biocombustível in the production of biodiesel. The case study is based on the theoretical arguments of the Entrepreneur State and on a case study accompanied by a qualitative analysis. The results indicate that the formation of agricultural research networks favored the construction of knowledge, research infrastructures and training of people, but did not innovated the production of biodiesel. Introduction The large share of oil in the composition of the world energy matrix has encouraged discussions on several fronts.They bring together, in particular, the debate around themes such as energy security, pollution, increase of greenhouse gas emissions and global warming.Among the ways pointed out are the possibilities and challenges for the production and use of biofuels, ethanol and biodiesel, as alternatives for reducing the consumption of fuels derived from oil, gasoline and diesel. In this debate are the discussions of Elliot (2000) that places biofuels as complementary or alternative to gasoline and diesel, and therefore immersed in the need for a large and continuous scale of production and the necessary construction and implementation of public incentives.Mowery et al. (2010), also highlight the role of the State in the construction of an environment favorable to the progress of biofuels, and emphasize fiscal and financial incentives as well as taxation of competing technologies and support for technological development as fundamental.In addition to the aspects related to market formation and new technologies.Is also considered the production of biofuels as an alternative for the development of agriculture through the production of raw materials.Similarly, Floray et al. (2012) shows that the use of new technologies, such as biofuels, requires an active state and capable of understanding its role in building spaces connected to the demands of society. These collected evidences are present in the National Program for the Production and Use of Biodiesel (Programa Nacional de Produção e Uso de Biodiesel -PNPB), a public policy aimed at encouraging the sustainable production of biodiesel in Brazil that seeks to form a new market, through social inclusion and regional and technological development.This program, implemented in 2005, structured in fiscal and financial incentives, compulsory consumption and the Social Fuel Seal (Selo Combustível Social), an instrument that links the access of companies to incentives and the promotion of social inclusion and regional development. The seal is granted to companies that buy raw materials from family farming present in certain regions of Brazil, especially the Northeast Region.This condition places the PNPB the challenge of the development of agricultural technologies in the production of oilseeds adapted to the characteristics of the familiar regional production.The architecture of the program and its objectives are the subject of debates that highlight the difficulties to be overcome, whether those evidencing the production reality of the Brazilian Northeast (Garcia, 2007) or those that expose the traps placed by the predominance of soybean in the production of biodiesel, strengthened by the limited technological knowledge of alternative raw materials (Abramovay & Magalhães, 2007).Despite the criticisms, the PNPB has advanced and completed twelve years of existence, mobilizing various actions in the different social segments that it seeks to articulate.The most important segment is soybean agroindustry, which accounts for 70% of biodiesel production in Brazil.This mature and widely established agroindustry in Brazilian agriculture is capable of supplying the advance of biodiesel production, at the same time reducing the interest and risk of the technological development of aletrnativas raw materials. The actions aimed at Brazilian biodiesel production involve the participation of Petrobras Biocombustível, a subsidiary of Petrobras S/A.The subsidiary's strategies are based on the construction of production infrastructure and, unlike the soybean industry, on research and development (R&D).The effort in the search for the technological development involved the organization of the Research Networks in Oilseeds of Petrobras Biocombustível and mobilized 19 public agricultural research centers.Petrobras' differentiated and ariscated position -an innovative company in its main business, oil exploration and refining -motivates the issues that lead to this article.How were research networks organized and conducted?What developments can be identified? The coping or incorporation of risks and uncertainties in the development of new technologies and new markets are elements that form an unattractive universe for companies.This instability has an important strategy in the formation of new businesses that characterize the new markets, such as renewable energy, such as the production of biodiesel in Brazil in the State of Entrepreneurship and its ability to reduce uncertainties (Mazzucato, 2014).Thus, the article is organized into four sections beyond this introductory one.The second section presents the contributions made by the Entrepreneurial State of Mazzucato (2014) and the structure of analysis composed by descriptive analysis and case study.Subsequently, the third section discusses the PNPB and its results.The fourth section discusses the elements present in the construction and results achieved with Petrobras R&D strategy for biodiesel production.Finally, the final section deals with conclusions and final considerations. Entrepreneurial Status: the risk of technological development This section is organized in two subsections.The first subsection presents and discusses the arguments and concepts that structure the theoretical approach of the State Entrepreneur, highlighting the importance of public policies to boost the development of new technologies and new markets, such as biodiesel.In the sequence, the second subsection incorporates the elements of the theoretical discussion to the study methodology that articulates a qualitative descriptive analysis to the case study. New products and new markets The development of technologies in renewable energies is fundamental for the transformation of the current energy matrix rooted in the technologies of the fossil standard.The permanence in the fossil pattern excludes the chance of the society to promote a truly transforming space.This transformation requires technologies for recyclable materials, advanced techniques of waste management, improvement of agricultural practices, among other innovations.The starting point, the path to be followed and where the arrival will be are quite nebulous variables of this high risk process (Mazzucato, 2014). The uncertainty and risk associated with these technologies is rooted in a diverse range of economic segments and in the deep domain of existing technologies.This environment marked by the necessary technological development places the State in the important role of formatting public policies with instruments that enable the execution of and R&D projects and encourage production (Mowery et al., 2010;Olmos, et al., 2010). For Mazzucato (2014) the role of the State lies in the articulation of the innovation process and also in entrepreneurial actions.The undertaking for the State is in the strategy of taking the risks of technological development and occupying a space of uncertainty that private initiative is not willing to face.This placement originates from arguments built from two fronts of discussion. The first one is aligned with the concepts and arguments of the evolutionary economy, punctuated, among other evidences, by the competition marked by innovation in products and processes and the search for technological evolution from scientific, technical and tacit knowledge, institutions, processes and organizational forms.As well as machines, equipment and products.In this search, companies are placed as the prime responsibility for innovation, but they are accompanied by governments, research organizations, universities, public policies and other components that form the so-called systems of innovation with repercussions for the development of nations.As for competitiveness in markets, both in industry and agriculture (Dosi & Nelson, 2009). The second front of discussion deals with the role of the State in economic development and has in the critics of neoliberalism the references for the construction of the arguments that support the vision of Entrepreneurial State.For Mazzucato (2014), the discourse that the public sector is bureaucratic, incompetent, intrusive and intrusive and too big to be dynamic, as opposed to the dynamism, efficiency, entrepreneurship and innovative nature of the private sector, has built the idea that the state must basic services and correcting any market failures.In this vision, it would be up to the State to facilitate the process of innovation and to provide conditions for the private initiative, always available to take risks and invest in R&D. This way of interpreting reality has emptied the debate about the role of the state in development and has resulted in the shaping of public policies in innovation that built myths.The first of these lies in the direct causality between R&D and innovation -just invest in R&D to innovate -when in fact a series of complementary assets is needed for innovation to occur.The second myth is that smaller firms are more apt to innovate.The evidence shows that the size of companies does not always justify their growth but rather productivity.Another myth is that the number of patents reflects growth in terms of innovation, which in fact in certain markets is much more related to legislation and competitive strategies.Next, the myth that business investment needs fewer taxes and bureaucracy when evidence does not prove that R&D tax credits actually contribute to its development.In addition to these, the myth that venture capital "worships" risk, when in fact it escapes risk and only appears when uncertainties are smaller (Mazzucato, 2014).Mazzucato (2014) points out that the risks and uncertainties associated with entrepreneurship and innovation linked to investments in R&D and technological change are not always absorbed by the private sector.This entrepreneurial role has in many cases been played by the state with its long-term investments in science and technologys (S&T) and R D, which are materialized in new products, new processes and new markets.The state assumes the risk of betting on fronts so unknown that even the uncertainties are identified, such as the development of the internet.Private initiative participates from the moment the risks and uncertainties of technological development can be identified.The author takes as an example the technologies touchscreen and GPS that gave support to the development and commercialization of iPhone and iPad and that are the result of many years of research financed by the State with the Department of Defense of the United States. In this sense, the arguments put forward put the State as an active participant in the process of technological development necessary for the adoption of new products and new processes and also for the creation of new markets.This construction has in the interaction between public and private action formatted in public policies the ways to reduce the risks and uncertainties tied to new markets and new technologies. Analysis structure The arguments of Mazzucatto ( 2014) emphasize the importance of state participation in technological development, in encouraging the formation of new markets.The public incentive to development is permeated by the view that the private initiative seeks to invest in businesses with risk and uncertainty foreseeable.These conditions are not always measurable in new technologies and markets.The difficulty of analyzing new investment scenarios for private enterprise is partly amortized by public action through policies and programs that support specific technologies and markets. The formatting of structured public policies in support mechanisms and incentives to new technologies and markets is present in the architecture of the PNPB.Thus, the methodology adopted initially seeks to explore the instruments and results achieved with the PNPB, and then, to guide the case study and to identify how the private initiative has internalized the incentives and objectives of the program. The descriptive analysis of the program was conducted from two stages of research.The first met and analyzed information contained in laws and decrees that regulate the activities and objectives of the PNPB.The second stage worked on statistical series and data on the dynamics of Brazilian biodiesel production from 2005 to 2016, made available by the Agência Nacional de Petróleo, Gás Natural e Biocombustíveis (ANP -National Agency of Petroleum, Natural Gas and Biofuels).The information collected and analyzed in the two stages of research were complemented with results of recent technical-scientific studies that dealt with the PNPB. The results of the descriptive analysis of the PNPB helped in the case study that, among the different strategies to overshadow the incentives and risks of biodiesel production in Brazil, explored Petrobras Biocombustível's R&D investment strategy, structured in Petrobras Biocombustível' Oilseeds Research Networks.In order to address this case study, in order to identify the company's strategies aimed at biodiesel production, the information provided by the company was gathered through its annual reports, considering the period from 2005 to 2016.The analysis is completed with the treatment of the development of technological development strategies, through interviews with researchers who coordinated research projects in two of the nine networks structured by Petrobras Biocombustível. The two research networks selected were those dedicated to research with jatropha.The choice of these nets is due to the fact that jatropha is placed as a rustic oleaginous and placed as promising to be produced by family farming in the Northeast Region of Brazil.To apprehend this and previous investments in research, still lacking knowledge about its genetics and breeding, production, harvesting and processing techniques, according to Martins (2010).The interviews were conducted from November 2015 to March 2016, through a script with 12 open questions organized in two categories: network operation and results achieved.The interviewees lead studies carried out at the following public agricultural research centers: Universidade Estadual Paulista (UNESP) Campus de Ilha Solteira, Instituto Agronômico (IAC), Empresa de Pesquisa Agropecuária de Minas Gerais (EPAMIG) and Universidade Estadual de Campinas (UNI-CAMP), Laboratory of Biotechnology. In the search to understand the unfolding of this initiative in R&D for the company, an interview was conducted through a virtual process during the month of April 2016 and a structured itinerary in 12 questions and three categories: network objectives, results achieved and future prospects.The interview was directed to the Agricultural Technology Management to the Petrobras Biocombustível. The production of biodiesel in Brazil The production of biodiesel in Brazil is linked to PNPB and Law 11,097/2005, that inserted biodiesel into the Brazilian energy matrix by mixing diesel oil and biodiesel (BX).Started in 2005 with the optional blend of 2% (B2) of biodiesel to diesel, it reaches 2017 with a mandatory blend of 8% (B8) and projections to 9% and 10% by 2019.The goal of the program is to implement production and the use of biodiesel in Brazil in a sustainable manner, promoting social inclusion, guaranteeing competitive prices, quality, supply and production from different sources of oilseeds in diverse regions.The actions have an interministerial structure, with emphasis on the Ministério de Minas e Energia (MME), Minstério de Desenvolvimento Agrário (MDA), currently transformed into the Secretaria Epecial da Agricultura Familiar e Desenvolvimento Agrário, Ministério da Ciência, Tecnologia e Inovação (MCTI), now Ministério da Ciência, Tecnologia, Inovação e Comunicação (MCTIC). To achieve its objective, this public policy brings together various instruments and organizations that accommodate a set of incentives and rules for production, commercialization and support for technological development.In the promotion of production are the Programa de Financiamento a Investimentos no Biodiesel, conducted by the Banco Nacional de Desenvolvimento (BNDES) and tax incentives.The commercialization, in a regulated market, is supported by a system of auctions of purchases and quality control, with the participation of the ANP in the organization of the auctions and edition of the technical specifications, and of Petrobras in the control of stock executed through negotiations involving the Purchase of biodiesel in the ANP auctions and the transfer to the diesel distributors that serve the national diesel market. The financial and tax incentives and the rules of the auctions are linked to the Social Fuel Seal, an identification component granted to the biodiesel producer who acquires a minimum percentage of raw material from family farmers included in the Programa Nacional de Fortalecimento da Agricultura Familiar (PRONAF), executed by the MDA.This instrument for the promotion of social inclusion and regional development, with emphasis on the Northeast Region and on the oil-producing area produced by family agriculture, castor bean, as Azevedo (2010) points out, was widely publicized in the media and its links and objectives built expectations in the solution of historical problems of this Brazilian region.The biodiesel producer that holds the seal is destined to participate in 80% of the auction volumes and special conditions in access to financing and tax incentives.In addition to these instruments, PNPB also incorporates support for technological development through the Rede Brasileira de Tecnologia de Biodiesel and the Câmara Setorial da Cadeia de Produtiva de Oleaginosas e Biodiesel, which brings together representatives of the various segments of the production chain and created Ministério de Agricultura, Pecuária e Abastecimento (MAPA). The discussion between the proposed objectives the instruments and the results are based from the beginning of the PNPB in works such as de Abramovay & Magalhães (2007) and Garcia (2007) who, as in García (2016), discuss the difficulties of implementing Development policies without understanding the local reality and joint mobilization of their assets.For example, the regional concentration of production, as in 2016, together, the Central-West and Southern regions of Brazil accounted for more than 80% of the national production, as opposed to the small Northeast Region (ANP, 2017).The regional distribution of production is based on regionalization of soybean production and processing, the main raw material for biodiesel production, responsible for more than 70% of the national production (ANP, 2017).This reality limited the effective participation of other oilseeds capable of promoting the regional distribution of production and the inclusion of family agriculture, especially the one that needs support to consolidate, according to Campos & Carmélio (2009). The debates about the regional distribution and its relations with the raw materials for biodiesel production intertwine with the questions about the participation of the biodiesel plants.The discussions involve the ample idle capacity and its unfolding for the market and the investments with public resources that were made (Mendes & Costa, 2010).Also, the participation of companies originally associated with the production and processing of soybeans is highlighted, with biodiesel being another business option, as a counterpoint to the formation of new enterprises for a new market (Sampaio & Bonacelli, 2015). The results built in front of the proposed objectives open space for questions about the scope of institutional arrangements formatted in the PNPB and its limitations to promote development in interaction with private initiative and connected to the needs of society (Pedroti, 2013).This discussion is reinforced when the evolution of the production is observed, accompanying the increase of the mixing percentages and the consumption of diesel.Brazilian biodiesel production jumps from 750 m3 in 2005 to 3.8 million m3 in 2016.According to the ANP (2017), in the period from 2013 to 2016, Petrobras Biocombustível accounted for 10% to 15% of annual production, among the main biodiesel producing companies in Brazil. In this sense, we have the success of PNPB in the proposal to include biodiesel in the Brazilian energy matrix.But, also, the realization that this capacity did not extend to the promotion of the innovations necessary to reach its objectives of social inclusion and regional development, especially of the Northeast Region of Brazil.These results indicate that the implementation of R&D actions and the construction of technologies, mainly in the production of raw materials, are fundamental for a scenario of enormous technological challenges and restricted indications of the paths to be followed.In this way, the experience of Petrobras Biocombustível, based on investments in R&D, positions itself as a strategy that seeks to break this obstacle built from the PNPB. Agricultural R&D at Petrobras Biocombustível The implementation of the PNPB and its development, as elaborated in the previous section, built a scenario based on the need for agricultural technological development in oilseeds or raw materials alternative to soy, in order to achieve the objectives of social inclusion and regional development.Under these circumstances, Petrobras Biocombustível created R&D initiatives.To present and discuss the results achieved, this section was structured in two subsections.The first deals briefly with Petrobras' move towards renewable energy and the production of biofuels, especially biodiesel.The second subsection analyzes investments in R & D in oilseed production, observing the actions directed to research with jatropha. The Petrobras Biocombustível The history of one of the main Brazilian companies is permeated by economic aspects inherent in the main market in which it operates -the oil market.But, also, for political and driving issues of the Brazilian economy that influence the choices and paths traced by Petrobras over more than 60 years.The creation of Petrobras takes place amidst polarized discussions that addressed, in particular, the issue of the monopoly on oil.At that time, the private initiatives in the construction and operation of refineries were being absorbed by the state as its knowledge was improved in a broad process of learning from oil exploration to refining and distribution of derivatives.The capacity developed by the company has been expanded with emphasis on investments in oil prospecting and exploration in offshore fields.This strategy expanded the company's oil production capacity and established a broad investment program in S&T and R&D, organized in partnership networks with universities, research institutions and supplier companies. In the last years the movement of Petrobras involves the generation of electricity, discussions and worldwide actions around sustainable development, biofuels and several other applications for biomass.In this context, the Petrobras System is consolidated with new subsidiaries and expansion of activities, with the production of ethanol, sugar, bioelectricity and biodiesel.In the same period, Brazil achieves selfsufficiency in oil production and Petrobras becomes a signatory of the ONU Global Pacto and announces the discovery of the new oil reserves -Pré-Sal. The expansion of Petrobras' activities from biofuels can also be seen in other oil companies operating in Brazil.It highlights the partner-ship between Shell and the Grupo Cosan, creating Raízen, which produces and markets sugar, ethanol and bioelectricity.The same strategy is carried out by BP Brasil with the participation in Tropical Bioenergy.Also, it is worth highlighting the Brazilian euphoria with the possibility of the country assuming a prominent position in the world production of biofuels. These conditions are important in the construction of the creation strategies in 2008 of Petrobras Biocombustível, which brings together Petrobras' activities related to the production and commercialization of ethanol and biodiesel.Particularly for biodiesel, the company's operations began in 2006 with the installation of an experimental plant in the Northeast region of Brazil for the production of biodiesel, associated to the intensification of R & D activities related to the production of biofuels and other applications, for example Biolubricants and the interactions between biomass and petroleum products (Petrobras, 2005). The creation of Petrobras Biocombustível brought together actions aimed at the construction of three biodiesel production plants.A located in Candeias, Bahia, where Petrobras operates the production terminal for the Refinaria Mataripe.The second plant located in Montes Claros, Minas Gerais, and the third in Quixadá, Ceará (Petrobras, 2006).The biodiesel production infrastructure was reinforced with the acquisition of 50% of the BSBios plant located in Marialva, Paraná.As well as 50% of the shares of BSBios Indústria e Comércio de Biodiesel Sul Brasil, Passo Fundo plant in Rio Grande do Sul and 50% of Bioóleo Industrial e Comercial SA, located in Feira de Santana, Bahia, by crushing oilseeds (Petróleo, 2010).This move of the company established the production structure for biodiesel, from three own plants located in the Northeastern Region and the semi-arid region of Brazil, in addition to two plants operated in partnership in the South Region.Petrobras Biocombustível also organized two clearly integrated strategies associated with instruments formatted in PNPB and its objectives of social inclusion and regional development.One of them, the o Programa de Apoio à Produção de Oleaginosas pela Agricultura Familiar and the Redes de Pesquisa em Oleaginosas da Petrobras Biocombustível, focus on this article and work on the next subsection. The Redes de Pesquisa em Oleaginosas da Petrobras Biocombustível In order to work on the strategy of creating the research networks of Petrobras Biocombustível, it is important to retrieve elements that deal with the management model of Petrobras R&D activities and the financing sources provided for in legislation.Since 2006, Petrobras' R&D management model has been organized in 49 thematic networks distributed in the following topics: 14 in oil and gas production, 13 in refining and petrochemicals, 6 in oil and gas exploration, 5 in materials, 4 In environment, 3 gas and energy, 2 for advanced computing.And an for technological management and another that opens up research on bioproducts. In the bioproducts research network, technologies are being developed in evaluation of glycerine injection for advanced oil recovery and tests for the production of second generation ethanol from sugarcane bagasse.Also in evaluation of new methodologies for emissions measurement and evaluation of the potential of different types of vegetation in CO 2 sequestration.The research network on bioproducts also aggregates studies for the production of microalgae in hypersaline waters, production of lubricant from castor biodiesel (biofuel), production of polystyrene from soybean oil, production of gasoline and diesel oil from of bio-oil of wood.Biodiesel production with tilapia oil, produced from the viscera and mixed with 10% of bovine tallow (Tecnologia Petrobras, 2011Petrobras, , 2012Petrobras, , 2013)).In addition, this research group is also part of the activities of the network in bioproducts the research areas of the Redes de Pesquisa em Oleaginosas da Petrobras Biocombustível Redes de Pesquisa em Oleaginosas da Petrobras Biocombustível were established in 2010 through agreements signed by the Centro de Pesquisa da Petrobras (CENPES), which applies financial resources and accompanies the execution of research projects, including subsidies such as Petrobras Biocombustível.Prior to the creation of Petrobras Biocombustível, there were already research projects with oleaginous plants in progress that were integrated into the Networks.These projects are those linked to agreements with public agricultural research centers, allocated to networks 6, 8 and 9, as shown in Table 1.The other research projects were signed as of 2010, except for the Jatropha Network, object of this study, which was started in 2009. According to information gathered from the interviews, the selection of related oilseeds followed different criteria.For castor beans the justifications were the existence of a defined production system, the Semiárido region and family farming as a traditional producer.The sunflower for having the potential to adapt to the crop in the Northeast, but dependent on technical-scientific knowledge for its production.The palm, traditional culture, cultivated in Pará and Southeast of the State of Bahia, with a system of cultivation suitable for both large extensions and family farming.For Jatropha, expectations about this little-known "new oilseeds", but with national and international research, were promising for production by the family farming of the Northeast.The macaúba, also a "new oilseeds", the rusticity and being a native plant in Brazil present in several biomes, offered the contours for its inclusion in the research networks.In addition to these oleaginous crops, other crops were also investigated prior to the formation of nets, such as soybean and canola, and prospectively the tucumã, inajá and faveleira.The results of the interviews indicate that, on the side of those who accessed the financial resources destined to network 5 "Technological development for jatropha", the contact with Petrobras occurred in a personal way, through relations between researchers who already developed researches with jatropha before the formation of the networks, and those responsible for the strategy with CENPES.In this process, the interested researchers and the related research organizations started the registration procedures with the ANP, a legally required step for any research project, as well as completing the forms and gathering the necessary documents to submit the research proposal Petrobras' evaluation criteria. After several adjustments, UNESP became the coordinator of the network, which hosted three projects: Basic and applied technology development for jatropha, executed by UNESP, Biotechnologies suitable for the genetic improvement of jatropha in order to obtain interspecific hybrids, under the control of the IAC, and jatropha as an alternative of raw material for the production of biofuels, linked to EPAMIG. During the execution of the research projects, from 2009 to 2015, meetings were held with CENPES to present the results achieved and discuss the steps to be taken.Likewise, the CENPES team also visited the research centers to follow up the activities and the experiments carried out.In this process, the EPAMIG project was disconnected from the jatropha network and financial resources were allocated to the IAC project.In addition to face-to-face control, there was the preparation of activity and financial reports related to the application of resources, as well as the control of publications and dissemination of the results achieved with the projects. In the investigation of the contribution achieved with the projects, the interviews allow to highlight the advances in the knowledge of the jatropha culture.At present, it is a consensus that the expected rusticity has not been confirmed, that there is potential for more than one crop per year and that the production of jatropha consorted with other crops is shown as a feasible and necessary way for the viability of the crop as an alternative Family farming.From the point of view of the genetic knowledge of the plant, the project allowed the increase of the germplasm bank with non-toxic materials, important in the use of oil byproducts, protein-rich bran, also the selection of small specimens, fusário tolerant, uniformity of maturation and conformation of the productive cycle. The researchers also highlighted the training of human resources, including foreigners.There were more than twenty research grants for students of scientific initiation, masters and doctors, as well as several publications, theses and dissertations with culture as focus.Likewise, the partnership with other research centers, especially in Mexico.Also, it is worth mentioning the development of research based on the results achieved with the GENDIESEL project and the partnership with researchers from the Oswaldo Cruz Foundation (FIOCRUZ).Another result mentioned was the improvement of the research infrastructure, through the acquisition of equipment, construction and adaptation of the facilities.In this regard, in particular at UNESP, laboratories and a pilot plant for the production of biodiesel were created and equipped, thus creating a favorable environment for new research to be carried out, as well as the formation of the largest experimental field of jatropha.Likewise, the accreditation of research centers is seen as an important element for new opportunities with Petrobras. The information gathered from the interview with gerencia de tecnologia agrícola by Petrobras Biocombustível indicates that the expected results with the networks have been achieved, but each agreement and project has its particularities.The practical or strategic knowledge was applied to the operations with the Programa de Apoio à Produção de Oleaginosas pela Agricultura Familiar.But, the results in basic research still require further efforts to be applied.When considering the operation of the networks as a whole, it was observed that there are more collaborative institutions and others less.Such as the macaúba research network, in which the works were carried out in an integrated way with good results, different from the network of "Development of systems ", which had a large number of research institutions, but with difficulties in its management. Considering the results achieved for jatropha, a perennial crop, the continuity of support for research projects is pointed out as fundamental for this raw material to effectively contribute to the production of biodiesel.This action by Petrobras Biocombustível was expected by the project coordinators.But, during the interviews it was pointed out that from 2012 there was a certain weakening of the actions gathered in the networks and the vision of closing the activities was present.Despite the discontinuity and interruption of the research, it was indicated that without the support of Petrobras Biocombustível, the research carried.This not only for the resources invested, but also for the important actions in the articulation of the research centers for the construction of new paths for the technological development of raw materials for biodiesel production in Brazil.As pointed out by Scoponi (2016), the articulation and cooperation among research institutions is fundamental for boosting agricultural innovation, constituting established and long-lasting research networks. At Petrobras Biocombustivel the research networks were finalized and the orientation is to discontinue the development of oilseed research for biofuels.At the time of the debutant, there was no prospect of resuming activities.It was commented, however, that the lessons learned in the period from 2008 to 2016 are being considered.That the company will continue to work, through its mills, in the promotion of the acquisition of raw materials from family farming aimed at participation in the purchase auctions of Biodiesel promoted by the ANP, with no plans to implement new projects. On the other hand, the research institutions that participated in the jatropha network seek two paths.The first one related to the continuity of the researches and maintenance of the experiments with the objective of avoiding losses, through new research projects submitted to the de-velopment agencies, for example, the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and others sources, in a strategy with no prospect of relying on the private initiative.The second path identified was the abandonment of research with jatropha and the directing of efforts to other crops, in the face of the disillusionment with the possibilities of jatropha participation in the production of biofuels Petrobras Biocombustível also invested in other fronts, such as the use of geoprocessing as a tool to support the management of the application of resources in areas with greater productive potential.In 2012, the company systematized its safety, environmental and health processes and created the Corporate Waste Management Plan.In 2013 and 2014, in partnership with Embrapa, it implemented twelve Unidade de Teste e Demonstração (UTDs) with castor bean in the Semiárido region, as well as researches on fish oil, algae and macaúba, as commented above.In 2015, it completed and implemented the Geographic Information System, which allows, through the Internet, geographic, environmental and agricultural performance analyzes, providing detailed information that improves management. Conclusions The search for new paths in energy generation highlights the production of biofuels and the need to format public policies.These public action instruments seek to encourage the formation of new markets and the development of these new technologies.In Brazil, the PNPB is an example of these initiatives that are based on the sustainable production of biodiesel, through economic viability, social inclusion and regional development.This program is structured on fiscal, financial and technological incentives that mitigate the risks of a new market and attract private initiative in the construction of strategies.Among the companies participating in the PNPB, the soybean agroindustry, the main raw material used, and Petrobras Biocombustível stand out. The company, a subsidiary of the oil company Petrobras, structured strategies that involved broad support for R&D activities, aiming at the development of alternative raw materials for soybeans, gathered in Petrobras Biocombustível Oilseed Research Networks, which is the object of the study.The arguments of the Entrepreneur State that highlight the importance of the State in the development of new markets and new technologies led to a descriptive research and case study to investigate the training, conduction and results achieved with the Networks. The results show the interest of Petrobras, an innovative company of international renown, in the development of research involving biomass in its various applications, including biofuels.For biodiesel, the company structured production and R&D strategies with a view to aligning PNPB instruments with their objectives.Experience with agricultural research and its public centers has resulted in important advances in knowledge with oilseeds, especially jatropha, building infrastructure and training of people, as well as the difficulties to continue the work in the face of the closing of investments.The results also show the fragility of the relationship between the research centers and the company.Exposes the company's distance from the timid application of the knowledge achieved and thus, highlight the discussions about R&D risks and private initiative in the production of Called the background research, to the same extent that it highlights the limits of the PNPB in involving companies in the development of new technologies.	2018-12-15T07:03:30.204Z	2018-05-02T00:00:00.000	{ "year": 2018, "sha1": "77133a0e3979ad3bd90f8fcf14065b3b710617fe", "oa_license": "CCBY", "oa_url": "https://scielo.conicyt.cl/pdf/jotmi/v13n1/0718-2724-jotmi-13-01-00066.pdf", "oa_status": "GOLD", "pdf_src": "ScienceParseMerged", "pdf_hash": "77133a0e3979ad3bd90f8fcf14065b3b710617fe", "s2fieldsofstudy": [ "Agricultural and Food Sciences", "Business", "Environmental Science" ], "extfieldsofstudy": [ "Business" ] }
186355393	pes2o/s2orc	v3-fos-license	PROBLEMS OF PLACING INDUSTRIAL CAPACITIES AND LOCALIZATION OF PRODUCTION This article discusses issues related to the location of production facilities. Industrial zones created in Uzbekistan are also discussed. The list of free economic zones created in Uzbekistan has been redesigned. At the end of the article the author proposed recommendations on the establishment of free trade zones with accessible infrastructure, provided information on the investment climate of free economic zones. Introduction Industry is one of the leading sectors of the economy, it is in this industry that localization is one of the key factors for achieving efficiency. Consequently, we can observe the widespread introduction and intensive implementation of localization in the economic development of a number of foreign countries recognized as countries with developed market economies, as well as a growing market economy. It is noteworthy that China has effectively organized localization in the automotive sector, in Brazil -in healthcare and pharmaceuticals, in Nigeria -in the oil and gas sector, and in Canada -in the electricity sector. Also, the program "Buy American products", which displays localization in the US, was developed. In practice, Uzbekistan localization is aimed at the production of finished products and components on the basis of industrial cooperation, with the achievement of a number of positive results. In particular, localization in such priority areas as the mining industry, automotive industry, pharmaceuticals, and the food industry is rapidly proceeding. In order to broadly implement this area, a number of programs were developed and implemented on the basis of 6 resolutions of the President of the country. In particular, Resolution PP-1236 on 01.12.2009 [1] serves as a guide to the development of localization programs, the adoption and implementation of projects. Proper organization and location of production in the process of localization is one of the key problems that determines the relevance of the topic. Literature review The issues of the location and location of production were first published in 1826 by Johann Thunen [2]. On the basis of this theory, Thunen conducted his own research and studied the stages of localization of production, location of industrial production such scientists as Wilhelm Laundhardt [3], Alfred Weber [4], August Lösch [5], Walter Kristaller [3]. In particular, in 1909 the scientific work of Alfred Weber "The Net Theory of the Location of Production" was presented to the public. This, in turn, was recognized as a continuation of the studies conducted by Thunen and Laundhardt. In this research, Weber dwelled on the issues of the location of production and to ensure minimum costs suggested reducing labor and transportation costs. Albert Vaziansky [6] and Vladimir Kondratiev [7] in their studies studied the foreign experience of managing localization processes. In the scientific works of a number of local scientists, such as Nizomiddin Khaidarov [8], Otabek Elov [9], Sherzod Mustafakulov [10], the place of internal investments in the localization of production enterprises influencing factors and principles of development has been investigated. Proper organization of production location is one of the main tasks in localization processes. Under the influence of scientific and technical and social development, production complexes can change not only the appearance, but also the nature of their location. Consequently, the introduction of innovative technologies and conveyor lines, automation and computerization and, as a result, a reduction in the number of employees and working time leads to a reduction in production, territorialplanned transformation of industrial facilities. The location of industrial production in cities played an important role in the development of world urban development. The need to ensure the power of the state at different times and determine the development strategy is one of the most important historical conditions for the emergence of industrial cities. The conducted studies make it possible to distinguish specific periods of time on the basis of the characteristics of integration, isolation and differentiation, which describe the specific methods for locating production areas in cities (Figure 1). the same time, the formation of territorial industrial structures depends on the architectural planning (master plan) of settlements. As a result, industrial zones, industrial nodes, industrial areas were formed and continuous interconnectedness of certain structures was formed. Analysis and results The industrial zone (cluster, industry, district) is a single territorial structure in the city, formed on the basis of technologically interconnected groups of industrial enterprises having common engineering communications. Source: Author's drawing based on literature. Figure 1. Stages of production location Industrial enterprises in urban areas. They are part of the city and at the same time. There are no functional and social relations with adjacent structures, except for a certain number of workers among the local population. Analyzes show that many cities emerged either spontaneously or as a result of planning, taking into account the allocation of good land for production areas with large balance reserves within the framework of intensive industrialization carried out during the Soviet period. Such a non-optimal location of industry in cities, in turn, has a number of negative consequences, including: -deterioration of the ecological situation; -transport problems; -Regional barriers to development; -Comprehensive destruction of architectural appearance, etc. Town-planning approaches to the location of industry lead to ensuring that different functional zones are not mixed while organizing production activities and ensuring their functional dependence. Initial period (from the beginning of the industrial revolution to the end of the XIX century). The settlements were formed as residential areas around the plant. Places of residence and industrial zones were created as a single system, and residential areas became a direct place of work. The period of rapid development of industrial cities (the end of the XIX century -70-80 years of the twentieth century). This period is associated with acute industrialization, production growth, construction of large enterprises (chemistry, oil refining, etc.), the emergence of sanitary and hygienic legislation. During this period, divisions between industrial and residential areas began to arise. The period of separation of industrial and residential areas in cities (70-80 years of the twentieth century The location of production in the regions is designed from the point of view of zoning, taking into account the sanitary characteristics of enterprises, technologies, professions, production capacities, the volume of the occupied pliad, the availability of railway transport, the number of workers and urban planning conditions. This period is characterized by the transformation of regional structures and the regulation of regulatory documents, taking into account market relations, technical process and acceleration of globalization. Industrial zones, which are the main component of urban architecture, have had a qualitative impact on the principles of urban organization At the same time, the main task of the city in relation to citizens was that labor conditions and housing conditions were contrasted. This found its expression in the form of a decisive functional-planned division of cities into industrial and residential areas. The practice of urban planning in the first half and middle of the XX century proved inadequate in terms of a multidimensional approach to human life. As the scientific analysis of the concept of urban construction shows, starting from the 1960s and 1970s, a purposeful search for options allowing the convergence and functional integration of industrial and residential areas was carried out to create a unified environment that unites work, rest, living, service, culture and other tasks. From the point of view of planning, urban industrial areas in most cases had a multifunctional and complex structure. Only new industry structures have been placed taking into account specific specifications, environmental conditions, economic structures and agglomeration effects. Regional industrial enterprises are divided into different categories according to certain indicators. For example, Enterprises of the first and second categories of production hazards. The level of harmfulness of enterprises makes it possible to determine the sanitary distance between the enterprise and housing units. This distance, in many cases, requires the location of production away from residential areas (outside the city). This includes enterprises with a large turnover of goods in rail transport. In accordance with the current sanitary standards, production with a low level of harmfulness (categories III and IV), requiring a distance of 300-500 m, should be located near the boundaries of the residential areas of the city (on the periphery of the locality). Industrial areas intended for those belonging to category V that do not throw industrial waste or are not associated with a fire-hazardous or explosive production, noiseless, with a small freight turnover, which do not require railway transportation of enterprises can be located within the city. The location of industrial centers largely depends on the characteristics of the industrial sectors. For example, chemical, metallurgical, oil refineries, industrial enterprises associated with obtaining raw materials from the ground, large cement plants with a capacity of more than 150,000 tons per year should be located at a distance of 10-15 km from populated areas. The next group of industrial zones can include various machine-building plants, textile factories, light and food industries. In the third group of industrial zones it is silly to include such enterprises as optics, printing houses, garment factories, local industry enterprises, consumer service centers, etc. Sanitary protection zone can be a green highway or boulevard, passing through the industrial area and residential areas. The area of the industrial zone should be sufficient to accommodate various industries and servicing farms (transport routes, marshalling yards, power stations, etc.) [9]. According to the analysis, the impact of regions on the environment in the organization of production and the availability of infrastructure is the main problem. Therefore, the localization of production in special industrial zones is an efficiency factor. At the same time, the issue of organizing production or service activities in free economic zones organized in the country is a solution to the problem. To date, 17 free economic zones (FEZs) have been created in 11 regions of Uzbekistan (Table 1). In these regions, the infrastructure necessary to organize production activities was formed, and investors were given a number of advantages. In particular, in paragraph 3 of the Decree of the President of the Republic of Uzbekistan UP-4853 of October 26, 2016 "On additional measures to activate and expand the activities of free economic zones" provides for the provision of benefits for a period of 3 to 10 years, depending on the volume of investment, including number in the equivalent: -from 300 thousand US dollars to 3 million US dollars -for a period of 3 years; -from 3 million US dollars to 5 million US dollars -for a period of 5 years; -from 5 million US dollars to 10 million US dollars -for a period of 7 years; -from 10 million US dollars and above for the period of 10 years, with application for the next 5 years, the income tax rate and a single tax payment of 50 percent below the current rates. These opportunities testify to the creation of a favorable investment climate in the country for wider introduction of processes of organization and localization of production and the formation of the necessary infrastructure. Conclusions and Suggestions Thus, it can be concluded that in the process of localization it is the organization of production in free economic zones that can lead to high productivity. When choosing the location of the production facility location, we recommend the following: -analysis of regional factors of location. Priority here is determined by the relative advantage over the number of problems that arise when choosing a region for a specific purpose; -development of alternative options for regional location; -determination of land plots meeting the established requirements; -examination of the availability of raw materials and labor in the organization of production; -evaluation of the alternative of placement and selection of the last version of localization.	2019-06-13T13:17:01.479Z	2018-05-30T00:00:00.000	{ "year": 2018, "sha1": "d0bea650f86a244eb52a0d36608aea7f5d65d55b", "oa_license": null, "oa_url": "https://doi.org/10.15863/tas.2018.05.61.28", "oa_status": "GOLD", "pdf_src": "MergedPDFExtraction", "pdf_hash": "6d702c71d668550ac52d29c3eaefd2adbbc00c09", "s2fieldsofstudy": [ "Economics" ], "extfieldsofstudy": [ "Computer Science" ] }
9073423	pes2o/s2orc	v3-fos-license	QCD sum rules at finite temperature We derive thermal QCD sum rules for the correlation function of two vector currents in the rho-meson channel. It takes into account the leading non-perturbative corrections from the additional operators, which appear due to the breakdown of Lorentz invariance at finite temperature. The mixing of the new operators has a drastic effect on their coefficients. The thermal average of all the operators can be related to that of the quark condensate and the energy density. The sum rules then yield the temperature dependence of the parameters of the $\rho$-meson, namely its mass and coupling to the vector current. Our result is that these parameters are practically independent of temperature at least up to a temperature of 125 MeV. I. INTRODUCTION The QCD sum rules [1], proposed about two decades ago, prove remarkably successful in addressing the nonperturbative problems of hadron phenomenology. In this approach one considers the product of two local operators, like the currents of the QCD theory. A sum rule is obtained by equating the dispersion relation for its vacuum matrix element at sufficiently large space-like momenta to the corresponding Wilson operator product expansion [2]. The higher dimension operators present in the expansion give rise to the non-perturbative, power corrections. The idea of extending these sum rules to finite temperature by replacing the vacuum matrix element with the thermal average naturally suggests itself. The original work establishing the thermal QCD sum rules is that of Bochkarev and Shaposhnikov [3]. They recognised the importance of the (low energy) continuum states in the spectral function to account for the effects of the medium. On the basis of these sum rules they discussed the temperature dependence of the resonance parameters and the existence of phase transition. However, there arise additional operators in the Wilson expansion at finite temperature [4], which were not correctly incorporated in their sum rules: In effect, they calculated these new operator contributions perturbatively, which cannot be justified, particularly at low temperature. The additional operators arise because of the breakdown of Lorentz invariance at finite temperature by the choice of the thermal rest frame, where matter is at rest at a definite temperature [5]. The residual O(3) invariance naturally brings in additional operators. The expected behaviour of the thermal averages of these Lorentz non-invariant (new) operators is somewhat opposite to those of the Lorentz invariant (old) ones: While the old operators start with non-zero values at zero temperature and decrease in magnitude with the rise of temperature, the new ones, on the other hand, are zero at zero temperature but grow rapidly with temperature. The importance of including these new operators in the thermal sum rules, particularly at not too low a temperature, is thus clear. Although a number of works on thermal QCD sum rules exist by now, these are flawed with respect to the new operators: Either some of these are missed [6] or their mixing, which changes their coefficients drastically, is not taken into account [7]. In a recent work [8] we applied a simple, configuration space method [9], [10] to evaluate the Wilson coefficients of these new operators (up to dimension four) which appear at finite temperature in the short distance expansion of the product of two quark bilinear operators. Here we make use of this result to write and evaluate the thermal QCD sum rules, incorporating correctly the contributions from all the dimension four operators. We consider the correlation function of the time ordered (T ) product of two vector currents in the ρ-meson channel. The use of the T -product, rather than the retarded (or advanced) product, is a little complicated in writing down the spectral representation but has the advantage in perturbative calculations, for which we can apply the conventional formalism. Throughout this work we shall employ the real time formulation of the thermal field theory [11] , which requires in general not only the physical fields but also the accompanying 'ghost' fields. Since, however, we work to lowest order in perturbation expansion, ghost fields do not show up. It is convenient to write difference sum rules by subtracting the vacuum sum rules from their finite temperature counterparts. For such sum rules the absorptive parts are expected to be saturated with the ρ-meson pole and the ππ-continuum [3], whose contributions we derive here for completeness. The thermal averages of the different operators reduce essentially to that of the quark condensate and the energy densities of quarks and gluons [12]. Chiral perturbation theory [13] has been used to calculate the temperature dependence of the quark condensate [14]. The difficulty with the energy densities is that while one of them is the total energy density, which can be obtained from a knowledge of the hadronic spectrum at low temperature, the other is an unphysical combination of the two densities. We need an additional input to relate this latter combination to the total energy density. Once the power corrections are known, the difference sum rules give the temperature dependence of the ρ-meson parameters, namely its mass and its coupling with the vector current. With our simple saturation scheme, the sum rules can be used up to a temperature of about 125 MeV. The numerical evaluation shows that these parameters have negligible dependence on temperature. In sec.II we write the kinematic decomposition for the thermal correlation function of two vector currents, derive the Landau representation [15] for the time ordered product and state the results of operator product expansion to derive finally the form of the thermal QCD sum rules. In sec.III we obtain the contributions of ρ and ππ intermediate states to the spectral function. In sec.IV we collect the information on thermal average of the operators present in the sum rules and evaluate the difference sum rules. In sec. V we discuss how to extend the sum rules to higher temperature. In the Appendix we give the details of the evaluation of a limit stated earlier [3]. II. SUM RULES We derive the QCD sum rules for the thermal average of the time ordered (T ) product of two currents, Here V a µ (x) is the vector current (in the ρ meson channel) in QCD theory, q(x) being the field of the u and d quark doublet and τ a the Pauli matrices. The thermal average of an operator O is denoted by O , where H is the QCD Hamiltonian, β is the inverse of the temperature T (coinciding with the time ordering symbol !) and T r denotes the trace over any complete set of states. A. Kinematics At finite temperature Lorentz invariance is broken by the choice of a preferred frame of reference, viz, the matter rest frame where temperature is defined. But the book-keeping with indices becomes simpler if we restore it formally by introducing the four-velocity u µ of the matter [5]. (In the matter rest frame u µ = (1, 0, 0, 0).) The time and space components of q µ are then raised to the Lorentz scalars, ω = u · q andq = ω 2 − q 2 . We shall, however, return to the matter rest frame while doing all actual computations. In such a Lorentz invariant framework, there are two independent conserved kinematic covariants [16], which we choose as The invariant decomposition of T ab µν is then given by where the invariant amplitudes T l,t are functions of q 2 and ω. Notice that the kinematic covariants P µν and Q µν are free from singularities at q 2 = 0 (and also atq 2 = 0 ). This is convenient at finite temperature as there are dynamical singularities extending up to q 2 = 0. With this choice of kinematic covariants, the dynamical singularities reside only in the invariant amplitudes. To extract the invariant amplitudes from T ab µν , it is converient to form the scalars Π 1,2 , Then the invariant amplitudes are given by In the limit q = 0, the amplitudes T l and T t are related. To see this we write the spatial components of T µν as , whereq i is the ith component of the unit vector along q. As \| q\| → 0, there cannot be any dependence on \| q\|, getting B. Spectral representation Let us obtain the spectral representation for the correlation function in q 0 at fixed \| q\|. First, evaluate the trace over a complete set of eigenstates of four-momentum, when it becomes a sum over forward amplitudes weighted by the corresponding Boltzmann factors. Then insert the same set of complete states between the currents to extract its x-dependence which is then integrated out. Introducing a δ-function in q 0 , the result can be written as where the expression for M ab µν with the double sum over states may be converted back to the form, Using the double sum representation, it is easy to show that The opposite sign of iǫ in the two terms in eqn.(2.6) is typical of T -products. As a result the imaginary part of T ab µν is given by the sum, 1 2 (M ab µν + M ba νµ ), while the principal value integral contains the difference, 1 2 (M ab µν − M ba νµ ). But the two are related by (2.8), We thus get the Landau representation for the T product at finite temperature [15], where, for brevity, we write we recover the representation for the invariant amplitudes T l,t . Further, using the symmetry properties, ImT l,t (−q µ ) = ImT l,t (q µ ) and going over to imaginary values of q 0 (q 2 0 = −Q 2 0 , Q 2 0 > 0), for which N µν vanishes, these become, It may actually require subtractions, but it does not affect the Borel transformed sum rules we shall write below. C. Operator product expansion The contributions of operators of dimension four to T l,t are obtained from the short distance expansion and improved upon by the renormalisation group equation in a previous work [8]. Including the unit operator (the perturbative contribution), T l is given for large Euclidean momenta by (Q 2 = −q 2 ), T t is also given by the same expression, except for an overall factor of −Q 2 and a factor of (1 − 2q 2 /Q 2 ) multiplying the term with Θ's. Herem is the degenerate mass of the u and the d quarks and 1 2 qq = ūu = d d . The operator G 2 is quadratic in the gauge field strength G a µν , (a = 1, · · · , 8), G 2 = (α s /π)G a µν G aµν , with α s = g 2 /4π, where g is the gauge coupling constant. Along with these two operators already contributing to zero temperature, we now have the linear combinations of two new ones, Θ f,g ≡ u µ Θ f,g µν u ν , where Θ f,g µν are the energy-momentum tensors for quarks of a single flavour and of gluons. The corresponding components for the total tensor is Θ = n f Θ f + Θ g , where n f is the number of effective quark flavours. The logarithmic Q 2 dependence of a(Q 2 ) arise due to the anomalous dimension d of the operator where µ(≃ 1 GeV) is the scale at which all renormalisations are carried out. Note the mixing of the operators Θ f and Θ g in (2.13) under the renormalisation group, which is well-known in the context of deep inelastic scattering [17]. In the operator product expansion of two quark currents, the Wilson coefficients of Θ f and Θ g are, to leading order, of zeroth and first order in α s , arising from Born and one-loop graphs respectively. However, due to operator mixing, the coefficients are drastically changed in that both Θ and (16Θ f /3 − Θ g ) have coefficients with leading terms of zeroth order in α s . In (2.13) we retain only these leading terms. D. Sum rules We now equate the spectral representation and the result of operator product expansion for the amplitudes T l and T t at sufficiently high Q 2 0 . Taking Borel transform we arrive at the thermal QCD sum rules [3]. For T l we get where O is the non-perturbative contribution of higher dimension operators, and a similar one for T t . Each is a two parameter sum rule, dependent not only on T but also on \| q\|. In the thermal rest frame the thermal average of the new operators are energy densities, which increase rapidly with temperature. Earlier works on thermal QCD sum rules were flawed, as these contributions were not properly included. III. ABSORPTIVE PARTS We work below the critical temperature, where hadrons constitute the physical spectrum. As with the vacuum sum rules, the dominant contribution to the spectral function is given by the ρ-meson. We also calculate the contribution of the non-resonant ππ-continuum. The question of other significant contributions will be discussed in Sec. IV. A. ρ-pole The coupling of the vector current to the ρ-meson is given by where ǫ µ is the polarisation vector of ρ of mass m ρ . The experimental value of F ρ as measured by the electronic decay rate of ρ 0 [18] is F ρ = 153.5 MeV. A simple way to calculate the absorptive part of the ρ-pole diagram is to note the field-current identity of V a µ (x) with the rho-meson field ρ a µ , which reproduces (3.1). Then the ρ-meson contribution is given essentially by its thermal propagator, where ∆ ρ 11 (q) is the 11-component of a scalar field propagator with mass m ρ , and n(ω q ) is the Bose distribution function, n(ω q ) = (e βωq − 1) −1 , ω q = q 2 + m 2 ρ . We then get Loop corrections at finite temperature will make m ρ and F ρ temperature dependent, modifying them, in general, differently in the longitudinal and transverse amplitudes. These modifications may be obtained by calculating the appropriate loop graphs. Here we shall find them by evaluating our sum rules. B. ππ-continuum The ππ-contribution to the amplitudes describes the interaction of the current with the particles in the medium, which are predominantly pions. Chiral perturbation theory [13] gives the contribution of the pion field φ a (x) to the vector current, which, to lowest order, is Then the pions contribute to the correlation function as where ∆ π 11 is again the 11-component of the scalar propagator (3.4) but with mass m π . Its imaginary part can be obtained by the cutting rules at finite temperature [19]. Here we obtain it directly for this simple amplitude. We express ∆ π 11 as ∆ π 11 (k) = {1 + n(ω k )}D(k) + n(ω k )D * (k), where D(k) is the zero temperature propagator, D(k) = i/(k 2 − m 2 π + iǫ), and carry out the k o -integration [20]. The imaginary part may then be read off as ImT ab µν (q) = δ ab {L µν (q) + L µν (−q)}, (3.8) where Here n 1 ≡ n(ω 1 ), n 2 ≡ n(ω 2 ) with ω 1 = k 2 + m 2 π , ω 2 = ( k − q) 2 + m 2 π . The time component of k µ in the tensor structure is understood to be given by k 0 = ω 1 . With the help of the δ-functions we can rerwite (3.9) to get N µν defined by (2.11) for q 0 > 0, (3.10) In this form the factors involving the density distributions can be interpreted in terms of pion absorption from and emission into the medium [21]. Since the first and the second δ-functions in (3.10) contribute to time-like (q 2 ≥ 4m 2 π ) and space-like (q 2 < 0) regions respectively, we write it as Thus in addition to the usual cut, 4m 2 π + \| q\| 2 ≤ q 2 0 ≤ ∞, the amplitude at finite temperature acquires a new short cut, 0 ≤ q 2 0 ≤ \| q\| 2 [21], [3]. The angular integration is carried out using the δ-function, when the constraint \| cos θ q, k \| ≤ 1 results in a θ- We thus get It is now simple to extract the absorptive parts of the invariant amplitudes by using (2.4). Changing the variable ω 1 to x given by ω 1 = 1 2 (q 0 + \| q\|x), we get for q 2 > 4m 2 π (3.14) The superscripts (±) on N denote time-like and space-like q µ respectively, where they are non-vanishing. The first term on the right of (3.14) arising from the unity in the factor (1 + n 1 + n 2 ) in (3.13) is the zero temperature contribution of the ππ state. Evaluated here in a non-covariant way it, of course, agrees with the covariant evaluation of the Feynman amplitude (3.6) with ∆ π 11 (k) replaced by the vacuum propagator D(k) [13]. C. Explicit sum rules Let us now write explicitly the sum rule (2.14) for T l . Saturating N l with the above contributions it becomes, As the temperature goes to zero, the two terms in bracket go to zero and the thermal average of the operators on the right become the vacuum expectation values, recovering the familiar vacuum sum rule. The integral on the left is the non-resonant 2π contribution and is small compared to the resonance contribution. As \| q\| → 0, the sum rule (3.17) simplifies considerably. The limit for the second integral in bracket is given in ref. [3]. Here we derive it in the Appendix. The sum rules for T l and T t then become, and with v ≡ v(s) = 1 − 4m 2 π /s. Observe that the sum rules (3.18-19) are not independent, in agreement with the relation (2.5): the second one is obtained by differentiating the first with respect to 1/M 2 . IV. EVALUATION OF SUM RULES In the above sum rules we have approximated the absorptive parts of the amplitudes by the ρ-meson pole and the 2π continuum, while we retain only the contributions of the unit operator (the perturbative result) and of all the operators of dimension four in the operator product expansion. To check this saturation scheme, let us compare the zero temperature limit of our sum rules with the corresponding vacuum sum rules [1]. The latter include, in addition, the rather large contribution from the high energy continuum beyond 1.5 GeV, as indicated by the experimental data, as well as the contribution of a quark operator of dimension six, which is also large because of its origin in Born (rather than loop) diagram. Since we do not include any of these contributions, we cannot expect the sum rules as written above to be well saturated. Rather than incorporate these contributions, we isolate the thermal effects by considering the difference sum rules, obtained by subtracting out the vacuum sum rules from the corresponding finite temperature ones. Then the contribution to the absorptive parts beyond 1.5 GeV, being temperature independent, cancels out in the difference. Thus it is the temperature dependent contributions of the ρ-meson and of the 2π continuum, which should dominate the absorptive parts of these sum rules. Also the dimension six quark operator, O 6 say, contributes an amount ∼ O 6 − 0\|O 6 \|0 , which is insignificant in the immediate neighbourhood of T = 0 and as the temperature rises, this contribution is overwhelmed by that of the two-gluon and other Lorentz non-invariant operators, as our estimates below for these operators show. The difference sum rules for the two invariant amplitudes allow us to calculate the temperature dependence of the ρ-meson parameters, where the bar on the operators indicates subtraction of their vacuum expectation values. Here we insert the experimental values for m ρ and F ρ , as these are well reproduced by the vacuum sum rules. We now collect information on the operator contributions. The vacuum expectation value of the chiral condensate 0\|qq\|0 is known from the PCAC relation of Gell-Mann, Oakes and Renner, 2m 0\|ūu\|0 = −F 2 π m 2 π , wherem = 1 2 (m u + m d ) and the pion decay constant F π , defined by, has the value F π = 93.3 MeV. Takingm = 7 MeV [22], we get 0\|ūu\|0 = −(225M eV ) 3 . The vacuum expectation value of the two-gluon operator, as determined from the QCD sum rules [1], is 0\|G 2 \|0 = (330M eV ) 4 . The operator G 2 is related to the trace of the energy momentum tensor Θ µν by the trace anomaly. Normalising it to zero vacuum expectation value and taking thermal average, it gives The trace at finite temperature is given by Θ µ µ = Θ − 3P, where Θ is the energy density and P the pressure. The temperature dependence of both ūu and Θ have been calculated in chiral perturbation theory [14]. Corrections due to nonzero quark masses as well as the contributions of the massive states (K, η, ρ, · · ·) have also been incorporated. However, as the authors point out, the validity of the perturbation theory and the use of dilute gas approximation to calculate the contribution of the massive states restrict these results to within a temperature of about 150M eV . Thus the critical temperature T c is, strictly speaking, beyond the range of validity of their calculation. Since, however, the order parameter ūu falls rapidly at the upper end of this range, one has only to extrapolate it a little further to get T c = 190M eV . Besides the total energy density Θ , there also occurs the thermal average, 16Θ f /3 − Θ g . The last one cannot be calculated without further input, at least in the hadronic phase. Now both naive counting of the degrees of freedom and empirical study of the pion structure function [4] suggest the quark fraction of the energy density to be about half of the total. So we assume As with the zero temperature sum rules, the results are expected to be stable in a region in M 2 , which is neither too high to make the continuum contribution large relative to the resonance contribution nor too low to emphasize the neglected power corrections of higher order. Since the high energy continuum contribution gets cancelled in the difference sum rules, the region of M 2 may be extended somewhat at the upper end. Figs. 1 and 2 show the evaluation for M 2 equal to 1GeV 2 and 4GeV 2 . The results for ∆m ρ and ∆F ρ are rather stable for temperatures up to about 125 MeV. At higher temperatures the results, particularly for ∆m ρ appear unstable. Closer observation reveals here a large cancellation between the 2π contribution and the leading power correction we have retained. Thus the non-leading power correction become important here, whose inclusion may restore the stability in M 2 to higher temperatures. V. DISCUSSIONS In this work we have written the thermal QCD sum rules for the two point correlation function of vector currents in the ρ-channel, including the leading power correction due to all the operators of dimension four. Because of the loss of Lorentz invariance at finite temperature, two new (Lorentz noninvariant) operators creep up in the Wilson expansion, in addition to the two old (Lorentz invariant) ones, already existing in the vacuum sum rules. Thus compared to the two numbers, 0\|qq\|0 and 0\|G 2 \|0 in the vacuum sum rules, we have now four temperature dependent quantities, qq , G 2 , Θ and 16Θ f /3 − Θ g in the thermal sum rules. All these thermal averages can be evaluated from chiral perturbation theory, supplemented by contributions from the massive states. An extra input is needed only for the last unphysical operator. The sum rules can then be used to determine the temperature dependence of the ρ-meson parameters. Our evaluation shows that the mass of the ρ-meson and its coupling with the vector current remain practically unaffected by the rise of temperature up to at least 125 MeV. The absence of the shift in the mass appears to agree with one set of results obtained in ref [23]. Unfortunately the sum rules, as they stand, cannot be extended up to the critical temperature. One reason contributing to this restriction has to do with the evaluation of the thermal average of the operators, as we already discussed in the last section. What restricts it further is their instability under a change of M 2 for temperatures above 125M eV . Even in this restricted temperature range, the numerical evaluation shows that the new operators are significant. In fact, for temperatures above say 70 MeV, the new contributions overwhelm the old ones in the difference sum rules. This situation necessitates reanalysis of all earlier results based on thermal QCD sum rules, where the new operators are not properly taken into account. We now discuss a possible way to extend the sum rules to higher temperatures. In the vacuum sum rules the operators of dimension four (and higher) provide corrections to the leading (perturbative) result. But in the difference sum rules it is these corrections which become the leading contribution. One thus expects that by including nonleading contributions from higher dimension operators along with those of dimension four already included, the sum rules would be stable against variations in M 2 over a wider range of temperature. This inclusion is all the more necessary for sum rules like the one for ∆m ρ , where the leading operator contribution cancels largely with that of ππ-continuum. The higher dimension operators will, of course, complicate the evaluation of the sum rules in that we have to know the temperature dependence of their thermal averages. Also there is more proliferation of operators than what is generally thought. The procedure in the literature [6] of including dimension six quark operators and excluding the Lorentz noninvariant gluon operators [24], because of the smallness of their coefficients by a factor of α s (M 2 ), is not justified. For, as we have seen, the quark and the gluon operators mix under a change of scale, so that after renormalisation group inprovement, both the coefficients are of the same order in α s (M 2 ). A way to proceed here is to write the entire set of sum rules by considering two-point functions of not only the vector quark bilinear but also the others, like the scalar, tensor, etc. All of these sum rules receive contribution from a few resonances and the operators from the same set. The simultaneous evaluation of all these sum rules is expected to provide a self-consistent check on the thermal average of the operators. Further, using quark bilinears of appropriate chiralities, one can get sum rules without any of the gluon operators [25]. These sum rules should prove easier to evaluate and would also check the saturation scheme in a simpler context. To get the second line we integrate by parts over u and interchange the order of integration in the remaining double integral. The limit of the corresponding integral for T t is zero, because of the presence of q 2 in the integrand.	2014-10-01T00:00:00.000Z	1997-11-12T00:00:00.000	{ "year": 1997, "sha1": "ad58c95eca1e9e23fef00754fc5f53d09150f79e", "oa_license": null, "oa_url": "http://arxiv.org/pdf/hep-ph/9711297", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "ad58c95eca1e9e23fef00754fc5f53d09150f79e", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
218763555	pes2o/s2orc	v3-fos-license	Some open book embeddings of higher dimensional smooth and contact manifolds We prove some open book embedding results in the smooth and contact category. We prove an open book version of the Haefliger--Hirsch embedding theorem by showing that every $k$-connected closed manifold $M^n$ has an open book embedding in $S^{2n-k} = \mathcal{O}b(D^{2n-k-1},id)$. We also give various examples of contact open book embeddings of a contact manifold $(M^{2n+1},\xi)$ in $(S^{4n+1},\xi_{std})$. Finally, we prove a smooth open book embedding result for Weinstein/Stein fillable contact manifolds and briefly discuss its relation to the fillability problem. introduction Embedding of manifolds is an old and much studied problem in topology. The simplest target manifold to embed a manifold is the Euclidean space R N or the Euclidean sphere S N . The celebrated Whitney embedding theorem [Wh] tells that every such n-manifold M n can be embedded in S 2n . The work of Massey [Ma], Haefliger-Hirsch [HH] and Hirsch [Hi] says that for orientable n-manifolds the minimal dimension of embedding can be further reduced to give embedding in S 2n−1 . In [HH], Haefliger and Hirsch generalized this theorem for k-connected manifolds and produced embeddings in S 2n−k−1 . Embedding questions can also be studied with extra geometric structures on the manifolds. One of the first geometric embedding problem, the C 1 -isometric embedding problem, was studied by John Nash [N]. This inspired the study of embedding problems for manifolds preserving other given geometric structures. An open book, roughly speaking, is a decomposition of a manifold into a co-dimension 2 submanifold and a mapping torus (see section 2 for details). The existence of an open book on a manifold follows from the works of Tamura [Ta], Winkelnkemper [Wi], Lawson [La] and others. It is known that all odd dimensional manifolds admit open book decomposition. For 4m-dimensional manifolds, the index is assumed to be zero In [Wi], Winkelnkemper gave one the first construction of open book decompositions on simply connected manifolds. Combining the open book decomposition of Winkelnkemper [Wi] and embedding results of Haefliger and Hirsch [HH] we get the following open book version of the Haefliger-Hirsch theorem. open book embedding were obtained by Mori [Mo] and Torres [To], using techniques from approximate holomorphic geometry. Using Gromov's h-principle type results for isosymplectic immersions and embeddings, we prove the following open book embedding result in the contact category. Here, dλ 0 denotes the standard symplectic form on the 4n-ball. As an application of Theorem 1.2, we find large classes of contact 2n + 1-manifolds that admit contact open book embedding in the trivial open book of (S 4n+1 , ξ std ). Note that Kasuya [Ka] has proved that every 2-connected contact (2n + 1)-manifold admits isocontact embedding in (S 4n+1 , ξ std ). Another class of contact manifolds admitting contact open book embedding is the following. Corollary 1.4. Let (W 2n , dλ W ) be a simply connected Weinstein domain which has the homotopy type of a CW complex that has no nonzero even dimensional cells. Then Ob (W 2n , dλ W , φ W ) contact open book embeds in (S 4n+1 , ξ std ) = Ob(D 4n , dλ 0 , id), for n ≥ 3. We also point out another class of examples of 2n + 1-dimensional contact manifolds that contact open book embed in (S 4n+1 , ξ std ). To state our results we need the following definition. Definition 1.5. Consider the canonical symplectic structure dλ M on the cotangent bundle of a manifold M . We call the contact open book Aob(V 2n , ω, φ) of type-1, if it satisfies the following properties. (1) (V 2n , ω) be an Weinstein domain symplectomorphic to Here, Mis and Njs are either n-sphere S n or a closed n-dimensional manifold. (2) The monodromy φ is generated by Dehn-Seidel twists along the S n s among Mis and Njs. Here # b and § denote boundary connected sum and plumbing, respectively. For the notion of symplectic boundary connected sum and plumbing we refer to [Ge] and [Ko]. The proof of Theorem 1.6 is essentially the same as that of Theorem 1.4 in [S]. We will only give an outline of it with the slight modifications needed. The proof of Theorem 1.7 relies on the existence of an abstract Weinstein Lefschetz fibration (see section 2.7 on a Weinstein domain (up to deformation equivalence) due to the work of Giroux and Pardon [GP]. Theorem 1.7 can be used to provide a topological obstruction to the existence of Weinstein or Stein filling of almost contact manifolds of sufficiently high dimension. We discuss such examples in section 6.1. We would like to remark that recent times have seen a lot of progress in the problem of smooth and contact open book embedding and isocontact embedding. In particular, much progress has been made on the question of co-dimension 2 isocontact embedding due to the works of Kasuya [Ka2], Etnyre and Furukawa [EF], Etnyre and Lekili [EL] and Pancholi and Pandit [PP]. Recently, the existence and uniqueness questions for co-dimension 2 isocontact embedding has been completely answered by the works of Casals, Pancholi and Presas [CPP], Casals and Etnyre [CE] and Honda and Huang [HoH]. Some explicit constructions of smooth open book embedding and isocontact embedding were also provided in the works of [EF], [EL], [PPS] and [S2]. While much is known regarding explicit isocontact or contact open book embeddings for 3-manifolds, similar problems for higher dimensional contact manifolds are yet to be studied in full generality. An open book is a decomposition of a manifold into a co-dimension 2 submanifold and a fibration over S 1 . Definition 2.1 (Open book decomposition ). An open book decomposition of a closed oriented manifold M consists of a co-dimension 2 oriented submanifold B with a trivial normal bundle in M and a locally trivial fibration π : M \ B → S 1 such that π −1 (θ) is an interior of a co-dimension 1 submanifold N θ and ∂N θ = B, for all θ. The submanifold B is called the binding and N θ is called a page of the open book. We denote the open book decomposition of M by (M, Ob(B, π)) or sometimes simply by Ob(B, π). Example 2.1. S n admits an open book decomposition with page D n−1 and monodromy the identity map. We call this open book, Ob(D n−1 , id), the trivial open book of S n There is another notion of an abstract open book that is equivalent to the above definition and in many cases is easier to work with. Definition 2.2. Let Σ be as before and let φ be a diffeomorphism of Σ that is identity on a collar neighborhood of ∂Σ. An abstract open book decomposition of M is a pair (Σ, φ) such that M is diffeomorphic to where id denotes the identity mapping of ∂Σ × S 1 . Here, MT (Σ, φ) = Σ × [0, 1]/ ∼ is the mapping torus, where ∼ is the equivalence relation identifying (x, 0) with (φ(x), 1). We will denote such an abstract open book by Ob(Σ, φ). [E] and [Ge]. Two abstract open books Note that the isotopy class of φ uniquely determines M , up to diffeomorphisms. The map φ is called the monodromy of the open book. The manifold obtained by identifying the boundary of MT (Σ, φ) with the boundary of ∂Σ × D 2 , as described in the Definition 2.2, will be denoted by Ob (Σ, φ). Here ∂Σ and ∂V could be empty sets. Example 2.4. By [Wu], any two embeddings of a closed manifold M m in R 2m+1 are isotopic. Let f : M → R 2m+1 be an embedding and let φ ∈ Dif f eo + (M ). Then by [Wu], f and f • φ are isotopic. Using the isotopy extension theorem one can then extend this isotopy to an ambient isotopy Φ t of R 2m+1 such that Φ 0 = id and Φ 1 • f = f • φ. Therefore, any embedding of M m in R 2m+1 is flexible. 2.2. Winkelnkemper open books for closed manifolds. In [Wi], Winkelnkemper showed existence of open book decomposition for simply connected manifolds of dimension ≥ 7. The prototype for Winkelnkemper's proof, as Winkelnkemper himself mentions in [Wi], is the following example. Let us quickly review Winkelnkemper's proof. As in [Wi], we assume that M n is a simply connected closed n-manifold and n = 2k + 1 for k ≥ 3. Take a minimal handle decomposition of M n . Let W 1 denote the handlebody constructed by attaching handles of dimension up to k. Let W 2 denote the handlebody constructed by attaching such handles of dimension up to k of the dual handle decomposition. Then, we know that M n = W 1 ∪ ∂ W 2 . Where, W 1 and W 2 are attached along their common boundary E = W 1 ∩W 2 = ∂W 1 = ∂W 2 via some diffeomorphism. There exist k-dimensional sub-complexes K 1 ⊂ W 1 and K 2 ⊂ W 2 whose inclusions are homotopy equivalences. The following lemma is the main step in Winkelnkemper's proof. Lemma 2.5 (Winkelnkemper). There exists a k-complex K ⊂ E ⊂ ∂W 1 = ∂W 2 such that both inclusions K ⊂ W j (j = 1, 2) are homotopy equivalences. Now, let V be a regular neighborhood of K in E. V -will be the candidate for page and ∂V will be the binding. Since K has co-dimension ≥ 3, V and ∂V are simply connected. Take a collar ∂V × I of V and regard W 1 and W 2 as a relative cobordism between V and the closure of the complement of V ∪ ∂V × I in E. The assertion then implies that both W 1 and W 2 are relative h-cobordisms. Thus, by the relative h-cobordism theorem W 1 = V × I = W 2 and by contracting the collar neighborhood ∂V × I we get the desired open book with binding ∂V and page V . We call this open book the Winkelnkemper open book. Note that if M 2k+1 is an l-connected manifold, then according to the above construction we get an open book with an l-connected page. This is because we can then start with a handlebody decomposition M 2k+1 = W 1 ∪ W 2 such that each W i has no handles upto dimension l. Therefore, the following holds. Lemma 2.6. If M 2k+1 is an l-connected manifold, then the Winkelnkemper open book has l-connected pages. Embedding and isotopy of manifolds in Euclidean In [HH], Haefliger and Hirsch proved the following generalizations of Whitney's embedding theorem [Wh] and Wu's isotopy theorem [Wu]. Theorem 2.8 (Theorem 2.2, [HH]). Assume 0 ≤ k ≤ 1 2 (n − 4). If M n is orientable there is a 1 − 1 correspondence between the isotopy classes of embeddings of M in R 2n−k and the regular homotopy classes of immersions of M 0 in R 2n−k with a normal vector field. The proof of Theorem 2.7 and Theorem 2.8 uses the following theorems of Hirsch and Haefliger [HH]. (1) If m ≥ 2n − k − 1, then M 0 can be immersed in R m , and any immersion is regularly homotopic to an embedding. (2) If m ≥ 2n − k, any two embeddings f and g of M 0 in R m are regularly homotopic. If G is a regular homotopy connecting f and g, there is a regular homotopy G t of G such that G 0 = G, G 1 is an isotopy, and for each t, G t connects f to g. Theorem 2.10 (Theorem 3.2, [HH]). Let X be an m-manifold and E an open n-disk. (1) Suppose 2m ≥ 3(n + 1) and X is (2n − m + 1)-connected. Let g : E → X be a proper map whose restriction to the complement of some compact set is an embedding. Then there is a homotopy, fixed outside of a compact set, which deforms g into an embedding. (2) Suppose 2m ≥ 3(n + 1) and X is (2n − m + 2)-connected. Let g 0 , g 1 : E → X be proper embeddings which which are connected by a homotopy fixed outside of a compact set. Then g 0 and g 1 are also connected by an isotopy g t , fixed outside of a compact set. By the Smale-Hirsch immersion theory M 0 has an immersion in R 2n−k−1 with a normal vector field. Using statement (1) of Theorem 2.9, one regularly homotopes this immersion to an embedding f . Now, using the normal vector field, the embedding is extended to a map g 0 : M → R 2n−k−1 such that g 0 coincides with f outside a small neighborhood of the deleted point. Then using statement (2) of Theorem 2.10, g 0 is homotoped to an embedding g with the homotopy identity outside a neighborhood of the deleted point. This shows the main steps in the proof of Theorem 2.7. For Theorem 2.8, a similar approach is taken. By statement (2) of Theorem 2.9, we can start with two regularly homotopic embeddings f 0 , f 1 of M 0 in R 2n−k with normal vector fields. Then one finds two homotopic maps h 0 and h 1 : M → R 2n−k extending f 0 and f 1 , respectively. Statement (1) of Theorem 2.10 allows to assume that h 0 and h 1 are embeddings and therefore statement (2) of Theorem 2.10 implies that h 0 and h 1 are isotopic as embeddings and the isotopy is fixed outside a neighborhood of the deleted point. The proof of Theorem 2.7 and Theorem 2.8 also holds for proper embeddings of a manifold (V n , ∂V n ) in an Euclidean disk (D N , ∂D N ). We will be using this relative versions of Theorem 2.7 and Theorem 2.8 in our proofs. 2.4. Contact manifold and isocontact embedding. A contact manifold is an odd dimensional smooth manifold M 2n+1 , together with a maximally non-integrable co-dimension 1 distribution ξ ⊂ T M . A contact form α representing ξ is a local 1-form on M such that ξ = Ker{α}. The contact condition is equivalent to saying that α ∧ (dα) n is a volume form. The 2-form dα then induces a conformal symplectic structure on ξ. If the line bundle T M/ξ over M is trivial, then the contact structure is said to be co-orientable. For a co-orientable contact structure ξ, one can define a contact form α representing ξ on all of M . We will only consider co-orientable contact structures on closed, orientable manifolds. We will denote a manifold M together with a contact structure ξ by (M, ξ) and use ξ std to denote the standard contact structure on an odd dimensional Euclidean space R 2N +1 , given by Ker{dz The dimension will be understood from the notation (R 2N +1 , ξ std ). Two contact manifolds (M 1 , ξ 1 ) and (M 2 , ξ 2 ) are equivalent if there is a diffeomorphism h between them such that Dh(ξ 1 ) = ξ 2 . We say, the two contact manifolds are contactomorphic to each other. For more details on contact manifolds see [Ge]. We now define the notion of embedding in the category of contact manifolds. It follows from the definition that if α is a contact form representing ξ and β is a contact form representing η. Then ι * (β) = h · α for some positive function h on M . Dι(ξ) is a conformal symplectic sub-bundle of (η\| ι(M ) , dβ). Gromov [Gr] reduced the existence of an isocontact embedding of a contact manifold (M 2n+1 , ξ) in a contact manifold (V 2N +1 , η), for N ≥ n + 2, to a problem in obstruction theory. In particular, Gromov proved the following contact analog of Whitney's embedding theorem. Remark 2.13. When the embedding co-dimension is ≤ dim(M )−1, there is a natural topological obstruction to contact embedding. It has the following description. If ι : (M 2n+1 , ξ) → (R 2N +1 , η) is a contact embedding, then the normal bundle ν(ι) = ι * (η)/ξ has an induced complex structure on it. So we have the following relation of total Chern classes. 2.5. Contact open book and embedding. We start with a discussion of the Thurston-Winkelnkemper construction [TW] of contact open book decomposition. Let (V, ∂V, dα) be an exact symplectic manifold which has a collar symplectomorphic to ((−1, 0]×∂V, d(e t · α)), where t ∈ (−1, 0]. The Liouville vector field Y for dα is defined by i Y dα = α. Near boundary this Figure 2. Functions for the contact form near binding vector field looks like ∂ ∂t and is transverse to ∂V pointing outwards. The 1-form e t · α induces a contact structure on ∂V . Let φ be a symplectomorphism of (V, dα) that is identity in a collar of the boundary. The following lemma, due to Giroux, shows that we can assume φ * α − α to be exact. Here h : V → R is a function well defined up to addition by constants. Note that dt + α is a contact form on R × V , where the t-co-ordinate is along R. Take the mapping torus MT (V, φ) defined by the following map. The contact form dt + α then descends to a contact form λ on MT (V, φ). Since φ is identity near ∂V , a neighborhood of the boundary of MT (V, φ) looks like (− 1 2 , 0) × ∂V × S 1 with the contact form e r · α\| ∂V + dt. Denote the annulus {z ∈ C \| r < \|z\| < R} by A(r, R). Define Φ as follows. Let σ t : T * S n → T * S n be a map defined as follows. For k ∈ Z >0 , let g k : [0, ∞) → R be a smooth function that satisfies the following properties. (2) Fix p 0 > 0. The function g k (\| y\|) decreases to 0 at p 0 and then remains 0 for all y with \| y\| > p 0 . See Figure 3. Now we can define the positive k-fold Dehn-Seidel twist as follows. The Dehn-Seidel twist is a proper symplectomorphism of T * S n . From Figure 3, we see that τ k has compact support. Therefore, choosing p 0 properly, τ k can be defined on the unit disk bundle (DT * S n , dλ can ), such that it is identity near boundary. In fact, we can choose the support as small as we wish without affecting the symplectic isotopy class of the resulting τ k . More precisely, let g 1 k and g 2 k be two functions similar to g k as above. Say, g 1 k has support p 1 and g 2 k has support p 2 . Then σ tg 1 k (\|·\|)+(1−t)g 2 k (\|·\|) gives a symplectic isotopy between σ g 1 k (\|·\|) and σ g 2 k (\|·\|) . Similarly, for k < 0, we can define the negative k-fold Dehn-Seidel twist. For k = 0, τ 0 is defined to be the identity map of DT * S n . Sometimes we may say just Dehn twist instead of Dehn-Seidel twist. 2.6.1. The standard contact open book of (S 2n+1 , ξ std ). An important open book decomposition of (S 2n+1 , ξ std ) is given with page (DT * S n , dλ n can ) and monodromy a positive Dehn-Seidel twist τ n . We call this open book the standard open book of S 2n+1 . Lefschetz fibrations on Weinstein domains. Here we briefly recall the notion of an abstract Weinstein Lefschetz fibration defined Giroux and Pardon in [GP]. We formally define what Weinstein manifolds and domains and avoid the technical details of it as we will not use them in our proof. For details on Weinstein manifolds/domains we refer to [CE]. Recall that a 1-form λ on a manifold V such that ω = dλ is symplectic, is called a Liouville form and the vector field X such that i X ω = λ, is called the Liouville field of λ. An exact symplectic manifold means a pair (V, λ) where λ is a Liouville form. Equivalently, an exact symplectic manifold can be described by a triple (V, ω, X) with i X ω = λ. Definition 2.17 (Liouville cobordism). A Liouville cobordism is an exact symplectic manifold (V, ω, X) such that X points outward along ∂ + V and points inward along ∂ − V . Definition 2.18 (Weinstein manifolds and Weinstein domains). A Weinstein manifold (W, ω, X, φ) is a symplectic manifold (W, ω) with a complete Liouville vector field X which is gradient-like for an exhausting Morse function φ : W → R. A Weinstein cobordism (W 1 , ω, X, φ) is a Liouville cobordism (W, ω, X) whose Liouville field X is gradient-like for a Morse function φ : W 1 → R which is constant on the boundary. A Weinstein cobordism with ∂ − W 1 = ∅ is called an Weinstein domain. Given an abstract Weinstein Lefschetz fibration W = (W 0 ; L 1 , . . . , L m ), one can construct a Weinstein domain W 2n+2 (its total space) by attaching critical Weinstein handles to the stabilization W 0 × D 2 along Legendrians Λ j ⊂ W 0 × S 1 ⊂ ∂(W 0 × D 2 ) near 2j m ∈ S 1 , obtained by lifting the exact Lagrangians L j . So the abstract Weinstein Lefschetz fibration is a sort of singular fiber bundle over D 2 with Weinstein pages and where the monodromy is given by composing the Dehn-Seidel twists along the Lagrangian spheres. Giroux and Pardon proved that every Weinstein domain can be deformed to an abstract Weinstein Lefschetz fibration. Theorem 2.21 (Giroux-Pardon, [GP]). Let W be a Weinstein domain. There exists an abstract Weinstein Lefschetz fibrationW = (W 0 ; L 1 , . . . , L m ) whose total space (which we again denote byW ) is deformation equivalent to W. It is clear that the existence of an isosymplectic homomorphism is a necessary condition for existence of an isosymplectic immersion. Gromov [Gr] proved the following h-principle for isosymplectic immersions to show that to some extent it is also sufficient. Theorem 2.24 (Isosymplectic immersion theorem [Gr]). Let (W, ω W ) and (V, ω V ) be symplectic manifolds of dimensions 2N and 2n respectively. Suppose a continuous map , and that f 0 is covered by an isosymplectic homomorphism F : T V → T W . If V is closed and 2N ≥ 2n + 2, then there exists a homotopy f t : V → W such that f 1 is an isosymplectic immersion and the differential Df 1 is homotopic to F through isosymplectic homomorphisms. The h-principle for isosymplectic immersion is also true for the relative and the parametric version (see Theorem 16.4.3 in [EM]). Following is Gromov's h-principle for isosymplectic embedding. (1) (Open case) If n ≤ q − 2 and the manifold V is open then there exists an isotopy f t : V → W such that the embedding f 1 : V → W is isosymplectic and the differential df 1 is homotopic to F 1 through isosymplectic homomorphisms. (2) (Closed case) If n ≤ q − 4 then the above isotopy f t exists even if V is closed. Moreover, one can choose the isotopy f t to be arbitrarily C 0 -close to f 0 . The above h-principle is also true for the relative and parametric case. In particular, if a family of proper isosymplectic immersions f t : (V, ∂V, dλ V ) → (W, ∂W, dλ W ) (satisfying the cohomology condition: is regularly homotopic to a family of embedding e t , depending smoothly on t, then by Theorem 2.25, f t can be isotoped to a family of proper isosymplectic embeddings f s t . Obstructions to isosymplectic homomorphism : Consider a symplectic vector space (X, ω). Let J be an ω-compatible almost complex structure (i.e., ω(Ju, Jv) = ω(u, v) and ω(u, Ju) > 0 for all u, v ∈ X \ {0}). If Y is a symplectic subspace of (X, ω), then Y has to be a J-subspace of (X, J) and vice-versa. An almost complex structure J ξ on the contact hyperplane bundle ξ = Ker{α} is called ξ-compatible, if it is compatible with the conformal symplectic structure on ξ induced by dα. An isosymplectic homomorphism takes (T V, ω V ) to a symplectic sub-bundle of (T W, ω W ). If J W is an ω W -compatible almost complex structure, then the isosymplectic homomorphism takes (T V, ω V ) to a J W -sub-bundle of (T W, ω W ). When W = D 2N and ω W = ω 0 , finding an isosymplectic homomorphism from (T V, ω V ) to (T W, ω W ) is equivalent to finding a U (n)-equivariant map from the complex n-frame bundle associated to (T V, ω V ) to St C (N, n). In other words, finding an isosymplectic homomorphism is equivalent to the existence of a section of the associated St C (N, n)-bundle of (T V, ω V ). Here, St C (N, n) denotes the complex Stiefel manifold. Thus, (T V, ω V ) has an isosymplectic homomorphism in (T D 2N , ω 0 ) if and only if all the obstruction classes in Two isosymplectic homomorphisms are homotopic via isosymplectic homomorphisms if the corresponding sections of the associated St C (N, n)-bundle of (T V, ω V ). Such homotopy obstructions lie in H i (V, ∂V ; π i St C (N, n)), for 1 ≤ i ≤ 2n. 3. Open book embedding of k-connected manifolds in S 2n−k We now prove Theorem 1.1. We want to use a relative version of Theorem 2.7 and Theorem 2.8, which can be stated as follows. Lemma 3.1. Let (V n , ∂V n ) be a k-connected manifold. Let V 0 denote the manifold obtained by removing an n-disk from the interior of V . (1) If V 0 can be immersed in R 2n−k−1 with a normal vector field, then V n admits a proper embedding in (D 2n−k , ∂D 2n−k ). (2) There is a 1 − 1 correspondence between the isotopy classes of proper embeddings of V in D 2n−k and the regular homotopy classes of proper immersions of V 0 in (D 2n−k , ∂D 2n−k ). (3) Any two proper embeddings f and g of V n in D 2n−k+1 are isotopic. The proof of Lemma 3.1 will become easy once we observe how the proofs of Theorem 2.7 and Theorem 2.8 works. We now review the main steps behind these proofs from [HH]. Proof of Theorem 2.7: Let f 0 : M n 0 → R 2n−k−1 be an immersion with a normal vector field v. By Theorem 2.9, f is regularly homotopic to an embedding. So we may assume f 0 to be an embedding. Let D i be an embedded closed disk of radius i around a point x 0 ∈ M n for i = 1, 2. Let M i = M \ int(D i ). The claim is that f (∂M 1 ) is an (n − 1)-sphere homotopic to zero in X = R 2n−k−1 \ f (M 2 ). Consider an -tubular neighborhood of f (M 1 ) in R 2n−k−1 . Let λ : M → [0, ] be a smooth function that has value on M 2 and is 0 on D 1 . Then f (∂M 1 ) bounds the image of M 1 under the map f v : M → R 2n−k−1 given by x → f (x) + λ(x)v(x). Thus, f (∂M 1 ) is null-homologous in X. By Poincare and Alexander duality H i (X) ∼ = H k+i+2−n (M ) (with Z-coefficient). Since M is k-connected, H i (X) = 0 for i ≤ n − 2. Thus, by Hurewicz isomorphism between π n−1 (X) and H n−1 (X) we get that that f (∂M 1 ) is null-homotopic in X. One can therefore, extend the map f \| M1 to a map g : M → R 2n−k−1 such that g(M 2 ) ∩ g(int(D 2 )) = ∅. An application of Theorem 2.10 then leads to an embedding of D 2 in X relative to boundary. Together with the embedding f \| M2 , this gives an embedding of M in R 2n−k−1 . Next we discuss the proof of Theorem 2.8. We shall continue with the notations used above. Proof of Theorem 2.8: First one shows that Given an embedding f : An argument similar to the previous proof shows that X is (n − 1)-connected and π n (x) = H n (X) = H n−k−1 (M 2 ). If v 1 and v 2 are two normal (to f (M 2 )) vector fields, then the difference class d(v 1 , v 2 ) ∈ H n−k−1 (M 2 ) corresponds to the homology class [f v1 (M )] − [f v2 (M )] ∈ H n (X). Also, the homotopy classes of normal vector fields on f (M 0 ) are in 1 − 1 correspondence with H n−k−1 (M 0 ) = H n (X). there is one and only one normal vector field V, up to is homologous to zero in X. Therefore, up to homotopy, there is only one vector field such that f v (M ) is null-homologous in X. Figure 5. Isotoping the embedding of M 1 by the vector field λ(x)·v(x). The region enclosed between the two red curves is X = M \ D 2 . The next step is to show that this correspondence is 1 − 1. We skip the proof of surjectivity and focus only on the proof of injectivity. Let h t be an isotopy joining f 1 \| M0 and f 2 \| M0 , and let u t , be a normal vector field of h t (M 0 ) joining v 1 and v 2 . An isotopy of h 0 (M 0 ) can be extended to an isotopy of R 2n−k−1 . So one can assume that f 1 and f 2 agree on M 1 and that v 1 = v 2 . Now observe that f v1 (M ) and f v2 (M ) are homologous in X. Therefore, by similar arguments as before f v1 (M ) and f v2 (M ) are homotopic in X. Thus, f 1 \| D1 and f 2 \| D1 are homotopic in X. Therefore, by Theorem 2.10 they are isotopic in X by an isotopy fixed on a neighborhood of ∂D 1 . Hence, f 1 and f 2 are isotopic. Proof of Lemma 3.1. (1) First we embed V in the boundary (2n − k − 1)-sphere of D 2n−k by Theorem 2.7. Let f 0 be the embedding. Now define a smooth function ψ : V → [0, ] such that ψ is zero in a neighborhood of ∂V . Let R be the radial unit vector field on D 2n−k pointing inward from boundary. Let r t denote the time t flow of the vector field ψR. r 1 • f 0 then gives the required embedding. Informally, we just pushed f 0 (V ) into the interior of D 2n−k relative to its boundary. (2) Let g 1 , g 2 be two proper embeddings of V in D n−k such that g 1 and g 2 agree on a neighborhood of ∂V . We assume that g 1 and g 2 are transverse to the boundary ∂D 2n−k . Recall the embedding f 0 from the proof of (1). Then ι 1 = f 0 ∪ g 1 and ι 2 = f 0 ∪ g 2 are embeddings of the double, D(V ), of V . Let v 1 and v 2 be two corresponding normal vector fields on ι 1 (D(V )) and ι 2 (D(V )). Since ι 1 = ι 2 in a neighborhood of ∂V , we may assume that v 1 = v 2 in a neighborhood N (∂V ) of ∂V . If g 1 and g 2 are regularly homotopic relative to boundary with homotopic normal vector fields then by Theorem 2.9 (ι 1 \| D(V )0 , v 1 ) and (ι 1 \| D(V )0 , v 2 ) can be assumed to be isotopic. Moreover, this isotopy can be fixed near f 0 (V ) ∪ g 1 (N (∂V )). We can proceed as in the proof of Theorem 2.8 to show that ι 1 and ι 2 are isotopic in D 2n−k via an isotopy Φ t . Note that during the construction of this isotopy we have not moved f 0 (V ) ∪ g 1 (N (∂V )). Therefore, Φ t \| V gives us the required isotopy between g 1 and g 2 . The surjectivity case can also be dealt with similarly. For details see [HH]. Let f 1 , f 2 be two such embeddings. By Smale-Hirsch immersion theory the obstruction to a regular homotopy between (f 1 \| M0 , v 1 ) and (f 2 \| M0 , v 2 ) lies in the group H i (M 0 ; π i St(2n − k + 1, n + 1)) for 1 ≤ i ≤ n. Since M 0 is homotopic to its (n − k − 1)-skeleton and St(2n − k + 1, n + 1) is (n − k − 1)connected, these obstructions vanish. Therefore, we can assume f 1 and f 2 to be isotopic on M 0 . Since H n (Y ) = 0, both f v1 (M ) and f v2 (M ) are null-homologous in Y . Arguing as in Theorem 2.8, we then see that f 1 \|D 1 and f 2 \| D1 are homotopic relative to boundary. Then by Theorem 2.10 we see that f 1 and f 2 are isotopic. Contact open book embedding in We find a suitable embedding of V 2n in D 4n to apply the h-principle for isosymplectic immersion. We recall that an embedding g : Lemma 4.1. Given an isocontact embedding g : (M 2m+1 , ker{α} = ξ) → (N 2n+1 , ker{β} = η), there exist a constant C > 0 and a positive functionh on M such that the following holds. The next lemma is essentially Proposition 4 in [Au], adapted to our setting. For a proof we refer to [S]. (1) The restriction of (T V, J V ) on ∂V is trivial as a complex vector bundle. Proof. The proof is essentially an application of a version of the Whitney sum formula for relative characteristic classes. It was first developed by Kervaire in [Ker]. Let f s be a proper isosymplectic immersion of (V 2n , dλ V ) in (D 4n , dλ 0 ). Note that the i-th Chern class is a characteristic class in the group H 2i (V 2n ; π 2i−1 St C (n, n − i + 1)), which is an obstruction to the existence of a section of the associated St C (n, n − i + 1)-bundle of (T V, J V ). Since the restriction (T V, J V ) on ∂V is a trivial complex bundle, there is also no obstruction to the existence of a section of that associated St C (n, n − i + 1)-bundle. Therefore, we can talk about relative Chern classes of (T V, J V ). Moreover, all the relative Chern classes of (T D 4n , dλ 0 , J 0 ) vanish. Therefore, the relative version of the Whitney sum formula gives the following for the normal bundle of immersion ν(f s ). φ t Figure 6. Using the isotopy φ t , we push DT * S 0 away from the zero section S 1 0 (denoted by red circles) of DT * S 1 . Then we apply Dehn twist along S 1 0 , with support in the shaded region. section, and then again bring DT * S n back to its initial position. Figure describes the situation for n = 0. Thus, by a similar argument as before, one can prove that Ob(DT * S n , τ l n ) contact open book embeds in Ob(DT * S 2n , τ k 2n ) for any l, k ∈ Z. In fact, this follows from the following stronger result. Theorem 5.4 (Theorem 1.1, [S]). For n ≥ 1 and k, l ∈ Z, Aob(DT * S n , dλ n can , τ k n ) contact open book embeds in Aob(DT * S n+1 , dλ n+1 can , τ 2n ). Since both Ob(DT * M n , id) and Aob(DT * S n , dλ n can , τ k n ) contact open book embed in Aob(DT * S 2n , dλ n+1 can , τ 2n = (S 4n+1 , ξ std ), any combination of these open books by taking plumbings and boundary connected sums of their pages also contact open books in (S 4n+1 , ξ std ). The supporting open book of (S 4n+1 , ξ std ) will then be changed by doing sufficient number of positive stabilizations. Apart from the higher co-dimension of embedding, the proof of Theorem 1.6 follows the proof of Theorem 1.4 in [S] verbatim. Note that all these results are also implicit in the work of Casals and Murphy [CM]. Smooth open book embedding of Weinstein fillable contact manifolds We now prove Theorem 1.7. The following lemma is our main ingredient. Proof. By Cieliebak-Eliashberg [CE], every Weinstein domain is deformation equivalent to a Stein domain. On the other hand, Eliashberg and Gromov [EG] have shown that every Stein manifold of complex dimension n admits a proper holomorphic embedding in C 3n 2 +2 . Therefore, W 2n admits a proper smooth embedding in D 2 3n 2 +4 . Let N = 2 3n 2 + 4. By the Lagrangian neighborhood Theorem, L ⊂ (W 2n , dλ W ) has a neighborhood symplectomorphic to (DT * S n , dλ n ). This gives a smooth embedding of (DT * S n , dλ n ) in S N . Let h 1 denote this embedding. As discussed in the proof of Theorem 1.6, one has a standard proper isosymplectic embedding of (DT * S n , dλ n ) in (DT * S n+k , dλ n+k ) for all k ≥ 0. Since Ob(DT * S n+k , τ n+k ) is the standard open book of S 2n+2k+1 , this also gives an embedding of DT * S n in S 2n+2k+1 . Note that there exists a flow Φ t on S 2n+2k+1 whose time 1 map Φ 1 maps its page DT * S n+k to itself by applying a Dehn-Seidel twist along the zero section of DT * S n+k . This induces another embedding of DT * S n in S N = ∂D N +1 . Let h 2 denote this embedding. This induces an open book decomposition on M 2n+1 with page W and monodromy φ = τ L1 • τ L2 • · · · • τ Lm . Identify the S 1 -interval of the mapping torus of this open book with [0, 1]/0 ∼ 1. Divide [0, 1] into subintervals I j = [ j m+1 , j+1 m+1 ] for j ∈ 0, 1, ..., m. Let N = 2 3n 2 + 4. On the interval I j , we apply Lemma 6.1 for the vanishing cycle L j to obtain embedding of the mapping cylinder of τ Lj : W → W in the mapping cylinder of the identity map (up to isotopy) of D N +1 , for 0 ≤ j ≤ m − 1. We then glue these mapping cylinders together in cyclic order to obtain embedding of the mapping torus of φ in D N +1 × S 1 . This gives us an open book embedding of M 2n+1 in S N +2 = Ob(D N +1 , id). Remark 6.2. One may consider a higher dimensional analogue of the 4-dimensional achiral Lefschetz fibration, which is like an abstract Weinstein Lefschetz fibration, but it may also have, in its monodromy, negative Dehn-Seidel twists along the vanishing cycles. Such an achiral abstract Weinstein Lefschetz fibration will naturally induce a contact open book structure on its boundary and in this case also the proof of Theorem 1.7 goes through. It will be interesting to know whether one can strengthen Theorem 1.7 to get a contact open book embedding. 6.1. Relation of Theorem 1.7 to fillability of contact manifolds. If an almost contact manifold M 2n+1 does not embed in R 2 3n 2 +6 , then by Theorem 1.7, it can not have a Weinstein or Stein filling. This can be used to show that certain almost contact manifolds does not admit any Weinstein/Stein fillable contact structure. For example, consider the manifold S 1 × CP n . This manifold admits almost contact structure. Now, it was shown by Sanderson and Schwarzenberger [SS] that CP n cannot be immersed in R 4n−2a(n)+ , where a(n) is the number of 1s in the binary expression of n and ∈ {0, 1, −1}. In particular, = 0 for n even and a(n) ≡ 1 (mod 4). Take n = 16. Then a(n) = 1 and = 0. Thus, CP 16 does not immerse in R 62 . Therefore, S 1 × CP 16 cannot embed in S 62 . Since 2 3×16 2 + 6 = 54 < 62, S 1 × CP 16 does not admit any Weinstein/Stein fillable contact structure. Similarly, one can show that S 3 × CP 32 does not embed in 126 > 2 3×33 2 + 6 = 105. Therefore, S 3 × CP 32 cannot admit any Weinstein/Stein fillable contact structure. 6.2. Embedding of Stein/Weinstein fillable manifolds. By a theorem of Eliashberg and Gromov [EG], every complex n-dimensional Stein manifold has a proper holomorphic embedding in C N for N = 3n 2 + 2. It is therefore natural to ask what can we say about the optimal dimension of contact open book embedding of a Stein fillable contact manifold of dimension 2n + 1 in some trivial open book. We end our discussion with the following question. Question 6.3. What is the minimal k such that every Stein fillable contact manifold of dimension 2n + 1 admits contact open book embedding in (S 2( 3n 2 )+k , ξ std )?	2020-05-22T01:01:04.939Z	2020-05-21T00:00:00.000	{ "year": 2020, "sha1": "4f719b1430a33303051ccea38a1a1cfb75bce2d8", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "4f719b1430a33303051ccea38a1a1cfb75bce2d8", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Mathematics" ] }
266580234	pes2o/s2orc	v3-fos-license	Exploring the Central Mechanisms of Botulinum Toxin in Parkinson’s Disease: A Systematic Review from Animal Models to Human Evidence Botulinum toxin (BoNT) is an effective and safe therapy for the symptomatic treatment of several neurological disturbances. An important line of research has provided numerous pieces of evidence about the mechanisms of action of BoNT in the central nervous system, especially in the context of dystonia and spasticity. However, only a few studies focused on the possible central effects of BoNT in Parkinson’s disease (PD). We performed a systematic review to describe and discuss the evidence from studies focused on possible central effects of BoNT in PD animal models and PD patients. To this aim, a literature search in PubMed and SCOPUS was performed in May 2023. The records were screened according to title and abstract by two independent reviewers and relevant articles were selected for full-text review. Most of the papers highlighted by our review report that the intrastriatal administration of BoNT, through local anticholinergic action and the remodulation of striatal compensatory mechanisms secondary to dopaminergic denervation, induces an improvement in motor and non-motor symptoms in the absence of neuronal loss in animal models of PD. In human subjects, the data are scarce: a single neurophysiological study in tremulous PD patients found that the change in tremor severity after peripheral BoNT administration was associated with improved sensory–motor integration and intracortical inhibition measures. Further clinical, neurophysiological, and neuroimaging studies are necessary to clarify the possible central effects of BoNT in PD. Introduction Botulinum toxin (BoNT) is an approved, safe, and effective drug for the symptomatic treatment of several neurological and non-neurological conditions.The main effect of BoNT therapy, consisting of a transient chemical paralysis of the treated muscles, stems from the blockage of the release of acetylcholine (Ach) from the motor axons of the neuromuscular junction [1].After the injection, through the binding with proteins, including synaptotagmin and synaptic vesicle protein 2, BoNT is internalised in the presynaptic terminal where it cleaves and deactivates the SNARE proteins (SNAP 25, VAMP, syntaxin) that are essential for the fusion of vesicles with the synaptic membrane and the release of Ach into the synaptic cleft [2].BoNT is commonly used in the treatment of many neurological disorders, including dystonia and spasticity, among others [3,4].In recent years, however, growing attention has been paid to the possible application of BoNT to treat motor and non-motor symptoms in Parkinson's disease (PD) [5,6].The appeal of BoNT stems from its targeted action, the absence of widespread side effects, and its compatibility with dopaminergic treatments, offering a promising avenue for treating symptoms that traditionally relied on systemically active medications.The symptoms in PD that are amenable to treatment with BoNT include motor symptoms such as tremors, focal dystonia, and dyskinetic movements, as well as non-motor symptoms, including sialorrhea, dysphagia, gastroparesis, constipation, bladder hyperactivity, and sweating dysfunction [6,7].The evidence derived from studies conducted on patients suffering from spasticity and dystonia suggests that BoNT acts not only on the neuromuscular junction but also at the level of intrafusal muscle fibres, neuromuscular spindles, the spinal cord, and suprasegmental structures [8][9][10][11][12][13][14][15][16][17][18][19][20].The action of BoNT at these levels could explain its wide and long-lasting effects [21,22].Unlike dystonia and spasticity, only a few studies have provided evidence regarding potential central effects of BoNT in PD. In this systematic review, we aimed to evaluate the studies conducted so far on both animal models and patients with PD that focused on examining and clarifying the possible central effects of BoNT in PD. Search Strategy This systematic review was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) flow diagram (https://www.bmj.com/content/339/bmj.b2700,accessed on 4 May 2023).The PRISMA flow diagram is presented in Figure 1 [23]. Literature searches in PubMed and SCOPUS were performed in May 2023 using the following search string: ((Botulinum toxin [Title/Abstract]) OR BoNT [Title/Abstract]) AND Parkinson's disease [Title/Abstract] OR PD [Title/Abstract].The searches were limited to publications in English.All articles published before May 2023 were included.All search results were aggregated in Excel for Windows, and duplicates were discarded, so only unique references were retained.This review was not registered. Study Selection The records were screened according to their title and abstract by two independent reviewers (CC and FM).The relevant articles were selected for full-text review.Additionally, the studies where the eligibility remained undetermined based solely on the title or abstract were also considered for a full-text review. Eligible studies for inclusion were as follows: (I) Firstly, studies exploring PD animal models where molecular, motor behavioural, and non-motor changes after central nervous system (CNS) injection of BoNT were assessed.(II) Secondly, studies focusing on PD human subjects that evaluated the central effect of BoNT treatment through clinical, neuroradiological, and neurophysiological assessments. In instances where disagreement arose between the reviewers, debates were conducted until a unanimous decision was achieved regarding the inclusion of the study.This systematic review was open to including both longitudinal and cross-sectional studies.We imposed no restrictions regarding the age of participants or the duration of their disease, as our goal was inclusivity.As we aimed to produce a comprehensive review of any evidence of the effect of BoNT in the CNS, no study quality threshold was set. • The density of striatal ChAT-ir neurons is higher in the injected striatum. • BoNT does not exert toxic effects on cholinergic neurons. • The density of ChAT-ir BiVs is higher in the twice-injected group. • In 6-OHDA rats, there is a constant trend towards higher binding to striatal D2R.This trend tends to return to normal values after BoNT-A administration. • BoNT-A induces a significant decrease in the D1R concentrations in contrast to sham-treated animals.6-OHDA is a molecule similar to dopamine that, when it binds to the dopamine transporter molecule, can reach neurons, causing strong oxidative stress in the cytosol and mitochondria.The injection of 6-OHDA into the median forebrain bundle (MFB) induces almost total dopaminergic denervation in the substantia nigra pars compacta (SNPc) in 2-3 days [43].In most of the studies included in our search, the 6-OHDA toxin was injected into the right MFB, obtaining a hemi-parkinsonian model.MPTP is a compound that is able to cause a selective degeneration of the SNPc after systemic administration and has been widely used in the last 30 years to obtain a PD animal model.In the only study we included in our selected studies that adopted this approach, the protocol consisted of an intraperitoneal injection four times per day for 7 days in C57BL/6 mice [25]. Molecular Evidence We identified eleven studies that employed immunohistochemistry, immunofluorescence, autoradiography, positron emission tomography (PET), and functional magnetic resonance imaging (fMRI) techniques to examine alterations in synaptic transmission and receptor systems after intracerebral administration of BoNT within the striatum. In the following sections, we will discuss separately the evidence regarding the effects of BoNT on cholinergic, dopaminergic, GABAergic, serotonergic, noradrenergic, and glutamatergic pathways in hemi-PD animal models. BoNT Effects on Cholinergic System SNAP-25 represents one of the pivotal targets through which BoNT interrupts cholinergic transmission at the presynaptic level [44,45].A study employing immunostaining against cleaved SNAP-25 in rats described that, after BoNT intrastriatal injection, there is an increase in cleaved SNAP-25 immunostaining limited to the ipsilateral striatum, and motor and somatosensory cortices, sparing the contralateral hemisphere and interhemispheric connection structures [46].In addition, the impact of BoNT was subtype-dependent, with BoNT-A2 exhibiting more pronounced effects [27]. Three studies investigated the impact of intrastriatal BoNT injections on local cholinergic transmission in animal models of PD.Two investigations highlighted the morphological modifications indicative of Ach accumulation at presynaptic terminals [26,36].In a 6-OHDA PD rat model, Wree et al. (2011) observed the development of varicosities, which were immunohistochemically positive for choline acetyltransferase (ChAT), along striatal neurons on the ipsilateral side of the intrastriatal BoNT injection [26].Analogous results were reported by Hawlitschka et al. in a study conducted on C57BL/6 mouse models [28].Importantly, both studies concluded that BoNT does not affect the count of ChAT-positive interneurons, implying it does not exert toxic effects on cholinergic neurons [26,28]. In a nine-month longitudinal study, Mann et al. used in vitro receptor autoradiography to delineate the temporal trajectory of BoNT-induced alterations in the densities of various muscarinic and nicotinic receptor subtypes within the striatum of 6-OHDA rats.The investigation uncovered distinct temporal patterns of receptor density alterations after the intrastriatal injection of BoNT.Specifically, the density of M1 receptors initially diminished three weeks post-intervention, followed by a progressive recovery over the ensuing nine months.Conversely, densities of M2 and M4 receptors, initially stable, showed a reduction over the subsequent nine months without apparent recovery.The M3 receptors consistently manifested a 10% reduction in density, which persisted throughout the study.Lastly, nicotinic receptors, especially the α4 and β2 subtypes, exhibited a substantial 50-60% decrease post-injection, with no indication of subsequent recovery [29]. In the studies mentioned above, potential correlations between BoNT-induced ultrastructural changes and motor behavioural measures were not explored. Overall, the identified morphological changes and modulation of receptor density shed light on the intricate, time-dependent, and eventually network-specific impacts of BoNT, underscoring its non-toxic impact on cholinergic neurons and suggesting potential therapeutic implications in PD models. BoNT Effects on Dopaminergic System In our systematic review, we identified five studies that focused on the effect of intrastriatal BoNT injections on dopaminergic neurotransmission. The dopaminergic denervation, reproduced experimentally in 6-OHDA rats and MPTP mice, triggers compensatory mechanisms, including increased expression of striatal dopaminergic receptors D1R and D2R, increased interhemispheric differences in dopamine receptor expression, and an increased acetylcholine/dopamine ratio in lysates of whole brain tissue [25]. By using immunohistochemistry and ELISA on lysates of the whole brain, Ham et al. observed that intrastriatal BoNT injections induce a recovery of dopamine levels and an increase in the expression of tyrosine hydroxylase-positive neurons in the SNpc, which are both reduced by 6-OHDA-or MPTP-induced lesions. The BoNT-induced effect on the compensatory modification of dopaminergic receptor expression has been studied both in vitro and in vivo, using autoradiography and positron emission tomography (PET) in animal models of PD [32,33].Three longitudinal studies showed a significant reduction in ipsilateral D1R expression and a reduction in interhemispheric differences in striatal binding of D2R in 6-OHDA rats treated with intrastriatal BoNT compared to sham-treated animals [26,28,30]. A correlation between motor behavioural measures and striatal changes in dopamine receptor expression has also been reported.In 6-OHDA rats, Wedekind et al. found a positive correlation between asymmetry in forelimb use, evaluated through the cylinder test (see below), and the inter-striatal difference in D2R binding [31].In a similar animal model, Mann et al. described a significant positive correlation between the difference in D2R/D3R availability in the right/left striatum and an increase in apomorphine-induced rotational behaviour (see below) [33]. Overall, these studies might suggest that striatal BoNT injections, by modulating compensatory mechanisms arising from dopaminergic denervation, can improve motor performance in animal models of PD. In our review, we only identified one study that focused on the assessment of D2/D3 receptors at the level of the olfactory bulb in 6-OHDA rats treated with intrastriatal BoNT using PET/TC [34].The authors showed that the administration of BoNT into the striatum increased both D2R and D3R availability within the olfactory bulb, accompanied by improved olfactory performance [34].The evidence coming from this study confirms that dopamine is a key transmitter for processing olfactory information in the olfactory bulb.The authors hypothesised that the increase in receptor availability in the OB after ipsilateral striatal BoNT injection parallels olfactory performance and occurs through indirect connections between the striatum and olfactory bulb via the dorsal raphe nucleus or the amygdala nuclear complex [34]. BoNT Effects on Glutamatergic and GABAergic Systems We only identified one study assessing BoNT's impact on the glutamatergic and GABAergic neurotransmitter systems.BoNT injections were administered into the entopeduncular nucleus (EPN) of 6-OHDA rats, a region analogous to the human globus pallidus internus (GPi), in an attempt to target presynaptic glutamatergic inputs from the subthalamic nucleus (STN) [35].An immunohistochemical analysis using markers specific to glutamatergic (vesicular glutamate transporter 2-vGlut2) and GABAergic (GAD-67) terminals was performed.While no significant modifications were observed in GABAergic immunoreactivity, a significant reduction was detected in glutamatergic immunoreactivity.Even though the chronological trajectory between BoNT-induced immunohistochemical and motor behavioural changes was similar, the presence of potential correlations between these measures was not investigated [35]. Overall, these findings indicate that BoNT might be able to modulate glutamatergic systems, leaving GABAergic systems unaffected.These observations might suggest the possibility of a neurochemical modulation of the STN through the local injection of BoNT, with a therapeutic target similar to what has been observed in PD patients treated with deep brain stimulation (DBS) of the STN. BoNT Effects on Serotoninergic and Noradrenergic Systems Our search identified only one study that focused on the effect of intrastriatal BoNT injections on the serotoninergic and noradrenergic systems.In this study, Mann et al. employed quantitative in vitro receptor autoradiography in the 6-OHDA PD animal model to measure BoNT-induced changes in alpha 1, alpha2, and 5HT2A receptors at the striatal level.The mean density and the relative interhemispheric right-left difference were assessed for each receptor [32]. No changes were observed for alpha1 and alpha 2 receptors at the striatal level after the BoNT injection.Conversely, there was an initial 50% reduction in the mean density of the 5HT2A receptor in 6-OHDA PD rats, which underwent a further reduction following the BoNT injection.Finally, no significant correlation was reported between these receptors' alterations and motor improvements, as assessed by the rotational test. Overall, these observations suggest that the noradrenergic and serotonergic systems do not represent preferential targets of BoNT at the striatal level in animal PD models [32]. Evidence for BoNT Effects on Motor Behaviour We identified eleven sham-controlled longitudinal studies investigating motor behaviour changes in PD animal models following the intracerebral injection of BoNT.While the majority of studies administered BoNT into the striatum, two studies injected BoNT into the EPN, which is equivalent to the human GPi [25][26][27]31,33,[35][36][37][38]40].The studies also varied in terms of the dosage of BoNT used and whether the injection was ipsilateral or contralateral to the lesioned side. Beyond the differences in the injection methodologies employed, the outcome measures used across these studies were also quite heterogeneous.Indeed, various motor behaviour measures were employed, including analysis of drug-induced motor behaviours and performance evaluations conducted through validated motor tasks.The type of motor tasks that were used are shown in Table 2. Buried pellet test Olfactory performance The rat is put in a cage with 3 cm of clean bedding and one pellet buried 0.5 cm below in one corner of the cage or in the surface of the bedding.The rat is placed in the centre of the test cage and the latency time is measured until the rat uncovers the pellet and begins eating it. Lehmkuhl et al., 2014 [61] Drug-induced motor behaviours were investigated using apomorphine and amphetamine, which induce rotational behaviours in animals either opposite (apomorphine) or on the same side (amphetamine) of the 6-OHDA-induced lesion.Apomorphine, a dopamine agonist, binds more to the supersensitive dopamine receptors (DRs) on the lesioned side than the contralateral normal DRs and induces an anticlockwise rotation away from the striatal lesion [47,48].The findings coming from studies employing apomorphine-induced rotational behaviour as an outcome measure consistently indicated that in 6-OHDA PD rats, intrastriatal injections of BoNT led to a reduction of the anticlockwise rotational behaviour contralateral to the injected striatum [25][26][27]30,31,33,[35][36][37]39,40].Moreover, the BoNT-induced effects in 6-OHDA PD rats were more intense and prolonged when a second injection was administered at the same site after 6 months [30].These effects were found to vary between BoNT subtypes, with BoNT-2A achieving the abolition of the apomorphine-induced rotational behaviour at a lower dosage than BoNT-1A [27].Conversely, three studies reported that intrastriatal BoNT injections resulted in a significant increase in ipsilateral amphetamine-induced rotations [33,36,37].Amphetamine acts as an indirect agonist of monoamines and administration to 6-OHDA lesioned rats causes a stronger delivery of dopamine to the contralateral striatum causing an ipsilateral clockwise rotation to the lesioned side.Although its application is simple, the amphetamine rotation test is a weak predictor of 6-OHDA-induced lesions and its interpretation may not be straightforward [62].The persistence of this rotational behaviour after BoNT striatal injections may be due to the loss of catecholaminergic afferents in the 6-OHDA PD rat that is not influenced by BoNT and the further increase in this rotational behaviour after injection could be explained by changes in the basal ganglia circuits that may involve non-dopaminergic neurotransmitter systems [36]. In addition to drug-induced behaviours, several authors used validated motor tasks to test BoNT's effects on motor function in animal models of PD, showing heterogeneous results. Only one study investigated the effects of BoNT injections in the striatum contralateral to the 6-OHDA-induced lesion [37].The authors reported a transient increase in rotations induced by apomorphine, accompanied by a transient reduction in amphetamine-induced rotations 2 weeks after the injection.In addition, contralateral BoNT injections induced a significant improvement in forelimb use preference symmetry and forelimb akinesia and neglect contralateral to the 6-OHDA-induced lesion from 2 weeks to 9 months after the BoNT striatal injection [37]. Two studies evaluated the effects of administration of BoNT into the EPN, equivalent to human internal globus pallidus, on measures derived by a quantitative gait analysis in 6-OHDA PD rats [35,40].In these studies, the assessment of static and dynamic gait parameters was obtained using the CatWalk apparatus, a video-based automated gait analysis system developed to evaluate footfall and gait changes in rodents.The BoNT injection into the EPN abolished the apomorphine-induced rotational behaviour, increased the gait speed and cadence, reduced gait speed variations, and improved dynamic gait parameters beginning 1 week after the injection, peaked 1 month later, and returned to basal values at 3 months [35,40]. Finally, our search identified one study in which a sample of a mouse model of PD was studied.In this study, Ham et al. found that BoNT striatal injections can improve forced motor activity, increase the latency to fall in the rotarod test, reduce bradykinesia (pole test), and improve gait parameters such as stride length and stance length in both 6-OHDA and MPTP PD mice [25]. Overall, the results coming from studies that have evaluated BoNT-induced effects on motor behaviour in PD animal models suggest its use as a potential therapeutic option in PD (see Table 3).CPu: caudate-putamen complex; EPN: entopeduncular nucleus; ipsi: ipsilateral to the lesioned striatum; contra: contralateral to the lesioned striatum; n.p.: not performed. Evidence for BoNT Effects on Non-Motor Behaviours In our systematic review, we identified two studies that evaluated the effects of BoNT intracerebral injections on non-motor symptoms in PD animal models. One study evaluated BoNT-induced effects on anxiety and depression in PD animal models. To evaluate the presence of behavioural manifestations of mood disorders in rats, several validated tests, including the open field test, the elevated plus maze test for anxiety-like behaviour, the forced swim test (FST), and the tail suspension test for depressive-like behaviour, were used (see Table 2).The authors observed that 6-OHDA PD rats did not show an increase in anxiety behaviours compared to naive rats, thereby preventing the authors from evaluating a potential effect of intrastriatal BoNT administration on these symptoms.Conversely, 6-OHDA PD rats had an increased depressive-like behaviour that significantly improved after BoNT treatment.In this paper, the authors failed to find a correlation between motor performance and depressive behaviour before and after the intrastriatal BoNT injections.This led them to conclude that the severity of the motor impairment might not influence improvements in affective symptoms in PD animal models [41]. Finally, a single study evaluated the olfactory performance changes in 6-OHDA PD rats after treatment with intrastriatal BoNT injections.Even though 6-OHDA rats had comparable olfactory performance with naive rats, the authors described an improvement in the execution of an orienting odour identification test in BoNT-treated compared to sham-treated 6-OHDA rats [34]. Evidence in Humans In our review, we did not identify any clinical or neuroimaging studies providing indirect evidence of a central effect of BoNT in patients with PD.However, a recent neurophysiological study provided novel evidence on possible central mechanisms of BoNT in PD patients [42].In a cohort of 12 "de-novo" and 7 "L-dopa-treated" tremulous PD patients, the authors used various transcranial magnetic stimulation (TMS) paradigms to evaluate intracortical inhibition/facilitation and sensory-motor integration before and 6 weeks after a BoNT injection in the forearm muscles.The TMS protocols were performed in both hemispheres before BoNT infiltration and 6 weeks after BoNT injection, extending over 42 weeks.Eight time points were considered for four cycles of BoNT injections.The inhibitory intracortical circuits were evaluated using a short intracortical inhibition (SICI) paradigm, in which a sub-threshold conditioning stimulus was followed by an above-threshold test stimulus with inter-stimulus intervals of 2 ms (SICI2) or 4 ms (SICI4).Additionally, prolonged intra-cortical inhibition (LICI) was assessed, where a supra-threshold conditioning stimulus with an interstimulus interval of 100 ms preceded the test stimulus.On the other hand, Short Afferent Inhibition (SAI) and Long Afferent Inhibition (LAI) paradigms were employed to assess sensory-motor integration involving the stimulation of a median nerve preceding the TMS pulses.This peripheral pulse inhibits the motor response from contralateral motor cortex stimulation, with specific interstimulus intervals of 23 ms for SAI and 200 ms for LAI [42,63]. The findings of this study demonstrated that intramuscular BoNT injections resulted in changes in cortical neurophysiological parameters.Specifically, de novo tremulous PD patients at baseline exhibited reduced SICI, LICI, and SAI on the tremulous/treated side compared to the non-tremulous side.However, the BoNT treatment led to an increase in LICI, SAI, and LAI and a reduction in ICF.By adopting a linear mixed model analytical approach, the authors found that the BoNT-induced changes in LICI, SICI, and LAI were significantly related to the changes in tremor severity, as assessed by a kinematic tremor analysis [42]. The evidence coming from this study might suggest that the efficacy of BoNT in the treatment of PD tremors is partially attributable to central mechanisms and to modulation of intracortical inhibitory circuits and sensory-motor integration mechanisms. Discussion In this systematic review, we aimed to comprehensively evaluate the findings derived from studies conducted on animal models and human patients that focused on elucidating the potential central effects of BoNT in PD.Most of the evidence we reviewed arose from studies involving animal models of PD.At the same time, limited data were available from human studies, highlighting a gap in our current understanding and application of BoNT in the clinical management of PD.The evidence coming from animal studies contributes to clarifying the multifaceted impact of BoNT on various neurotransmitter systems within the central nervous system.At the level of the cholinergic system, intrastriatal BoNT injections induce the expected blockage of Ach release as well as morphological changes in cholinergic neurons and a timedependent modulation of cholinergic receptor density [26,29,46].In the striatum, under normal physiological conditions, a complex and bidirectional interaction exists between the cholinergic and dopaminergic systems [64].Multiple lines of evidence suggest that in PD, the loss of dopaminergic input leads to a reduced inhibition of tonically active cholinergic striatal interneurons [65,66].This results in a state of relative cholinergic hyperactivation, which contributes in part to the motor symptoms of the disease [67].Therefore, BoNT's ability to modulate cholinergic transmission may represent one of the central mechanisms by which BoNT induces motor improvement in animal models.This hypothesis is, however, speculative, given that the animal studies did not investigate the potential relationship between BoNT-induced effects on the cholinergic system and motor and non-motor clinical improvement. Similarly, BoNT administration at the striatal level modulates compensatory mechanisms arising from dopaminergic denervation.Indeed, BoNT reduces the overexpression of dopaminergic receptors and normalises dopamine release levels [31][32][33].The modulation of dopaminergic receptors seems clinically relevant given that two studies reported a correlation between the pattern of receptor expression and motor impairment in 6-OHDA PD rats [31][32][33].The mechanism by which BoNT reduces dopamine receptor expression remains unknown, but cytotoxicity is ruled out, as the number of striatal neurons remains unchanged post-BoNT injection [26].The regulation of dopaminergic receptor expression after BoNT injections may be due to the complex interplay between dopaminergic and cholinergic interneurons at the striatal level, as supported by observations in a PET study in monkeys [25]. In regard to glutamate, in our search, only the group of Tsang et al. targeted the EPN, i.e., the rodent equivalent of the GPi, and showed that BoNT can reduce glutamatergic transmission from the STN to EPN.Intriguingly, the authors observed that the chronological trajectory of BoNT-induced glutamatergic modulation was similar to that of BoNT-induced motor behavioural changes.The authors observed that, by blocking the glutamatergic influences from the STN, BoNT improved the gait parameters evaluated through the gait analysis [35].Clinical and neurophysiological studies have described that dopaminergic denervation in PD induces an increased influence of STN efferents to the GPi and that the neuronal fibres that project from the STN to GPi are mainly glutamatergic [68][69][70].This evidence is in contrast to what we observe in human PD patients treated with STN DBS, in which, the presence of axial symptoms, imbalance, and gait disturbances are considered a contraindication to implantation [71].BoNT's ability to modulate the abnormally increased glutamatergic transmission in PD animal models may contribute to BoNT's clinical effects. Conversely, BoNT treatment does not appear to significantly influence the GABAergic, noradrenergic, and serotonergic systems in PD animal models, suggesting that its therapeutic effects might not be mediated through these systems. Are BoNT's Central Mechanisms Clinically Relevant in Animal Models of PD? A few authors evaluated the clinical effects of BoNT injections at the central nervous system level in animal models of PD.Drug-induced motor behaviours and validated motor tasks were used to evaluate BoNT-induced motor changes.Regarding BoNT's effects on drug-induced motor behaviours, the most consistent finding is that BoNT injections into the striatum of 6-OHDA PD rats induces a reduction in apomorphine-induced rotations [26,27,30,31,33,36,37,39]. The apomorphine-induced rotation test is considered a good predictor of the effectiveness of 6-OHDA-induced lesions in PD animal models.After dopaminergic denervation, apomorphine, a dopamine receptor agonist, stimulates the supersensitive DR1 and DR2 of the dopamine-depleted hemisphere more than those in the contralateral hemisphere, causing an anticlockwise rotation away from the lesion site [47].The abolishment of or reduction in this rotatory behaviour after BoNT treatment suggests the action of BoNT in counteracting the striatal compensation mechanisms for the dopaminergic denervation due to DR expression changes [36].However, using the apomorphine rotation test in other animal models of PD has shown less reproducible results [72].Antipova and Mann described an enhanced amphetamine-induced rotation behaviour in 6-OHDA PD rats treated with BoNT injections [33,36,37]. In addition to the study of drug-induced motor behaviours, the analysis of spontaneous behaviours of PD animal models after BoNT striatal injections has been carried out using different validated tests and showing variable results [25,30,31,[35][36][37]39,40].The observed variability, which significantly complicates the comparison of evidence across various research groups, arises from the utilisation of diverse animal models, variations in sample sizes, differences in methodologies, varying drug doses, distinct outcome measurement tests, and disparities in the latency between the intervention and measurement. In terms of the effect of BoNT treatment on non-motor symptoms, only mood disorders and hyposmia have been explored so far.BoNT can induce a reduction in depressionlike behaviours in 6-OHDA PD rats [41].The improvement in depressive-like behaviour is independent of motor improvement after BoNT treatment, suggesting the presence of distinct pathophysiological mechanisms.These observations align with our current knowledge of non-motor symptoms in PD.It has been largely described that depression can precede the motor onset by up to months, is independent of the motor symptom severity, and has distinct pathophysiological mechanisms [73]. Focusing on hyposmia, Alberts et al. found that hemi-PD rats showed no olfactory deficits.BoNT striatal injections significantly improve olfactory performance and increase DR expression in the OB.A connectomics study demonstrated the presence of indirect connections between the striatum and the olfactory bulb, which could explain the modulation of DR expression in the OB after BoNT administration into the caudate-putamen complex [34].These findings prompted the authors to posit that this influence could occur through anterograde and retrograde transportation of BoNT, a phenomenon previously described in the optic pathway of other animal models [74,75].Nevertheless, even in the context of olfactory function, employing an animal model that mimics the symptoms of the disease without replicating the specific etiopathogenetic mechanism raises uncertainties about the extrapolation of these observations to human subjects with PD. Can BoNT Exert Central Effects in Patients with PD? Despite the predominance of animal model-based findings, only one study provided compelling evidence that BoNT, when intramuscularly injected, also exerts its action on the central nervous system and leads to significant time-dependent modifications in several neurophysiological measures of intracortical inhibitory circuits and sensory-motor integration in patients with PD [42].In this study, the authors demonstrated that in PD patients with tremors, BoNT induces a reduction in tremor severity that may be related to the reorganisation of intra-cortical inhibitory activity, secondary to the action of BoNT on muscular afferent inputs and to an improvement in sensory-motor integration, as tested by the TMS parameters SAI and LAI.In line with this hypothesis, previous observations showed that an effective treatment for PD tremors is subthalamic DBS which also improves both SAI and LAI [76,77]. These preliminary findings not only highlight the potential of BoNT to modulate central neural pathways beyond its known peripheral effects, broadening our comprehension of its therapeutic impact in PD, but they also emphasise the need for further research to elucidate the central mechanisms of BoNT, aiming to refine and enhance the therapeutic strategies in the management of PD. Limitations and Gaps of the Current Literature Regarding the use of animal models to investigate BoNT's effects on motor and nonmotor symptoms, the translatability of evidence arising from animal models to humans is limited by several factors.First, the experimental design involves directly injecting BoNT into the central nervous system, while in clinical practice, BoNT is administered by intramuscular injections.Although retrograde and anterograde BoNT transportation from the muscle to the central nervous system has been hypothesised in animals, the experimental counterpart of these mechanisms in humans is still lacking [78,79].Second, the animal studies investigating BoNT-induced behavioural changes generated a unilateral lesion of the striatum, failing to precisely replicate the etiopathogenetic and pathophysiological mechanisms of PD.Rather, they primarily capture the behavioural phenomenology resulting from a unilateral striatal lesion and may not comprehensively mirror the full spectrum of motor symptoms observed in human PD patients.Third, a significant limitation of animal studies assessing BoNT's effects on mood disorders is represented by the challenge of identifying and assessing anxiety and depression in rats.To address this limit, the authors employed validated tasks capable of uncovering anxiety-like and depression-like behaviours in rats.Nevertheless, these tasks fall short of fully capturing the complex phenomenology of mood disorders associated with PD in humans.In addition, further investigations are needed to validate the existing evidence and to elucidate the impact of BoNT on other PD non-motor symptoms, which have not yet been examined in PD animal models but are currently under consideration for BoNT-based treatment.Fourth, the lack of investigation of possible clinical correlates mainly limits animal studies investigating BoNT effects on PD central pathways.Indeed, studies assessing BoNT's effects on the cholinergic system did not explore the potential correlation between the molecular mechanisms and clinical changes.Conversely, the evidence on the clinical effects of BoNT's action on the dopaminergic and glutamatergic systems is promising but still limited. To date, the evidence in humans is represented by only one TMS study.Further neurophysiological and neuroimaging studies are, therefore, needed to clarify if central mechanisms are involved in the BoNT-induced clinical effects in patients with PD. Conclusions In conclusion, the evidence from PD animal models suggests that the intracerebral injection of BoNT leads to motor improvement by modulating the interactions between the cholinergic and dopaminergic systems at the striatal level.In humans, the evidence suggesting any potential central effects of BoNT is quite limited, with the only neurophysiological study we reviewed suggesting that the improvement in tremors following the peripheral administration of BoNT may be partly due to central mechanisms.The central mechanism through which BoNT may alleviate motor as well as non-motor symptoms of PD certainly requires further exploration and must be confirmed through additional neuroimaging, clinical, and neurophysiological studies in human subjects. Elucidating BoNT's central mechanism of action could provide novel insights into the pathophysiological mechanisms underpinning the various motor and non-motor symptoms of PD.Finally, this research could also improve PD management, enhancing the quality of life for patients and proving critical evidence for advancing personalised therapy by tailoring treatments to individual needs. Figure 1 . Figure 1.Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) on central effect of botulinum toxin (BoNT)-A in PD animal models and patients.Reprinted with permission from reference [24]. Figure 1 .Figure 1 . Figure 1.Preferred R eporting Item s for System atic R eview s and M eta-A nalysis (P R ISM A ) on C entral botulinum toxin (B oN T )-A in PD anim al m od els and patients.R ep rinted w ith perm ission of reference 4. 1 . How Does BoNT Interact with PD Pathways?Evidence from Animal Models Table 1 . Studies included in our review divided by type of study in animal models (molecular evidence, evidence on motor behaviour and non-motor behaviour) and studies on human PD patients. Table 2 . Validated tasks used to assess motor and non-motor behaviours in PD animal models. Table 3 . Variability in motor behaviour testing after intrastriatal BoNT injection in PD animal models.	2023-12-29T16:22:32.890Z	2023-12-23T00:00:00.000	{ "year": 2023, "sha1": "f3364c99df19f0c10e69da2d585a6d59aa535206", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2072-6651/16/1/9/pdf?version=1703311796", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "3771a1491837680a018cea198e55193489869048", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
6816702	pes2o/s2orc	v3-fos-license	The asymptotic number of planar, slim, semimodular lattice diagrams A lattice L is slim if it is finite and the set of its join-irreducible elements contains no three-element antichain. We prove that there exists a positive constant C such that, up to similarity, the number of planar diagrams of these lattices of size n is asymptotically C times 2 to the n. Introduction and the result A finite lattice L is slim if Ji L, the set of join-irreducible elements of L, contains no three-element antichain. Equivalently, L is slim if Ji L is the union of two chains. Slim, semimodular lattices were heavily used while proving a recent generalization of the classical Jordan-Hölder theorem for groups in [4]. These lattices are planar, that is, they have planar diagrams, see [4]. Hence it is reasonable to study their planar diagrams, which are called slim, semimodular (lattice) diagrams for short. The size of a diagram is the number of elements of the lattice it represents. Let D 1 and D 2 be two planar lattice diagrams. A bijection ϕ : D 1 → D 2 is a similarity map if it is a lattice isomorphism preserving the left-right order of (upper) covers and that of lower covers of each element of D 1 . If there is a similarity map D 1 → D 2 , then these two diagrams are similar, and we will treat them as equal ones. Let N ssd (n) denote the number of slim, semimodular diagrams of size n, counting them up to similarity. Our target is to prove the following result. Theorem 1.1. There exists a constant C such that 0 < C < 1 and N ssd (n) is asymptotically C · 2 n , that is, lim n→∞ N ssd (n)/2 n = C. Note that there are two different methods to deal with N ssd (n). The present one yields the asymptotic statement above, while the method of [1] gives the exact values of N ssd (n) up to n = 50 (with the help of a usual personal computer). Also, [1] determines the number N ssl (n) of slim, semimodular lattices of size n up to n = 50 while we do not even know lim n→∞ N ssl (n)/N ssl (n − 1) , and it is only a conjecture that this limit exists. Lattice theoretic lemmas A minimal non-chain region of a planar lattice diagram D is called a cell, a four-element cell is a 4-cell ; it is also a covering square, that is, cover-preserving four-element Boolean sublattice. We say that D is a 4-cell diagram if all of its cells are 4-cells. We shall heavily rely on the following result of G. Grätzer and E. Knapp In what follows, we always assume that 4 ≤ n ∈ N = {1, 2, . . .}, and that D is a slim, semimodular diagram of size n. Let w ℓ D be the smallest doubly irreducible element of the left boundary chain BC ℓ (D) of D, and let rank ℓ (D) be the height of w ℓ D .The left-right duals of these concepts are denoted by w r D and rank r (D). See Figure 1 for an illustration, where w ℓ D and w r D are the black-filled elements. By D. Kelly and I. Rival [10, Proposition 2.2], each planar lattice diagram with at least three elements contains a doubly irreducible element = 0, 1 on its left boundary. This implies the following statement, on which we will rely implicitly. For a ∈ D, the ideal {x ∈ D : x ≤ a} is denoted by ↓a. Suppose, for a contradiction, that the lemma fails, and let u be the smallest join-reducible element belonging to BC ℓ (D) ∩ ↓w ℓ D . By D. Kelly and I. Rival [10, Proposition 2.2], there is a doubly irreducible element v of the ideal ↓u = {x ∈ D : x ≤ u} such that v ∈ BC ℓ (↓u); notice that v also belongs to BC ℓ (D). Clearly, v < u and v is join-irreducible in D. Therefore, since v < u ≤ w ℓ D and w ℓ D is the least doubly irreducible element of BC ℓ (D), v is meet-reducible in D. Hence there exist a p ∈ D such that v ≺ p and p / ∈ ↓u. Denote by u 0 the unique lower cover of u in BC ℓ (D). Since v < u, we have that v ≤ u 0 . By semimodularity and p ≤ u 0 , we obtain that u 0 = u 0 ∨ v ≺ u 0 ∨ p = u. Hence u 0 has two covers, u and u 0 ∨ p. Next, we prove the following lemma. Proof. The set of slim, semimodular diagrams of size n is denoted by SSD(n). Let Since we can omit the least element and the least three elements, respectively, and the remaining diagram is still slim and semimodular by Lemma 2.1, we conclude that \|SSD 00 (n)\| = N ssd (n − 1) and \|SSD 11 (n)\| = N ssd (n − 3). This implies (2.1). For D ∈ SSD ++ (n), we define We know from By D. Kelly and I. Rival [10, Proposition 2.2], mentioned earlier, that This together with the fact that D ∈ SSD ++ (n) is not a chain yields that Let w ℓ D − denote the unique lower cover of w ℓ D in D. Since each meet-reducible element has exactly two covers by [5, Lemma 2], we conclude from Lemma 2.3 that It follows from Lemma 2.1 that D * ∈ SSD(n − 1). From (2.5) we obtain that (2.6) D * ∈ SSD(n − 1) determines D. Next, let This is the "wrong" set from our perspective since W (n) = ∅, which is far from reality, would turn inequality (2.2) into an equality. Fortunately, this set is relatively small by the following lemma. The upper integer part of a real number r is denoted ⌈x⌉, for example, ⌈ √ 2 ⌉ = 2. Proof. First we show that (2.4), and (2.5). These facts together with Lemma 2.1 also imply the reverse inclusion since by adding a new cover to w ℓ E , to be positioned to the left of BC ℓ (E), we obtain a slim, semimodular diagram D such that D * = E. It follows from Lemma 2.3 that no down-going chain starting at BC ℓ (E) can branch out. Thus (2.8) ↓w ℓ E ⊆ BC ℓ (E) and ↓w ℓ E is a chain. Since w ℓ E is a coatom, we have that (2.9) with the notation E ◭ = E \ ↓w ℓ E , \|E ◭ \| = \|E\| − length E. Clearly, E ◭ is a join-subsemilattice of E since it is an order-filter. To prove that (2.10) E ◭ is a slim, semimodular diagram, assume that x, y ∈ E ◭ − {1}. We want to show that x ∧ y, taken in E, belongs to E ◭ . Let x 0 and y 0 be the smallest element of BC ℓ (E) ∩ ↓x and BC ℓ (E) ∩ ↓y, respectively. Since x 0 , y 0 ∈ BC ℓ (E) ∩ ↓w ℓ E − {w ℓ E } , (??) implies that x 0 and y 0 are meet-reducible. Hence they have exactly two covers by [5,Lemma 2]. Let x 1 and y 1 denote the cover of x 0 and y 0 , respectively, that do not belong to BC ℓ (E), and let x + and y + be the respective covers belonging to BC ℓ (E). By the choice of x 0 , we have that is a chain and the case x 0 = y 0 will turn out to be trivial, we can assume that x 0 < y 0 . We know that x 1 ≤ y 0 since otherwise x 1 would belong to BC ℓ (E) by (2.8). Using semimodularity, we obtain that x 1 ∨ y 0 ≻ y 0 . Since y 0 has only two covers by [5, Lemma 2] and x 1 ≤ y + would imply x 1 ∈ BC ℓ (E) by (2.8), it follows that x 1 ∨ y 0 = y 1 . Hence x 1 ≤ y, x 1 ≤ x, and x 1 ∈ E ◭ implies that x ∧ y belong to (the order filter) E ◭ . Thus E ◭ is (to be more precise, determines) a sublattice of (the lattice determined by) E. The semimodularity of E ◭ follows from Lemma 2.1. This proves (2.10). We conclude this section by the following lemma. With the auxiliary steps made so far, we are ready to start the final argument. We have reasons (but no proof) to believe that 0.023 ≤ C ≤ 0.073, see the Maple worksheet (version V) available from the authors's home page.	2012-06-16T17:33:55.000Z	2012-06-16T00:00:00.000	{ "year": 2016, "sha1": "6abf9453874f008e1a9d0a6952113a3b7402e081", "oa_license": null, "oa_url": "https://publicatio.bibl.u-szeged.hu/14537/1/czedli_the-asymptotic-number-of-slimsemimodularplanar-diagrams.pdf", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "6abf9453874f008e1a9d0a6952113a3b7402e081", "s2fieldsofstudy": [ "Mathematics" ], "extfieldsofstudy": [ "Mathematics", "Computer Science" ] }
225833804	pes2o/s2orc	v3-fos-license	Technical Analysis of Airtightness of Low-Rise Steel Structure Assembled Ultra-Low-Energy Building Fabricated steel structure ultra-low-energy building is a new type of building, which is the integration of many new technologies. The air-tightness treatment of low-rise fabricated steel structure ultra-low-energy buildings is one of the key technologies for buildings to achieve ultra-low energy consumption. Through technical analysis of each airtight weak part, the technical measures adopted by the nodes are determined, which provides a reference basis for the popularization and application of low-rise fabricated steel structure ultra-low-energy buildings. Introduction With the acceleration of China's urbanization process, the construction industry has been actively developed. Under the background of sustainable development strategy, it has become a new goal of the construction industry to meet the demand of housing construction while reducing the energy consumption of buildings and protecting the ecological environment. Ultra-low energy consumption buildings can greatly reduce building energy consumption, steel structure buildings have strong tensile and compressive performance and can be recycled, and prefabricated buildings can greatly reduce construction pollution on the construction site. The combination of ultra-low energy consumption building, steel structure building and prefabricated building has become a new development direction of the construction industry. Low-rise steel structure prefabricated ultra-low energy consumption building The structural stability of the steel structure is strong, the construction period is short, the wind resistance performance is good, plus the style is flexible, low carbon, environmental protection. The steel structure is composed of prefabricated i-beams, with the upper and lower columns and transverse beam joints bolted and welded. Prefabricated building is a building with standardized design, industrial production, assembly construction, integrated decoration, information management, intelligent application, and support for standardized parts and components. Ultra-low energy consumption building is to establish a reliable envelope structure system through reasonable and effective measures, and select reasonable envelope structure parameters to achieve the goal of ultra-low energy consumption. The advantages of low-rise steel structure prefabricated ultra-low energy consumption buildings lie in: from the perspective of the whole life cycle of buildings, the design phase adopts ultra-low energy consumption design technology, adopts efficient assembly and energy-saving envelope system, and pays attention to reducing building energy consumption, saving materials and reducing construction period. The key of low-rise steel structure prefabricated ultra-low energy consumption buildings is to minimize the heat loss of the building. The key technologies include efficient heat preservation and insulation system, non-thermal bridge construction technology, efficient air tightness technology and efficient heat recovery system, etc. The difficulty lies in the overall air tightness treatment technology of the building. Low-rise steel structure prefabricated ultra-low energy consumption building The overall air tightness of low-rise steel structure prefabricated ultra-low energy consumption building should be dealt with against the building's external walls, doors and windows, roofing, ground and other weak air tightness links. Air tightness of roof panels The connection method between prefabricated building roof slabs and walls, beams, and columns is similar to that of walls, airtight layer treatment on the basis of ensuring thermal insulation bridge. Holes should be reserved for pipes that go through roof holes, and air tightness treatment should be made at the junction of roof casing and roof. Parapet wall waterproof roll back, forming a tight air tight layer, insulation materials should be wrapped parapet wall, forming a continuous insulation layer, as shown in figure 1. During the installation of the roof casing, ventilation should be avoided. The waterproof steam diaphragm should be used on the indoor side to realize the continuity of the air-tight layer between the casing and the structure, while the waterproof roll material should be inverted on the outdoor side to achieve the double-layer air-tight layer. Non-combustible heat preservation material is filled between the pipe and the casing, and both ends of the casing are sealed with sealant to achieve the gas tightness of the casing itself. Through the roof ventilation pipe and roof board contact, the outside paste with aluminum film of self-adhesive modified asphalt waterproof steam insulation coil, the inside paste crack resistant glass fiber and waterproof steam insulation film. The roof panel hole is filled with aerogel to form a tight double air-tight layer, as shown in figure 2. Air tightness treatment of exterior wall panels ASA board, ALC board and other prefabricated wall boards can be used for prefabricated buildings with low steel structure. The gap of the assembled outer wall is mostly located between the wall board and the position of the wall board connection with the foundation. The gap of the wall panel can be filled with foamed polyurethane material. If the gap is too large, polystyrene board material can be used. If the gap is small, the sealant can be used. Waterproof and breathable film can be pasted on the outside to effectively prevent water vapor from penetrating into the wall panel. When connecting the prefabricated wall panels, attention should be paid to the staggered joints of the wall panels, and special binder should be used for scraping and squeezing to avoid the occurrence of open joints and ensure the overall air tightness of the building. Outside wall board still should use passive room special waterproof separate vapor film, permeate vapor film. The indoor side shall paste the waterproof vapor barrier film, and the outdoor side shall paste the waterproof vapor barrier film, as shown in figure 3.After the airtight layer is completed, the finish layer is processed. In addition to the special bonding mortar connection in the gap, the connection between the wall board and the floor board should be extended upward for 300~350mm and to the floor board side for 300~350mm to form a complete air-tight layer, as shown in figure 4. Windows is less than 1.0W/ (m 2 •K).For traditional external doors and windows, the load-bearing capacity of assembled wall panels is poor, which is difficult in actual construction, and it is easy for cracks, air leaks, and water leaks in wall panels. In view of this phenomenon, before installing passive doors and Windows, install C section steel in advance at the entrance of wall board doors and Windows to ensure the bearing capacity of wall board. Paste the waterproof vapor barrier film along the indoor side of the door and window frame, paste the waterproof vapor barrier film along the outdoor side, squeeze the pre-pressure expansion sealing rubber strip into the middle area, to ensure that the cleanliness and flatness of the reserved holes meet the construction requirements. In order to avoid hot bridge phenomenon, the connector can be isolated by rubber sheet, the door and window openings can be fixed by collision anchor bolt, and foaming polyurethane can be filled in the window, as shown in figure 5. Surface air tightness treatment In the passive building ground system, heat preservation, air tightness, waterproof and other performance are very critical. After the treatment of the ground base, 4mm SBS modified asphalt waterproof roll should be laid to ensure a lap width of more than 100mm, as shown in figure 6. Air tightness treatment at the outside wall In order to avoid the damage of the air-tight layer caused by the pipe going through the outer wall, the hole through the wall is enlarged to a certain size to be reserved or holed on site, and the casing is installed. The wall panel and casing are filled with aerogel and other sealant materials to ensure air tightness. The interior side wall board and casing are pasted with waterproof vapor insulation film, while the exterior side is pasted with waterproof breathable film. The outside of the vapor insulation layer is pasted with anti-crack mortar glass fiber, and the insulation layer is pasted with rock wool, forming a double-layer air-tight layer and preventing the diffusion of indoor water vapor to the outdoor insulation layer, as shown in figure 7. Non-combustible insulation material should be filled in the middle of the wall casing and pipeline, and sealant should be filled in both ends of the casing to form the seal of the casing itself, so as to ensure the overall air tightness of the assembled ultra-low energy consumption building. Conclusion Low-rise steel structure prefabricated ultra-low energy consumption building is to improve construction efficiency and construction quality, reduce building energy consumption, to achieve the purpose of building energy conservation. Air tightness not only affects building energy consumption, but also affects indoor air quality and building comfort. It is one of the key factors to realize ultra-low energy consumption building. Good air tightness treatment is an important measure to realize low-rise steel structure prefabricated ultra-low energy consumption building, providing a reference for the promotion and application of low-rise steel structure prefabricated ultra-low energy consumption building.	2020-07-02T10:32:21.186Z	2020-06-01T00:00:00.000	{ "year": 2020, "sha1": "3a9ccc53d5e1312ecb4b2e3987dc1da76a0df9f6", "oa_license": null, "oa_url": "https://doi.org/10.1088/1742-6596/1549/4/042152", "oa_status": "GOLD", "pdf_src": "IOP", "pdf_hash": "0126c63f221dd77516be61aa69eb8c9e4d758f29", "s2fieldsofstudy": [ "Engineering" ], "extfieldsofstudy": [ "Materials Science" ] }
219313184	pes2o/s2orc	v3-fos-license	Peroxisomal oxidation of erucic acid suppresses mitochondrial fatty acid oxidation by stimulating malonyl-CoA formation in the rat liver Feeding of rapeseed (canola) oil with a high erucic acid concentration is known to cause hepatic steatosis in animals. Mitochondrial fatty acid oxidation plays a central role in liver lipid homeostasis, so it is possible that hepatic metabolism of erucic acid might decrease mitochondrial fatty acid oxidation. How-ever, the precise mechanistic relationship between erucic acid levels and mitochondrial fatty acid oxidation is unclear. Using male Sprague – Dawley rats, along with biochemical and molecular biology approaches, we report here that peroxisomal b -oxi-dation of erucic acid stimulates malonyl-CoA formation in the liver and thereby suppresses mitochondrial fatty acid oxidation. Excessive hepatic uptake and peroxisomal b -oxidation of erucic acid resulted in appreciable peroxisomal release of free acetate, which was then used in the synthesis of cytosolic acetyl-CoA. Peroxisomal metabolism of erucic acid also remarkably increased the cytosolic NADH/NAD 1 ratio, suppressed sirtuin 1 (SIRT1) activity, and thereby activated acetyl-CoA carboxylase, which stimulated malonyl-CoA biosynthesis from acetyl-CoA. Chronic feeding of a diet including high-erucic-acid rapeseed oil diminished mitochondrial fatty acid oxidation and caused hepatic steatosis and insulin resistance in the rats. Of note, administration of a specific peroxisomal b -oxidation inhibitor attenuated these effects. Our findings establish a cross-talk between peroxisomal and mitochondrial fatty acid oxidation. They suggest that peroxisomal oxidation of long-chain fatty acids suppresses mitochondrial fatty acid oxidation by stimulating malonyl-CoA formation, which might play a role in fatty acid – induced hepatic steatosis and related metabolic disorders. Feeding of rapeseed (canola) oil with a high erucic acid concentration is known to cause hepatic steatosis in animals. Mitochondrial fatty acid oxidation plays a central role in liver lipid homeostasis, so it is possible that hepatic metabolism of erucic acid might decrease mitochondrial fatty acid oxidation. However, the precise mechanistic relationship between erucic acid levels and mitochondrial fatty acid oxidation is unclear. Using male Sprague-Dawley rats, along with biochemical and molecular biology approaches, we report here that peroxisomal b-oxidation of erucic acid stimulates malonyl-CoA formation in the liver and thereby suppresses mitochondrial fatty acid oxidation. Excessive hepatic uptake and peroxisomal b-oxidation of erucic acid resulted in appreciable peroxisomal release of free acetate, which was then used in the synthesis of cytosolic acetyl-CoA. Peroxisomal metabolism of erucic acid also remarkably increased the cytosolic NADH/NAD 1 ratio, suppressed sirtuin 1 (SIRT1) activity, and thereby activated acetyl-CoA carboxylase, which stimulated malonyl-CoA biosynthesis from acetyl-CoA. Chronic feeding of a diet including high-erucic-acid rapeseed oil diminished mitochondrial fatty acid oxidation and caused hepatic steatosis and insulin resistance in the rats. Of note, administration of a specific peroxisomal b-oxidation inhibitor attenuated these effects. Our findings establish a crosstalk between peroxisomal and mitochondrial fatty acid oxidation. They suggest that peroxisomal oxidation of long-chain fatty acids suppresses mitochondrial fatty acid oxidation by stimulating malonyl-CoA formation, which might play a role in fatty acid-induced hepatic steatosis and related metabolic disorders. It is well-known that high-erucic-acid rapeseed oil feeding develops transient cardiac lipidosis in animals as well as in humans, and the imbalance between the input and oxidation of erucic acid was proposed to be a critical cause for the acute lipid deposition in heart (1)(2)(3). High-erucic-acid rapeseed oil feeding also caused lipid deposition in liver; however, the effect of erucic acid on hepatic steatosis was progressive and irreversible (4,5); therefore, the mechanism that led to lipid deposition in liver was distinct from that in heart. As mitochondrial fatty acid oxidation plays a central role in liver fatty acid metabolism, it is rational to assume that metabolism of erucic acid might negatively regulate mitochondrial fatty acid oxidation and lead to hepatic steatosis. However, the precise mechanism linking erucic acid and liver mitochondrial fatty acid oxidation is not clear. To explore the potential mechanism, we focused on peroxisomal b-oxidation system, a fatty acid oxidation (FAO) system that acted exclusively on very long-chain and branched-chain fatty acids (6). As a very long-chain fatty acid, erucic acid was reported to be preferentially metabolized by the peroxisomal b-oxidation system (7,8). Interestingly, PPARa was activated, and peroxisomal fatty acid oxidation is extensively induced in animals fed a high-erucic-acid rapeseed oil diet (9)(10)(11), which led to accelerated turnover of erucic acid as well as long-chain fatty acids in peroxisomes. It has been suggested that the peroxisomal b-oxidation system and mitochondrial fatty acid metabolism system are mutually competitive; inhibition of peroxisomal b-oxidation stimulated mitochondrial b-oxidation in a previous report (12). We therefore hypothesized that excessive oxidation of erucic acid by peroxisomes might negatively regulate mitochondrial fatty acid oxidation and lead to lipid deposition in liver. This study investigated the effect of peroxisomal oxidation of erucic acid on mitochondrial fatty acid oxidation as well as the potential mechanism by which metabolism of erucic acid leads to hepatic steatosis in animals. Results Erucic acid was mainly metabolized by the peroxisomal b-oxidation system To determine the cellular compartmentation of erucic acid oxidation, the kinetic parameters of key enzymes involved in mitochondrial or peroxisomal FAO were determined (Fig. 1A). For carnitine palmitoyltransferase-1 (CPT1), the enzyme responsible for the transport of long-chain fatty acids into mitochondria, the V max value for C22:1-CoA (erucyl-CoA) is nearly 1 order of magnitude lower than that for C16-CoA (palmitoyl-CoA), whereas its K m value was 1 order of magnitude higher, which was in accordance with previous reports that CPT1 or CPT2 showed no obvious activity toward very long-chain fatty acid (.C22) (13,14). For acyl-CoA dehydrogenases, which catalyze the first step of mitochondrial b-oxidation, short-chain (SCAD) and medium-chain (MCAD) acyl-CoA dehydrogenases are completely inactive with C22:1-CoA. As for longchain acyl-CoA dehydrogenase (LCAD), a significantly lower V max value was observed along with a higher K m value for C22:1-CoA as compared with C16-CoA, indicating that C22:1-CoA was not preferentially oxidized by mitochondria. Acyl- ‡ These authors contributed equally to this work. * For correspondence: Jia Zeng, zengj@hnu.edu.cn. CoA oxidase-1 (ACOX1), the rate-limiting enzyme of peroxisomal b-oxidation, showed a relatively high activity for C22:1-CoA with V max and K m values at the same order of magnitude compared with C16-CoA. Moreover, the capacities of subcellular fractions for erucic acid oxidation were determined using isolated peroxisomes or mitochondria (Fig. 1B). Compared with its mitochondrial counterpart, the peroxisomal oxidation system showed relatively high activity for C22:1-CoA, which was strongly enhanced by treatment with clofibrate (CFB), a classical PPARa activator (15), and suppressed by the addition of 10,12-tricosadiynoic acid (TDYA)-CoA, a specific inhibitor for ACOX1 (12). Furthermore, the metabolism of erucic acid by liver homogenate led to dose-dependent generation of hydrogen peroxide, a byproduct of peroxisomal b-oxidation, as inhibited by TDYA-CoA (Fig. 1C). All of the results supported the hypothesis that erucic acids are preferentially metabolized by peroxisomal FAO system, which was in agreement with previous reports (7,8). Peroxisomal b-oxidation was significantly enhanced in livers of the rats fed high-erucic-acid rapeseed oil (HRO) To selectively increase the liver oxidation of erucic acid, CFB was used to strongly induce peroxisomal b-oxidation (15). TDYA, a specific inhibitor for peroxisomal b-oxidation, was applied to inhibit liver peroxisomal b-oxidation (12). Activation of PPARa triggers downstream transcription of genes involved in peroxisomal b-oxidation (6,16,17). Expression of genes involved in peroxisomal b-oxidation were upregulated by HRO feeding, suggesting that erucic acid was a potential endogenous ligand for PPARa, and CFB treatment caused robust induction of peroxisomal b-oxidation in higholive-oil diet (HOO)-and HRO-fed rats ( Fig. 2A). Peroxisomal FAO was significantly induced in livers of the rats fed the HRO diet, and administration of CFB strongly stimulated peroxisomal b-oxidation in HOO-and HRO-fed rats, whereas peroxisomal b-oxidation was suppressed by treatment with TDYA (Fig. 2B). HRO feeding increased hepatic hydrogen peroxide level (by 357 and 272% versus the HOO and LRO groups, respectively), CFB treatment further elevated hydrogen peroxide generation in the HOO-fed (by 386% versus the HOO group) and HRO-fed groups (by 123% versus the HRO group), and TDYA treatment reduced hydrogen peroxide generation in the liver (Fig. 2C). These results suggested that the presence of erucic acid in the diet activated PPARa and accelerated liver erucic acid oxidation, as strongly induced by CFB. Peroxisomal b-oxidation of erucic acid led to hepatic lipid deposition Liver long-chain acyl-CoAs increased significantly in HROfed rats (by 133 and 90% versus the HOO and LRO groups, respectively), and CFB treatment further elevated longchain acyl-CoAs in HRO-fed rats, as reduced by the treatment with TDYA (Fig. 3A). HRO feeding led to triacylglyceride (TAG) accumulation in rat liver (by 109 and 95% versus the HOO and LRO groups, respectively), as further elevated after treatment of CFB (increased by 105% versus the HRO group) and reduced by TDYA (Fig. 3B); CFB treatment also caused significant elevation in liver TAG in HOO-fed rats (by 70% versus the HOO group). HRO feeding increased the density of fat droplets in liver sections (23.5 6 4.4/1000 mm 2 versus 3.4 6 0.6/1000 mm 2 and 3.9 6 0.7/1000 mm 2 in the HOO and LRO groups, respectively). Lipid deposition in the HOO1CFB group was even more serious, where the size of lipid droplets was significantly increased (with average diameters of 5.06 6 1.41 mM) compared with the LRO and HRO groups (with average diameters of 1.54 6 0.12 and 3.49 6 0.28 mM, respectively). TDYA treatment significantly reduced lipid deposition in HRO-fed rats (Fig. 3C). HRO feeding also led to increased liver index (by 29 and 26% versus the HOO and LRO groups, respectively), and CFB treatment further elevated liver index in HOO-as well as HROfed rats, as decreased after treatment of TDYA (Fig. 3D). Plasma TAG was significantly higher in HRO-fed rats compared with the HOO and LRO groups (by 68 and 46%, respectively), as further elevated by the treatment of CFB and lowered by TDYA (Fig. 3E). Daily body weight gain increased by 17% in the HRO group compared with the LRO group, and TDYA significantly decreased body weight gain (by 18% versus the HRO group) (Fig. 3F). HRO feeding led to a significant increase in homeostasis model assessment of insulin resistance (HOMA-IR) index (by 118% versus LRO), and administration of TDYA to the rats fed HRO caused a significant decrease in the HOMA-IR index (Fig. 3G). Oral glucose intolerance was evident in HRO-fed rats, as shown by a significantly higher glucose curve compared with the Figure 1. Erucic acid was mainly oxidized by peroxisomes. A, kinetic parameters for key enzymes involved in peroxisomal and mitochondrial b-oxidation with palmitoyl-CoA (C16-CoA) and erucyl-CoA (C22:1-CoA) as substrates. B, liver peroxisomal and mitochondrial b-oxidation activities with C16-CoA and C22:1-CoA as substrates. C, the addition of C22:1-CoA into liver homogenate from normal or CFB-treated rats generated hydrogen peroxide dosedependently, which was completely abolished by pretreatment of TDYA-CoA. N, normal diet; N1CFB, normal diet treated with CFB. Results shown are mean 6 S.E. (error bars); , p , 0.05 by t test between paired conditions. normal and LRO-fed rats, which was exacerbated after treatment of CFB and improved by TDYA (Fig. 3H). These results suggested that excessive oxidation of erucic acid in peroxisomes led to significant lipid deposition in the liver and insulin resistance in rats. Peroxisomal b-oxidation of erucic acid suppressed mitochondrial b-oxidation by stimulating malonyl-CoA formation Excessive peroxisomal b-oxidation of erucic acid led to lipid deposition in rat liver, suggesting a potential cross-talk between Figure 2. HRO feeding enhanced liver peroxisomal b-oxidation. A, gene expression of the enzymes involved in peroxisomal b-oxidation of rat liver was up-regulated by HRO feeding, and CFB treatment strongly induced peroxisomal b-oxidation in HOO-and HRO-fed rats. B, liver peroxisomal b-oxidation activity was elevated in rats feeding HRO, as further enhanced by the treatment of CFB and inhibited by TDYA. C, HRO feeding increased hydrogen peroxide generation in rat liver, as further elevated by the treatment of CFB and reduced by TDYA. CFB treatment also significantly increased hydrogen peroxide generation in HOO-fed rats. Results are mean 6 S.E. (error bars); , p , 0.05 by t test between paired conditions. Figure 3. HRO feeding led to hepatic steatosis and insulin resistance in rats. A, liver long-chain (LC) acyl-CoA was significantly higher in HRO-fed rats, as further elevated in the CFB-treated rats and reduced by TDYA. B, liver TAG was significantly higher in the HRO group, which was further increased by the treatment of CFB and reduced by TDYA. CFB treatment also led to a significant increase in liver TAG in HOO-fed rats. C, significant hepatic steatosis was observed in HRO and HOO 1 CFB groups, CFB treatment exacerbated and TDYA relieved hepatic steatosis in HRO-fed rats. Magnification: 3200. D, liver index was increased by HRO feeding, which was further elevated by CFB treatment, and decreased by TDYA. CFB treatment also significantly increased the liver index in HOO-fed rats. E, plasma TAG level was significantly higher in the rats fed HRO, as further elevated by CFB and reduced by TDYA. F, HRO treatment increased body weight gain, as further elevated by CFB and reduced by TDYA. G, HOMA-IR was significantly higher in HRO-fed rats, as further enhanced by the treatment of CFB and lowered by TDYA. H, oral glucose intolerance was evident in HRO-fed rats, as exacerbated by the treatment of CFB and improved by TDYA. Results are mean 6 S.E. (error bars); , p , 0.05 by t test between paired conditions. mitochondria and peroxisome in which mitochondrial FAO might be suppressed because the mitochondrial FAO system plays a central role in liver lipid homeostasis. A previous report (12) also indicated that the peroxisomal and mitochondrial fatty acid metabolism systems are mutually competitive. Mitochondrial fatty acid oxidation in isolated hepatocytes was first measured by 14 CO 2 formation from [1-14 C]palmitate, and the results suggested that the capacity of mitochondrial b-oxidation was significantly lowered in the liver of the HRO-fed rats (by 45 and 42% versus the HOO and LRO groups, respectively), which was recovered by TDYA (Fig. 4A). Mitochondrial b-oxidation was also significantly lower in the HOO1CFB group compared with the HOO group (decreased by 44%). The rate of ketogenesis using palmitate as a substrate was significantly lower in isolated hepatocytes from the HRO-fed rats (decreased by 42 and 39% versus the HOO and LRO groups, respectively), as further suppressed by CFB (by 37% versus the HRO group) and elevated by TDYA, a specific inhibitor for peroxisomal b-oxidation. CFB treatment caused diminished ketogenesis rate in HOO-fed rats, as shown in Fig. 4B. When octanoate was used as a substrate, there was no difference in the ketone body production (Fig. 4B). The results suggested that peroxisomal oxidation of erucic acid inhibited mitochondrial b-oxidation by blocking the entry of cytosolic longchain fatty acids into mitochondria. To verify this, liver malonyl-CoA was measured, and HRO treatment remarkably increased liver malonyl-CoA content (by 162 and 119% versus the HOO and LRO groups, respectively), as further increased by the treatment of CFB and reduced by TDYA. Liver malonyl-CoA was also significant higher in the HOO1CFB group compared with the HOO group (by 127% versus the HOO group) (Fig. 4C). The results confirmed that the diminished mitochondrial FAO in livers of the rats fed HRO was due to elevated malonyl-CoA level, which specifically inhibited carnitine palmitoyltransferase-1a (CPT1a) and restricted the transport of long-chain fatty acid into mitochondria (18,19). Liver malonyl-CoA decarboxylase (MCD) activity was not statistically significantly different among all the groups (Fig. 4D), suggesting that the elevated malonyl-CoA formation as caused by erucic acid oxidation was due to increased generation of malonyl-CoA in liver. In the meantime, hepatic citrate content was not significantly altered by HRO feeding or CFB treatment, suggesting an extramitochondrial source of acetyl-CoA for malonyl-CoA synthesis (Fig. 4E). The results indicated that peroxisomal b-oxidation of erucic acid suppressed mitochondrial b-oxidation by stimulating malonyl-CoA formation, whereas increased oleic acid flux through peroxisomal b-oxidation in the HOO1CFB group exhibited similar effects as in the HRO group, which further suggested that stimulation of malonyl-CoA formation and suppression of mitochondrial fatty acid oxidation were a general phenomenon occurring when the fatty acid flux through peroxisomal b-oxidation was increased. Peroxisomal b-oxidation of erucic acid generated acetate as a precursor for cytosolic acetyl-CoA synthesis Because erucic acid is mainly oxidized in peroxisomes, the product of peroxisomal b-oxidation of erucic acid was analyzed. The addition of C22:1-CoA into liver homogenate led to dosedependent generation of acetate, as completely blocked by pretreatment of TDYA-CoA, whereas the addition of C6-CoA, a . Peroxisomal oxidation of erucic acid suppressed mitochondrial FAO by stimulating malonyl-CoA formation. A, mitochondrial FAO was suppressed in the isolated hepatocytes of the rats fed HRO, as further decreased in hepatocytes of CFB-treated rats and recovered by TDYA. B, ketogenesis from palmitate was suppressed in the isolated hepatocytes of the rats fed HRO, which was further reduced by CFB treatment and increased by TDYA, whereas ketogenesis from octanoate was not affected. C, liver malonyl-CoA was significantly higher in the HRO-fed group, as further increased by CFB treatment and lowered by TDYA. CFB treatment also significantly increased liver malonyl-CoA in HOO-fed rats. D, liver MCD activity was not statistically significantly different among all of the groups. E, liver citrate content was not significantly altered among all of the groups. Results are mean 6 S.E. (error bars); , p , 0.05 by t test between paired conditions. substrate for mitochondrial b-oxidation, contributed very little to acetate formation in liver homogenate (Fig. 5A). Incubation of purified peroxisomes with C22:1-CoA led to the release of acetate dose-dependently, as reduced by pretreatment of TDYA-CoA (Fig. 5B). The generation of acetate in peroxisomes was due to a specific acetyl-CoA hydrolase (ACOT12) in liver peroxisomes. Gene expression of ACOT12 was up-regulated in livers of HRO-fed rats (by 105% versus LRO-fed rats) (Fig. 5C). Peroxisomal carnitine acetyltransferase (CAT) and ACOT12 activities were assayed (Fig. 5D). ACOT12 activity was markedly induced by HRO or CFB, and the activity of ACOT12 was much higher than that of CAT (Fig. 5D), which suggested that acetate rather than acetyl-carnitine was the predominant ultimate product of erucic acid subjected to peroxisomal b-oxidation. HRO feeding significantly increased peroxisomal acetyl-CoA content, which was further elevated by CFB and reduced by the treatment of TDYA (Fig. 5E). Liver acetate content was significantly higher in HRO-fed rats (increased by 64% versus LRO-fed rats) and further elevated by CFB treatment, and administration of TDYA reduced acetate formation in rat liver (Fig. 5F). Therefore, peroxisomal b-oxidation of erucic acid generated considerable free acetate in rat liver, which provided substrate for the generation of acetyl-CoA in cytosol. Peroxisomal oxidation of erucic acid enhanced acetyl-CoA synthetase (ACS) and acetyl-CoA carboxylase (ACC) activity by elevating cytosolic NADH/NAD 1 ratio and inhibiting SIRT1 activity The addition of C22:1-CoA into liver homogenates elevated the NADH/NAD 1 ratio dose-dependently, as lowered by TDYA-CoA, whereas there was no alteration for C6-CoA (Fig. 6A). High NADH/NAD 1 ratio suppressed the activity of SIRT1, a NAD 1 -dependent deacetylase that plays a critical role in regulating fatty acid oxidation and synthesis, including the enzymes in cytosolic acetyl-CoA and malonyl-CoA generation. SIRT1 activity was then measured, and the addition of C22:1-CoA to rat liver homogenate significantly inhibited SIRT1 activity, as recovered by pretreatment of TDYA-CoA (Fig. 6B). Cytosolic NADH/NAD 1 ratio was significantly higher in the livers of HRO feeding rats (by 135%, 87% versus normal and LRO-fed rats, respectively), as robustly elevated by CFB treatment (by 38% versus HRO control), and lowered by TDYA (Fig. 6C). SIRT1 activity was diminished in liver of the rats fed HRO (decreased by 27% versus LRO feeding) and further suppressed by the treatment of CFB (Fig. 6D). ACS expression was up-regulated in livers of the HRO-fed rats, as strongly induced by CFB treatment and lowered by TDYA (Fig. 6E). Liver ACS activity was significantly higher in rats fed HRO and further increased by CFB and decreased by TDYA (Fig. 6F). Cytosolic acetyl-CoA concentration was elevated by HRO feeding or CFB treatment as reduced by TDYA (Fig. 6G). Hepatic AMP-activated protein kinase (AMPK) activity was significantly reduced by HRO feeding or CFB treatment as elevated by TDYA (Fig. 6H). Liver ACC activity was significantly higher in HRO-feeding rats, as further increased by CFB and decreased by TDYA treatment (Fig. 6I). The results indicated that elevated cytosolic NADH/NAD 1 ratio in HRO-fed rats suppressed the activity of SIRT1 and AMPK, which subsequently increased ACS and ACC activity, and stimulated Figure 5. Peroxisomal oxidation of erucic acid generated appreciable acetate in liver. A, the addition of C22:1-CoA to liver homogenate led to dose-dependent generation of acetate, as inhibited by TDYA-CoA, and no significant generation of acetate with C6-CoA. B, incubation of isolated peroxisomes with C22:1-CoA or acetyl-CoA generated acetate dose-dependently, as blocked by pretreatment of TDYA-CoA. C, mRNA expression level of ACOT12 was up-regulated in livers of the rats fed HRO and strongly induced by CFB treatment. D, peroxisomal ACOT12 activity increased in livers of the rats fed HRO and was further enhanced by CFB treatment, whereas peroxisomal CAT activity was very low in rat liver. E, peroxisomal acetyl-CoA content increased significantly in livers of the rats fed HRO and was further elevated after treatment of CFB and reduced by TDYA. F, liver acetate content increased significantly in the rats fed HRO, as further elevated by CFB, and was reduced by the treatment with TDYA. Results are mean 6 S.E. (error bars); , p , 0.05 by t test between paired conditions. hepatic malonyl-CoA formation from cytosolic acetate, thereby suppressing mitochondrial fatty acid oxidation. Discussion Chronic uptake of high-erucic-acid rapeseed oil causes hepatic steatosis in animals and possibly in humans (4, 5); however, for a long time, the mechanism linking erucic acid metabolism and lipid deposition has not been clear. The results in this study indicate that oxidation of erucic acid by peroxisomes increased malonyl-CoA generation in rat liver and suppressed mitochondrial fatty acid oxidation, and chronic intake of higherucic-acid rapeseed oil led to diminished mitochondrial fatty acid oxidation and hepatic steatosis, which revealed a mechanism linking erucic acid and hepatic steatosis. The proposed mechanism is shown in Fig. 7. Excessive uptake of erucic acid in liver resulted in activation of PPARa, and the gene expressions involved in peroxisomal b-oxidation were extensively induced. Liver oxidation of erucic acid by peroxisomes led to increased generation of free acetate and elevated cytosolic acetyl-CoA. Peroxisomal oxidation also significantly elevated the redox state of cytosolic NADH/NAD 1 and activated ACC via suppression of SIRT1, which stimulated the synthesis of malonyl-CoA from acetyl-CoA and led to diminished mitochondrial fatty acid oxidation and hepatic steatosis. This study established a cross-talk between peroxisomal and mitochondrial fatty acid oxidation in liver through the formation of malonyl-CoA, and peroxisomal oxidation of very longchain fatty acid stimulated malonyl-CoA formation, thereby suppressing mitochondrial fatty acid oxidation. As the core molecule in fatty acid synthesis, malonyl-CoA was identified to be the molecule responsible for the inhibition of CPT1a, the critical enzyme in the transfer of long-chain fatty acids into mitochondria, thereby suppressing mitochondrial fatty acid oxidation and stimulating fatty acid synthesis (18,19). Although the peroxisomal b-oxidation system was discovered more than 40 years ago, the intrinsic relationship between the Figure 6. HRO feeding enhanced the activity of liver ACS and ACC through elevating the cytosolic NADH/NAD 1 ratio and inhibiting SIRT1 activity. A, the addition of C22:1-CoA into liver homogenate increased NADH/NAD 1 ratio dose-dependently, whereas there was no alteration for C6-CoA. B, SIRT1 activity in liver homogenate was suppressed by the addition of C22:1-CoA, as recovered by TDYA-CoA. C, cytosolic NADH/NAD 1 ratio was significantly higher in livers of the rats fed HRO, elevated by CFB, as lowered by TDYA. D, liver SIRT1 activity was significantly lower in the rats fed HRO and was further decreased by CFB and elevated by treatment with TDYA. E, ACS expression was up-regulated in livers of the HRO-fed rats and further induced after exposure to CFB, as decreased after TDYA treatment. F, liver ACS activity was significantly higher in HRO-fed rats and further enhanced after treatment of CFB, as reduced by TDYA. G, cytosolic acetyl-CoA was significantly higher in livers of the HRO-fed rats, as further increased after treatment of CFB and lowered by TDYA. H, liver AMPK activity was significantly lower in HRO-fed rats compared with the LRO group, as further reduced by the treatment of CFB and recovered by TDYA. I, liver ACC activity was significantly higher in the rats fed HRO, as further increased by CFB treatment and lowered by TDYA. Results are mean 6 S.E. (error bars); , p , 0.05 by t test between paired conditions. peroxisomal and mitochondrial fatty acid oxidation systems is not fully established (20). It was proposed that this metabolism system was to handle excessive fatty acids that were left by mitochondrial fatty acid oxidation, which transferred the acetyl-CoA to mitochondria for final burning (21). However, there was evidence that peroxisomal b-oxidation did not contribute to significant ketone body formation. For example, the addition of palmitic acid to hepatocytes stimulated peroxisomal b-oxidation, whereas ketone bodies were not significantly increased (22). Our results suggested that peroxisomal b-oxidation of erucic acid suppressed mitochondrial fatty acid oxidation through increasing liver malonyl-CoA level, and specific inhibition of peroxisomal b-oxidation partly abolished the effects on malonyl-CoA generation and mitochondrial fatty acid oxidation as caused by erucic acid. We therefore proposed that one of the physiological functions of peroxisomal b-oxidation was to stimulate the formation of malonyl-CoA in liver through metabolism of endogenous substrates, thereby suppressing mitochondrial b-oxidation. Very long-chain fatty acids have been well-accepted to be exclusively metabolized in peroxisomes (23), and our results confirmed that erucic acid was oxidized preferentially by the peroxisomal b-oxidation system. Mitochondria showed very low oxidative activity toward C22:1-CoA, which was in good agreement with previous reports (7,8). Interestingly, the results indicated that the presence of erucic acid resulted in an adaptive elevation in peroxisomal b-oxidation capacity through activation of PPARa, which suggested that erucic acid might act as a potential ligand for PPARa because long-chain fatty acids were identified to be endogenous ligands for PPARa and triggered downstream transcription of the target genes, including the genes involved in peroxisomal b-oxidation (17,24,25), thereby accelerating peroxisomal turnover of erucic acid. It was reported that administration of PPARa activator stimulated hepatic fatty acid synthesis (26). Our results suggested that a regulatory mechanism existed for the control of mitochondrial fatty acid oxidation through peroxisomal metabolism of very long-chain fatty acids that was induced by PPARa. Peroxisomes are not permeable to acetyl-CoA (27). It is wellaccepted that there are two pathways for acetyl-group transfer from peroxisome to the cytosol. One way is to hydrolyze acetyl-CoA to acetate via peroxisomal acetyl-CoA thioesterase, and the other way is to transform the acetyl-CoA to acetyl-carnitine via CAT (28). We analyzed the final product of C22:1-CoA that was subjected to peroxisomal b-oxidation, and the results clearly indicated that peroxisomal oxidation of C22:1-CoA generated free acetate as the predominant product, as further confirmed in vivo, which was in good agreement with previous reports that peroxisomal fatty acid metabolism generated free acetate (29,30). The formation of acetate from acetyl-CoA was attributed to high-level expression and activity of ACOT12 in rat liver. On the other hand, the activity of CAT was very low in rat liver, whereas its expression was high in heart and muscle (31). Cytosolic free acetate can barely be metabolized in liver because it can hardly be transformed to acetyl-CoA in mitochondria due to lack of a specific acetyl-CoA synthetase in liver mitochondria (32). The acetate that was released from peroxisomal b-oxidation of erucic acid was then used for the synthesis of cytosolic acetyl-CoA because a specific acetyl-CoA synthetase existed in the cytosol of rat liver (33,34). The acetyl-CoA generated from peroxisome-released acetate would then be further transformed to malonyl-CoA by acetyl-CoA carboxylase (35). It is interesting to note that an acetyl-CoA synthetase was in the mitochondria of rat heart, suggesting that the acetate generated in heart may be used as an energy source (35). In this circumstance, we noted the fact that acetate generated from ethanol oxidation stimulated liver fatty acid synthesis and led to significant hepatic steatosis (36). Unlike mitochondria, a respiration chain is absent in peroxisomes; however, it was reported that the redox state of pyridine nucleotide within the peroxisome and the cytosol are in equilibrium through lactate/pyruvate and glycerol phosphate shuttles (27,37). Therefore, elevated erucic acid oxidation by peroxisomes would lead to increased NADH release from the peroxisomes to the cytosol and significantly increased cytosolic NADH/NAD 1 ratio. High NADH/NAD 1 ratio resulted in diminished activity of SIRT1, a NAD 1 -dependent deacetylase (38). The decreased SIRT1 activity may reduce the deacetylation of LKB1 and inhibit this kinase, which in turn inhibits AMPK (39,40). SIRT1 and AMPK are known fuel-sensing molecules, and activation of the SIRT1/AMPK pathway plays a central role in regulating hepatic fatty acid metabolism (39). The diminished AMPK activity may further lead to activation of ACC, the key enzyme for malonyl-CoA synthesis. Inhibition of ACOX1, the rate-limiting enzyme in peroxisomal b-oxidation by a specific inhibitor TDYA significantly decreased the liver NADH/NAD 1 ratio, which resulted in activation of AMPK and suppression of ACC via decreasing the deacetylation level to LKB1 and PGC-1a (12). It was reported that PPARa agonist clofibrate treatment significantly increased the NADH/NAD 1 ratio in rats (41), indicating that peroxisomal b-oxidation was critical for the control of cytosolic NADH/NAD 1 ratio. We also noted that ethanol oxidation in liver also caused a significant increase in the liver NADH/NAD 1 redox state and suppressed mitochondrial b-oxidation and led to hepatic lipid deposition, as completely abolished by pyrazole, an inhibitor of alcohol dehydrogenase (36). Therefore, suppression of AMPK and activation of ACC by erucic acid were attributed at least in part to the elevation of the cytosolic NADH/NAD 1 ratio and the diminished SIRT1 activity through peroxisomal oxidation of this very long-chain fatty acid. Mitochondrial dysfunction has been well-accepted to play a critical role in the development of nonalcoholic fatty liver disease and related metabolic disorder (42). The results suggested that excessive oxidation of erucic acid in liver peroxisomes suppressed mitochondrial fatty acid oxidation, and specific inhibition of peroxisomal b-oxidation significantly attenuated erucic acid-induced depression in mitochondrial fatty acid oxidation and hepatic steatosis. Accumulation of hepatic lipid has been well-accepted to be a critical cause of insulin resistance (43). Therefore, the results in this research had clinical significance; excessive oxidation of erucic acid or endogenous very longchain fatty acids by peroxisomes might turn out to be a pathological cause that leads to fatty liver and insulin resistance in our modern life. High-erucic-acid rapeseed oil as an edible oil is widely consumed in South China and India (44), and more than 150 million people in China suffer from fatty liver and diabetes (45). Although the clinical connection between high-erucicacid rapeseed oil intake and metabolic diseases has not yet been established, we propose that chronic intake of high-erucic-acid rapeseed oil might lead to a high risk of liver steatosis and insulin resistance and strongly suggest low-erucic-acid rapeseed oil or olive oil as an edible oil instead of high-erucic-acid rapeseed oil in daily life (46)(47)(48). This might benefit the liver as well as the heart by reducing the harmful metabolites released from peroxisomal b-oxidation upon mitochondrial fatty acid oxidation. On the other hand, this mechanism is not confined to erucic acid, as long-chain fatty acids were ideal substrates for the peroxisomal b-oxidation system, and peroxisomal oxidation of long-chain fatty acids is significantly elevated under the conditions of obesity and diabetes (6). We therefore further propose that peroxisomal oxidation of exogenous or endogenous longchain fatty acids might act as a common mechanism for fatty acid-induced hepatic steatosis and insulin resistance. Small molecules that specifically inhibit peroxisomal b-oxidation might be promising agents for treating fatty liver and related metabolic diseases as caused by excessive fatty acid metabolism through peroxisomal b-oxidation. Animal studies Male Sprague-Dawley rats were purchased from Slac Laboratory Animal Co. Ltd. (Changsha, China). Standard rodent diet (12% fat by calories), a high-rapeseed-oil diet containing 15% (w/w) rapeseed oil (55% fat by calories), and HOO containing 15% (w/w) olive oil (55% fat by calories) were supplied by Slac Laboratory Animal Co. Ltd. The rapeseed oil used in HRO contained 35% (w/w) erucic acid, whereas the rapeseed oil in LRO contained 2% (w/w) erucic acid. All animals were housed in single cage with free access to food and water under controlled temperature (22°C) and light (12 h of light and 12 h of dark). Male Sprague-Dawley rats at the age of 8 weeks (200-220 g) were exposed to standard, HOO, LRO, or HRO diet for 4 weeks. CFB (200 mg/kg) or TDYA (50 mg/kg) was administered to the indicated groups by gavage, once per day at 5 p.m. All rats were weighed every day at 5 p.m. An oral glucose tolerance test was performed on day 25. Glucose at 3 g/kg was introduced by gavage for each rat, with blood samples collected at the indicated time from tail vein, and blood glucose was determined by a glucometer (Lifescan, Johnson and Johnson). After the experiments, all of the rats were bled from the eyes and then sacrificed. Livers were removed quickly, weighed, and stored in liquid nitrogen immediately. The HOMA-IR index was calculated as described previously (51). All of the animal studies were approved by the Animal Care Committee of Hunan University of Science and Technology. Histological analysis Same parts of the right lobe were cut quickly from the livers of the killed rats and immediately fixed with 4% paraformaldehyde. Paraffin sections were prepared and cut into 5-7-mmthick sections and stained by hematoxylin-eosin. Hepatic steatosis was observed by an optical microscope, and four samples were used for each group to observe the lipid droplets in liver tissues. Fatty acid oxidation by isolated hepatocytes Hepatocytes of treated rats were prepared as described before (52). Cell viability was assessed by trypan blue exclusion and estimated to be more than 90%. Ketogenesis and CO 2 formation, as specific indicators for mitochondrial b-oxidation, were performed as described before with newly isolated intact hepatocytes after minor modification. Hepatocytes (1 3 10 6 ) were incubated under an atmosphere of O 2 /CO 2 (19:1) in Krebs-Henseleit bicarbonate medium, pH 7.4, containing 0.4 mM [1-14 C]palmitate or 0.8 mM [1-14 C]octanoate, 0.34 mM defatted BSA, and 1 mM carnitine. After incubation at 37°C for 15 min, the reaction was terminated by the addition of ice-cold perchloric acid. After injection of hyamine hydroxide, the flask was shaking for another 45 min to trap 14 CO 2 for the radioactivity assay. The rate of mitochondrial fatty acid oxidation determined by the 14 CO 2 formation was expressed as nmol of labeled carbon atoms/min/g of cell (wet weight). After neutralization and centrifugation, ketone bodies (sum of b-hydroxybutyrate and acetoacetate) in the supernatant were determined enzymatically (53). Isolation of liver subcellular fractions Mitochondria were isolated by differential centrifugation in 0.25 M sucrose as described previously (54), the mitochondrial pellet was washed three times and resuspended in the same medium at a concentration of ;40 mg/ml. The integrity of isolated mitochondria was examined by a commercial mitochondrial staining kit (Sigma). For the isolation of peroxisomes, the light mitochondrial (L) fraction after differential centrifugation was further isolated by a Percoll gradient as described before (55). 15 mg of L fraction sample was layered on 5 ml of 50% (v/v) solution of Percoll containing 250 mM sucrose, 2 mM Mops, 1 mM EGTA, and 0.1% (v/v) ethanol at pH 7.2. After centrifugation at 85,000 3 g for 30 min on a Beckman Optima MAX-XP ultracentrifuge with an MLN80 rotor, the fractions were collected for the catalase activity assay. The pooled peak fractions were diluted with 250 mM sucrose and centrifuged at 35,000 3 g for 15 min to recover sediment containing purified peroxisomes. For the preparation of cytosolic fraction, the post-light mitochondrial supernatant was first centrifuged at 27,000 3 g for 15 min to pellet residual large organelles and finally centrifuged at 100,000 3 g for 60 min to obtain a cytosolic fraction for the metabolite and enzyme assay. The purity of isolated organelle was determined by marker enzyme activity assay: catalase for peroxisomes and cytochrome c oxidase for mitochondria (55). Purity of mitochondria and peroxisomes was more than 92%, whereas mitochondria or peroxisome contamination was less than 1% in purified peroxisome or mitochondria, facilitating reliable measurement of peroxisomal or mitochondrial FAO. Characteristics of the enzymes involved in liver b-oxidation Mitochondrial SCAD, MCAD, LCAD, and peroxisomal ACOX1 were expressed in Escherichia coli and purified by nickel metal affinity chromatography. Mitochondrial CPT1 was expressed in Saccharomyces cerevisiae and purified as described (56). Acyl-CoA dehydrogenases were measured using 2,6-dichloroindophenol as the final electron receptor (57). ACOX1 activity was assayed spectrophotometrically by directly measuring H 2 O 2 formation (12). CPT1 activity was assayed by measuring the formation of free CoA by a DTNB method (56). Measurement of peroxisomal and mitochondrial b-oxidation Mitochondria b-oxidation was determined spectrophotometrically by the method of Osmundsen and Bremer (58), with 100 mM palmitoyl-CoA (C16-CoA) or erucyl-CoA (C22:1-CoA) as a substrate. Peroxisomal b-oxidation was assayed by acyl-CoA-dependent NAD 1 reduction in the presence of KCN as reported by Lazarow (59), with 100 mM C22:1-CoA as a substrate. Biochemical analysis Plasma TAG was determined by a commercial kit according to the manufacturer's instructions (Wako, Osaka, Japan). Liver long-chain acyl-CoAs were extracted and determined by the method of Tubbs and Garland (61). Liver TAG were extracted by the method of Bligh and Dyer (62) and determined with a commercial kit (Wako, Osaka, Japan). Liver hydrogen peroxide, NADH/NAD 1 ratio, citrate, and acetate were determined by commercial kits (Sigma). Peroxisomal and cytosolic acetyl-CoA content was measured by a fluorometric assay kit from Sigma, and cytosolic concentration of acetyl-CoA was calculated according to the water space of the cytosolic fraction (63). The cytosolic NADH/NAD 1 ratio was determined by an indirect method by analyzing the concentration of lactate and pyruvate, which are in equilibrium with free NADH and NAD 1 in a lactate dehydrogenase system (64). Liver malonyl-CoA was analyzed by HPLC as described previously (65). Plasma insulin was measured by a rat/mouse insulin ELISA kit from Merck Millipore (Billerica, MA, USA). Protein concentration was measured by a Bio-Rad DC protein assay kit (Hercules, CA, USA). Peroxisomal acetyl-CoA thioesterase (ACOT12) was measured by a DTNB assay using isolated peroxisomes with 100 mM acetyl-CoA (66). Peroxisomal CAT activity was assayed as described before using 100 mM acetyl-carnitine (67). Activity of malonyl-CoA decarboxylase was measured enzymatically by a coupled reaction with malate dehydrogenase and citrate synthase (68). The reaction solution contained 0.1 M Tris-HCl (pH 8.0), 0.5 mM DTT, 0.6 mM NAD 1 , 1 mM malate, malate dehydrogenase (74 units), 300 mM malonyl-CoA, citrate synthase (1.7 units), and 0.3 mg of liver homogenate in a total volume of 1 ml. SIRT1 deacetylase activity of rat liver was evaluated by the Cyclex SIRT1/Sir2 deacetylase fluorometric assay kit (CycLex, Nagano, Japan) according to the manufacturer's guidance. SIRT1 activity was expressed as relative fluorescence/mg of protein (arbitrary units). ACS activity was determined spectrophotometrically as described previously (32). Liver AMPK activity was measured spectrophotometrically by a commercial kit (Genmed Scientific Inc., Arlington, MA, USA). Liver ACC activity was measured by a commercial kit (Solarbio, Beijing, China). Statistical analysis Data are presented as mean 6 S.E. n = 6-8 for all of the groups. The significance of differences was evaluated using Student's t test by SPSS 18.0. p , 0.05 was considered statistically significant. Data availability All data are contained within the article.	2020-06-04T09:07:56.703Z	2020-06-03T00:00:00.000	{ "year": 2020, "sha1": "5d93db9b8b0068f6aa351a8c7a5c4e58c18707ee", "oa_license": "CCBY", "oa_url": "http://www.jbc.org/article/S0021925817501301/pdf", "oa_status": "HYBRID", "pdf_src": "ScienceParsePlus", "pdf_hash": "71c2d015400dd1830794f1a230d17ce76a3299d1", "s2fieldsofstudy": [ "Biology", "Chemistry" ], "extfieldsofstudy": [ "Chemistry", "Medicine" ] }
89385704	pes2o/s2orc	v3-fos-license	Vernalization and Growing Degree-day Requirements for Flowering of Thalictrum delavayi `Hewitt's Double' Vernalization and growing degree-day (GDD) requirements of Thalictrum delavayi Franch. 'Hewitt's Double' were investigated by exposing crowns to cold storage for 0, 3, 6, 9, 12, or 15 weeks at 8 °C, and subsequently planting in a heated greenhouse under long-day conditions. Cumulative vernalization of crowns was complete after 6 weeks of cold storage at 8 °C. The time to flower, including time at 8 °C, was 3338 GDD (base temperature of 0 °C) without vernalization and 2802 GDD after complete vernalization. Commercial recommendations for rapid and predictable flowering of T. delavayi 'Hewitt's Double' should include cold storage of crowns for a minimum of 6 weeks at 8 °C as part of the 2802 GDD during vernalization and forcing. Two-year-old crowns of 'Hewitt's Double', lifted in Auckland, N.Z., in late April (fall), were shipped to Palmerston North, N.Z., before treatments were applied. Crowns, covered with moistened bark, were exposed to 8 ± 1 °C in a dark cooler for 0, 3, 6, 9, 12, or 15 weeks at the Plant Growth Unit, Massey Univ., Palmerston North (40°21´S). After cold storage, the crowns were planted in a glasshouse heated to 15 °C and vented at 20 °C. The first planting commenced on 2 May 1994. The glasshouse received a natural photoperiod (11 h increasing to 13 h) plus 4 h of night lighting between 2200 and 0200 HR (incandescent lamps with a mean light intensity of 2.4 µmol·m -2 ·s -1 at the height of the growing medium during the experiment). The crowns were planted individually in black polythene bags (27 × 27 cm, 1.2-L volume, Epic Ltd., Auckland, N.Z.) containing 100% pine bark plus 3.0 kg·m -3 each of agricultural lime and dolomite, 0.5 kg·m -3 iron sulfate, and 4.0 kg·m -3 Osmocote 16N-3.5P-10K. Water was provided by a mixture of continuous capillary and once-weekly overhead applications. After storage, treated plants were arranged in the greenhouse in a nested unbalanced experimental design with two blocks. One block contained large crowns (>6.9 cm in diameter) with two replicates, while the second block had small crowns (4.2-6.9 cm in diameter) with five replicates. Each replicate consisted of three plants. The experiment comprised six treatments with 126 plants in all. The experiment was blocked for crown size to evaluate any possible influence of size on flower yield and timing (Corr and Widmer, 1991). Greenhouse air temperatures, measured with a shaded sensor at a height of 1.3 m, were recorded at 30-min intervals during the study with a Squirrel 1200 Digital Meter/Logger (Grant Instruments Ltd., Barrington, Cambridge, U.K.). Daily mean air temperatures in the cooler during vernalization and in the greenhouse during growth were used for the calculation of GDD. Actual air temperatures within the greenhouse rarely exceeded 25 °C over the course of the study, ranging between the set points of 15 and 20 °C. Flowering was defined by the stage when ≈5% of the buds were open in the upper 45 cm of the primary inflorescence of each plant. The mean time to flower was calculated using both calendar days (CD) and GDD for the first inflorescence harvested. A linear GDD model (Roberts and Summerfield, 1987;Wang, 1960) with a base temperature of 0 °C (Roberts and Summerfield, 1987) was used. Saturation of the vernalization response was judged to have occurred when the cold storage treatment did not further advance GDD to flower, based on total forcing time including duration in cold storage. Stem number per plant and stem length were recorded at flowering. Data were subjected to analysis of variance, and trend analysis using the GLM procedure of SAS (SAS Institute, Cary, N.C.). Mean separation was by Fisher's least significant difference. to cold temperature. Some species require a period of cold temperature for flowering for either vernalization or breaking of bud dormancy (Iversen and Weiler, 1994;White et al., 1989;Whitman et al., 1996). Vernalization typically proceeds within the range of -5 to 15°C , with the most effective range typically 5 to 9°C (Vince-Prue, 1975; Wiebe, 1990). The cold requirement for flowering may be qualitative or quantitative. If plants remain vegetative without exposure to cold, the vernalization response is qualitative (Iversen and Weiler, 1994;Shedron and Weiler, 1982;White et al., 1989). A quantitative response occurs where exposure to cold hastens flower induction and differentiation. When the response is quantitative, the date of flowering, total number of leaves, and/or flower stalk length may differ with the extent of vernalization (Wiebe, 1990). The flowering response of T. delavayi to cold temperatures has not been previously reported in the literature. However, a qualitative vernalization requirement has been reported in the genus Aquilegia (Shedron and Weiler, 1982;White et al., 1989), a close relative of Thalictrum (Hoot, 1991). Combinations of temperatures and durations ranging between 0 and 8 °C for between 4 and 10 weeks satisfied the vernalization requirement for various cultivars of Aquilegia. Given that T. delavayi both originates from a temperate climate and has a high potential market value, the cold requirement of 'Hewitt's Double' was considered worthy of investigation. The photoperiodic requirement for flowering of T. delavayi has not been reported. We forced plants of 'Hewitt's Double' in a longday environment to approximate the 10.5-to 13.5-h photoperiods that occur during growth and flowering in the parent species' region of origin (Huxley et al., 1992). Forcing in a longday environment allowed us to focus on investigating the influence of cold treatment on flowering of 'Hewitt's Double' and to determine the GDD requirement for flowering. Thalictrum delavayi is a herbaceous perennial native to west China (Durand and Jackson, 1901;Huxley et al., 1992). The storage organ is a crown, which produces slender stems up to 1.2 m in height, with long petiolate leaves comprising two to three terminate or pinnately decompound leaflets (Huxley et al., 1992). In summer the stems terminate in an inflorescence comprising a loose panicle of lilac to white flowers. 'Hewitt's Double' is commonly grown as a garden display plant (Huxley et al., 1992) or as a medicinal plant (Gao et al., 1990). In recent years 'Hewitt's Double' has been viewed by members of the floriculture industry as a potential new commercial cut flower (I. Ivey, personal communication). As predictable production is required, producers are interested in understanding the flowering requirements of this species and developing production schedules. A common method of scheduling uses the accumulation of heat units or sum of growing degree-days (GDD) to estimate the expected time to flowering (McKay et al., 1981;Whitman et al., 1997). Production scheduling of some herbaceous perennials must also account for their response Received for publication 9 Feb. 1998. Accepted for publication 2 Sept. 1998. We thank Flower Enterprises (New Zealand) Ltd. for the supply of plant materials. Thanks also to the Ministry of Foreign Affairs and Trade (New Zealand) and the Chinese government: Guanqxi State Farms Bureau, and the State Education Commission of the People's Republic of China, for providing funding for Ning Huang. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked advertisement solely to indicate this fact. 1 Graduate Student. 2 To whom reprint requests should be addressed; e-mail: K.Funnell@massey.ac.nz Results All plants flowered in all treatments. Linear and quadratic trends were significant for all calculations of CD and GDD (i.e., with and without storage duration included) with increasing duration of storage (P < 0.0001). Both CD and GDD, including the duration of the cold storage, declined as a result of 3 and 6 weeks of cold storage in a linear trend (P < 0.0001) from 196 CD and 3338 GDD to 187 CD and 2848 GDD, respectively (Fig. 1 a and c). After 6 weeks of cold storage, the number of CD necessary for flowering followed a positive linear trend with increasing storage duration up to 215 d (P < 0.0001) (Fig. 1a), while GDD remained relatively unchanged (P > 0.05). Between 6 and 15 weeks of storage, GDD needed for flowering ranged between 2760 and 2848 (Fig. 1c), with an average of 2802. Excluding cold storage duration, the time to flower decreased linearly with increasing duration of cold storage for both CD and GDD (P < 0.001) (Fig. 1 b and d). Flower stem yield averaged between three and five stems per plant, with stem lengths ranging from 140 to 200 cm (data not presented). Storage duration did not affect flower yield significantly (P > 0.05). All stems produced were longer than required to meet current commercial standards. While stem length was reduced to between 141 and 148 cm for plants receiving 6 and 9 weeks of storage, vs. 176-200 cm for all other storage durations (P < 0.05), these changes were not considered commercially important. Discussion Crowns of 'Hewitt's Double' exhibited a quantitative vernalization requirement. Vernalization reduced the time to flower (Fig. 1 a and c), but exposure to cold temperatures was not an obligate requirement for flowering. After at least 6 weeks of vernalization, the time to flower (including duration of the cold storage) was similar with respect to GDD (Fig. 1c), but increased with respect to CD (Fig. 1a). Thus, the vernalization response became saturated at 6 weeks of cold storage, with longer periods in cold storage merely extending the time in CD to flower. The vernalization response is similar to that previously reported for Triticum aestivum L. and Hordeum vulgare L., in which the reduction in the time to flower following low-temperature treatment resulted from concurrent accumulation of GDD during that period (Brooking, 1996;Ellis et al., 1989). In the current experiment, sprouting of buds on crowns was detected after 15 weeks of cold storage (data not presented), indicating that growth occurred during cold storage. Therefore, 8 °C is effective for both vernalization and GDD accumulation (i.e., plant development) in this species. Satisfying the vernalization requirement after 6 weeks in the current experiment is within the 1-to 20-week range reported as optimal for various crops (Iverson and Weiler, 1994;Whitman et al., 1997;Wiebe, 1990), and is similar to that reported for Aquilegia (Shedron and Weiler, 1982;White et al., 1989). Depending on cultivar, the vernalization re-quirement for Aquilegia ranged between 4 and 10 weeks at 4.5 °C (Shedron and Weiler, 1982). Similarly, White et al. (1989) reported that Aquilegia required up to 6 weeks at temperatures between 0 and 1 °C to satisfy the vernalization requirement. The most effective temperature range for vernalization for many species is between 5 and 8 °C (Vince-Prue, 1975;Wiebe, 1990). The temperature used for vernalization in this study (8°C), is higher than the temperatures found to be most effective with other herbaceous perennials (Iverson and Weiler, 1994;Shedron and Weiler, 1982;White et al., 1989;Whitman et al., 1997). Further research is required to determine if 8 °C is the most effective temperature for meeting the vernalization requirement in 'Hewitt's Double'. Comparison of time to flower using CD and GDD summation illustrated that time to flower for 'Hewitt's Double' was predominantly temperature-dependent, once the vernalization requirement was satisfied (Fig. 1 a and c). This result confirmed the suitability of using GDD for describing the time to flower for this crop, as reported for other herbaceous perennials (McKay et al., 1981;Whitman et al., 1997). For gladiolus, the GDD requirements from planting to flowering, after cold temperature requirements have been met, varied from 1300 to 2300, depending on cultivar (McKay et al., 1981). For Delphinium 'Blue Mirror' 1353 GDD were required for flowering after saturation of the vernalization response (Whitman and Heins, personal communication), and Whitman et al. (1997) reported that only 909 GDD were required for flowering of Campanula carpatica Jacq. 'Blue Clips'. In the current experiment, the vernalization and forcing phases could not be separated with accuracy. However, if the 336 GDD required to satisfy vernalization (i.e., 6 weeks at 8°C) were considered to be separate from that for forcing, ≈2466 GDD would be required for forcing of 'Hewitt's Double'. A total of 2466 GDD for forcing indicates that this species requires a greater number of GDD for flowering than do the better-known herbaceous perennials noted above. These results can be used to develop schedules for flowering of commercial plantings of 'Hewitt's Double', with expected improvements in reducing both time to flower and variability in timing between plantings. As an example, if vernalized for 6 weeks at 8 °C and forced at a daily average temperature of 17.5°C , flowering would occur 141 d after planting (i.e., 2466 ÷ 17.5 = 141). This would reduce the time to flower by ≈30 d compared with nonvernalized plants. Recommendations for rapid and predictable timing of flowering of 'Hewitt's Double' for commercial production include using cold storage of crowns for a minimum of 6 weeks at 8 °C as part of the 2802 GDD during vernalization and forcing.	2019-04-01T13:13:07.822Z	1999-02-01T00:00:00.000	{ "year": 1999, "sha1": "7c9b23ea0b449ca1e65e2fbfb4c9e46d543dfb81", "oa_license": null, "oa_url": "https://journals.ashs.org/downloadpdf/journals/hortsci/34/1/article-p59.pdf", "oa_status": "GOLD", "pdf_src": "Adhoc", "pdf_hash": "1d2b2fc81e22789707173722531c5abedf513817", "s2fieldsofstudy": [ "Agricultural And Food Sciences" ], "extfieldsofstudy": [ "Biology" ] }
227151858	pes2o/s2orc	v3-fos-license	Health-Focused Optimal Power Flow In this paper, we propose a novel Health-Focused Optimal Power Flow (HF-OPF) to take into account the equipment health in operational and physical constraints. The health condition index is estimated based on the possible fault characteristics for generators and batteries. The paper addresses the need for understanding the relationship between health condition index and the operational constraints in OPF problems. Such a relationship is established through Finite Element Method (FEM)-based simulations. The simulations for generator and battery faults indicate the presence of an inflection point after which effect of fault becomes severe. A microgrid system is used to illustrate the performance of the proposed HF-OPF. The results show that health condition inflicts high cost of generation and can lead to infeasibility even with less critical faults. I. INTRODUCTION The optimal power flow (OPF) is a fundamental problem for power system operation. It is solved for finding an economical operating point within a physical network and operational constraints [1]. In recent years, the development of various microgrid architectures leads to several OPF formulations, including various sources and load types [2]. The microgrid hold conventional generators, renewable generators and as well as battery energy storage systems (BESS) [3]. All these formulations consider all network components to be working perfectly. To incorporate the effect of equipment failures, an extension of classical OPF has been proposed which considered the constraints arising due to operation under a fixed list of postulated contingencies [4]. All these formulations handle equipment failure contingencies in binary fashion i.e., either perfectly working or completely failed. Due to this discrete handling of equipment health status, these formulations cannot be used to examine and incorporate the effects of various types of faults in generators or BESS. Further, Energy Management System (EMS) for on-board microgrid of an aircraft [5], maritime systems on ships and submarines [6] and remote areas without grid connection [7] are required to work reliably for long and critical duration. These microgrid systems may not be able to shut down a power source as they do not have access to extra power most of the times. Also, the maintenance cannot be done on-site in most of the cases, and system may need to work properly for long-duration before it is safe to shut down for maintenance. In addition to these concerns, the advancements in online health monitoring have enabled EMS to access health conditions remotely. Hence EMS of microgrid can act upon these for preventive as well as corrective actions. Therefore, it is imperative to incorporate the effect of equipment health, in a non-discrete manner, in OPF. This will help in maintaining the system reliability with the least cost infliction. In this paper, we propose a novel Health-Focused Optimal Power Flow (HF-OPF) incorporating the effect of equipment health condition as a continuous function. This work attempts to understand the relationship between incipient faults and OPF constraints. A health condition index is defined for generator and BESS to reflect various fault conditions uniformly. Further, these health condition index have been converted into OPF constraints. The HF-OPF is solved using state-of-art Semi-Definite Programming (SDP) relaxation. The main contributions of this paper are: • Proposing a novel Health-Focused Optimal Power Flow (HF-OPF) formulation to incorporate equipment health by modelling OPF constraints as functions of health condition index. • Defining health condition index for generator and battery for the establishment of a relationship between health and OPF constraints via FEM analysis. • Analysing the variation OPF constraints with health condition and cost inflicted by health deterioration of equipment. II. HEALTH CONDITION MONITORING For a generator, various individual faults in generator such as Turn-Turn Short Circuit (TTSC) [8], demagnetization fault [9], eccentricity fault have been studied independently, and individual health condition have been established. Yet, the insights into the overall, combined health condition of the generator or battery system are not well studied. This paper develops the overall health of generator and battery as a function of fault characteristics. Further, the relationship between the health condition index and OPF constraints has been established through FEM simulations. Below we elaborate the notion of health condition index in more detail. A. Health Condition Index For a machine, the health condition index is estimated based on the fault characteristics established in this context as fault severity, fault factor, and fault weightage. In general, calculating the health condition index of a machine is nontrivial. In this paper, we leverage the component simulations with high fidelity modelling to estimate a machine's health condition. In particular, Finite Element Method (FEM) will be performed The generator and battery health condition index is quantified in this subsection. The key emphasis is on the definition alone, so the design aspect and their parameters are not discussed as the health condition index are normalized to their base rating. The health condition index of the generator is simulated through a generator drive system with the electromagnetic field solved in ANSYS Maxwell, and the control action is established in MATLAB-SIMULINK environment to control the fluctuating DC load voltage. The co-simulation is carried out in Twin Builder/Simplorer for the continuous exchange of data between the FEA ANSYS and MATLAB and is shown in Figure 1. The electrical faults in generator The TTSC faults are generally initiated by insulation breakdown caused by overheating or mechanical breakage [10]. TTSC faults, when undetected in early-stage, may lead to other severe winding short faults due to high fault loop current. The TTSC fault produces flux in opposite direction due to the reactive nature of the fault, which increases the eddy current and hysteresis loss in the machine. The TTSC fault is simulated in ANSYS Maxwell by splitting the total conductors in coil A as a separate rectangular conductor. The separate conductors are taken externally to the Twin Builder to model it as an extra winding to carry the fault loop current. Two levels of fault severity are simulated accounting for 8.33%, and 25%. The demagnetization fault occurs due to either TTSC fault or prolonged overloading in the flux weakening region of the machine. In severe cases, the demagnetization and TTSC fault may lead to the saturation of the core resulting in asymmetrical flux distribution in the machine. The demagnetization fault is simulated by increasing the temperature of magnet using the BH curve. Two different fault severity of one magnet and three magnets with fault factor of 100 • C and 150 • C is simulated. The various causes for the mechanical faults are misalignment due to manufacturing defects, unbalanced magnetic pull, or mechanical resonance at critical speed. The eccentricity fault creates high-frequency components based on the switching frequency as well as the length of the cable. The low-frequency component is also produced in torque due to other faults in machine leading to the increase in shaft voltage and consequently to the bearing fault in the machine. In ANSYS Maxwell, the SE is introduced by shifting the stator and the coils to 72% and 88.8% from normal position. Similarly, DE is implemented by shifting the rotor and the magnets by 17.7%, and 22.2% from their initial position. Various faults in asset exhibit different characteristics features. Common platform to weigh these features for formulating the health index of the generator is necessary. Figure 2 shows the block diagram for generator health condition index, real power, and voltage formulation. The total harmonic Table I. The relationship between the health condition index of the component and the constraint will not be linear. This is due to the definition of index and their corresponding fault characteristics. As different fault behaves differently, and their effect on system parameters vary based on the fault type and their severity. So, this non-linearity in parameters tend to occur with incipient fault cases. The battery electrochemistry is solved in CFD based AN-SYS Fluent. The three common faults in battery are external short circuit, internal short circuit, and thermal runaway. The internal short circuit is the incipient fault case and can lead to thermal runaway in long run. The leading causes for internal short circuit fault are lithium plating decomposition, electrode or electrolyte decomposition, or various external factors contributing to localized heat spots in the battery. The number of steps or cycles for healthy and various fault cases will be different for the same c-rate and are represented as endof-discharge (F ) factor over time. The external short circuit fault is simulated by introducing an external resistance with the battery terminal for 50% fault severity of 0.5Ω. The various SOC condition for c-rate equal to one is formulated to get B HCI as shown in Table II. The internal short circuit fault and thermal runaway are introduced by patching the center of the battery to a resistance of 5 × 10 −7 Ω and 5 × 10 −8 Ω respectively. The temperature contour for healthy and faulty cases with SOC of 90% is shown in Figure 4. III. HEALTH-FOCUSED OPF AND ITS SDP RELAXATION The conventional OPF is a constraint optimization problem for minimization of an objective while working over a set of equality and inequality constraints as: Here, x is variable vector having control and state variables, f i (x) is equality constraint function while x u k represents the upper limit of variable x k . The upper bound vector x u is considered constant if the equipment is connected. The proposed general HF-OPF formulation is given as: It is essential to understand that health condition index cannot be determined with certainty in various conditions. Therefore, we propose to use the probability distribution of health, β k (h k ), in defining the bounds on x. This way, the HF-OPF formulation (2) will have uncertain limits which needed to take care by the chance-constrain or robust optimization tools. Currently, for better understanding of the effects of health on OPF, we use β k (h k ) = h k , which can be interpreted as a mean of the probability function. We define network nodeset as N , generator node-set G ⊆ N and the set of branches as L. We define B as a set of nodes having BESS and use k as node index. Other system parameters are defined as: • S G k = P G k + jQ G k := Apparent power generation by generator connected to k ∈ G. • S L k = P L k + jQ L k := Apparent power load at k ∈ N . • V k = V x k +jV y k := Complex node voltage at node k ∈ N . • S kl = P kl +jQ kl := The apparent, real, and reactive power flow in branch from node k to l where (k, l) ∈ L. • C(P G k ):= Quadratic cost function for real power generation at node k ∈ G. • Y:= Admittance matrix of size N × N with elements y kl where (k, l) ∈ L The ACOPF model is studied in detail by various works [1], [11]- [13]. The vital difference in proposed HF-OPF is that limits are a function of health which are considered as constant in conventional ACOPF formulation. The HF-OPF for the real power generation cost minimization objective can be expressed as [14]: Here, it is essential to understand that HF-OPF considers a continuous health condition index h k , which affects the capabilities of the devices connected with the network. Thus, it takes a very realistic situation into account instead of event-based analysis where only binary conditions (on/off) are considered. In the proposed formulation, the BESS is treated as a generator with lower real power limits being negative and function of health during the discharging. The additional constraint on BESS are: Here, E k (t) represents state-of-charge (SOC), in works related OPF with BESS, P u k is has been taken as rated power capacity and E u k /E l k is a fixed number defining upper/lower limit of energy stored [15]. In the proposed HF-OPF, the complexity arises in the non-convex power flow equation (3g) solely due to the product term of voltage (V k V l ). In literature, various models have been proposed for the convex relaxation of the ACOPF problem. One of the initial model was presented in [14] using a lifting variable matrix W which is equal to the voltage product term as W kl = V k V l , (k, l ∈ N ). In this work, we use the model described in [13], [14] with W ∈ R 2n b ×2n b and V ∈ R 2n b . The HF-OPF problem (3a-3g), with matrix variable W can be expressed as: Here, the real and reactive power generation and V are: The definition of the constant matrices Y k , Y k , Y kl , Y kl and M k is similar the one presented in [14] and has not presented here to avoid repetition. This non-convexity of power flow constraint (3f) is now transferred into a single equality constraint (5f). For relaxed HF-OPF the equality constraint (5f) has been replaced by: The exactness of the HF-OPF has been sacrificed by removing the non-convex rank-one constraint of W . The details of exactness conditions can be found in [14] for similar relaxation. Thus, the SDP relaxed HF-OPF is minimization of objective (5a) constrained to BESS SOC constrain (4b) and relaxed ACOPF constrains (5b)-(5e), (6a), (6b) and (7) abd. The constrain (5b) is obtained from (4a) for BESS modeled as real power generator. IV. TEST RESULTS For testing the proposed HF-OPF and analysis of the effect of health on cost, we use a 3-Bus test microgrid system as given in Fig. 5. The G1 is a costly healthy generator while different fault conditions are simulated on G2 and battery. First, we consider nominal loading condition. The relative cost change, with respect to ACOPF solution having with perfectly healthy generator, is calculated. Except for the variation in P u 2 , all other system and solver parameters are kept constant to observe only the effect of health on ACOPF solution. P G2 decreases sharply when the maximum output is limited due to Due to the effect on voltage limit, the system may not have any solution as it will violate the voltage tolerance band (considered as 0.9 − 1.1 (pu) (5d)). Therefore, results of relative cost variations, including health condition's effect on voltage, are given in Figure 6. Considering V u 2 as a function of generator health makes a more significant impact on the HF-OPF solution cost in comparison to taking only P u 2 (G HCI ) into account. These results also emphasize the requirement of HF-OPF as the effect on cost is pronounced very rapidly with a decrease of G HCI . We also perform the test using three different scaling factors (SF ). The nominal loading condition shows the same results when only P u 2 (G HCI ) is considered in Figure 7. Subsequently, at higher loading, the effect on cost is very high with even small health decrease. These results indicate that for critical microgrid systems, the health monitoring and HF-OPF solution is essential. Similarly, we analyze the HF-OPF results with the health condition of the battery. The cost variation is observed and step change will be similar to the generator. This variation is due to non-linearity in the effects of faults on the real power discharge capacity of the battery. These results have been able to highlight the fact that the HF-OPF solution differs from OPF solution significantly. V. CONCLUSION The contingency-based OPF methods consider the health of power system equipment discretely. The critical microgrid systems need to have better incorporation of equipment health to ensure high availability and reliability. To facilitate this, we propose a novel Health-Focused OPF formulation, which includes constraints as a function of health condition index. The FEM based analysis is used to establish the relationship of health condition index with constraints. The test results indicate that health issues will cause severe costs on real power generations, let alone the problems of infeasibility. The proposed HF-OPF can work with online health monitoring systems, and EMS of remote microgrid will provide improved reliability. Future works include the establishment of the nonlinear relationship between health condition index and OPF constraints as well as solving HF-OPF for uncertain health condition index and constraints.	2020-11-25T02:00:59.649Z	2020-11-24T00:00:00.000	{ "year": 2020, "sha1": "3443910470832182fb8f853c40c297c3d962c60a", "oa_license": null, "oa_url": "http://arxiv.org/pdf/2011.11849", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "3443910470832182fb8f853c40c297c3d962c60a", "s2fieldsofstudy": [ "Engineering" ], "extfieldsofstudy": [ "Mathematics", "Computer Science", "Engineering" ] }
235709396	pes2o/s2orc	v3-fos-license	Concentrated Raw Fibers Enhance the Fiber-Degrading Capacity of a Synthetic Human Gut Microbiome The consumption of prebiotic fibers to modulate the human gut microbiome is a promising strategy to positively impact health. Nevertheless, given the compositional complexity of the microbiome and its inter-individual variances, generalized recommendations on the source or amount of fiber supplements remain vague. This problem is further compounded by availability of tractable in vitro and in vivo models to validate certain fibers. We employed a gnotobiotic mouse model containing a 14-member synthetic human gut microbiome (SM) in vivo, characterized a priori for their ability to metabolize a collection of fibers in vitro. This SM contains 14 different strains belonging to five distinct phyla. Since soluble purified fibers have been a common subject of studies, we specifically investigated the effects of dietary concentrated raw fibers (CRFs)—containing fibers from pea, oat, psyllium, wheat and apple—on the compositional and functional alterations in the SM. We demonstrate that, compared to a fiber-free diet, CRF supplementation increased the abundance of fiber-degraders, namely Eubacterium rectale, Roseburia intestinalis and Bacteroides ovatus and decreased the abundance of the mucin-degrader Akkermansia muciniphila. These results were corroborated by a general increase of bacterial fiber-degrading α-glucosidase enzyme activity. Overall, our results highlight the ability of CRFs to enhance the microbial fiber-degrading capacity. Introduction Diets prevalent in industrialized countries are characterized not only by high amounts of protein and fat, but also by a deficiency of plant-derived fibers [1]. These so-called "Western-style" nutritional habits are linked to altered and potentially disease-promoting properties of the intestinal microbiome [2], further suggesting that supplementation of such diets with prebiotic fibers might be beneficial for the host. The intestinal microbiome has a remarkable impact on susceptibility and progression of various intra-and extra-intestinal pathologies [2]. Thus, the targeted manipulation of the host's microbiome may alleviate this risk and has recently received considerable attention [3]. In this context, plant-derived fibers are considered to be promising host-beneficial dietary supplements for microbiota modulation [4]. Health-beneficial impacts of fibers are either mediated by general physiological influences, maintaining the integrity of the mucus layer or by microbial fermentation into host-beneficial metabolites, such as short-chain fatty acids (SCFAs). SCFAs play a crucial role in maintaining barrier integrity and immune homeostasis [5], and soluble fibers represent a major source of these microbially produced metabolites [2]. Previously, we reported a causal role of fiber deprivation, increasing susceptibility towards enteropathogenic infections in a gnotobiotic mouse model containing a 14-member synthetic human gut microbiome (14SM) [6]. We demonstrated that a lack of dietary fiber resulted in a bloom of mucin-degrading commensals, such as Akkermansia muciniphila, leading to the excess degradation of the intestinal mucus layer, and subsequently, facilitated infection with Citrobacter rodentium [6]. These results further strengthen the connection between dietary fiber and gut microbial modulation. Moreover, our 14SM gnotobiotic model provides an attractive approach to validate the modulation of the gut microbiota with fiber supplementation using the basal fiber-free diet [6]. However, due to the complexity of the intestinal microbiome and the resulting individual responses, general recommendations on quantity, source or combinations of fiber supplements for consumption for humans remain vague [7,8]. Plant-derived fibers come in different chemical forms and structures, therefore providing distinct access for intestinal microbes to hydrolyze structure-specific glycosidic linkages. Here, we used dietary "concentrated raw fiber" (CRF) preparations from pea, oat, psyllium, wheat and apple to evaluate the detailed effects of fiber supplementation under strictly controlled conditions in our 14SM gnotobiotic mouse model. In contrast to purified fibers [9], CRFs are fiber concentrates, which are extracted and isolated from skeletal substances in a non-chemical, thermophysical process, thus providing a diverse polysaccharide composition. Of note, the wheat CRF used was previously shown to increase fecal bulking in a randomized controlled human study [10], while the psyllium CRF was associated with an increased overall SCFA production [11] in an in vitro system. We evaluated the in vivo effects of these CRFs on the relative abundances of the 14SM constituent strains, the emerging activities of bacterial glycan-degrading enzymes and the associated concentrations of different SCFA. Furthermore, we performed extensive correlation analyses to evaluate potential inter-microbial influences in response to fiber supplementation and thus better understand community-shaping properties of such dietary modulation. Experimental Setup to Study the Specific Effects of Concentrated Raw Fibers on Composition and Function of A 14-Member Microbial Community in Mice Germ-free (GF) C57BL/6N mice were raised and maintained under gnotobiotic conditions on a standard mouse chow (SC). At the age of six to eight weeks, mice were colonized via intragastric gavage with a synthetic microbiota consisting of 14 human commensals (14SM), as described previously [6]. Strains of this 14SM community represent the five dominant phyla of the human intestinal microbiota and provide important core metabolic function [6]. Five to sixteen days after the initial gavage, mice were either switched to a fiberfree (FF) diet or a fiber-supplemented (FS) diet ( Figure 1a) containing CRFs (VITACEL ®, J. Rettenmaier und Söhne (JRS, Rosenberg, Germany)) derived from pea, oat, psyllium, wheat and apple. As controls, seven mice were maintained on a SC diet. Before the diet switch, we confirmed the proper colonization of all animals with the 14SM community by strain-specific qPCR from fecal samples, as described previously [6]. Twenty days after the diet switch (feeding period), mice fed all three different diets were sacrificed and contents of the cecum and colon were harvested for downstream analyses. As we aimed to determine the direct impact of fiber supplementation on microbiota composition and function in a tightly controlled gnotobiotic setting, we designed the FF and FS diets with the aim of providing an isocaloric composition as well as an identical formulation among these two diets, with the exception of the non-cellulose complex fiber amount (Figure 1b). Experimental outline to study effects of concentrated raw fibers on a 14-member microbial community. (a) Experimental outline. Germ-free (GF) C57BL/6N (n = 15), raised and maintained on a standard mouse chow (SC) were colonized with the 14SM community. Five to sixteen days after colonization, mice were either continued to be fed a SC diet (n = 7) or switched to a fiber-free (FF; n = 4) or a fiber-supplemented (FS, n = 4) diet. Twenty days after diet switch, cecal and fecal samples were harvested for analyses. (b) Composition in % (w/w) of the diets; MVA = Minerals, vitamins, and ash; NFE = Nitrogen-free extracts. Thus, to generate the FS diet, we reduced the dextrose content in the FF diet by an amount corresponding to 10% of the total weight and replaced it by the same amount of a concentrated raw fiber mix. The fiber mix in the FS diet consisted of equal amounts of CRF preparations obtained from pea, oat, psyllium, wheat and apple (2% (w/w) each). The five different CRF preparations contained an average fiber length within a two-digit µm range and the fiber content (w/w) in these preparations ranged from 55% (apple) to 97% (wheat) ( Table 1). Importantly, the ratio of insoluble-to-soluble fibers (I/S-ratio) differed significantly among the different preparations, with an I/S ratio of 34 in the case of the pea preparation to an I/S ratio of 0.2 for the psyllium preparation. Of note, the apple preparation contained 9% (w/w) pectin. The SC diet contained 3.9% fibers from naturally milled fibers, while contents of protein and fat were considerably lower compared to both, the FF and FS diets (Figure 1b). Increase in Relative Abundance of Certain Fiber-Degrading Commensals in Response to Dietary Fiber Supplementation At the end of the 20-day feeding period with the three diets, the microbiota composition was analyzed in fecal samples using 16S rRNA gene sequencing, revealing different clustering of each of the three groups ( Figure 2a). These findings not only highlight the overall impact of diet on the microbiota composition, but also the specific and considerable effect of CRF supplementation. On a phylum level, we determined a significant increase in the abundance of Bacteroidetes (p = 0.0046; t-test) and a decrease in Firmicutes (p = 0.0149; t-test) (Figure 2b) in FS-fed mice compared to their FF-fed counterparts. On a strain-level, we detected significantly different relative abundances of 8 of the 14 community members in FS-fed mice compared to the FF-fed control mice (Figure 2c,d). Specifically, we detected significantly lower abundances of A. muciniphila and M. formatexigens (see Figure 2c for strain abbreviations) in FS-fed mice compared to their FF-fed counterparts, while the relative abundances of E. coli, D. piger, E. rectale, B. ovatus, R. intestinalis and B. thetaiotaomicron were significantly increased (Figure 2d) in response to CRF supplementation. Inter-Bacterial Relations in Relative Abundance within the 14-Member Microbial Community To further investigate such potential inter-microbial influences and dependencies in response to CRF supplementation, we performed pairwise correlation analyses of all strains within each individual and across all groups ( Figure 3a). All correlation analyses were performed using the "rcorr" function within the R package "Hmisc" and visualized using the "corrplot" package. While the relative abundances of some strains, such as B. intestinihominis, F. prausnitzii and B. thetaiotaomicron, provided little to no correlation with any of the other community members, certain bacteria, such as A. muciniphila, B. caccae, B. ovatus, B. uniformis, D. piger or E. rectale, significantly correlated with multiple other strains ( Figure 3a). These findings indicate a high inter-microbial dependency of A. muciniphila, B. caccae, B. ovatus, B. uniformis, D. piger or E. rectale with other strains within the 14SM community, suggesting that the relative abundance of these strains was either strongly dependent on the overall microbiota composition or, conversely, are major influencers of the remaining microbiota in response to certain environmental changes, such as dietary However, we also detected significant differences in the relative abundances of nine strains when comparing the FS-fed to the SC-fed mice. Since the SC diet also contains natural fiber, albeit in a non-concentrated form and in lower amounts, these differences could not be rooted in presence of fibers alone and might be a result of different sources of fibers or the distinct protein and fat content. Since we were particularly interested in strain-specific changes in response to fiber supplementation and the emerging effects on microbiota function, we associated strain-specific changes between the FS-and FF-fed mice with the metabolic potential of the respective strains to grow on a suit of monoand polysaccharides as determined previously using carbohydrate in vitro growth as-says [6] (Figure 2e). This association revealed a decreased relative abundance of a mucin specialist, A. muciniphila (Figure 2d), which is in line with previous findings that fiber deprivation results in overgrowth of this particular strain [6] leading to decreased mucosal barrier integrity. This finding further highlights the inverse correlation between relative A. muciniphila abundance in the colon and dietary fiber intake. Of note, increased abundances of A. muciniphila were associated with various pathologies in human studies [12][13][14][15][16][17], supporting the idea that, in addition to promoting microbiota-mediated SCFA production, fiber supplementation considerably contributes to the maintenance of mucosal barrier integrity by preventing excess mucus degradation. Furthermore, fiber supplementation resulted in significantly increased relative abundances of B. thetaiotaomicron, E. rectale, R. intestinalis and B. ovatus (Figure 2d), which share the capability to metabolize a broad variety of plant-derived polysaccharides, such as starch, cellobiose and αand/or β-glucans ( Figure 2e) as previously confirmed with a carbohydrate in vitro utilization assay [6]. Thus, the ability to degrade αand/or β-glucans promoted commensal growth under CRF-supplemented conditions, probably due to such glucans being major components of the CRF preparations [18]. Importantly, not all of the strains capable of metabolizing complex polysaccharides, such as B. uniformis, were increased in response to supplementation with the selected fiber formulation (Figure 2d), indicating strain-specific effects of supplementation with the chosen CRF supplements. In general, intensified fiber consumption by the microbiome is associated with increased intestinal H 2 levels, which can exhibit disadvantageous effects on the host [19]. Thus, the increased abundance of D. piger in FS-fed mice might have a counter-regulating effect given the H 2 -consuming properties of this bacterium [20]. Since E. coli is not a fiber fermenter [6], its increased abundance is very likely a secondary effect due to changed abundances of microbes that are directly affected by fiber supplementation, resulting in altered microenvironments or nutrient availability. Inter-Bacterial Relations in Relative Abundance within the 14-Member Microbial Community To further investigate such potential inter-microbial influences and dependencies in response to CRF supplementation, we performed pairwise correlation analyses of all strains within each individual and across all groups ( Figure 3a). All correlation analyses were performed using the "rcorr" function within the R package "Hmisc" and visualized using the "corrplot" package. While the relative abundances of some strains, such as B. intestinihominis, F. prausnitzii and B. thetaiotaomicron, provided little to no correlation with any of the other community members, certain bacteria, such as A. muciniphila, B. caccae, B. ovatus, B. uniformis, D. piger or E. rectale, significantly correlated with multiple other strains (Figure 3a). These findings indicate a high inter-microbial dependency of A. muciniphila, B. caccae, B. ovatus, B. uniformis, D. piger or E. rectale with other strains within the 14SM community, suggesting that the relative abundance of these strains was either strongly dependent on the overall microbiota composition or, conversely, are major influencers of the remaining microbiota in response to certain environmental changes, such as dietary supplementation. In addition to B. thetaiotaomicron, all other α-and β-glucan metabolizing microbes (B. ovatus, B. uniformis, E. rectale and R. intestinalis) provided significantly positive correlations with each other (Figure 3a; Pearson correlation coefficient R > 0), suggesting that there was no nutrient competition for these polysaccharides between these strains. Interestingly, B. caccae, which is able to metabolize pectin but not αand β-glucans, provides a strong negative correlation to all of the αand β-glucan degraders. Given the overall pectin concentration in the FS diet of roughly 0.2% (w/w) ( Table 1), this suggested a potential competition for pectin with those αand β-glucan degraders, which share the ability to metabolize pectin. Correlations between relative abundances of 14SM community members. (a) Pairwise correlation between relative abundances of all strains as determined by using 16S rRNA gene sequencing data from all mice across all groups; Analysis performed using the "rcorr" function of the R package "Hmisc" and visualized using the "corrplot" function. Colored circles depict statistically significant correlations (p < 0.05); empty squares represent non-significant correlation independent of the determined correlation coefficient R; color intensity and circle size vary depending on the Pearson correlation coefficient R, with R = 1 (positive correlation) and R = −1 (negative correlation) displayed with maximal circle size and color intensity. To better illustrate such potential inter-microbial correlations, we performed a correlation network analysis (Figure 3b), employing the "network_plot" function in the R "rcorr" package to highlight clusters of the correlations shown in Figure 3a. In this plot, variables undergo multidimensional clustering using the absolute values of the correlations, where tightly clustered variables exhibit similar relationships with the other variables ( Figure 3b). Figure 3b demonstrates that strains metabolizing αand β-glucans are clustering strongly together. Interestingly, B. caccae (pectin degrader) and A. muciniphila (mucin degrader) also fall into the same correlation cluster (Figure 3b) through their significant negative correlation with the glucan degraders (Figure 3a), suggesting that the decreased relative abundance of A. muciniphila under fiber-supplemented conditions (Figure 2d) is a secondary effect due to the bloom of fiber-fermenting microbes, while B. caccae remains unaffected in its relative abundance in FS-fed mice (Figure 2d), while it is strongly decreased in SC-fed mice. Concentrated Raw Fiber Supplementation Is Associated with Changes in Fecal Bacterial Glycan-Degrading Enzymes Given these fiber supplementation-mediated changes of microbial abundances (Figures 2 and 3), we evaluated the functional outcomes of these compositional alterations. Thus, we determined the enzymatic activity of certain bacterial enzymes in fecal pellets that are involved in either the fermentation of fiber-derived polysaccharides or the degradation of host-secreted mucin glycans, which were previously reported to be inversely associated with the amount of dietary fiber consumed [6]. The enzymes β-glucosidase (GLUC) and α-galactosidase (GAL) primarily target glycosidic linkages present in plant fiber-derived polysaccharides, with β-glucosidase being a crucial enzyme for hydrolyzing linkages in β-glucans [6]. Conversely, α-fucosidase (FUC), sulfatase (SULF) and β-Nacetylglucosaminidase (NAG) catalyze reactions involved in mucin glycan degradation [6]. While fecal activities of SULF and NAG remained unaffected by fiber supplementation, we detected significantly increased activities of GLUC and GAL in FS-fed mice compared to FF-fed mice, albeit FS-fed mice provided significantly lower GAL and GLUC activities as compared to SC-fed controls (Figure 4a). This indicates that the source of fibers and their fine-scale composition seems to be more important to mediate functional outcomes of the microbiome than the amount of CRFs alone. Surprisingly, we also detected a significant increase in FUC activities in FS-fed mice (Figure 4a). This may be due to the presence of certain glycans in the FS diet harboring an alpha-1,6-linked fucose residue joined to the reducing end of an N-acetylglucosamine moiety, which is absent in the other diets. (a) Tukey boxplots of enzymatic activities of bacterial glycan-degrading enzymes in fecal samples, normalized on the amount of total fecal protein; FUC: α-fucosidase, GLUC: β-glucosidase, GAL: α-galactosidase, NAG: β-N-acetlyglucosaminidase, SULF: sulfatase; statistics: Wilcoxon rank sum test performed with the "compare_means" function within the R package "ggpubr". *: p < 0.05. (b) PCA plot of the glycan-degrading enzyme activity pattern of FF-, FS-and SC-fed mice as calculated with the "prcomp" function within the R package "stats", using data sets shown in (a) and based on a Euclidian distance matrix. Visualization using the "autoplot" function. (c) Pairwise correlation between glycan-degrading enzyme activities and the relative abundance of 14SM strains from all mice across all groups. Analysis performed using the "rcorr" function of the R package "Hmisc" and visualized using the "corrplot" function. Colored circles depict statistically significant correlations (p < 0.05); empty squares represent non-significant correlation; color intensity and circle size vary depending on the Pearson correlation coefficient R, with R = 1 (positive correlation) and R = −1 (negative correlation) displayed with maximal circle size and color intensity. The overall glycan-degrading enzyme activity pattern, as determined by principal components analysis (PCA) using activity data of all determined enzymes, revealed a different clustering between FF-and SC-fed mice only, while FS-fed mice provided an intermediate activity pattern (Figure 4b). The activity of GLUC and GAL exhibited a strong positive correlation with the relative abundance of the fiber-degrading strains B. ovatus, B. uniformis and E. rectale, but also with C. aerofaciens (Figure 4c), which might benefit from released monosaccharides from polysaccharide degradation catalyzed by other strains. As expected, FUC activity exhibited a strong positive correlation with the relative abundance of mucin-glycan-degrading A. muciniphila, but also with D. piger, B. caccae and E. coli (Figure 4c). While B. caccae is mucin generalist, meaning that it is also capable of mucin degradation (Figure 2e), based on our previous work [6], E. coli is neither a mucin glycandegrading nor a fiber-degrading commensal (Figure 2e). Although correlation analyses suggest that increased FUC activity in FS-fed mice are associated with E. coli, we could, so far, not confirm FUC expression in E. coli, and this finding might be a non-causal, correlative artifact due to secondary effects. Changes of Bacterial Glycan-Degrading Enzyme Activities Are Interlinked with a Specific Short-Chain Fatty Acid Production Profile In addition to other features, microbe-mediated fiber degradation results in the production of short-chain fatty acids (SCFAs) [21]. Since SCFAs provide important beneficial effects for the host [5,22], we next investigated whether the fiber degradation-associated enzyme activity pattern in FS-fed mice resulted in altered SCFA production. While cecal concentrations of acetate and formate did not differ significantly between the FF-and FS-fed mice, propionate concentrations in cecal contents were significantly lower in the FS-fed mice compared to their FF-fed counterparts (Figure 5a). In line with this, PCA analysis of the overall SCFA production pattern between the three groups revealed that CRF supplementation did not result in a significantly different SCFA production compared to FF-fed mice, while the SC-mediated SCFA production was significantly different from the FF diet (Figure 5b). In contrast to the SC-fed control group, the concentration of the main host-modulatory SCFA, butyrate [23], did not increase in FS-fed mice compared to FF-fed mice (Figure 5a). The most important butyrate producers within the 14SM community are F. prausnitzii, R. intestinalis, C. symbiosum and E. rectale [6] (Figure 2e). Although the relative abundances of R. intestinalis and E. rectale were significantly higher in the FS-fed mice compared to the FF-fed mice (Figure 2d), the abundances of these strains were significantly lower compared to SC-fed control mice, which provided the highest butyrate concentrations among the three groups. Thus, the relative abundance of R. intestinalis and E. rectale appear to be predictors of butyrate concentration in 14SMcolonized mice, due to their strong positive correlation with corresponding butyrate levels ( Figure 5c) and their relative abundances in FS-fed mice was probably not elevated enough to translate into significant increases in butyrate and propionate concentrations compared to FF-fed mice. Additionally, the relative abundance of B. ovatus also correlated positively with butyrate concentrations (Figure 5c). However, this correlation is probably rooted in non-butyrate related inter-microbial interactions, since this strain is not known to be a main butyrate producer within the 14SM community. Furthermore, while butyrate concentrations exhibited positive correlation with GLUC and GAL activities, propionate only correlated positively with GLUC and formate with FUC activities (Figure 5d). In summary, given the strong effects of fiber supplementation on the relative abundances of certain fiber degraders (Figure 2c,d) and the associated increase in bacterial fiber-degrading enzyme activities (Figure 4a), the non-significant SCFA levels compared to FF-fed mice (Figure 5a) were somewhat unexpected but in line with the relatively decent increase of GLUC and GAL activities compared to the FF-fed mice. Combining the data from Figures 2-5 suggests the presence of two independent functional correlation pathways connecting the 14SM community with glycan-degrading enzyme activities and SCFA production ( Figure 6). While we found a strong positive correlation between GLUC and GAL activity with butyrate and propionate levels, FUC activity correlated with the production of acetate and formate. In addition to the ability of the host to metabolize certain amino acids into formate, it can also be produced as a by-product of metabolic activities of intestinal commensals [24]. Importantly, elevated concentrations of formate were previously reported to be a signature feature of inflammation-associated microbiome dysbiosis in a mouse model of colitis [25] and was associated with increased abundances of commensal E. coli strains, which is in line with our correlation analyses (Figure 5c or Figure 6). Supplementation of the FF diet with the chosen mix of CRFs derived from pea, oat, psyllium, wheat and apple did result in an increased relative abundance of some, but not all strains that were found to correlate with the GLUC/GAL-associated pathway. Meanwhile, D. piger and E. coli, which correlate with the FUC-associated pathway, were increased. It is worth noting, that such identified correlations do not necessarily equate with causal connections within these pathways. For example, B. uniformis strongly correlated with GLUC and GAL activities as well as with cecal butyrate concentrations, although this species is not a known butyrate producer. Consequently, this strain might be important to support the butyrate production of other strains via yet unknown mechanisms. Discussion A lack of fiber intake is commonly associated with decreased microbial diversity in the gut [26] as well as with increased concentrations of metabolites that can be harmful to the host [27]. Thus, supplementing a fiber-deprived, Western-style diet [2] appeared as a reasonable strategy to restore or even boost host-beneficial properties of a given individual's indigenous microbiome. Although this approach seems trivial at first glance, it launches several challenges concerning the dose or source of fiber to be consumed by a certain individual, fitting the preconditions and needs of a specific microbiome composition. Although various human studies demonstrated beneficial effects of general fiber supplementation in most, but not all, study participants (reviewed in [28]), personalized and more tailored approaches are rare. Additionally, in-depth analyses of microbiome-specific effects of fiber supplementation on inter-microbial interactions and the resulting functional outcomes are difficult to conduct due to the complexity of the microbiome and the multitude of potential inter-microbial influences. Thus, we aimed to investigate such interconnections and functional outcomes in a gnotobiotic mouse model with a standardized microbiome consisting of 14 human commensal bacteria strains. These commensals comprise the five most abundant bacterial phyla in the human host and provide all core metabolic functions. Importantly, their ability to consume certain poly-and monosaccharides, as well as their capacity to produce SCFAs has been assessed previously [6]. We could demonstrate that most, but not all, fiberdegrading commensals within this community provided increased relative abundances in response to supplementation of a fiber-deprived diet with a mix of CRFs obtained from pea, oat, psyllium, wheat and apple. This was particularly the case for αand βglucan degrading commensals, while commensals capable of hydrolyzing pectin, which is prevalent in the added apple preparation, were not positively affected. Although the relative abundances of fiber-degrading commensals and the activities of enzymes involved in bacteria-mediated fiber degradation increased significantly in response to fiber supplementation, this did not translate into a more host-beneficial SCFA production pattern. Our correlation network analyses suggest dynamic interconnections in response to fiber supplementation between the 14 constituent strains and reveal the importance of yet unidentified inter-microbial interactions to exhibit beneficial properties. Previous studies have routinely used purified fiber supplements in rodent systems to show a positive impact on the generation of SCFAs [29]. However, one needs to be cautious about the high amount of fibers used in the rodent diets and the translatability of such amounts to human hosts. Given the far more complex microbiome composition and associated microbial interconnections and dependencies in the human gut, this highlights the challenges in designing personalized dietary recommendations for the benefit of the host. A pioneering study to address these points investigated the effects of four different fiber supplements on microbial diversity and emerging SCFA production in a human trial [30]. Among other findings, applied dietary fiber interventions were specific and limited to a few taxa within each participant, which, however, translated into a relatively consistent SCFA production pattern across the participants receiving the same supplements [30]. However, only ten participants were recruited for each cohort, which seems insufficient to make generalized statements on suitability of these supplements for a larger pool of individuals. In summary, our findings demonstrate that supplementation of a fiber-free diet with a mix of CRFs resulted in significant changes to the intestinal microbiome structure and activity. While some, but not all, fiber-degrading commensals provided increased abundances in response to fiber supplementation, abundances of the mucin-specialist A. muciniphila were significantly decreased. Interestingly, increased abundances of this species were detected in multiple sclerosis patients [12][13][14]31] and were implicated in the increased susceptibility towards enteropathogenic infections in the same 14SM mouse model as used in this study [6]. However, other studies report on strong host-beneficial effects of this species [17,32], in some cases classifying A. muciniphila as "probiotic" [33]. These findings, which appear contradictory at first, might be rooted in different microbiome-mediated mechanisms of disease pathology, the considerable diversity among different commensal A. muciniphila strains [34] or in the complex inter-microbial influences within a given microbiome, which we observe even in a reduced community of only 14 strains. Thus, these factors might determine either the health-beneficial or disease-promoting properties of A. muciniphila. Given the specialization of A. muciniphila on degrading mucin-associated glycans, increased abundances of this species might result in excess mucus layer degradation under certain circumstances. Interestingly, impairment of the intestinal mucus layer integrity was already suggested to be involved in the pathology of ulcerative colitis [35,36]. The mucus layer represents a key component of the intestinal mucosal barrier, and increased mucosal barrier permeability is supposed to be a contributing factor to pathophysiology of autoimmune diseases [37]. However, the specific role of microbiome-mediated mucus degradation in this process is yet unclear and a potential pivotal contribution of particular commensal species remains to be elucidated. Either way, we demonstrate that dietary habits crucially impact the activity of bacterial mucin glycan-degrading enzymes, possibly resulting in altered mucosal barrier integrity. Thus, over-focusing on SCFA production should be avoided when assessing host-influencing properties of a diet-modulated microbiome. On the other hand, other beneficial properties of a fiber-modulated microbiome, such as regulation of the mucus turnover, could be taken into account. Mouse Experiments Germ-free (GF) female C57BL/6N mice were originally purchased from Taconic Biosciences, Germany. The animals were bred and housed inside the local germ-free facility of the University of Luxembourg. Aerobic and anaerobic microbial culturing of fecal samples was used to confirm the GF status of mice. For ethical aspects of the performed animal experiments, see "Institutional Review Board Statement" below. Mice were raised and maintained under gnotobiotic conditions on a standard mouse chow (SC). The animals were kept in ISO-cages with a maximum of 5 mice per cage and colonized at the age of six to eight weeks via intragastric gavage with a synthetic microbiota consisting of 14 different human commensals (14SM), as described previously [6]. Five to sixteen days after initial gavage, after 14SM colonization confirmation via qPCR, mice were either switched to a fiber-free (FF) diet, a fiber-supplemented (FS) diet or remained on the SC diet as a control group. All diets and water were provided in sterile conditions ad libitum. The well-being of all animals was evaluated, and fecal samples were collected once per week. Twenty days after diet switch, mice fed all three different diets were sacrificed and contents of the cecum and colon were harvested for downstream analyses. Culturing and Colonization of Germ-Free Mice with Synthetic Microbiota Culturing of all 14 bacterial strains of the synthetic microbiota (SM) and subsequent colonization of germ-free C57BL/6 mice was performed as described in detail previously [38]. In brief, all strains were cultured in a modified yeast-and short-chain fatty acid-containing culture medium (mYCFA), which was based on a previously published recipe [39]. However, its composition was adapted to fit the specific needs of the bacterial strains used in this study. Thus, mYCFA did not contain maltose and cellobiose, but contained N-acetyl-D-glucosamine to support growth of the mucin-specialist A. muciniphila. Furthermore, the concentration of sulfate ions was increased 46-fold and sodium lactate was added to support growth of Desulfovibrio piger [38]. Culturing of all strains was started 3 d before initial gavage by inoculation of 50 µL cryo-preserved bacterial cultures into 3 mL of oxygenreduced mYCFA. Cultures were diluted daily by factor 100 in mYCFA if OD 600 was higher than 0.4. The final gavage mix consisted of equal volumes of each bacterial culture, which were grown to an OD between 0.5 and 2.0 [38]. As described in detail elsewhere [38], this way of preparing the gavage mix results in reproducible colonization of GF mice with comparable relative abundances of a given strain across different experiments. Isolation of Bacterial DNA from Mouse Fecal Samples Collected mouse fecal samples were stored at -20 • C until processing for bacterial DNA extraction. Isolation of bacterial DNA from these fecal samples was performed using a phenol:chloroform:isoamyl alcohol (25:24:1)-based approach, followed by purification of DNA with the QIAGEN DNeasy Blood & Tissue kit, as previously described in detail [6,38]. In brief, 500 µL of "Buffer A" (0.2 M NaCl, 0.2 M Trizma base, 20 mM EDTA pH 8), 210 µL 20% (w/v) SDS (pH 5.2) and 500 µL phenol:chloroform:isoamyl alcohol (25:24:1) (pH 8.0) were added to one fecal sample of approx. 20-40 mg. After adding 250 µL of acid-washed glass beads (212-300 µm) to this mixture, samples were subjected to bead-beating on the highest frequency (30 Hz) for 3 min using a bead mill. After centrifugation at 18,000× g and 4 • C for 3 min, the aqueous phase was harvested and 500 µL of a phenol:chloroform:isoamyl alcohol (25:24:1) mix was added. After mixing by tube inversion, samples were centrifuged again at 18,000× g and 4 • C for 3 min. The aqueous phase was harvested and 500 µL of 100% chloroform was added to the harvested aqueous phase followed by mixing through tube inversion. Samples were centrifuged at 18,000× g and 4 • C for 3 min, followed by another aqueous-phase harvesting. Next, 60 µL 3 M NaCl (pH 5.5) and 600 µL 100% iso-propanol were added and incubated for 1 h at −20 • C for DNA precipitation. After centrifugation at maximum speed and 4 • C for 20 min, the supernatant was discarded, and the pellet was resuspended in 1 mL 70% ethanol. After centrifugation for 3 min at max speed, the supernatant was removed, and the pellet was dried. The dry pellet was resuspended in 100 µL nuclease-free water and subjected to further DNA purification using the QIAGEN DNeasy Blood & Tissue kit according to the manufacturer's instructions. Illumina 16S rRNA Gene Sequencing and Data Analysis This protocol uses dual-index primers to amplify the V4 region of the 16S rRNA gene [40]. For each plate, ZymoBIOMICS™ Microbial Community DNA Standard (D6305) and an internal 16S mock bacterial community control (DNA QC 16S) from 10 genomic DNAs obtained from DSMZ (Lot No: 2019-1) were also run in quadruplicate. Libraries were prepared using Quick-16S™ NGS Library Prep Kit (Zymo Research, Irvine, CA), according to the manufacturer protocol. The final pooled library was quantified with Qubit ® and the amplicons were sequenced on an Illumina MiSeq with MiSeq ® Reagent Kit v2 (500-cycle) (Illumina, USA). The raw sequencing data have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under the study accession number PRJEB45381. Sequences were processed with the program mothur (v1.44.3) [41] according to the MiSeq SOP, which can be found on the mothur website (https://mothur.org/wiki/miseq_sop/, accessed on 23 June 2021) [40,42]. Intestinal Fatty Acid Analysis Thirty to one hundred mg of flash-frozen cecal content was homogenized using 1.4 mm ceramic beads (5 beads per tube). Per 50 mg of cecal content, 500 µL of stock solution (2-Ethylbutyric acid, 20 mmol/L) was used (VK05 Tough Micro-Organism Lysing Kit). Cecal content was homogenized for 30 sec at 4500× g at 10 • C (Precellys24 Homogenizer) and centrifuged at 21,000× g for 5 min and 4 • C. Sample homogenate was further processed and measurements of SCFAs was performed as previously described using high-performance liquid chromatography (HPLC) [43]. Detection of Glycan-Degrading Enzyme Activities in Fecal Samples Enzymatic activities of β-glucosidase, α-galactosidase, α-fucosidase, β-N-acetylgluco saminidase and sulfatase were detected from fecal samples stored at −20 • C as described previously [44]. In brief, bacterial glycan-degrading enzymes were solubilized from the fecal samples by incubation in a lysozyme-, DNase I-and Triton X-100-containing lysis buffer on ice followed by sonification and removal of unsolubilized material by centrifugation. Supernatants were collected and protein concentrations in these supernatants was measured. For detection of enzymatic activities, equal amounts of protein were incubated with enzyme-specific p-nitrophenol-coupled substrates, and the substrate turnover (pnitrophenol release) was monitored by kinetic measurements of optical density at 405 nm. For details on buffers, substrates and final computation of enzymatic activities from optical density data, refer to [44].	2021-07-03T06:16:57.144Z	2021-06-14T00:00:00.000	{ "year": 2021, "sha1": "6e78d60e08438f66aa0db479dd1be42f878558e6", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/1422-0067/22/13/6855/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "a6be1490d32de526685382067b07e9d377675836", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
239009400	pes2o/s2orc	v3-fos-license	Non-Terrestrial Networks for UAVs: Base Station Service Provisioning Schemes with Antenna Tilt By focusing on unmanned aerial vehicle (UAV) communications in non-terrestrial networks (NTNs), this paper provides a guideline on the appropriate base station (BS) service provisioning scheme with considering the antenna tilt angle of BS. Specifically, two service provisioning schemes are considered including the inclusive-service BS (IS-BS) scheme, which makes BSs serve both ground users (GUs) and aerial users (AUs) (i.e., UAVs) simultaneously, and the exclusive-service BS (ES-BS) scheme, which has BSs for GUs and BSs for AUs. By considering the antenna tilt angle-based channel gain, we derive the network outage probability for both IS-BS and ES-BS schemes, and show the existence of the optimal tilt angle that minimizes the network outage probability after analyzing the conflict impact of the antenna tilt angle. We also analyze the impact of various network parameters, including the ratio of GUs to total users and densities of total and interfering BSs, on the network outage probability. Finally, we analytically and numerically show in which environments each service provisioning scheme can be superior to the other one. I. INTRODUCTION Due to an increasing demand for novel and high-quality mobile services, it becomes more difficult to provide reliable communications by the existing terrestrial networks only, up to the level required by future mobile services. To address these issues, non-terrestrial networks (NTNs) have been considered as a promising solution to complement terrestrial networks by providing ubiquitous and global connectivity [2], [3]. Conventional 2D ground space in terrestrial networks is now expanded to 3D aerial space in NTNs with supporting communications for unmanned aerial vehicles (UAVs), high altitude platform systems (HAPS), and satellites [4]. Among them, UAV communications have been in the spotlight because UAVs have more flexible mobility and can locate closer to ground users and base stations (BSs) in terrestrial networks, compared to HAPS and satellites. Therefore, many applications and services based on UAV communications have appeared such as working as a relay in hotspot and a data collector in largescale networks [5]- [7]. However, the integration of UAVs into existing terrestrial networks brings a lot of challenges such as resource and interference management since UAV communications usually use the frequency band as well as BSs of terrestrial networks. In this context, many works have been presented for reliable UAV communications. At the beginning of studies, the wireless channel modeling of UAV networks has been studied in [8]- [11], which is different from that of terrestrial networks. Specifically, according to the height of the UAV, the distancedependent path loss model for the cellular-to-UAV channel and the line-of-sight (LoS) probability between the UAV and the ground device were modeled in [8], [9] and [10], [11], respectively. Based on the wireless channel modeling of UAV networks, the works in [12]- [19] studied to present the optimal location of UAVs for various environments and applications. The deployment and the power allocation for the UAV jointly optimized to minimize the outage probability in [12], [13]. The height of the UAV and the antenna beamwidth jointly optimized to maximize the data rate [14] and the coverage probability [15]. The joint optimization of the UAV trajectory and the spectrum allocation were considered to maximize the throughput [16] and minimize the mission completion time [17]. The outage probability was presented by considering the effect of the UAV height and the channel environment in [18]. In [19], multi-layer aerial networks have been considered and designed optimally to maximize the successful transmission probability and the area spectral efficiency. However, the works in [13], [14], [16], [17] made a strict assumption that UAV-to-ground communications channels are dominated by LoS links only without considering the location-dependent probability of having LoS links. Furthermore, all of those aforementioned works did not consider a BS antenna tilt angle, which significantly affects the communication performance between the ground BS and the UAV. Especially, the antenna tilt angle of the ground BS has been conventionally designed for ground devices only, so the UAV can actually receive the signal from these BSs with considerably small power [20], [21]. To overcome these issues, the efficient design of the BS antenna tilt angle for UAV communications has been considered in recent works [22]- [28]. The vertical antenna gain was considered for analyzing the successful transmission probability of UAV communications in [22]. The BS antenna tilt angle was optimized to maximize the coverage probability according to the heights of the UAV and the BS in [23], [24], and also to maximize the successful content delivery probability in massive multiple-input multiple-output (MIMO) systems in [25]. The BS association probability and signal-tointerference-plus-noise ratios (SINRs) were studied for two different association policies such as nearest-distance based and maximum-power based associations by considering the antenna gain, determined by the tilt angle in [26]. The handover rate as well as the coverage probability were analyzed by considering the practical antenna configuration [27] and also for the coordinated multi-point (CoMP) transmission [28]. However, the aforementioned works considered limited scenarios and parameters of UAV networks in the design of the antenna tilt angle. For instance, in [22], [27], [28], a simple UAV network, where ground users (GUs) do not exist, was considered in spite of using ground BSs. In [22]- [28], they considered either the down tilt angle or the up tilt angle although both should be considered to support aerial users (AUs) together with GUs. In [23]- [25], [28], a simple constant power gain model was used for the antenna main lobe although it can be changed according to the BS antenna tilt angle as well as the elevation angle of the communications link [29]. Furthermore, only the inclusive-service BS (IS-BS) scheme that makes each BS serves both GUs and AUs was explored as in [22], [23], [25]. However, the exclusive-service BS (ES-BS) scheme that makes BSs serve GUs or AUs exclusively might be a better scheme for certain UAV network environments. Therefore, in existing works, it failed to present or analyze the performance of UAV communications with more realistic tilt angle-based antenna gains as well as existing both GUs and AUs in the networks. Therefore, in this paper, we provide a framework to explore an appropriate BS service provisioning scheme to support both GUs and AUs with considering the tile angle-based antenna gain. First, the network outage probabilities of the ES-BS scheme as well as the IS-BS scheme are analyzed. We then explore how the optimal antenna title angles of BSs that minimize the network outage probability are determined for different service provisioning schemes as well as different types of BSs. The impact of various network parameters such as the spatial densities of total BSs and interfering BSs on the performance of service provisioning schemes are also discussed. The main contributions of this paper are summarized as follows. • We newly derive the network outage probability for two BS service provisioning schemes, i.e., IS-BS and ES-BS schemes, by considering the tilt angle-based antenna gain in both general environment (where the interference exists) and noise-limited environment. • We analytically show that changing the antenna tilt angle gives conflicting impacts on the network outage proba-bility. Specifically, as the absolute value of the tilt angle decreases, the service area with the main lobe becomes wider (i.e., positive impact), but the link distance between the serving BS and the user increases (i.e., negative impact). From these results, we show that there exists the optimal BS antenna tilt angle that minimizes the network outage probability. • We show the impact of various network parameters on the optimal antenna tilt angle that minimizes the network outage probability including the BS height, the UAV height, the ratio of GU, as well as densities of the total BSs and the interfering BSs. For instance, we show that the optimal antenna tilt angle increases as the ratio of GUs increases, and the absolute value of the tilt angle increases, as the total BS density increases. • We also explore which service provisioning scheme can be better in terms of the network outage probability in various environments. Specifically, in the noise-limited environment, we analytically show the superiority of the ES-BS scheme to the IS-BS scheme for high BS density regime. In the general environment, we numerically show the superiority of the IS-BS scheme for low interfering BS density regime and that of the ES-BS scheme for high interfering BS density regime. The rest of this paper is organized as follows. In Section II, we represent the BS service provisioning schemes to serve both types of users and describe the channel model and the BS antenna power gain, which is affected by the BS antenna tilt angle. We then describe the BS association rule. In Section III, we derive the network outage probability for two service provisioning schemes in the general environment and the noise-limited environment, respectively. In Section IV, we evaluate the network outage probability according to the various network design parameters. We then compare the communication performance of the IS-BS scheme and that of the ES-BS scheme for network parameters. Finally, conclusions are presented in V. Notation: The notation used throughout the paper is listed in Table I. II. SYSTEM MODEL In this section, we introduce the non-terrestrial network model by mainly focusing on UAV networks. Moreover, we describe the antenna power gain and the BS association rules. A. Network Model We consider a NTN for UAVs, where BSs, ground users (GUs), and aerial users (AUs) (i.e., UAVs) are randomly distributed in the spatial domain. The locations of users are modeled by homogeneous Poisson point process (HPPP) Φ U,i with density λ i , where i ∈ {G, A} denotes the type of users, i.e., i = G for GUs and i = A for AUs. 1 The height of the kth user is h k , where h k = h G for k ∈ U G and h k = h A for k ∈ U A . Here U G and U A are the user index sets of GUs and AUs, respectively. In this paper, as shown in Figure 1, we consider the two types of BS service provisioning schemes as follows. • Inclusive-service BS (IS-BS) scheme: In this scheme, BSs serve both GUs and AUs simultaneously. Hence, the antenna tilt angle of the BS has to be designed to serve both GUs and AUs efficiently. The locations of BSs are modeled by HPPP Φ B,O with density λ B . Since there is only one type of BS, the BS density for GUs, λ IS B,G , and the BS density for AUs, λ IS B,A , are the same as the total BS density (i.e., λ IS B,G = λ IS B,A = λ B ). Note that this scheme is the one, generally used in prior works such as [22], [23], [25]. BSs are divided into two groups: 1) a BS for GUs (BS for GUs (GBS)) and 2) a BS for AUs (BS for AUs (ABS)). The GBSs and ABSs exclusively serve GUs and AUs, respectively. Therefore, the antenna tilt angles of GBSs and ABSs need to be designed respectively to serve aimed users efficiently. We assume the distributions of GBSs and ABSs also follow HPPPs, Φ B,G and Φ B,A , with densities λ ES B,G = ρ B,G λ B and λ ES B,A = (1 − ρ B,G )λ B , respectively, where ρ B,G is the portion of GBSs among all BSs. Regardless of BS types, for all BSs, the antenna height is h B and the transmission power is P t . B. Channel Model In UAV communications, both LoS and NLoS environments can be considered for the links between a BS and a GU as well as between a BS and an AU. The probability of forming LoS link between the BS at x = (x B , y B , h B ) and the kth user at (x k , y k , h k ) is given by [32] where 2 ) dt is the Q-function and the horizontal distance between the BS and the kth user is given by Here, µ, ν and ξ are environment parameters determined by the density and the height of obstacles. Since the NLoS environment is a complementary event of the LoS environment, the NLoS probability between the BS and the kth user is given by Based on the LoS probability, we consider different path loss exponents and channel fading models for LoS and NLoS links. The path loss exponent for LoS and NLoS links are denoted by α L and α N , respectively. The channel fading is modeled by Nakagami-m fading, so the distribution of the channel gain is given by where Γ(x) = ∞ 0 t x−1 e −t dt and v ∈ {L, N} is the channel environment, i.e., v = L for LoS links and v = N for NLoS links. In addition, we assume that m L > 1, and m N = 1, which means Rayleigh fading, i.e., Ω N ∼ exp(1). From (2), we denote the channel fading between the kth user and the BS as C. Vertical Antenna Gain The antenna power gain of the BS is determined by two types of power gains: horizontal and vertical directional antenna gains. We consider an omnidirectional antenna in the horizontal direction, so the horizontal directional antenna gain remains constant regardless of the direction of the antenna. Therefore, we assume the horizontal directional antenna gain is equal to a unit gain [33]. In this paper, we focus on the design of the vertical antenna tilt angle for AUs as well as GUs, and we consider the directional antenna in the vertical direction. As shown in Fig. 2, the vertical directional antenna gain is determined by the vertical antenna tilt angle, −90 • < θ t < 90 • , which is the angle tilted upward or downward relative to the horizontal plane. 3 Here, we define that the BS antenna tilt angle is a negative value when the BS antenna tilt angle is up-tilted, i.e., tilting upwards with respect to the horizontal plane of the BS antenna. On the other hand, the BS antenna tilt angle is defined as a positive value when the BS antenna tilt angle is down-tilted, i.e., tilting downwards with respect to the horizontal plane of the BS antenna. Based on the 3rd generation partnership project (3GPP) specification [29], for given r k,x , the BS antenna power gain G(r k,x , θ t ) can be represented by a function of the tilt angle as where θ 3dB = 10 • is the 3dB beamwidth and η is the minimum power leaking to the side lobe besides the main lobe, which is commonly 20dB. In (4), θ (r k,x ) is the elevation angle between the BS antenna and the kth user, which is given by where h k −h B is the height difference between the BS and the kth user. In this work, without loss of generality, we assume that the height of AUs is higher than that of BSs (i.e., h A − h B > 0), while the height of GUs is lower than that of BSs (i.e., h G − h B < 0). From (4), for given θ t , the user can be served with the main lobe when 12 Here, we define the boundary of horizontal distance between a BS and the kth user that the user is served by the main lobe as where r lb k (θ t ) and r ub k (θ t ) are the lower and upper boundaries. Since all GUs and all AUs have the same height, h G and h A , respectively, the boundaries are determined by the user types not user's specific location, i.e. r ub k (θ t ) = r ub i (θ t ) and r lb k (θ t ) = r lb i (θ t ) for k ∈ U i , and given as follows where θ th = θ 3dB η/12. In (6)-(9), the boundaries r ub i (θ t ) and r lb i (θ t ) are defined to be positive when θ t satisfies each conditions. For the convenience of analysis, we rewrite G (r k,x , θ t ) in (4) according to the boundaries in (6)-(9) as is the antenna side lobe gain and G 2 (r k,x , θ t ) is the antenna main lobe gain, which is given by From (11), we can see that G 2 (r k,x , θ t ) is an increasing function of θ t for −θ th < θ t ≤ −θ (r k,x ), and G 2 (r k,x , θ t ) is a decreasing function of θ t for −θ (r k,x ) ≤ θ t < θ th . This is because as the antenna tilt angle θ t approaches the elevation angle between the BS and the user, the effect of the main lobe becomes dominant and it is maximized when the antenna tilt angle is equal to the elevation angle (i.e., θ t = −θ (r k,x )). D. BS Association Rule In conventional networks, the BS association is determined by the mean channel fading gain and the distance-dependent path loss, considering the LoS probability [35]. However, in more realistic UAV networks, the antenna gain G (r k,x , θ t ) affected by the horizontal distance between the serving BS and the kth user should also be considered in the BS association. To analyze BS association rules, we first denote BSs which belong to Φ B,l , l ∈ {O,G,A}, forming LoS and NLoS links as Φ L B,l and Φ N B,l , respectively. We then divide each of Φ L B,l and Φ N B,l into three groups according to the BS antenna power gain where j∈J is the index of BS groups which is determined by r k,x , and J={1, 2, 3}. Note that from (10) and (12), we know that BSs in Φ v1 B,l or Φ v3 B,l transmit with the antenna side lobe gain, and BSs in Φ v2 B,l transmit with the antenna main lobe gain. First of all, we examine the distribution of the distance between the user and the BS in Φ vj B,l . The horizontal distance to the nearest BS among the BSs in Φ vj B,l is denoted by X vj k . Here, depending on the LoS probability, the density function of Φ vj B,l is given by 2πλ s B,k tp v (t). Therefore, for BSs in Φ vj B,l , the complementary cumulative distribution function (CCDF) of X vj k can be obtained as where (a) is from the void probability [36] and u k,j (x, θ t ) is given as , otherwise. λ s B,k is the density of BSs that can serve the kth user, i.e., λ s B,k = λ s B,i when k ∈ U i . Here, s ∈ {IS, ES} is the index of the BS service provisioning scheme. By differentiating (13), we can obtain the probability distribution function (PDF) of X vj k as where f s We denote a ∈ {na, sa} as the index of the BS association criterion. Here, a = na and a = sa indicate the nearest BS association rule and the strongest BS association rule, respectively. 1) Nearest BS Association Rule: In the nearest BS association rule (a = na), the horizontal distance between the kth user and the serving BS is smallest. Therefore, the probability that the serving BS exists in Φ vj B,l , and the horizontal distance between the serving BS and the kth user is smaller than r is given by where x τ denotes the location of the serving BS and (a) is from the fact that for given j and v, the horizontal distance between the serving BS and the user is shorter than all other candidates. 2) Strongest BS Association Rule: In the strongest BS association rule (a = s), the main link has the strongest average received power. The probability that the serving BS exists in Φ vj B,l and the horizontal distance between the serving BS and the kth user is smaller than r is given in (16), shown at the top of this page. In (16), (a) is from the fact that for given j and v, the average power of the serving BS should be greater than all other candidates. From (15) and (16), given a ∈ {na, sa}, we can obtain the association probability A a vj as Therefore, when the kth user is associated with a BS in j-group under the channel environment v, the cumulative distribution function (CDF) of the horizontal distance between the BS and the user, r vj k,xτ , is given by By differentiating (18) 6 Note that for the strongest association, f s,sa r vj k,xτ (r) cannot be presented due to the complicated form of (16). However, in Section IV, we show that the performance of the strongest association and that of the nearest association have similar trends. This means we can use the analysis of the nearest association to design the case of the strongest association as well. III. OUTAGE PROBABILITY ANALYSIS In this section, for both IS-BS and ES-BS schemes, we derive the network outage probability in the presence of GUs and AUs. The outage probability is presented for two cases: the general environment in Section III-A and the noise-limited environment in Section III-B as a special case. A. General Environments We assume that the available frequency resource is divided into N sub-bands, and the interfering BSs are the ones that use the same sub-band. Hence, in the IS-BS scheme, the distribution of the interfering BSs is modeled as a HPPP Φ I,O with density λ IS I,O = λ B /N such as in [37]. In the ES-BS scheme, the interference from GBSs and ABSs needs to be defined differently as they use different tilt angles. The distributions of interfering GBSs and ABSs are also modeled as HPPPs, Φ I,G and Φ I,A , with densities λ ES I,G = λ ES B,G /N and λ ES I,A = λ ES B,A /N , respectively. For the case that a BS communicates with the kth user, the SINR at the user can be given by where N}, is the distance-dependent path loss between the kth user and the BS at x for LoS and NLoS links, and σ 2 is the noise power. In (20), I IS = I IS O and I ES = I ES G + I IS A , where I s l is given by Using the SINR in (20), when the user associates to a jgroup BS with the distance r k,xτ and the tilt angle θ t under the channel environment v, the outage probability is given by where γ t = 2 Ro W − 1 is the target SINR. Here, R o is the target data rate and W is the bandwidth allocated to each user [38]. In the following theorem, we derive the network outage probability. For readability, instead of notation θ t , when scheme s is used, we denote antenna tilt angles of the GBS and the ABS as θ s t,G and θ s t,A , respectively. Note that in the IS-BS scheme, since all BSs serve both GUs and AUs, we have a single antenna tilt angle Theorem 1: For IS-BS (s = IS) and ES-BS (s = ES) schemes, the network outage probability can be presented as a function of BS antenna tilt angles θ s t,G , θ s t,A as where P s no,i (θ s t,i ) is the network outage probability of i-type user for s ∈ {IS, ES}, and ρ i = λ i / (λ G + λ A ) is the ratio of the density of i-type users to that of total users, i ∈ {G,A}, and f s,a r vj k,xτ (r) is given in (19). In (23), P v o,j (r, θ s t,i ) is given by . In (24), is the Laplace transform of the interference from l-type BSs, l ∈ {O,G,A}, for the BS service provisioning scheme s, given in (25), shown at the top of next page. In (25), c k,j (r, θ s t,l ) = min b k,j+1 (θ s t,l ), max(r, b k,j (θ s t,l )) . Proof: See Appendix A. From Theorem 1, in the general environment, we can obtain the network outage probabilities for two types of service provisioning schemes, which consider different channel fadings for LoS and NLoS environments. Here, we can see that the network outage probability is affected by the main lobe service area that the BS can serve with the strong main lobe gain, i.e., the area with distance r lb i (θ s t,i )(= b k,2 ) to r ub i (θ s t,i )(= b k,3 ) from a BS (see Fig. 2). The main lobe service area is determined by the antenna tilt angle, and the effect of the antenna tilt angle on is presented in the following corollary. t,G and θ s t,A approach θ th and −θ th , respectively. Proof: From (6) and (7), we obtain the first derivative of for θ th < θ s t,G < π 2 , where ψ(θ) = csc 2 (θ + θ th ) − csc 2 (θ − θ th ). In (26), the inequality is obtained since ψ(θ s t,G ) < 0 and h B − h G > 0. From (8) and (9), the first derivative of r ub A (θ s t,A ) − r lb A (θ s t,A ) with respect to θ s t,A is given by for − π 2 < θ s t,A < −θ th . In (27), the inequality is obtained since ψ(θ s t,A ) > 0 and h A − h B > 0. Therefore, we can see that r ub G (θ s t,G ) − r lb G (θ s t,G ) and r ub A (θ s t,A ) − r lb A (θ s t,A ) are monotonically decreasing function and increasing function of θ s t,G and θ s t,A , respectively. Remark 1: From (6)- (9) and Corollary 1, we can see that , and r ub i (θ s t,i ) increases, as θ s t,G or θ s t,A approaches θ th or −θ th , respectively. This means the main lobe service area becomes wider as θ s t,G or θ s t,A approaches θ th or −θ th , respectively, as also shown in Fig. 2. However, as both r lb i (θ s t,i ) and r ub i (θ s t,i ) increases, the link distance between a BS and a user, located in the main lobe service area, becomes larger, as shown in Fig. 2. Hence, the change of the antenna tilt angle gives conflicting impacts on the network outage probability, so we need to carefully determine the antenna tilt angle to improve the network performance. B. Special Case: Noise-Limited Environments In this subsection, we consider the NTN for UAVs where the noise power is dominant over the interference power, i.e., the noise-limited environment. In the noise-limited environment, for given θ t , the outage probability at the kth user is defined aŝ whereγ s v (r k,x , θ t ) is obtained from (20) by substituting I s = 0. In the following lemma, we derive the network outage probability depending on the ratio of GUs and AUs. Lemma 1: In the noise-limited environment, the network outage probability can be presented by a function of BS antenna tilt angles θ s t,G , θ s t,A aŝ Proof: By substituting I s =0 and applying the CDF of the Gamma distribution in (39), we obtain (30). By replacinĝ P v o,j (r k,xτ , θ s t,i ) in (42) intoP v o,j (r k,xτ , θ s t,i ) and using (38), we obtain (29). Let the optimal values of the BS antenna tilt angle for the IS-BS and ES-BS schemes that minimizeP IS no (θ t,0 ) and P ES no θ ES t,G , θ ES t,A be θ * t,O and θ * t,i , i ∈ {G, A}, respectively. For given the optimal tilt angles, in the following corollary, we compare the network outage probabilities of the IS-BS and ES-BS schemes, i.e.,P IS no θ * t,O andP ES no θ * t,G , θ * t,A . Corollary 2: When the density of BSs approaches to infinity (i.e., λ B → ∞) and the optimal tilt angles are used for each scheme, the network outage probability of the ES-BS scheme is smaller than or equal to that of the IS-BS scheme, i.e., Proof: When λ B approaches to infinity, λ s B,G and λ s B,A also approach to infinity, respectively. Hence, in (19), regardless of the service provisioning scheme, the PDFs of the horizontal distance between the kth user and the serving BS become similar, i.e., f r vj (42), and using the optimal antenna tilt angles, the network outage probabilities of i-type users for the IS-BS scheme and the ES-BS scheme,P IS no,i (θ * t,O ) andP ES no,i (θ * t,i ), can be represented aŝ In (32), we can always obtainP IS no,i θ * t,O ≥P ES no,i θ * t,i , ∀i ∈ {G, A} because θ * t,G and θ * t,A in the ES-BS scheme are optimized ones for GUs and AUs, respectively, while in the IS-BS scheme, θ * t,O is optimized one for both type users to minimize the total network outage probability. Therefore, from (38), we can conclude as (31). From Corollary 2, we can see that when the density of BSs is sufficiently large, the ES-BS scheme outperforms the IS-BS scheme in terms of the network outage probability. Therefore, when the number of BSs is large enough, it is beneficial to exclusively serve GUs and AUs by independently optimizing the BS antenna tilt angles. This is also verified in Section IV, through the simulation results. In noise-limited environments, to obtain the insight of network parameters on the network outage probability, we simplify the antenna gain model in (10) as whereG 1 =G 3 is the constant antenna side lobe gain and G 2 is the constant antenna main lobe gain. We then obtain the network outage probability as in the following corollary. Corollary 3: When p N (r k,x ) = 1 and α N = 4, the network outage probability with the simplified antenna gain model in (33) is given bỹ whereh i = \|h B − h i \|, ω = γtσ 2 Pt , and g j (b) is given by Proof: From (30), by substituting p N (r k,xτ ) = 1, α N = 4, and G(r k,xτ , θ s t,i ) =G(r k,xτ , θ s t,i ),P o,j (r k,xτ , θ s t,i ) is represented bỹ Similar to (42), after averagingP o,j (r k,xτ , θ s t,i ) over r k,xτ , we obtain the network outage probability of i-type user, P no,i (r k,xτ , θ s t,i ) as wheref s r k (r) = 2πλ s B,i r exp −πλ s B,i r 2 andf s r k (r) =f s ri (r) for k ∈ U i . From (37), by using result in [39, eq. (3.322)] and (38), we obtain (34). In Corollary 3, we obtain the network outage probability in the noise-limited environment as a closed form. IV. NUMERICAL RESULTS In this section, we evaluate the effect of the BS antenna tilt angle, the BS density, the interfering BS density, and the network parameters on the network outage probability. We first show the network outage probability on each of the IS-BS and ES-BS schemes. We then compare the performance of the service provisioning schemes. In the numerical results, for the convenience of explanation, we denote the total interfering density as λ I regardless of the scheme, i.e., λ I = λ IS I,O = λ ES I,G + λ ES I,A . Unless otherwise specified, we use the simulation parameters given in Table II based on the 3GPP specification and consider the dense urban environment parameters µ, ν and ξ [40], [41]. Figure 3 presents the network outage probability of AUs for the cases of the fixed heighth A and the uniform distribution height (i.e., h A ∼ u[h A − δ,h A + δ]). As shown in this figure, the trends of network outage probability with the random height are similar to that with the fixed height only. Therefore, from this result, we show that only the performance of the fixed height case in the following figures. Even though there is a gap between the performance of the random height and that of the fixed height, the optimal height that minimizes network outage probability is almost the same. A. Network Outage Probability of Ground and Air Users In this subsection, we analyze the impact of the BS antenna tilt angle on the network outage probabilities of GUs and AUs. Figure 4 presents the network outage probability of i-type user, P s no,i (θ s t,i ), as a function of the BS antenna tilt angle, θ s t,i , for different channel environments and BS association rules. Here, we use λ I = 0.5λ B . From Fig. 4, for GUs (i = G) in the general environment, we can see that as θ s t,i increases, P s no,i (θ s t,i ) first increases up to a certain value of θ s t,i , and then decreases. This is because as θ s t,i increases, the number of interfering BSs that form the antenna main lobe gain to the GU increases i.e., the GU receives larger interference. However, for relatively large θ s t,i (e.g., 0 • < θ s t,i < 15 • ), the desired BS can transmit the signal with the antenna main lobe gain to the GU mostly, while the number of interfering BSs with the antenna main lobe gain to the GU decreases. Therefore, P s no,i (θ s t,i ) decreases with θ s t,i . Furthermore, when θ s t,i is much large (e.g., θ s t,i > 20 • ), as θ s t,i increases, the desired BS transmits the signal with the antenna side lobe gain to the GU with high probability. In this case, the performance loss of the main link is dominant, so P s no,i (θ s t,i ) increases again. For AUs (i = A), the trend becomes opposite, but the reason is the same as the case of GUs. In the noise-limited environment, the main link channel's quality, which is affected by the antenna gain, mainly determines the network performance. Hence, we observe that as θ s t,i increases, P s no,i (θ s t,i ) first decreases and then increases. This is because as θ s t,i increases, the main lobe of serving BS is first closer to the user, and then get further away. We can also see that our analysis is well matched with the simulation results. Furthermore, the network outage probability with the strongest association rule has a similar trend to that with the nearest association rule. The network outage probability of the nearest association is always higher than that of the strongest association. Hence, in the following figures, we present the numerical results of the nearest association only. Figure 5 presents the network outage probability of i-type user, P s no,i (θ s t,i ), as a function of the BS antenna tilt angle, θ s t,i , for different values of the interfering BS density, λ I . From Fig. 5, we can see that as λ I increases, the absolute value of the optimal tilt angle for i-type user, θ * t,i , which is marked by the dashed circle in the figure, increases. This is to ensure that the number of interfering BSs with the antenna main lobe gain to the GU or AU decreases, as the number of interfering BSs increases. Moreover, when λ I is much small (e.g., λ I ≤ 0.01λ B ), we can also observe that the network outage probability in the general environment approaches that in the noise-limited environment. B. Results of IS-BS Scheme In this subsection, we analyze the impact of the BS antenna tilt angle on the network outage probability with the IS-BS scheme. Figure 6 presents the network outage probability of the IS-BS scheme, P IS no (θ IS t,O ), as a function of the BS antenna tilt angle, θ IS t,O , for different values of the BS height, h B , and the AU height, h A . Here, we use λ I = 0.01λ B (i.e., similar to the noise-limited environment). From Fig. 6, we can see that for the fixed height of AUs (e.g., h A = 50 m), as the height of the BS increases (e.g., h B = 20 ∼ 40 m), the optimal value of the BS antenna tilt angle, θ * t,O , which is marked by the dashed circle in the figure, increases. For AUs, as h B increases, the LoS probability between the BS and the AU increases and the distance-dependent path loss decreases. Hence, the performance of AUs can be significantly improved by the high LoS probability and low path loss. On the other hand, for GUs, as h B increases, the LoS probability between the BS and the GU increases, while the distance-dependent path loss increases due to the increased distance from the BS to the GU and it is harmful to the GU. Consequently, as h B increases, since GUs experience relatively worse channel condition compared to AUs, the optimal values of the BS antenna tilt angle increases to downward to compensate the performance loss of GUs. In this figure, we can also observe that for the fixed height of BSs (e.g. h B = 30 m), as the height of the AU increases (e.g. h A = 40 ∼ 60 m), the minimum network outage probability, which is a value of the dashed circle in the yaxis, increases and the optimal value of the BS antenna tilt angle, which is a value of the dashed circle in the x-axis, decreases. As h A increases, the LoS probability between the BS and AU and the distance-dependent path loss increases. However, since the effect of the path-loss increasing is greater, the outage probability of AU increases. On the other hand, the performance of GUs is not affected by h A . Therefore, the optimal antenna tilt angle decreases to be compensated for the performance loss of AUs. Figure 7 presents the network outage probability of the IS-BS scheme, P IS no (θ IS t,O ), as a function of the BS antenna tilt angle, θ IS t,O , for different values of the BS height, h B , and the AU height, h A , similar to Fig. 6. Here, we use λ I = 0.5λ B . From Fig. 7, we can see that the optimal values of the BS antenna tilt angle, θ * t,O , exist in the considerably down tilted regions compared to Fig. 6. As shown in Fig. 5, the difference of the optimal antenna tilt angles for GUs and AUs increases as λ I increases. Consequently, in terms of the network performance of the IS-BS scheme, it is worth optimizing the antenna tilt angle toward a certain type of users, i.e., GUs and AUs. Specifically, for a given configuration, AUs are more affected by interference due to high LoS probability than GUs, hence BSs transmit the signal to AUs with the side lobe to reduce interfering signal power. On the other hand, to increase the main link power, BSs transmit the signal to GUs with the main lobe. Therefore, to minimize network outage probability, the BS antenna needs to be tilted downwards. We can also see that for the fixed height of AUs (e.g., h A = 50 m), as the height of the BS increases (e.g., h B = 20 ∼ 40 m), the optimal value of the BS antenna tilt angle increases. This is to reduce the number of interfering BSs which has the antenna main lobe gain to GUs and ensure that most serving BS transmits the signal with the antenna main lobe gain to GUs. On the contrary, for the fixed height of BSs (e.g., h B = 30 m), there is no change in the value of optimal tilt angle according to h A , because AUs are served by the side lobe. Figure 8 presents the optimal value of the BS antenna tilt angle, θ * t,O , according to the ratio of GUs to total users, ρ G , for different values of the total BS density, λ B , with the IS-BS scheme. Here, we use λ I = 0.1λ B . From Fig. 8, we can see that as ρ G increases, the optimal value of the BS antenna tilt angle, θ * t,O , also increases. Since the interference is not significant in this environment, the main link channel's quality mainly determines the network performance dominantly. Hence, as the portion of GUs increases, the BS needs to tilt its antenna downward. For large λ B (e.g., λ B ≥ 2 × 10 −5 ), we can also observe that the value of the optimal antenna tilt angle is either downwards (e.g., θ * t,O = 18 • ) or upwards (e.g., θ * t,O = −13 • ). Because of the significant difference of the optimal antenna tilt angles for GUs and AUs, it is worth optimizing the antenna tilt angle toward a certain type of users, as also explained in Fig. 7. C. Results of ES-BS Scheme In this subsection, we analyze the impact of the BS antenna tilt angle on the network outage probability with the ES-BS scheme. Note that, in the ES-BS scheme, since the GUs and AUs are exclusively served by the BSs, the antenna tilt angles for GUs, θ ES t,G , and AUs, θ ES t,A , are independently designed to minimize the network outage probability. Furthermore, in the ES-BS scheme, the ratio of GBSs affects the optimal BS tilt angles and hence, we optimize the BS tilt angles in accordance with the ratio of GBSs to total BSs, ρ B,G . Figure 9 shows the optimal ratio of GBSs to total BSs, ρ * B,G , that minimizes the network outage probability, according to the GU ratio to total users, ρ G . We consider different values of the total BS density, λ B , and we use λ I = 0.1λ B . Here, for given ρ G , λ B , and ρ B also optimized to minimize the network outage probability. In Fig. 9, as ρ G increases, ρ * B,G also increases because it is beneficial to have more GBSs when the portion of GUs is large. We can also see that for large ρ G (e.g., ρ G > 0.5), ρ * B,G becomes smaller as λ B increases. This is because as there is more the number of BSs, we can have a sufficient number of GBSs, so we can assign a larger portion of BSs as the ABSs. On the contrary, when ρ G is small (e.g., ρ G < 0.5 ), ρ * B,G becomes larger as λ B increases for a similar reason. Figure 10 presents the optimal value of the BS antenna tilt angles, θ * t,G and θ * t,A , according to the ratio of GUs to total users, ρ G , for different values of the total BS density, λ B , with ES-BS scheme. Here, we use λ I = 0.1λ B . From Fig. 10, we can see that as ρ G increases, the absolute values of θ * t,G and θ * t,A also increase. In the ES-BS scheme, as ρ G increases ρ * B,G also increases, as shown in Fig. 9. Therefore, as the number of BSs increases, to reduce the number of interfering BSs giving the large interference with the antenna main lobe gain, the antenna is tilted more downwards or upwards. For the same reason, we can observe that for given ρ G , as λ B increases, the absolute values of θ * t,G and θ * t,A also increase. D. Comparison between IS-BS Scheme and ES-BS Scheme In this subsection, we compare the performance of the BS service provisioning schemes in terms of the network outage probability according to the ratio of GUs to total users, ρ G . As a baseline scheme, we also plot the service provisioning scheme that the BS antenna is tilted toward GUs without considering AUs as in conventional cellular networks. In this baseline scheme, the BS optimizes the antenna tilt angle to minimize the outage probability of GUs. For the comparison of the IS-BS scheme and the ES-BS scheme, the antenna tilt angle of the IS-BS scheme (θ IS t,O ), that of the ES-BS scheme (θ ES t,G , θ ES t,A ), and the ratio of GBSs (ρ B,G ) are optimized, respectively. Figure 11 presents the network outage probability, P s no (θ s t,G , θ s t,A ), as a function of the ratio of GUs, ρ G , for different values of λ I and service provisioning schemes. From Fig. 11, we can see that when λ I is large (e.g., λ I ≥ 0.05λ B ), the ES-BS scheme outperforms the IS-BS scheme. This is because, for the ES-BS scheme, most of the interference from other types of BSs mostly transmits the signal to user with antenna side lobe gain. On the other hand, for small λ I (e.g., λ I ≤ 0.01λ B ) and the noise-limited environment, the IS-BS scheme performs better than the ES-BS scheme. This is because the effect of the interference is relatively small, so more serving BSs candidates (i.e., λ IS B,i > λ ES B,i ) improve the performance of the main link. Figure 12 presents the network outage probability in noiselimited environments,P s no (θ s t,G , θ s t,A ), as a function of the ratio of GUs ρ G for different values of the total BS density λ B and different service provisioning schemes. From Fig. 12, we can see that when the total BS density is small (e.g., λ B ≤ 10 −5 ), the IS-BS scheme outperforms the ES-BS scheme. On the contrary, for the large total BS density (e.g., λ B ≥ 2 × 10 −5 ), the ES-BS scheme provides better performance than the IS-BS scheme in terms of the network outage probability. From these observations, we can find that when there exist . We can also see that regardless of λ B and ρ G , the service provisioning schemes outperform the baseline scheme because the schemes design the BS antenna tilt angle by considering AUs as well as GUs. In Corollary 2 and Fig. 12, we show that when λ I is very small (i.e., noise-limited environments), the ES-BS scheme outperforms the IS-BS scheme for large λ B , but the IS-BS scheme provides better performance than the ES-BS scheme for small λ B . Therefore, there exist the value of λ B that makes the performance of two service provisioning schemes to be equal such as P IS no (θ IS t,O ) = P ES no (θ ES t,G , θ ES t,A ), and we define this value of λ B as the critical density of BSs, λ c B . That means in the region of λ B < λ c B , the IS-BS scheme is superior to the ES-BS scheme in terms of the network outage probability and vice versa. Figure 13 presents the critical density of the BS, λ c B , as a function of the BS height, h B , for the different values of the AU height, h A . In this figure, we can see that as the distance between the BS and the AU becomes closer, (i.e., h B increases for given h A or the h A decreases for given h B ), λ c B increases. In this case, since the performance of the AUs is good enough due to the relatively short distance, the BS in the IS-BS scheme mainly tilt the antenna for GUs to enhance the network performance. Therefore, the IS-BS scheme can provide better performance than the ES-BS scheme. In contrast, for the case that the BS is far from the AUs, the BS in the IS-BS scheme has to properly tilt the antenna by considering the performance of both GU and AU. Therefore, in this case, the ES-BS scheme can be more efficient as it can be independently optimized the antenna tilt angles for GUs and AUs, respectively. V. CONCLUSION This paper explores an appropriate BS service provisioning scheme to serve both GUs and AUs by considering tilt angle-based antenna gain. We first derive the network outage probability for two types of provisioning schemes, i.e., IS-BS scheme and ES-BS scheme (in Theorem 1). We then explore the conflict impact of the antenna tilt angle on the network outage probability, i.e., as the absolute value of the tilt angle decreases, the main lobe service area becomes wider, but the main link distance increases (in Corollary 1 and Remark 1). From this relation, we numerically show that there exists the optimal BS antenna tilt angle that minimizes the network outage probability. Moreover, we show the impact of the ratio of GUs, the BS height, the UAV height, and densities of the total BSs and the interfering BSs on the optimal tilt angle as well as network outage probabilities for two service provisioning schemes. Finally, for given network parameters, we present which service provisioning scheme is more appropriate. Specifically, in Corollary 3, we show that the ES-BS scheme is better than the IS-BS scheme when BSs are densely deployed. In contrast, the IS-BS scheme performs better than the ES-BS scheme for low BS density or interfering BS density. The outcomes of this work can be useful for the optimal antenna tilt angle design and the BS provisioning service scheme determination in the networks, where both GUs and AUs exist. A. Proof of Theorem 1 For the given ratio of GUs and AUs, ρ G and ρ A , the network outage probability can be presented by =ρ G P s no,G (θ s t,G )+ρ A P s no,A (θ s t,A ), s ∈{IS, ES}, (38) where P s no,i (θ s t,i ) is the network outage probability of i-type users and (a) is from the law of total probability. From (20) and (22), P v o,j (r vj k,xτ , θ s t,i ) can be presented by P v o,j (r vj k,xτ , θ s t,i ) = P   Ω k,xτ < γ t (I s + σ 2 ) P t l v r vj k,xτ G j (r vj k,xτ , θ s t,i ) In (24) 1 + z mL P t l L (r k,x )G j (r k,x , θ s t,l ) mL + p N (r k,x ) 1 + zP t l N (r k,x )G j (r k,x , θ s t,l ) , where x τ is the location of the serving BS, and (a) is from the Laplace transforms of the Gamma distribution and the exponential distribution. From (41), by applying the probability generating functional (PGFL) [42], we obtain (25 where (a) is from the definition of Φ vj B,l in (12). In (42) f s,a r vj k,xτ (r) = f s,a r vj i (r), and b k,j (θ s t,i ) = b i,j (θ s t,i ) as k ∈ U i . By substituting (42) into (38), we obtain (23).	2021-04-15T01:15:51.709Z	2021-04-14T00:00:00.000	{ "year": 2021, "sha1": "9ac6ee052c71cc8af45963de00b16149e47813a7", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "9ac6ee052c71cc8af45963de00b16149e47813a7", "s2fieldsofstudy": [ "Engineering", "Computer Science" ], "extfieldsofstudy": [ "Computer Science", "Engineering" ] }
220646630	pes2o/s2orc	v3-fos-license	Relative Pose from Deep Learned Depth and a Single Affine Correspondence We propose a new approach for combining deep-learned non-metric monocular depth with affine correspondences (ACs) to estimate the relative pose of two calibrated cameras from a single correspondence. Considering the depth information and affine features, two new constraints on the camera pose are derived. The proposed solver is usable within 1-point RANSAC approaches. Thus, the processing time of the robust estimation is linear in the number of correspondences and, therefore, orders of magnitude faster than by using traditional approaches. The proposed 1AC+D solver is tested both on synthetic data and on 110395 publicly available real image pairs where we used an off-the-shelf monocular depth network to provide up-to-scale depth per pixel. The proposed 1AC+D leads to similar accuracy as traditional approaches while being significantly faster. When solving large-scale problems, e.g., pose-graph initialization for Structure-from-Motion (SfM) pipelines, the overhead of obtaining ACs and monocular depth is negligible compared to the speed-up gained in the pairwise geometric verification, i.e., relative pose estimation. This is demonstrated on scenes from the 1DSfM dataset using a state-of-the-art global SfM algorithm. Source code: https://github.com/eivan/one-ac-pose depth predictions together with ACs to estimate the relative camera pose from a single correspondence. Exploiting ACs for geometric model estimation is a nowadays popular approach with a number of solvers proposed in the recent years. For instance, for estimating the epipolar geometry of two views, Bentolila and Francos [10] proposed a method using three correspondences interpreting the problem via conic constraints. Perdoch et al . [38] proposed two techniques for approximating the pose using two and three correspondences by converting each affine feature to three points and applying the standard point-based estimation techniques. Raposo et al . [41] proposed a solution for essential matrix estimation from two correspondences. Baráth et al . [3,6] showed that the relationship of ACs and epipolar geometry is linear and geometrically interpretable. Eichhardt et al . [14] proposed a method that uses two ACs for relative pose estimation based on general central-projective views. Recently, Hajder et al . [19] and Guan et al . [18] proposed minimal solutions for relative pose from a single AC when the camera is mounted on a moving vehicle. Homographies can also be estimated from two ACs as it was first shown by Kser [24]. In the case of known epipolar geometry, a single correspondence [5] is enough to recover the parameters of the homography, or the underlying surface normal [4,24]. Pritts et al . [39] used affine features for the simultaneous estimation of affine image rectification and lens distortion. In this paper, we propose to use affine correspondences together with deeplearned priors derived directly into minimal pose solvers -exploring new ways of utilizing these priors for pose estimation. We use an off-the-shelf deep monocular depth estimator [30] to provide a 'relative' (non-metric) depth for each pixel (examples are in Fig. 1) and use the depth together with affine correspondences for estimating the relative pose from a single correspondence. Aside from the fact that predicted depth values are far from being perfect, they provide a sufficiently strong prior about the underlying scene geometry. This helps in reducing the degrees-of-freedom of the relative pose estimation problem. Consequently, fewer correspondences are needed to determine the pose which is highly favorable in robust estimation, i.e., robustly determining the camera motion when the data is contaminated by outliers and noise. We propose a new minimal solver which estimates the general relative camera pose, i.e., 3D rotation and translation, and the scale of the depth map from a single AC. We show that it is possible to use the predicted imperfect depth signal to robustly estimate the pose parameters. The depth and the AC together constrain the camera geometry so that the relative motion and scale can be determined as the closed-form solution of the implied least-squares optimization problem. The proposed new constraints are derived from general central projection and, therefore, they are valid for arbitrary pairs of central-projective views. It is shown that the proposed solver has significantly lower computational cost, compared to state-of-the-art pose solvers. The imperfections of the depth signal and the AC are alleviated through using the solver with a modern robust estimator [2], providing state-of-the-art accuracy at exceptionally low run-time. The reduced number of data points required for the model estimation leads to linear time complexity when combined with robust estimators, e.g. RANSAC [15]. The resulting 1-point RANSAC has to check only n model hypotheses (where n is the point number) instead of, e.g., n 5 which the five-point solver implies. This improvement in efficiency has a significant positive impact on solving large-scale problems, e.g., on view-graph construction (i.e., a number of 2-view matching and geometric verification) [45,46,51] which operates over thousands of image pairs. We provide an evaluation of the proposed solver both on synthetic and real-world data. It is demonstrated, on 110 395 image pairs from the 1DSFM dataset [56], that the proposed methods are similarly robust to image noise while being up to 2 orders of magnitude faster, when applied within Graph-Cut RANSAC [2], than the traditional five-point algorithm. Also, it is demonstrated that when using the resulting pose-graph in the global SfM pipeline [11,56] as implemented in the Theia library [50], the accuracy of the obtained reconstruction is similar to when the five-point algorithm is used. Constraints from Relative Depths and Affine Frames Let us denote a local affine frame (LAF) as (x, M), where x ∈ R 2 is an image point and M ∈ R 2×2 is a linear transformation describing the local coordinate system of the associated image region. The following expression maps points y from a local coordinate system onto the image plane, in the vicinity of x. Local Affine Frames and Depth through Central Projection Let q : R 2 → R 3 be a function that maps image coordinates to bearing vectors. R ∈ SO(3) and t ∈ R 3 are the relative rotation and translation of the camera coordinate system, such that x ∈ R 2 is the projection of X ∈ R 3 , as follows: That is, a unique depth λ corresponds to X, along the bearing vector q (x). When image coordinates x are locally perturbed, the corresponding X and λ are also expected to change as per Eq. (2). The first order approximation of the projection expresses the connection between local changes of x and X, i.e., it is an approximate linear description how local perturbations would relate the two. Differentiating Eq. (2) along image coordinates k = u, v results in an expression for the first order approximation of projection, as follows: Observe that t is eliminated from the expression above. Note that, considering the k-th image coordinate, ∂ k X is the the Jacobian of the 3D point X, and ∂ k λ is the Jacobian of the depth λ. Using the differential operator ∇ = ∂ u , ∂ v it is more convenient to use a single compact expression, that includes the local affine frame M, All in all, Eqs. (2) and (4) together describe all constraints on the 3D point X and on its vicinity ∇X, imposed by a LAF (x, M). Affine Correspondence and Depth Constraining Camera Pose Assume two views observing X under corresponding LAFs (x 1 , M 1 ) and (x 2 , M 2 ). Without the loss of generality, we define the coordinate system of the first view as the identity -thus putting it in the origin -while that of the second one is described by the relative rotation and translation, R and t, respectively. In this two-view system, Eqs. (2) and (4) lead to two new constraints on the camera pose, depth and local affine frames, as follows: where the projection functions q 1 (x 1 ) and q 2 (x 2 ) assign bearing vectors to image points x 1 and x 2 of the first and second view, respectively. The first equation comes from Eq. (2) through the point correspondence, and the second one from Eq. (4) through the AC, by equating X and ∇X, respectively. Note that depths λ 1 , λ 2 and their Jacobians ∇λ 1 , ∇λ 2 are intrinsic to each view. Also observe that 3D point X and its Jacobian ∇X are now eliminated from these constraints. In the rest of the paper, we are resorting to the more compact notations using a, b, A and B, as highlighted above, i.e., the two lines of Eq. (5) take the simplified forms of a = Rb + t and A = RB, respectively. Relative depth. In this paper, as monocular views are used to provide relative depth predictions, λ 1 and λ 2 are only known up to a common scale Λ, so that Eq. (5), with the simplified notation, is modified as follows: These constraints describe the relationship of relative camera pose and ACs in the case of known relative depth. Relative Pose and Scale from a Single Correspondence Given a single affine correspondence, the optimal estimate for the relative pose and scale is given as the solution of the following optimization problem. To solve the problem, the first order optimality conditions have to be investigated. At optimality, differentiating f (Λ, R, t) by t gives: that is, t can only be determined once the rest of the unknowns, R and Λ, are determined. This also means that above optimization problem can be set free of the translation, by performing the following substitution t ← ΛRb − a. The optimization problem, where the translation is replaced as previously described, only contains the rotation and scale as unknowns, as follows: Below, different approaches for determining the unknown scale and rotation are described, solving Eq. (9) exactly or, in some manner, approximately. 1AC+D (Umeyama): An SVD solution. In the least-squares sense, the optimal rotation and scale satisfying Eq. (9) can be acquired by the singular value decomposition of the covariance matrix of A and B, as follows: Using these matrices, the optimal rotation and scale are expressed as where D is a diagonal matrix with its diagonal being 1, 1, det(UV T ) , to constrain det R = 1. Given R and Λ, the translation is expressed as t = a − ΛRb. This approach was greatly motivated by Umeyama's method [53], where a similar principle is used for solving point cloud alignment. 1AC+D (Proposed): Approximate relative orientation solution. Matrices A and B, together with their 1D left-nullspaces, define two non-orthogonal coordinate systems. By orthogonalizing the respective basis vectors, one can construct matrices R A and R B , corresponding to A and B. The relative rotation between these two coordinate systems can be written as follows: R A and R B can be determined, e.g., using Gram-Schmidt orthonormalization [47]. For our special case, a faster approach is shown in Alg. 1. Using this approach, the solution is biased towards the first columns of A and B, providing perfect alignment of those two axes. In our experiments, this was rather a favorable property than an issue. The first axis of a LAF represents the orientation of the feature while the second one specifies the affine shape, as long as the magnitude of ∇λ i is negligible compared to λ i , which is usually the case. The orientation of a LAF is usually more reliable than its shape. As R is now known and t has been ruled out, Λ is to be determined. Differentiating g (R, Λ) by Λ gives: that is, once R is known, Λ can be determined as follows: Finally, the translation parameters are expressed as t = a − ΛRb. The complete algorithm is shown in Alg. 1. Note that, although, we have tested various approaches to computing the relative rotation between the frames A and B, such as the above described SVD approach or the Gram-Schmidt process [47], the one introduced in Alg. 1 proved to be the fastest with no noticeable deterioration in accuracy. Algorithm 1 The 1AC+D (Proposed) algorithm for relative pose computation. Experimental results In this section, experiments and complexity analyses compare the performance of the two versions of the proposed 1AC+D solver, i.e., 1AC+D (Umeyama) and 1AC+D (Proposed), 2AC [6] and 5PT [48] methods for relative pose estimation. Processing time In Table 1, we compare the computational complexity of state-of-the-art pose solvers used in our evaluation. The first row consists of the major steps of each solver. For instance, 5×9 SVD + 10×20 EIG means that the major steps are: the SVD decomposition of a 5×9 matrix and eigendecomposition of a 10×20 matrix. In the second row, the implied computational complexities are summed. In the third one, the number of correspondences required for the solvers are written. The fourth row lists example outlier ratios. In the fifth one, the theoretical numbers of iterations of RANSAC [20] are written with confidence set to 0.99. The last row reports the total complexity, i.e., the complexity of one iteration multiplied by the number of RANSAC iterations. The proposed methods have significantly smaller computational requirements than the five-point solver, 5PT, or the affine correspondence-based 2AC method [6] when included in RANSAC. Note that while the reported values for the 1AC+D methods are the actual FLOPs, for 5PT and 2AC, it is in practice about an order of magnitude higher due to the iterative manner of various linear algebra operations, e.g., SVD. In Fig. 2, the theoretical number of RANSAC iterations -calculated as in [20] -and the number of floating point operations are plotted as the function of the outlier ratio (horizontal axes), displayed on logarithmic scales (vertical axes). The proposed solvers lead to fewer iterations compared to the solvers solely based on point or affine correspondences. In summary of the above analysis of the computational complexity, the proposed approach speeds up the robust estimation procedure in three ways. First, it requires at least an order of magnitude fewer operations to complete a single iteration of RANSAC than by using the five-point algorithm. Second, it leads to significantly fewer iterations due to the fact that 1AC+D takes only a single correspondence to propose a solution to pose estimation. Third, 1AC+D returns a single solution from a minimal sample, in contrast to the five-point algorithm. Therefore, each RANSAC iteration requires the validation of a single model. Synthetic evaluation The synthetic evaluation was carried out in a setup consisting of two cameras with randomized poses represented by rotation R i ∈ SO (3) and translation t i ∈ R 3 (i = 1, 2). The cameras were placed around the origin at a distance sampled from [1.0, 2.0], oriented towards a random point sampled from [−0.5, 0.5] 3 . Both cameras had a common intrinsic camera matrix K with focal length f = 600 and principal point [300, 300], representing pinhole projection π : R 3 → R 2 . For generating LAFs (x i , M i ), depths λ i and their derivatives ∇λ i , randomized 3D points X ∼ N (0, I 3×3 ) with corresponding normals n ∈ R 3 , n 2 = 1 were projected into the two image planes using Eqs. (15) and (16), for i = 1, 2. where R i \| (3,:) is the 3rd row of R i , ∇X = nulls (n) simulates the local frame of the surface as the nullspace of the surface normal n. Note that [14] proposed the approach for computing local frames M i , i.e., Eq. (15). Their synthetic experiment are also similar in concept, but contrast to them, we also had to account for the depth in Eq. (16), and we used the nullspace of n to express the Jacobian ∇X. The 2nd part of Eq. (16), the expression for the Jacobian of the depth, ∇λ i , was derived from the 1st part by differentiation. In this setup λ i is the projective depth, a key element of perspective projection. Note that the depth and its derivatives are different for various other camera models. Finally, zero-mean Gaussian-noise was added to the coordinates/components of x i , M i , λ i and ∇λ i , with σ, σ M , σ λ and σ ∇λ standard deviation (STD), respectively. The reported errors for rotation and translation both were measured in degrees ( • ) in this evaluation. The rotation error was calculated as follows: whereR is the measured and R is the ground truth rotation matrix. Translation error t is the angular difference between the estimated and ground truth translations. For some of the tests, we also show the avg. Sampson error [20] of the implied essential matrix. Solver stability. We randomly generated 30 000 instances of the above described synthetic setup, except for adding any noise to the LAFs and depths, in order to evaluate the stability of the various solver in a noise-free environment. Figs. 3(a) shifted lower compared to the other methods. However, all methods are quite stable having no peaks on the right side of the figures. Noise study. We added noise to the 30 000 instances of the synthetic setup. The estimated poses were evaluated to determine how sensitive the solvers are to the noise in the data, i.e., image coordinates, depth, and LAFs. As expected, the noise in the depth or parameters of LAFs has no effect on the 5PT [48] solver. This can be seen in Fig. 5(b), where 5PT [48] is only affected by image noise. Increasing noise on either axes has a negative effect on the output of the proposed method as seen on Fig. 5(a). The effect of noise on rotation and translation estimated by 1AC+D and 5PT [48] are visualized on diagrams Fig. 4(a)-(f). The curves show that the effect of image noise -on useful scales, such as 2.5 px -in itself has a less significant effect on the degradation of rotation and translation estimates of the proposed method, compared to 5PT [48]. However, adding realistic scales of depth or affine noise, e.g. Fig. 4(a), has an observable negative effect on the accuracy of the proposed ones. Although 2AC [6] is unaffected by the noise on depth, it is moderately affected by noise on the affinities of LAFs. Its rotation estimates are worse than that of any other methods in the comparison. The provided translation vectors are of acceptable quality. Summarizing the synthetic evaluation, we can state that, on realistic scales of noise, the accuracy of the rotation and translation estimates of 5PT [48] and both versions of 1AC+D are comparable. 2AC is the worst performer among all. Real-world evaluation We tested the proposed solver on the 1DSfM dataset [56] 5 . It consists of 13 scenes of landmarks with photos of varying sizes collected from the internet. Plots (a-f) compare the two proposed 1AC+D solvers, 2AC [6] and 5PT [48]. In the 1st row (a-c), the rotation error is shown. In the 2nd one (d-f) translation errors are plotted. All errors are angular errors in degrees. In each column, different setups are shown, where we fixed two of the three sources of noise -image, affine or depth noise -to analyse the negative effect of the third as its level increases. 1DSfM provides 2-view matches with epipolar geometries and a reference reconstruction from incremental SfM (computed with Bundler [45,46]) for measuring error. We iterated through the provided 2-view matches, detected ACs [32] using the VLFeat library [54], applying the Difference-of-Gaussians algorithm combined with the affine shape adaptation procedure as proposed in [9]. In our experiments, affine shape adaptation only had a small ∼10 % extra time demand, i.e., (0.31 ± 0.25) s per view, over regular feature extraction. This extra overhead is negligible, compared to the more time-consuming feature matching. Matches were filtered by the standard ratio test [32]. We did not consider image pairs in the evaluation with fewer than 20 corresponding features between them. For the evaluation, we chose scenes Piccadilly, NYC Library, Vienna Cathedral, Madrid Metropolis, and Ellis Island. To get monocular depth for each image, we applied the detector of Li et al . [30]. In total, the compared relative pose estimators were tested on a total of 110 395 image pairs. As a robust estimator, we chose the Graph-Cut RANSAC [2] algorithm (GC-RANSAC) since it is state-of-the-art and has publicly available implementation 6 . In GC-RANSAC, and other locally optimized RANSACs, two different solvers are applied: (i) one for fitting to a minimal sample and (ii) one for fitting to a larger-than-minimal sample when improving the model parameters by fitting to all found inliers or in the local optimization step. For (i), the main objective is to solve the problem using as few points as possible since the overall wallclock time is a function of the point number required for the estimation. For (ii), the goal is to estimate an accurate model from the provided set of points. In practice, step (ii) is applied rarely and, therefore, its processing time is not so crucial for achieving efficient robust estimation. We used the normalized eightpoint algorithm followed by a rank-2 projection to estimate the essential matrix from a larger-than-minimal sample. We applied GC-RANSAC with a confidence set to 0.99 and inlier-outlier threshold set to be the 0.05 % of the image diagonal size. For the other parameters, the default values were used. Fig. 6 reports the cumulative distribution functions -being accurate or fast is interpreted as a curve closer to the top-left corner -calculated on all image pairs from each scene which was connected by an edge in the provided pose graph made by incremental SfM. The left plot shows the processing time, in seconds, required for the full robust estimation. The pose estimation is more than an order of magnitude faster when using the 1AC-D solver than the 5PT algorithm. The right two plots show the rotation and translation errors calculated using the reference reconstructions of the 1DSfM dataset [56]. 1AC-D leads to accuracy similar to that of the 5PT algorithm, both in terms of rotation and translation. Note that, in practice, multiple solvers are used for creating the initial pose graph, considering homography, fundamental and essential matrix estimation simultaneously, e.g., by QDEGSAC [16], to improve the accuracy and avoid degenerate configurations. It is nevertheless out of the scope of this paper, to include 1AC+D in state-of-the-art SfM pipelines. However, in the next section we show as a proof-of-concept that the proposed solver can be used within global SfM pipelines and leads to similar accuracy as the widely-used 5PT method [48]. Table 2. The results of a global SfM [50] algorithm, on scenes from the 1DSfM dataset [56], initialized with pose-graphs generated by the 5PT [48] and 1AC+D solvers. The reported properties are: the scene from the 1DSfM dataset [56] (1st column), relative pose solver (2nd), total runtime of the global SfM procedure given an initial pose-graph (3rd), rotation error of the reconstructed global poses in degrees (4th), position error in meters (5th) and focal length errors (6th). Applying global SfM algorithm. Once relative poses are estimated for camera pairs of a given dataset, along with the inlier correspondences, they are fed to the Theia library [50] that performs global SfM [11,56] using its internal implementation. That is, feature extraction, image matching and relative pose estimation were performed by our implementation either using the 1AC+D or the 5PT [48] solvers, as described above. The key steps of global SfM are robust orientation estimation, proposed by Chatterjee et al . [11], followed by robust nonlinear position optimization by Wilson et al . [56]. The estimation of global rotations and positions enables triangulating 3D points, and the reconstruction is finalized by the bundle adjustment of camera parameters and points. Table 2 reports the results of Theia initialized by different solvers. There is no clear winner in terms of accuracy or run-time (of the reconstruction). Both solvers perform similarly when used for initializing global structure-from-motion. Conclusions In this paper, we propose a new approach for combining deep-learned non-metric monocular depth with affine correspondences (ACs) to estimate the relative pose of two calibrated cameras from a single correspondence. To the best of our knowledge, this is the first solution to the general relative camera pose estimation problem, from a single correspondence. Two new general constraints are derived interpreting the relationship of camera pose, affine correspondences and relative depth. Since the proposed solver requires a single correspondence, robust estimation becomes significantly faster, compared to traditional techniques, with speed depending linearly on the number of correspondences. The proposed 1AC+D solver is tested both on synthetic data and on 110 395 publicly available real image pairs from the 1DSfM dataset. It leads to an ac-curacy similar to traditional approaches while being significantly faster. When solving large-scale problems, e.g., pose-graph initialization for Structure-from-Motion (SfM) pipelines, the overhead of obtaining affine correspondences and monocular depth is negligible compared to the speed-up gain in the pairwise geometric verification. As a proof-of-concept, it is demonstrated on scenes from the 1DSfM dataset, via using a state-of-the-art global SfM algorithm, that acquiring the initial pose-graph by the proposed method leads to reconstruction of similar accuracy to the commonly used five-point solver. would only need to perform 112 iterations. That is equivalent to only about 14 896 FLOPs when using 1AC+D, not considering the few additional local optimization steps. Consequently, even though the bad quality of the relative depth, the proposed solver is able to recover the sought pose parameters. For more details on the theoretical number of RANSAC iterations and the performance of our proposed method, the Reader is referred to Fig. 2 and Table 1 of our paper. Summary. This brief evaluation suggests that, given the noisy correspondences obtained using the deep-learned depth priors and extracted affine features, 1AC+D produces a fairly low percentage of true inliers. Note that given higher-quality data, e.g. when using the metric depth from a depth camera, the above numbers are only expected to improve. Even though, the proposed 1-point RANSAC scheme easily handles such a high outlier ratio, resulting in low number of iterations and accurate results.	2020-07-21T01:01:15.518Z	2020-07-20T00:00:00.000	{ "year": 2020, "sha1": "bb4bdf690a6fcb66cdd798e5df5fc6ab14e73991", "oa_license": null, "oa_url": "http://eprints.sztaki.hu/10045/1/Barath_627_31798467_ny.pdf", "oa_status": "GREEN", "pdf_src": "Arxiv", "pdf_hash": "bb4bdf690a6fcb66cdd798e5df5fc6ab14e73991", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Computer Science" ] }
267863647	pes2o/s2orc	v3-fos-license	Escherichia coli and their potential transmission of carbapenem and colistin-resistant genes in camels Background Camels harbouring multidrug-resistant Gram-negative bacteria are capable of transmitting various microorganisms to humans. This study aimed to determine the distribution of anti-microbial resistance among Escherichia coli (E. coli) isolated from the feces of apparently healthy camels in Egyptian abattoirs. Additionally, we sought to characterize Shiga toxin-producing E. coli (STEC) strains, assess their virulence potential, and investigate the possibility of camels spreading carbapenem- and colistin-resistant E. coli. Methods 121 fecal swaps were collected from camels in different abattoirs in Egypt. Isolation and identification of E. coli were performed using conventional culture techniques and biochemical identification. All isolates obtained from the examined samples underwent genotyping through polymerase chain reaction (PCR) of the Shiga toxin-encoding genes (Stx1 and Stx2), the carbapenemase-encoding genes (blaKPC, blaOXA−48, blaNDM, and blaVIM), and the mcr genes for mcr-1 to mcr-5. Result Bacteriological examination revealed 75 E. coli isolates. PCR results revealed that one strain (1.3%) tested positive for Stx1, and five (6.6%) were positive for Stx2. Among the total 75 strains of E. coli, the overall prevalence of carbapenemase-producing E. coli was 27, with 7 carrying blaOXA48, 14 carrying blaNDM, and 6 carrying blaVIM. Notably, no strains were positive for blaKPC but a high prevalence rate of mcr genes were detected. mcr-1, mcr-2, mcr-3, and mcr-4 genes were detected among 3, 2, 21, and 3 strains, respectively. Conclusion The results indicate that camels in Egypt may be a primary source of anti-microbial resistance (AMR) E. coli, which could potentially be transmitted directly to humans or through the food chain. Supplementary Information The online version contains supplementary material available at 10.1186/s12866-024-03215-6. Background Camels play a vital role in the socioeconomic growth of many nations, particularly in Africa and the Middle East [1].They provide essential resources and services that benefit local communities and economies, such as milk and meat production, transportation, and entertainment, particularly in Egypt's tourism sector [2]. The impact of climate change and the rise in drought conditions have shifted livestock preferences in various regions worldwide, leading to a notable increase in the abundance of camels [3]. Camels in sub-Saharan Africa serve as a reservoir for various potentially zoonotic diseases and pathogens [4].Carcass microbiological contamination primarily occurs during processing and handling stages, such as skinning, evisceration, preparation, storage, and distribution at abattoirs and retail shops [5]. Escherichia coli, or E. coli, is a rod-shaped bacterium in the intestinal tracts of humans and warm-blooded animals.While many strains of E. coli are harmless and coexist naturally, certain strains, such as Shiga toxin-E.coli (STEC), can result in foodborne illnesses.Some E. coli strains can also cause infections in the urinary and respiratory systems and other diseases [6]. In contrast, STEC in camels, capable of causing gastrointestinal illnesses such as non-bloody or bloody diarrhoea, haemorrhagic colitis (HC), and haemolytic uremic syndrome (HUS), has been rarely documented.However, STEC is responsible for approximately 2,801,000 cases of acute illnesses each year, posing a substantial global health burden [7]. E. coli produces numerous highly virulent genes, with Shiga toxin-producing E. coli (STEC) being the most significant serotype regarding public health toxins.The virulence factors of STEC are primarily derived from Shiga toxin genes (Stx1 and Stx2), which play a significant role in the manifestation of clinical symptoms [8].Furthermore, Stx1 and Stx2 can have sequence variants, and a single STEC bacterium can produce multiple variants of these toxins [9]. The primary mode of transmission of STEC to humans is through consuming contaminated foods, including raw or undercooked ground meat products and unpasteurized milk.Additionally, cross-contamination during food preparation is another significant mode.Hand-to-mouth transfer involving direct contact with farm animals is also identified as a substantial transmission mode [10]. Antibiotic resistance (AMR) poses a significant threat to human and animal health, making the treatment of bacterial infections increasingly challenging.One of the essential AMR mechanisms in the Enterobacteriaceae family involves the production of extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs), which can inactivate a wide range of antibiotics, including carbapenems, considered last-line therapies [11]. Gram-negative bacteria can resist antibiotics in several ways, such as by producing enzymes that destroy antibiotics, making it harder for antibiotics to enter the cell, or changing the cell membrane's structure to prevent antibiotic penetration.These changes have been seen in many bacteria resistant to multiple drugs [12]. Carbapenems, important in human medicine as broadspectrum beta-lactam antibiotics, are considered the lastline therapies for severe infections.The five most crucial carbapenemase enzymes are KPC, NDM, IMP, VIM, and OXA.These enzymes can break down carbapenems, conferring antibiotic resistance [13]. E. coli can resist many different types of antibiotics; the most common is beta-lactam resistance, including cephalosporins, aminoglycosides, and tetracyclines.E. coli achieves this by producing enzymes called beta-lactamases associated with genes such as bla TEM and bla CTX , which code for beta-lactamases [14]. Colistin, a potent antibiotic used in treating severe infections and often considered a last-resort antibiotic, faces the challenge of resistance, which can spread to other bacteria through mobile genetic elements, rapidly spreading this resistance within bacterial populations.A plasmid known as mcr-1, capable of transmitting colistin resistance to other bacteria [15,16], can also be found on plasmids carrying other antibiotic-resistance genes, including those encoding carbapenemases and extendedspectrum beta-lactamases [17].Many studies have demonstrated that using colistin as an antibiotic growth promoter (AGP) in livestock contributes to the emergence and spread of plasmid genes, conferring resistance to polymyxins, including colistin itself [18]. This study aimed to update our understanding of the prevalence of E. coli bacteria in camels in Egypt, characterize the strains of E. coli causing STEC infections, assess the potential of camels to spread E. coli bacteria resistant to carbapenem and colistin antibiotics, and evaluate the potential risk to human and animal health arising from the transmission of these strains to the environment. Methods Sample preparation: A total of 121 faecal swaps were collected from camels aged 3-5 years in different abattoirs in Cairo and Giza governorates (Al Waraq and Al Basateen abattoirs) during the period from January 2022 to June 2022.Subsequently, the swabs were placed in 2 ml of sterile saline (0.9% NaCl) and stored in an ice box until transported to the laboratory. Bacterial isolation and identification: All samples were inoculated into brain-heart infusion broth tubes and incubated at 37 °C for 24 h.A loopful from the previously incubated tubes was streaked on eosin methylene blue agar (EMB) and incubated aerobically at 37 °C for 24-48 h.Suspected colonies were purified through subculture on EMB agar plates and subjected to traditional biochemical tests, including indole, methyl red, Voges-Proskauer, citrate utilization, and urease tests [19].The isolates were stored at − 20 °C until further molecular analysis. Molecular detection of virulence and antibiotic resistance genes in E. coli: All isolates obtained from the examined samples underwent genotyping using polymerase chain reaction (PCR), according to the protocol described by [20].The template DNA used consisted of boiled lysates prepared from the isolates.In brief, a loopful of culture was suspended in 100 µl of sterile TE buffer, boiled for 10 min at 100 °C, and centrifuged for 5 min at 6000×g.The extracted DNA was stored at -20 °C until use. All genomic DNA of the identified E. coli strains underwent PCR testing for Shiga toxin-encoding genes (Stx1 and Stx2) using multiplex PCR assays.The target genes, oligonucleotide primer sequences, and the expected product size in different PCR assays are outlined in Table 1. To detect the carbapenemase-encoding genes (bla KPC , bla OXA−48 , bla NDM , and bla VIM ), multiplex PCR was performed using specific oligonucleotide primers for detecting bla KPC and bla NDM (Table 2).The PCR mixtures had a total reaction volume of 25 mL.All reaction mixtures were subjected to 30 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min.Subsequently, 5 mL of the PCR product was electrophoresed on a 1% agarose gel to determine the size of the product [21].Uniplex PCR was also conducted using a specific oligonucleotide primer to detect bla VIM (Table 2).The PCR mixtures had a total reaction volume of 25 mL.All reaction mixtures were subjected to 35 cycles at 94 ºC for 30 s, 55 ºC for 30 s, 72ºC for 1 min, and a final elongation at 72 ºC for 10 min.Then, 5 mL of the PCR product was electrophoresed on a 1% agarose gel to determine the size of the product [22]. The plasmid DNA served as the template for PCR.The primer pair used to detect the bla OXA−48 gene consisted The thermal cycling process consisted of initial denaturation at 94 °C for 10 min, denaturation at 94 °C for 40 s, annealing at 60 °C for 40 s, extension at 72 °C for 1 min, and a final extension at 72 °C for 7 min.In total, 30 cycles were run.The amplified products were then subjected to gel electrophoresis [22]. Multiplex PCR was also performed using oligonucleotide primers for mcr-1 to mcr-5 (Table 3).The PCR conditions included denaturation at 94 °C for 5 min, followed by 25 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 90 s, elongation at 72 °C for 60 s, and a final cycle of elongation at 72 °C for 10 min. Isolation and identification of E. coli strains: The bacteriological examination of 121 camels' faecal swaps showed the presence of 75 E. coli isolates (Table 4).Using multiplex PCR for the detection of Shiga toxin-encoding genes (Stx1 and Stx2) (Table 5), one E. coli strain (1.3%) tested positive for Stx1, and five strains (6.6%) were positive for Stx2. Molecular findings of virulence and antibiotic resistance genes in E. coli: In Table 5, the total prevalence of carbapenemaseproducing E. coli was 27 strains: 7 carrying bla OXA−48 , 14 carrying bla NDM , and 6 carrying bla VIM , while no strain carried bla KPC .Additionally, the total prevalence of colistin resistance genes in E. coli isolates was 29 strains, with G C T T G A T C G C C C T C G A T T 283 [50] G A T T T G C T C C G T G G C C G A A A VIM A G T G G T G A G T A T C C G A C A G 261 [51] A T G A A A G T G C G T G G A G A C mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5 being 3, 2, 21, 3, and 0, respectively. Discussion Camels are susceptible to several infectious diseases, meaning eating camel meat or coming into contact with camels can pose a significant risk of zoonotic disease transmission [23].In this study (Table 4), special consideration aligns with Jones et al. [24], who concluded that eating raw camel meat often leads to outbreaks of diarrheagenic E. coli, a type of bacteria that can cause diarrhea.This typically occurs due to rough handling procedures during slaughter and transportation.Additionally, many countries worldwide have reported a high incidence of pathogenic E. coli strains in fresh camel milk [25]. STEC is a type of bacteria that can cause food poisoning.It is a zoonotic pathogen responsible for mild to severe diarrhea, hemorrhagic colitis (bloody diarrhea), and hemolytic uremic syndrome (HUS) [26].The distinguishing feature of STEC is the presence of one or more Shiga toxin (Stx) genes, which code for proteins that can damage the cells in the lining of the intestines.There are two main types of Shiga toxins: Stx1 and Stx2 [27]. Most E. coli bacteria live in the intestines of humans and animals without causing any harm.However, some E. coli bacteria produce toxins, leading to food poisoning.STEC infections are most common in ruminants.These animals can carry STEC bacteria in their intestines without getting sick.However, these bacteria can be spread to people through food or water contaminated with animal feces [28,29].This transmission occurs because the lining of their intestines lacks vascular receptors, preventing the toxins from being absorbed into the bloodstream and transported to other organs.As a result, the STEC bacteria can colonize the large intestine without causing symptoms [30]. The prevalence of Shiga toxin-encoding genes (Stx1 and Stx2) detected in this study was closely consistent with those reported by Erickson and Doyle [31] and Kintz et al. [32].These studies focused on uncovering the source and transmission of STEC infections in the food chain and humans. STEC can be transmitted to humans in several ways, including eating undercooked ground beef or other raw foods, such as lettuce, sprouts, or spinach, from manured gardens, drinking contaminated water or unpasteurized milk or juice, coming into direct contact with animal feces, or being infected with STEC [33]. Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) are bacteria equipped with enzymes that can break down a wide range of beta-lactam antibiotics, including penicillin and cephalosporins.However, they are not resistant to carbapenem antibiotics [34].ESBL-E bacteria pose a serious public health threat as they can be difficult or impossible to treat, leading to Table 3 List of primers used for the mcr 1-5 genes Target Primer sequence (5`→3`) Band size Reference more extended hospital stays, more severe illnesses, and even death [35].Infections caused by ESBL-producing Enterobacteriaceae (ESBL-E) are concerning for various reasons, including increased hospital costs, length of hospital stays, and mortality rates.ESBL-E infections can also be more deadly than other infections, necessitating treatment with last-resort antibiotics like carbapenems [36].Recently, the effectiveness of carbapenems has been disposed of globally by the emergence of carbapenemresistant bacteria.The resistance of Enterobacteriaceae to carbapenems includes numerous mechanisms, the most significant being the production of carbapenemases. In Table 5, the prevalence of carbapenemase-producing E. coli was 27 strains: 7 carried bla OXA , 14 carried bla NDM , and 6 carried bla VIM , while no strain carried bla KPC .This result agrees with Tzouvelekis et al. [37], who concluded that the clinical intake of carbapenems has increased, leading to a rise in the number of pathogen isolates producing carbapenemases.The increased prevalence of bacterial species carrying ESBL genes, as reported worldwide, included community-acquired Escherichia coli isolates with the ability to produce ESBLs [38].Moreover, this result agrees with Cantón et al. [39], who discussed that KPC, OXA, and NDM involve three of the 'big five' carbapenemases associated with nosocomial contagions.The global increase in carbapenemase-producing Enterobacteriaceae (CPE) has led to the overuse of colistin.This overuse has raised concerns about the emergence of colistin resistance (mcr) genes in bacteria [40], which are already resistant to many other antibiotics.Additionally, data on colistin-resistant E. coli and mcr genes in camels are lacking.Given the increasing use of camels for meat and milk production, this is a concern, raising the possibility that camels play a role in transmitting colistinresistant bacteria to humans. the present study showed a surprising occurrence of mcr genes in E. coli isolates where the total prevalence of colistin resistance genes in E. coli isolates was 29 (Table 5) and these findings was much higher than those obtained by Rhouma et al., who found no colistin resistance in E. coli isolated from camel feces in southern Tunisia [41]. Veterinarians working with camels face a significant challenge because there is no approved anti-microbial to treat bacterial infections in these animals.Anti-microbials approved for ruminants, horses, or other animal species to treat sick camels have proven ineffective due to the unique physiology of camels [42]. Evidence shows that camels could be a significant source of mcr gene contamination for Egypt's local population and tourists.This is because camels and tourists often come into close contact, potentially spreading resistant bacteria globally. New plasmid-mediated mcr genes have rapidly emerged in the past four years, compromising the therapeutic effectiveness of colistin, a last-resort antibiotic used to treat multidrug-resistant bacterial infections [44]. The mcr-1 and mcr-2 genes have engrossed worldwide consideration, heralding the polymyxin gap.However, in this study, the mcr-3 gene exhibited a prevalence of 28% compared with the mcr-1, mcr-2, mcr-4, and mcr-5 genes, which had prevalence rates of 4%, 2.6%, 45%, and 0%, respectively.These results agree with Yin et al. [45], who examined a colistin-resistant Escherichia coli isolate.The study yielded negative results for mcr-1 and mcr-2 and discovered a novel mcr-3.They found wide-ranging mcr-3 between Enterobacteriaceae and Aeromonas species initiating from clinical infections and environmental specimens across twelve countries on four continents. E. coli, normal inhabitants of the intestines of humans and mammals, potentially represent a significant reservoir of AMR and play a vital role in gaining and propagating AMR mechanisms.Since colistin is widely used in veterinary medicine and is increasing in use in human medicine, it is crucial to continuously monitor the spread of mcr genes in both the agricultural and healthcare sectors.This can be achieved by tracking the presence of mobile colistin resistance determinants in colistin-resistant Gram-negative bacteria [46]. Conclusion STEC is a significant foodborne zoonotic bacterium, and camels may play a role in transmitting E. coli, which resists many antibiotics to humans.These results recommend the need for careful veterinary practice of beta-lactams in the camel industry.For the first time in Egypt, camels could become a source of the mcr-3 gene.Therefore, the search for the mcr-3 gene should be immediately encompassed in investigating colistin-resistant Gram-negative bacteria from animals, humans, and the environment. Table 1 List of primer pairs used for the stx1 and stx2 genes in this study Table 2 Primer sequences used for PCR amplification of carbapenemase encoding genes Table 4 Occurrence rate of E-coli isolates from apparent healthy camels in Egyptian abattoirs	2024-02-25T14:16:31.894Z	2024-02-24T00:00:00.000	{ "year": 2024, "sha1": "c77afa3a3c785048aeae5cf020f6eb1970d16b78", "oa_license": "CCBY", "oa_url": null, "oa_status": null, "pdf_src": "PubMedCentral", "pdf_hash": "801c6be6fcdbc82f0ed99ff1cfa1a1cc581e100e", "s2fieldsofstudy": [ "Medicine", "Environmental Science" ], "extfieldsofstudy": [ "Medicine" ] }
219451997	pes2o/s2orc	v3-fos-license	Research on the Current Situation and Development of Chaozhou Opera in Thailand Thailand is an important country on the maritime Silk Road. Chaozhou Opera has a long history in Thailand. At present, it plays an important role in the transmission of national spirit, but it also faces new opportunities and challenges. Therefore, this paper analyzes the current situation of Chaozhou Opera in Thailand from the perspective of cultural communication, and puts forward corresponding solutions and innovative paths for the shortcomings in the communication process. Introduction Chaozhou Opera, one of the many variants of Chinese opera, has a time-honored history that is much longer than Beijing Opera and Yue Opera. Chaozhou Opera, also known as Chao opera, is one of the ten major operas in China, which is among the first batch in the National Intangible Cultural Heritage Protection List. It is popular in the Chaoshan, Guangdong province and is an ancient local opera sung in Chaozhou dialect. In 2015, The History of Chaozhou Opera, co-authored by Wu Guoqin and Lin Chunjun, revealed that Chaozhou Opera was the result of long-time exchanges of Nanxi Opera and the local art of Chaozhou [1], confirming that the traditional Chaozhou Opera was the combination of the local culture and vocal tunes of East Guangdong and South Fujian, rather than originating from ancient Chinese music of Guanxitong (an ancient Chaozhou witchcraft of inviting the patron saint of Chinese regional opera)or Zhengzi Opera. From the above, the origin of Chaozhou Opera has been clearly stated. Chaozhou Opera has now become an important local opera in China and is growing mature through innovative practices. Due to the rise of ancient Maritime Silk Road, Chaozhou Opera has become one of the most influential local operas in China. It has gone abroad together with Chaozhou people, serving as an emotional support of many overseas Chinese, and has become one of the most influential Chinese operas overseas. Chaozhou Opera is very popular in Southeast Asia, especially in Thailand, which is known as its second hometown. Chaozhou Opera was spread to Thailand hundreds of years ago with Chaoshanese people, serving as a cultural bond connecting Chaoshan and other countries in the world. The Spreading History of Chaozhou Opera in Thailand As early as the middle of the 17th century, Chaozhou opera was introduced into Thailand with immigrants. Before the 1920s, Thailand's Chaozhou Opera was basically a traditional Chinese opera. From the 1920s to the 1940s, Chaozhou Opera set off a climax in Thailand. [2] Great pressure also came from Thai policies. In 1947, Chinese schools at all levels were required to be registered by Thailand's Ministry of Education in accordance with the Regulations of Private Schools, so new Chinese schools and existing Chinese middle schools were not allowed. In 1948, a strict repression of Chinese education was initiated, and 53 people from the Thai Chinese Education Association and Nanyang Middle School were arrested by the military government on June 15th of the same year (commonly known as the Anti-Chinese Repression). Consequently, more Chinese schools were closed and there were only 230 Chinese schools (4-year lower primary schools) in the whole Thailand in 1951 [3]. A series of restrictions on Chinese education by Thai government resulted in the decreasing number of Chaozhou-dialect speakers in the younger generation of Thai Chinese and local people, as well as those fond of Chaozhou Opera. As a consequence, the development of Chaozhou Opera fell into a serious depression in Thailand. Later in the 1950s and 1960s, performances of Chinese Chaozhou Opera troupes were almost canceled in Thailand due to political reasons of the two countries. On July 1st, 1975, since Thailand and China established formal diplomatic relations, the two countries have developed an increasingly mature relationship, laying a solid foundation for enhancing economic and trade exchanges, cultural interconnectivity and mutual understanding between the two peoples. The Significance of Chaozhou Opera in Thailand With the development of global economy and political multipolarization, the world is moving towards cultural diversity. Cultural exchange has gradually become an important issue for all countries. The initiative of "One Belt, One Road" is the best way to create a cooperative, peaceful and harmonious external cooperation environment for China at present. The initiative of "One Belt, One Road" clearly pointed out that we should strengthen exchanges and cooperation in regional culture on the "maritime Silk Road", enhance exchanges, strengthen cooperation in cultural fields and enhance centripetal force of cooperation. The current economic development of China has exerted significant influence on the economy of ASEAN countries and the world at large. Moreover, China is a major trade partner of Thailand and plays an important role in Thailand's tourism development. The harmonious relations between China and Thailand promote the development of domestic undertakings of the two countries. Chaozhou Opera has a profound cultural heritage and unique artistry. Through the communication and exchange of Chaozhou Opera in Thailand, Thai people can deeply understand the core values of Chinese traditional culture, which is conducive to further promoting the international recognition of Chaozhou Opera culture and enhancing the soft power and international status of Chinese culture. Since cultural communication is the key for people's communication, Chaozhou Opera should follow the trend of times and play an active role in promoting mutual communication and trust between the people of China and Thailand. In addition, the spread of Chaozhou Opera in Thailand is conducive to the recognition and closeness of Thai overseas Chinese and other overseas people to China, further enhancing national self-esteem and self-confidence, and actually playing a role of cultural communication messenger. Lack of Diversification of Communication Methods Most of Chaoshanese people adhere to the folk customs. Even if they move to Thailand, they will still keep their native customs. On the important days, they will continue to worship their ancestors and gods in the temple, and at the important worship activities, they will hire a drama team to perform to show their respect. Such traditional performance methods have been unable to meet the needs of the development of reality. Communication effect usually depends on the content of communication, and the effect of communication content through different communication methods is not the same. Due to various reasons, the current communication mode of Chaozhou Opera in Thailand is still lack of diversity. The relevant cultural institutions of China and Thailand should carry out various forms of exchange, such as Chaozhou Opera lectures, course teaching, drama performance, etc. On the one hand, they can improve the artistic quality of local Chaozhou Opera workers in Thailand, on the other hand, they can exchange academic information of Chaozhou Opera culture with each other, so as to promote the development of Chaozhou Opera in Thailand. Limited Audience and Shrinking Market The innovation and development of science and technology have had a tremendous impact on society and people's lifestyle. With the rapid development of mobile network, the world has ushered in a new era of we-media, and the way people consume and entertain has also changed. Chaozhou Opera, one of the treasures of traditional Chinese culture, has also been greatly impacted. Its audience at home and abroad is decreasing day by day, and young audience is even fewer. In this sense, Chaozhou Opera is almost in an embarrassing situation. Since the spread of Chaozhou Opera can be seen as a process of symbolic communication, its spreading effect is closely related to the media and the acceptance mode of audience. In Thailand, with the passing of older generation of Chaozhou audience, the third and fourth generation of Chaozhou immigrants have a strange sense of Chaozhou Opera Culture. In addition, the spread of Chaozhou Opera in Thailand is also faced with some serious practical difficulties, such as language barriers, lack of successors of Chaozhou Opera actors, and the market is gradually shrinking. Poor Feedback Channel and Unsatisfactory Effect The five basic elements of communication---subject, contents, audience, channel and effect---are interacted in the communication process. Feedback and evaluation are important links to test the effect of communication. At present, the construction of communication evaluation system matching with the communication of Chaozhou Opera Culture in Thailand is still in a state of serious lag or even absence. It is an important problem to establish a comprehensive evaluation feedback mechanism including audience, professional actors, copyright brokers, overseas troupes, media, etc. Innovate the Content and Form of Chaozhou Opera From the perspective of cultural communication, in order to impress the Thai audience or readers, works of Chaozhou Opera should not only express the major concerns of human beings, but also show the unique aesthetic taste of the nation. Therefore, it is necessary to intensify efforts to cultivate Chaozhou Opera writers in Thailand. Based on telling "China's stories" well, these writers, who are familiar with the rules of creating Chaozhou Opera and Thai history and culture, have adapted and created Chaozhou Opera works according to Thai folk stories, historical allusions, classical films and TV works that conform to the common values of the two peoples. These cultural works of Chaozhou Opera in the new era are both innovative and well-established. They integrate the classics in ancient and modern times both at home and abroad, and will attract more and more Thai audiences. Integrate Resources and Promote Communication in an All-Round Way To further promote Chaozhou Opera in Thailand, it is not enough to rely on government sectors, publishing houses or opera troupes of China. Instead, it is necessary to connect all relevant parties at home and abroad, and establish a multi-dimensional and diversified international spreading channel. It is thought to be an effective way to promote Chaozhou Opera through the Confucius Institutes and Confucius Classrooms in Thailand. The Confucius Institute is a non-profit educational institution advocated by China, and also an important platform to deepen the understanding of Chinese culture and strengthen cultural cooperation and exchanges among people in the world. By 2019, there were 16 Confucius Institutes and 11 Confucius Classrooms in Thailand. The number of Confucius Institutes in Thailand ranked first among Asian countries [4]. The in-depth cooperation between the relevant parties and Confucius Institutes and Confucius Classrooms in Thailand will effectively promote the publication and spread of Chaozhou Opera. At the same time, the domestic "brand marketing" of Chaozhou Opera should also be paid attention in the same time. Chaozhou Opera can be effectively promoted through classical culture appreciation clubs and traditional cultural forums to Thai students, Thai embassies and consulates in China, as well as Thai scholars and journalists in China, so that they can spontaneously help to spread Chaozhou Opera and build a bridge of cultural communication between China and Thailand. Strengthen International Cooperation and Exchange to Achieve Win-Win Cooperation China and Thailand should attach importance to and strengthen the cultural exchange of Chaozhou Opera, absorb nutrition from many sides, so as to enhance its vitality. Through official and non-governmental organizations, we will strengthen the close exchanges and interaction between Chaozhou Opera performance groups, professional colleges and full-time teachers in the two countries. Thailand's Chaozhou Opera troupe should pay attention to introducing professional forces from China as the main creators, and also employ professional Chaozhou Opera actors and creators to engage in local teaching and research through a long-term and short-term combination in teaching. The cultural exchanges between China and Thailand not only promote the spread of Chaozhou Opera in Thailand, but also make the development of Thai opera in the international vision system. Conclusion Chaozhou Opera is a famous local opera art in China, which has an important influence not only in China, but also at abroad especially in Southeast Asia. The inheritance and development of Chaozhou Opera in Thailand has made outstanding contributions to its development. China and Thailand should further strengthen their exchanges of Chaozhou Opera art, publicizing and protecting traditional Chaozhou Opera, as well as learning from other arts and getting innovative. Therefore, Chaozhou Opera will make new contributions to promoting friendly exchanges between the two peoples.	2020-05-28T09:15:48.999Z	2020-05-18T00:00:00.000	{ "year": 2020, "sha1": "488989a22737e1dba6f58f1009daa3beb5ba6a1d", "oa_license": "CCBY", "oa_url": "http://www.clausiuspress.com/assets/default/article/2020/05/18/article_1589832817.pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "057b520436258bb5e2749e8e5003d433672547d9", "s2fieldsofstudy": [ "Business" ], "extfieldsofstudy": [ "Political Science" ] }
203689371	pes2o/s2orc	v3-fos-license	An Embedded Platform for Testbed Implementation of Multi-Agent System in Building Energy Management System : This paper presents a hardware testbed for testing the building energy management system (BEMS) based-on the multi agent system (MAS). The objective of BEMS is to maximize user comfort while minimizing the energy extracted from the grid. The proposed system implements a multi-objective optimization technique using a genetic algorithm (GA) and the fuzzy logic controller (FLC) to control the room temperature and illumination setpoints. The agents are implemented on the low cost embedded systems equipped with the WiFi communication for communicating between the agents. The photovoltaic (PV)-battery system, the air conditioning system, the lighting system, and the electrical loads are modeled and simulated on the embedded hardware. The popular communication protocols such as Message Queuing Telemetry Transport (MQTT) and Modbus TCP / IP are adopted for integrating the proposed MAS with the existing infrastructures and devices. The experimental results show that the sampling time of the proposed system is 16.50 s. Therefore it is suitable for implementing the BEMS in a real-time where the data are updated in an hourly or minutely basis. Further, the proposed optimization technique shows better results in optimizing the comfort index and the energy extracted from the grid compared to the existing methods. Introduction An energy management system is one of the popular and challenging topics in the electrical power system.In recent modern Smart Grid technology, the research topics in the energy management system field have increased significantly, especially in the areas of home energy management systems (HEMSs) and building energy management systems (BEMSs).The authors in [1] propose a method to reduce the energy consumption by switching on/off the air conditioning (AC) and adjusting the temperature setpoint.The objectives are to reduce the electricity consumption of the AC unit in a way that the users do not feel any changes in temperature comfort.In the experiments, they change the temperature setpoint of the AC during a certain time interval and observe whether the comfort changes are felt or not by the users.The temperature setpoints are changed remotely using a centralized server. The fuzzy logic controller (FLC) is employed in [2] to adjust the temperature setpoint of the AC units for energy management in residential buildings.The temperature setpoint is adjusted by a fuzzy inference system that considers four parameter inputs, i.e., (a) initial temperature setpoint; (b) outdoor temperature; (c) home occupancy; (d) electricity price.The system consists of two optimization units for handling the hot temperature setpoint and the cold temperature setpoint.An energy management system to control the AC unit and the electrical loads using control logic is proposed in [3].The control logic covers six functions i.e., (a) comfort, (b) economy, (c) emergency, (d) energy, (e) power, (f) thermal storage.The comfort function is used to ensure that the AC units and the electrical loads can be supplied with maximum comfort.The economy function optimizes the configuration of the AC unit and the electrical loads to minimize the cost.The emergency function is used during grid failures and allows the priority loads to be supplied by a battery system.The energy function is used to allocate energy consumption at a predefined time.The power function is used to ensure that the active power consumption does not exceed a fixed threshold.The thermal storage function is used to change the temperature setpoint of AC unit when the generated PV energy is greater than the consumption. The authors in [4] employed a fuzzy system in their BEMS as the control strategy and prediction tool.In the control strategy, the FLC is used to control the solar thermal air system and the window-related use such as controlling the indoor temperature and light.The fuzzy prediction system is used to predict the energy demand and solar energy.The prediction system improves the energy efficiency due to the ability to predict the behavior of building in advance.A system to predict the energy demand of the building using the Artificial Neural Network (ANN) is proposed in [5].The ANN model is trained using the dataset of monthly historical energy consumption of the building. Due to the distributed components (sensors, actuators, generators, loads) of the HEMS/BEMS, an intelligent multi-agent system (MAS) is widely adopted [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23].The MAS is a distributed control system where each agent works autonomously and coordinates with each other to achieve the global goal.The implementation of MAS in the HEMS is proposed in [6][7][8].The MAS in [6] consists of the permanent agent, the temporary agent and the coordinator agent.The permanent agent is used to control the permanent loads, i.e., the appliances which run in a whole-time such as the refrigerator the air conditioner, the water heater.The temporary agent controls the temporary loads, which are divided into two categories: (a) the must run loads such as the lighting, the television, the cooking appliances, etc.; (b) the shiftable loads such as the washing machine, the dishwasher, etc.The coordinator agent is used for the message coordination, controlling and the decision making among the agents.The fuzzy logic controller (FLC) is employed by each agent to manage energy consumption. The agents in [7] are grouped into three main agents, i.e., management agents, electrical supply system agents, and home appliance agents.The management agents consist of a supply side management (SSM) agent, which manages the electrical flow from the generator system, a demand side management (DSM) agent, which manages the electrical flow to the appliances, and the HEMS agent, which manages both SSM and DSM agents.The electrical supply system agents consist of a solar panel and storage system agent, main grid agent, and electric vehicle agent.The electric vehicle agent controls the charging/discharging of the battery of electric vehicle.Under normal conditions, the battery of electric vehicles will be charged.However under power shortage conditions, the battery may be discharged to supply the energy.The home appliance agents consist of the standing fan agent, rice cooker agent, air condition agent, television agent, etc. A different MAS architecture of the HEMS is proposed in [8], where the agents are divided into four categories: (a) control and monitoring agents (CMAs), which are used to control and monitor the actuators and sensors; (b) information agents (IAs), which is used to handle the data related to the home devices; (c) application agents (AAs), which are used for prediction, scheduling and feedback functions; (d) management and optimization agents (MOAs), which are used for the optimization tasks.To manage the energy, the HEMS adopts four optimization strategies consisting of the comfort for user satisfaction, the reduction costs, the green energy efficiency, and the smart demand response. The agents of the MAS employed in the BEMS [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] can be classified into four main agents, i.e., load agents, generator agents, central agents, and other agents as given in Table 1.The load agent handles the electrical loads in the buildings.The generator agent controls the generation system which supplies the electrical energy to the building.The central agent controls or coordinates the load and the generator agents.The MASs in [9,10,20] do not have a control agent, thus in [9], the load agent and the storage agent coordinate with the generator agent directly.Similarly, the heating agent and the cooling agent are connected to the electricity agent directly [10], while in [20], each local agent such as the local temperature agent is controlled by a load agent, then the load agents were connected to an intelligent coordinator. Reference Load Agent Generator Agent Central Agent Other Agent [9] Load agent Generator agent -Storage agent [10] Heating agent, Cooling agent Electricity agent -- The energy management system proposed by [26] allows the users (Smarthome or Smartbuilding) to exchange the local jointly renewable energy resources.This approach is based on the decentralized algorithm to optimize the energy from the renewable resource, i.e., to be exchanged with the neighbors, and to optimize the energy of the distribution network, i.e., to be delivered to the network or extracted from the network.The similar approach is proposed by [27], in which renewable energy is shared among the users.The users may lend/borrow the renewable energy to/from the neighbors. As discussed previously, the BEMS, especially MAS-based BEMSs, are still rarely implemented on a hardware platform (more specifically an embedded platform), especially when AI techniques are adopted for solving the optimization problems.In this paper, we propose a hardware testbed implementation of the MAS-based BEMS.The novelty of our proposed system is the implementation of a GA-based optimization technique on an embedded platform to optimize the energy cost and user comfort in the building using only a few optimized parameters.Our proposed hardware testbed is focused on the electronics and communication parts for the real-time implementation of the algorithm.Our proposed MAS consists of the central control agent which is implemented on a Raspberry Pi module [37], the generator agent and the load agents which are implemented on Wemos modules [38].The main contributions of our hardware testbed system are fivefold: (a) It implements the genetic algorithm (GA) technique on the embedded hardware for real-time optimization of the BEMS; (b) It emulates the generator system and the loads on the embedded hardware; (c) It adjusts the room temperature and illumination setpoints according to the optimized power; (d) It implements the popular industrial communication protocol, i.e., Modbus protocol [39] for interfacing between the agents and the devices; (e) It implements the state of the art communication protocol in the Internet of Things (IoT) technology, i.e., the Message Queuing Telemetry Transport (MQTT) protocol [40] for communicating between the agents. To the best of our knowledge, there are no prior works related to the first and second contributions or they are very rare.Furthermore, our proposed optimization technique, which is used to minimize the energy cost while maximizing the user comfort, utilizes a few parameters for calculating the objective function.Instead of using both the energy cost and the comfort parameters in the objective function explicitly [18][19][20][21][22][23]28,29], our method uses the energy cost parameter only, since the comfort parameters could be represented in the term of energy cost parameter as described in the following.The power consumption in each room, which is calculated by the load agent, reflects the user comfort (the thermal comfort and the illumination comfort) of the room, in the sense that high power consumption represents high comfort, and low power consumption represents low comfort.Then the total power consumptions of all rooms are considered as the energy cost that should be minimized.The advantage of using this approach is that only the power consumption data should be sent to the central control agent for the optimization calculation.The comfort data are handled by each load agent. Related to the third contribution, compared to [2] where the method to adjust temperature setpoint is used to minimize the energy cost only, our method considers both the energy cost and user comfort.While the adoption of Modbus protocol provides a wide range implementation without replacing the existing sensor and actuator devices. The selection of the MQTT protocol rather than the Lightweight Machine to Machine (LWM2M) protocol [41] is discussed as follows.Both the MQTT and the LWM2M protocols are lightweight protocols, which are suitable for the IoT applications.While the LWM2M protocol has a well-defined data and device management model, the communication data of the MQTT protocol must be developed from scratch.However, in the case of our testbed, we may have more flexibility to define the structure of communication data to fulfill the requirement of our proposed BEMS, when the MQTT protocol is employed. The rest of the paper is organized as follows: Section 2 describes our proposed system.Section 3 presents the experimental results and discussion.Conclusions are covered in Section 4. System Overview The architecture of the proposed system is depicted in Figure 1.The proposed BEMS adopts the MAS to manage the energy and user comfort in a building, more specifically a university building.The testbed consists of the central control agent, the generator agent, and the load agents.In this work, the load agents consist of three load agents which are located in the classroom, the laboratory room, and the office room.Each load agent controls three kinds of loads, namely the lighting, the air conditioning (AC), and the electrical load such as the computer and the printer.The generator agent controls the utility grid, the photovoltaic (PV) and the battery systems.Meanwhile, the central control agent is used to control all the agents. As described in [8], the MAS-based BEMS can participate in a demand response program, i.e., the user can change the power consumption to response the changes in the electrical price or the incentive tariff introduced by the utility grid, due to the availability of the smart metering and intelligent control system of the BEMS.As illustrated in Figure 1, the load agent, the generator agent, and the central control agent provide the functionality of smart metering and intelligent control.Further, the MAS-based BEMS could be extended to interact with the larger Smart Grid system [13].In the case of our proposed system, the integration with the Smart Grid could be done easily by extended the functionality of the central control agent to exchange information with the Smart Grid system such as the environment and the weather data, the generator, the storage, the loads, and the electrical networks. In a more complex system such as the smart city [42,43], there are five energy-related activities, i.e., the generation, the storage, the infrastructure, the facilities and the transport [42].In this context, our proposed BEMS takes part in the facility activity that consumes the energy.The energy management of smart city as proposed in [43] employs the hierarchical decision control where each subsystem may have different control decision scheme.Due to the decentralized scheme of our proposed BEMS, it is suitable to be adopted in such hierarchical decision control architecture. System Overview The architecture of the proposed system is depicted in Figure 1.The proposed BEMS adopts the MAS to manage the energy and user comfort in a building, more specifically a university building.The testbed consists of the central control agent, the generator agent, and the load agents.In this work, the load agents consist of three load agents which are located in the classroom, the laboratory room, and the office room.Each load agent controls three kinds of loads, namely the lighting, the air conditioning (AC), and the electrical load such as the computer and the printer.The generator agent controls the utility grid, the photovoltaic (PV) and the battery systems.Meanwhile, the central control agent is used to control all the agents. As described in [8], the MAS-based BEMS can participate in a demand response program, i.e., the user can change the power consumption to response the changes in the electrical price or the incentive tariff introduced by the utility grid, due to the availability of the smart metering and intelligent control system of the BEMS.As illustrated in Figure 1, the load agent, the generator agent, and the central control agent provide the functionality of smart metering and intelligent control.Further, the MAS-based BEMS could be extended to interact with the larger Smart Grid system [13]. In the case of our proposed system, the integration with the Smart Grid could be done easily by extended the functionality of the central control agent to exchange information with the Smart Grid system such as the environment and the weather data, the generator, the storage, the loads, and the electrical networks. In a more complex system such as the smart city [42,43], there are five energy-related activities, i.e., the generation, the storage, the infrastructure, the facilities and the transport [42].In this context, our proposed BEMS takes part in the facility activity that consumes the energy.The energy management of smart city as proposed in [43] employs the hierarchical decision control where each subsystem may have different control decision scheme.Due to the decentralized scheme of our proposed BEMS, it is suitable to be adopted in such hierarchical decision control architecture.Since our proposed system divides a whole system into agents, it provides the flexibility to add the new agents to fit the requirements, for instance, to be extended to multi-commodity smart energy systems [44], where the hybrid energy systems (the heat and the electricity) are controlled.It Since our proposed system divides a whole system into agents, it provides the flexibility to add the new agents to fit the requirements, for instance, to be extended to multi-commodity smart energy systems [44], where the hybrid energy systems (the heat and the electricity) are controlled.It is also possible to extend the agents to form the group of interconnected users/buildings with the shared Energies 2019, 12, 3655 7 of 29 generator agents as proposed in [45].It is worthy to note that several MAS-based BEMS as illustrated in Figure 1 could be interconnected and coordinated in the energy district system as proposed in [46], where the several central control agents appoint a coalition coordinator to manage the energy in the district. As shown in Figure 1, agents are implemented on the embedded systems in which the load agents and the generator agent are implemented on Wemos modules, while the central control agent is implemented on a Raspberry Pi module.A lighting simulator, AC simulator, and electrical load simulator are used to simulate the lighting, the AC, and the electrical load in the classroom, respectively.To provide a flexible implementation, a popular Modbus TCP/IP protocol is employed to communicate between the load agent and the devices.Fortunately, the Wemos is equipped with a built-in WiFi module, thus the Modbus TCP/IP protocol could be implemented easily via WiFi communication.A PV-battery simulator is developed to simulate the PV-battery system.Similar to the classroom, the simulator is implemented on Wemos modules and communicates with the generator agent using the Modbus TCP/IP protocol.The simulators in the laboratory room and the office room are developed in a rather different way.Instead of implementing the simulators on the separate modules, they are implemented in the same Wemos module with the load agent. The objective of our proposed BEMS is to minimize the energy extracted from the grid while maximizing the user comforts.It deals with the multi-objective optimization problem.Instead of using the scheduling techniques [47,48], that manages the operation time of the controllable loads, our method controls the amount of power required by the loads.Thus it offers better control, in the sense that rather than switching-on/off the loads in a specific time interval (one-hour [47,48]), our method can adjust the power consumption of the loads in real-time, i.e., in one-minute intervals. In this work, we propose a GA technique to solve the optimization problem and implement it on the control central agent.To provide a real-time implementation of the BEMS, the control central agent is implemented on a Raspberry Pi module.The Raspberry Pi communicates with the other agents (Wemos) via WiFi communication.Further, the MQTT protocol, a lightweight IoT protocol, is employed as the communication protocol.Compared to the method in [49] that combining the SCADA system and the Matlab software for implementing the optimization technique, our proposed method offers a simple and flexible approach due to the embedded hardware implementation.Thanks to the Raspberry Pi module that provides a small and powerful embedded computer.Further, our proposed testbed implements the Modbus protocol which is commonly used in the SCADA system. Multi Agent Systen To provide an easy explanation, the variables used in the proposed MAS-based BEMS are listed in Table 3. Central Control Agent The central control agent is the main agent that is responsible to manage the energy in the building by solving the multi-objective optimization problem as described in the following.The objective is to minimize power consumption, while maximizing the user comforts (temperature and lighting).Our multi-objective optimization problem is formulated using four objective functions and four constraints.The objective functions are expressed below Energies 2019, 12, 3655 9 of 29 Maximize (p 2 ) (3) Equation ( 1) represents the objective for minimizing the total power consumed by the classroom, the office room and the laboratory room, while Equations ( 2)-( 4) represents the objective for maximizing the user comfort in each room.It is noted here that instead of using the comfort index directly, we use the power consumption to represent user comfort.Therefore maximizing the user comfort could be defined by maximizing the power consumption of each room (Equations ( 2)-( 4)).This approach could be realized due to the fact that when the temperature and the illumination comforts to be increased, the power consumption will increase.The lower user comfort requires lower power consumption.This approach will reduce the number of parameters to be optimized.The objective functions in Equations ( 1)-( 4) only require three variables, i.e., p 1 , p 2 , p 3 that should be provided in the central control agent.Thus it offers an efficient data exchange between the agents. The constraints are formulated using the inequalities as expressed by Equations ( 5)-( 8).The constraints in Equations ( 5)-( 7) are the lower and upper bounds of the power consumptions which are used to ensure that the optimized parameters p 1 , p 2 , p 3 fall in the allowable range of the user comfort.While the constraint in Equation ( 8) ensures that the power consumption of the building could be supplied by the generator systems (the renewable energy resources and/or the utility grid). To solve the above multi-objectives optimization problem, we adopt the Multi-Objective Genetic Algorithm (MOGA), more specifically the NSGA-II as proposed by [50].The MOGA is implemented on the central control agent to find the optimal power required by the building based on the current power consumptions and the generated power as illustrated in Figure 2. As shown in the figure, the inputs of the central control agent are the minimum and maximum power consumptions of the classroom, the office room, and the laboratory room, the power produced by the battery and the PV.Then these values are used by the MOGA to solve the multi-objective optimization as expressed in Equations ( 1)- (8).The outputs of the central control agent are the optimal values of power required by the classroom, the office room, and the laboratory room which are sent to the respective agents. The power required by the classroom, the office room, and the laboratory room are the optimal values that should be consumed by the respective loads.Then the respective load agent control its loads to satisfy the power requirement using the strategy as described in the following section. Energies 2019, 12, x FOR PEER REVIEW 9 of 29 directly, we use the power consumption to represent user comfort.Therefore maximizing the user comfort could be defined by maximizing the power consumption of each room (Equations ( 2)-( 4)).This approach could be realized due to the fact that when the temperature and the illumination comforts to be increased, the power consumption will increase.The lower user comfort requires lower power consumption.This approach will reduce the number of parameters to be optimized.The objective functions in Equations ( 1)-( 4) only require three variables, i.e., p1, p2, p3 that should be provided in the central control agent.Thus it offers an efficient data exchange between the agents.The constraints are formulated using the inequalities as expressed by Equations ( 5)-( 8).The constraints in Equations ( 5)-( 7) are the lower and upper bounds of the power consumptions which are used to ensure that the optimized parameters p1, p2, p3 fall in the allowable range of the user comfort.While the constraint in Equation ( 8) ensures that the power consumption of the building could be supplied by the generator systems (the renewable energy resources and/or the utility grid). To solve the above multi-objectives optimization problem, we adopt the Multi-Objective Genetic Algorithm (MOGA), more specifically the NSGA-II as proposed by [50].The MOGA is implemented on the central control agent to find the optimal power required by the building based on the current power consumptions and the generated power as illustrated in Figure 2. As shown in the figure, the inputs of the central control agent are the minimum and maximum power consumptions of the classroom, the office room, and the laboratory room, the power produced by the battery and the PV.Then these values are used by the MOGA to solve the multi-objective optimization as expressed in Equations ( 1)- (8).The outputs of the central control agent are the optimal values of power required by the classroom, the office room, and the laboratory room which are sent to the respective agents. The power required by the classroom, the office room, and the laboratory room are the optimal values that should be consumed by the respective loads.Then the respective load agent control its loads to satisfy the power requirement using the strategy as described in the following section. Load Agent The load agents in the classroom, the office room and the laboratory room are similar, in the sense of the control function.However in our testbed they have the different device protocols, where the load agent in the classroom communicates with the load devices using the Modbus TCP/IP Load Agent The load agents in the classroom, the office room and the laboratory room are similar, in the sense of the control function.However in our testbed they have the different device protocols, where the load agent in the classroom communicates with the load devices using the Modbus TCP/IP protocol via the WiFi communication, while the data exchange between the load agents and the devices in the office room and the laboratory room are performed directly in the Wemos module. Figure 3 illustrates the input and output data of the load agent.As shown in the figure, the inputs are the required power, the outdoor temperature, and the outdoor illumination.These data are used by the fuzzy logic controllers (FLCs) to determine the optimal room temperature and illumination setpoints.There are two FLCs, one for controlling the room temperature setpoint (called as FLC-T), and another one for controlling the room illumination setpoint (called as FLC-I).The inputs of FLC-T are the required power and the outdoor temperature, while the output is the room temperature setpoint.The membership functions of the required power, the outdoor temperature, and the room temperature setpoint are shown in Figure 4, where each variable has three linguistic values, i.e., Low (LOW), Medium (MED), and High (HIGH).The values of the required power (658 W-1137 W), the outdoor temperature ( 26 The fuzzy rules of FLC-T are given in Table 4, where the rules are determined by considering the objectives as follows.It is worthy to note that the AC system discussed here is applicable for the hot season where the AC system is controlled to decrease the outdoor temperature (i.e., the cooling The fuzzy rules of FLC-T are given in Table 4, where the rules are determined by considering the objectives as follows.It is worthy to note that the AC system discussed here is applicable for the hot season where the AC system is controlled to decrease the outdoor temperature (i.e., the cooling system) to the desired comfortable room temperature.Therefore the consumed power of the AC system will increase when the room temperature setpoint is decreased and vice versa.The fuzzy rules of FLC-T are given in Table 4, where the rules are determined by considering the objectives as follows.It is worthy to note that the AC system discussed here is applicable for the hot season where the AC system is controlled to decrease the outdoor temperature (i.e., the cooling system) to the desired comfortable room temperature.Therefore the consumed power of the AC system will increase when the room temperature setpoint is decreased and vice versa. FLC -When the required power is low (LOW), then it is better to set the room temperature setpoint follows to the level of the outdoor temperature to minimize the consumed power of the AC. Thus the fuzzy rules given in Table 4 show that when the required power is LOW, the room temperature setpoint is set to LOW when the outdoor temperature is LOW, it is set to MED when the outdoor temperature is MED, and it is set to HIGH when the outdoor temperature is HIGH.-When the required power is high (HIGH), it allows the AC to consume high power.Thus the room temperature setpoint could be set to LOW for all outdoor temperature conditions.-When the required power is medium (MED), it is better to set the room temperature setpoint to the medium (MED) regardless of the outdoor temperature conditions.The inputs of FLC-I is the required power and the outdoor illumination, while the output is the room illumination setpoint.The membership functions of the required power, the outdoor illumination, and the room illumination setpoint are shown in Figure 5, where each variable has three linguistic values, i.e., Low (LOW), Medium (MED), and High (HIGH).The values of the required power (658 W-1137 W), the outdoor illumination (1000 lx-10,000 lx), and the room illumination setpoint (300 lx-500 lx) are normalized to 0-1. The fuzzy rules of FLC-I are given in Table 5, where the rules are determined by considering the objectives as follows.It is noted that the property of the comfort value of the illumination is the opposite from the one of the temperature, in the sense that the temperature comfort increases when the room temperature setpoint is decreased, while the illumination comfort increases when the room illumination setpoint is increased.Therefore the fuzzy rules of the FLC-I are defined below: -When the required power is low (LOW), then it is better to set the room illumination setpoint follows to the level of the outdoor illumination to minimize the consumed power of the lighting. Thus the fuzzy rules given in Table 5 show that when the required power is LOW, the room illumination setpoint is set to LOW when the outdoor illumination is LOW, it is set to MED when the outdoor illumination is MED, and it is set to HIGH when the outdoor illumination is HIGH.-When the required power is high (HIGH), it allows the lighting to consume high power.It means that the room illumination setpoint could be set to the HIGH for all outdoor temperature conditions.-When the required power is medium (MED), it is better to set the room illumination setpoint to the medium (MED) regardless of the outdoor illumination condition. Required Power The inputs of FLC-I is the required power and the outdoor illumination, while the output is the room illumination setpoint.The membership functions of the required power, the outdoor illumination, and the room illumination setpoint are shown in Figure 5, where each variable has three linguistic values, i.e., Low (LOW), Medium (MED), and High (HIGH).The values of the required power (658 W-1137 W), the outdoor illumination (1000 lx-10,000 lx), and the room illumination setpoint (300 lx-500 lx) are normalized to 0-1.The fuzzy rules of FLC-I are given in Table 5, where the rules are determined by considering the objectives as follows.It is noted that the property of the comfort value of the illumination is the opposite from the one of the temperature, in the sense that the temperature comfort increases when the room temperature setpoint is decreased, while the illumination comfort increases when the room Generator Agent The generator agent is used to manage the battery charging/discharging according to the state of charge (SOC) of the battery as illustrated in Figure 6.Besides controlling the battery, the generator agent receives the generated power from the PV-battery system and sends the data to the central control agent as shown in Figure 1.The battery charging/discharging is controlled using the simple strategy as expressed by Equations ( 9)- (11).Using these rules, the battery could be effectively operated to supply the load (discharging) and/or saving the PV power based-on the SOC of the battery.Equation ( 9) is used to prevent the over-discharge, while Equation ( 11) is used to prevent the over-charge.Equation ( 10 AC Simulator The model of an air conditioning (AC) system is a modified version of the model in [51].Since the model in [51] is the heater system, we modify it to become the cooling system to fulfill with our proposed system.The important contribution of our work is that the model is simulated in the embedded system (Wemos module) for real-time implementation, especially for the electronic/communication aspects.The AC simulator consists of two models: the AC (cooling system) and the thermodynamic model of a room.The AC system is expressed by Equation (12), while the thermodynamic model of a room is expressed by Equation (13).The electrical power consumed by the room is simplified by a linear function of the difference between the outdoor temperature and the room temperature as expressed by Equation ( 14): The block diagram of AC simulator is illustrated in Figure 7, where it has two inputs, i.e., the outdoor temperature which is predefined data stored in the Wemos module, and the room temperature setpoint which is received from the load agent (see Figure 1).The AC simulator sends the power consumption, the room temperature and the outdoor temperature to the load agent. AC Simulator The model of an air conditioning (AC) system is a modified version of the model in [51].Since the model in [51] is the heater system, we modify it to become the cooling system to fulfill with our proposed system.The important contribution of our work is that the model is simulated in the embedded system (Wemos module) for real-time implementation, especially for the electronic/communication aspects.The AC simulator consists of two models: the AC (cooling system) and the thermodynamic model of a room.The AC system is expressed by Equation (12), while the thermodynamic model of a room is expressed by Equation (13).The electrical power consumed by the room is simplified by a linear function of the difference between the outdoor temperature and the room temperature as expressed by Equation ( 14): The block diagram of AC simulator is illustrated in Figure 7, where it has two inputs, i.e., the outdoor temperature which is predefined data stored in the Wemos module, and the room temperature setpoint which is received from the load agent (see Figure 1).The AC simulator sends the power consumption, the room temperature and the outdoor temperature to the load agent. AC Simulator The model of an air conditioning (AC) system is a modified version of the model in [51].Since the model in [51] is the heater system, we modify it to become the cooling system to fulfill with our proposed system.The important contribution of our work is that the model is simulated in the embedded system (Wemos module) for real-time implementation, especially for the electronic/communication aspects.The AC simulator consists of two models: the AC (cooling system) and the thermodynamic model of a room.The AC system is expressed by Equation ( 12), while the thermodynamic model of a room is expressed by Equation (13).The electrical power consumed by the room is simplified by a linear function of the difference between the outdoor temperature and the room temperature as expressed by Equation ( 14): The block diagram of AC simulator is illustrated in Figure 7, where it has two inputs, i.e., the outdoor temperature which is predefined data stored in the Wemos module, and the room temperature setpoint which is received from the load agent (see Figure 1).The AC simulator sends the power consumption, the room temperature and the outdoor temperature to the load agent.To provide real-time implementation, we adopt the Modbus TCP/IP protocol for interfacing between the AC simulator and the load agent, where the load agent acts as the master device and the AC simulator is the slave device.The Modbus data such as the register address and the command type are listed in Table 6.To show that the adopted devices are available commercially, the name of the manufacturer is given in the table.As shown in the table, the Modbus data for the AC power To provide real-time implementation, we adopt the Modbus TCP/IP protocol for interfacing between the AC simulator and the load agent, where the load agent acts as the master device and the AC simulator is the slave device.The Modbus data such as the register address and the command type are listed in Table 6.To show that the adopted devices are available commercially, the name of the manufacturer is given in the table.As shown in the table, the Modbus data for the AC power consumption is provided by the power meter [52], while Modbus data for the room temperature, the temperature setpoint and the outdoor temperature are provided by the Modbus thermostat device [53]. Lighting Simulator The power consumption of the lighting in the room is calculated by dividing the luminous flux to the luminous efficacy as given in Equation ( 15): In the experiment, the LED lamp is used, where the luminous efficacy is 100 lm/W, while the daylight factor (DF) of the room is 2%, which means that the room illumination is 2% of the outdoor illumination. The block diagram of lighting simulator is illustrated in Figure 8.It has two inputs: the outdoor illumination which is a predefined data stored in the Wemos module, and the room illumination setpoint which is received from the load agent.The lighting simulator sends the data of the power consumption, the room and the outdoor illumination to the load agent. Lighting Simulator The power consumption of the lighting in the room is calculated by dividing the luminous flux to the luminous efficacy as given in Equation ( 15): In the experiment, the LED lamp is used, where the luminous efficacy is 100 lm/W, while the daylight factor (DF) of the room is 2%, which means that the room illumination is 2% of the outdoor illumination. The block diagram of lighting simulator is illustrated in Figure 8.It has two inputs: the outdoor illumination which is a predefined data stored in the Wemos module, and the room illumination setpoint which is received from the load agent.The lighting simulator sends the data of the power consumption, the room illumination and the outdoor illumination to the load agent.The Modbus data of the simulator is given in Table 7. Similar to the AC simulator, the Modbus data for the lighting power consumption is provided by the power meter [52].The Modbus data for the room illumination is provided by the daylight sensor [54].The LED dimmer [55] provides the Modbus data for the room illumination setpoint.The outdoor light sensor [56] provides the Modbus data for outdoor illumination.The Modbus data of the simulator is given in Table 7. Similar to the AC simulator, the Modbus data for the lighting power consumption is provided by the power meter [52].The Modbus data for the room illumination is provided by the daylight sensor [54].The LED dimmer [55] provides the Modbus data for the room illumination setpoint.The outdoor light sensor [56] provides the Modbus data for outdoor illumination. Electrical Load Simulator The block diagram of electrical load simulator is in Figure 9.The electrical load is modeled with the load profile stored in the Wemos module.The simulator sends the data of power consumption to the load agent using the Modbus data as given in Table 8, where it only contains the power consumption data provided by the power meter [52]. Electrical Load Simulator The block diagram of electrical load simulator is illustrated in Figure 9.The electrical load is modeled with the load profile stored in the Wemos module.The simulator sends the data of power consumption to the load agent using the Modbus data as given in Table 8, where it only contains the power consumption data provided by the power meter [52]. PV-Battery Simulator The relationship between current and voltage (I-V) of the PV is expressed by Equations ( 16)-( 18) [57], where NS = 4, NP = 65 and VPV = 48 V.The battery is modeled by its SOC as expressed by Equation ( 19) [58]: ) )) 432 /( 1 ( SOC( ) SOC( 1) The block diagram of the PV-battery simulator is illustrated in Figure 10.The PV-battery simulator receives the battery charging/discharging control signal from the generator agent.The predefined data of the solar irradiation is stored in the Wemos module which used to produce the PV-Battery Simulator The relationship between current and voltage (I-V) of the PV is expressed by Equations ( 16)-( 18) [57], where N S = 4, N P = 65 and V PV = 48 V.The battery is modeled by its SOC as expressed by Equation ( 19) [58]: i d = N P e −9 (e ((v PV /(36N S )+0.05i out /N P )/26e −3 )−1) ) ( 17) Energies 2019, 12, 3655 16 of 29 The block diagram of the PV-battery simulator is illustrated in Figure 10.The PV-battery simulator receives the battery charging/discharging control signal from the generator agent.The predefined data of the solar irradiation is stored in the Wemos module which used to produce the PV power as defined by Equations ( 16)- (18).Then the PV power, the battery power, and the battery SOC are sent to the generator agent using the Modbus data as given in Table 9.The Modbus data of the PV power is provided by the Gridtie inverter device [59].The battery power, the battery SOC and the battery charging/discharging control are provided by the battery charger controller [60]. Communication Protocol As described in the previous section, the MQTT protocol is adopted as the communication protocol between the central control agent and the other agents (the load agents and the generator agent).MQTT is lightweight messaging protocol that runs on the Transmission Control Protocol/Internet Protocol (TCP/IP).It works based on the publish-subscribe mechanism, where the MQTT broker is required to establish a connection between the publisher and the subscriber. The configuration of MQTT protocol is illustrated in Figure 11.In the system, the MQTT broker and the central control agent are installed on same Raspberry Pi module.Thus the central control agent communicates with the broker via a localhost connection.As shown in the figure, each agent publishes and subscribes the specific topics as described in the following. In Figure 11, an arrow with "Publish-C2CC" means that the load agent in the classroom publishes the "C2CC" topics, while an arrow with "Subscribe-CC2C" means the load agent subscribes the "CC2C" topics.The list of the topics is given in Table 10.As shown in the table, all topics that are published by the load agents and the generator agent are subscribed by the central control agent, thus all data sent by the load agents and the generator agent will be received by the central control agent.Meanwhile, the topics that are subscribed by load agents and the generator agent are published by the central control agent, thus the data that is required by the load agents and the generator agent will be provided by the central control agent. Communication Protocol As described in the previous section, the MQTT protocol is adopted as the communication protocol between the central control agent and the other agents (the load agents and the generator agent).MQTT is lightweight messaging protocol that runs on the Transmission Control Protocol/Internet Protocol (TCP/IP).It works based on the publish-subscribe mechanism, where the MQTT broker is required to establish a connection between the publisher and the subscriber. The configuration of MQTT protocol is illustrated in Figure 11.In the system, the MQTT broker and the central control agent are installed on same Raspberry Pi module.Thus the central control agent communicates with the broker via a localhost connection.As shown in the figure, each agent publishes and subscribes the specific topics as described in the following. In Figure 11, an arrow with "Publish-C2CC" means that the load agent in the classroom publishes the "C2CC" topics, while an arrow with "Subscribe-CC2C" means the load agent subscribes the "CC2C" topics.The list of the topics is given in Table 10.As shown in the table, all topics that are published by the load agents and the generator agent are subscribed by the central control agent, thus all data sent by the load agents and the generator agent will be received by the central control agent.Meanwhile, the topics that are subscribed by load agents and the generator agent are published by the central illumination setpoint, except at the time during the previous periods.Further, the power consumption shows the proper behavior, i.e., it increases when the difference between the illumination setpoint and the natural illumination is increased.temperature, and the power consumption during the simulation into the Wemos memory.The recorded data is illustrated in Figure 12.The time sampling of the simulator is one second, and the hourly data is simulated in one-minute interval.It is noted here that from 0 to 59 seconds and from 300 to 359 seconds, the classroom is not occupied and the AC is switched off.Therefore during these time intervals, the power consumptions are zero and the room temperature is the same as the outdoor temperature.As shown in the figure, the AC simulator works properly, in the sense that the room temperature follows the temperature setpoint, and the power consumption changes proportionally to the difference between the outdoor temperature and the temperature setpoint.The power consumption increases when the difference between the outdoor temperature and the temperature is increased.The profile of illumination and power consumption of the lighting simulator is illustrated in Figure 13.Similarly to the AC simulator, the lighting system is switched-off during 0 to 59 seconds and 300 to 359 seconds.In the figure, the natural illumination is the room illumination which is caused by the outdoor lighting (sunshine).As shown in the figure, the room illumination is able to follow the illumination setpoint, except at the time during the previous periods.Further, the power consumption shows the proper behavior, i.e., it increases when the difference between the illumination setpoint and the natural illumination is increased.To assess the sensitivity of the parameters of our proposed FLC, we conduct the sensitivity analysis as described in the following.Two sensitivity analysis methods, i.e., the elementary effect (EE) method [61], and the Fourier Amplitude Sensitivity Testing (FAST) method [62] are employed.The EE method is used to find the influential parameters of the model, while the FAST method is used to find the sensitivity indices of the parameters.The EE and FAST methods are calculated using the SAFE, a Matlab Toolbox for global sensitivity analysis provided by [63]. The mean and standard deviation of EEs of the FLC inputs of the FLC-T and the FLC-I are illustrated in Figure 14a,b respectively.In Figure 14a, the red dot and the blue dot represent the normalized required power and the normalized outdoor temperature respectively.As shown in the figure, the mean and the standard deviation of the required power are higher than the outdoor temperature.Thus it could be concluded that the required power input is more influential than the outdoor temperature input.This result could be understood from the observation of the fuzzy rules of FLC-T shown in Table 4, where the changes of linguistic values (LOW, MED, HIGH) of the outdoor temperature do not change the output when the linguistic values of required power is MED or HIGH.The changes of the outdoor temperature affect the output, only when the value of required power is LOW.Therefore it is clearly shown that the influential effect of the required power is higher than the outdoor illumination.In Figure 14b, the red dot and the blue dot represent the normalized required power and the normalized outdoor illumination respectively.As shown in the figure, the mean and the To assess the sensitivity of the parameters of our proposed FLC, we conduct the sensitivity analysis as described in the following.Two sensitivity analysis methods, i.e., the elementary effect (EE) method [61], and the Fourier Amplitude Sensitivity Testing (FAST) method [62] are employed.The EE method is used to find the influential parameters of the model, while the FAST method is used to find the sensitivity indices of the parameters.The EE and FAST methods are calculated using the SAFE, a Matlab Toolbox for global sensitivity analysis provided by [63]. The mean and standard deviation of EEs of the FLC inputs of the FLC-T and the FLC-I are illustrated in Figure 14a,b respectively.In Figure 14a, the red dot and the blue dot represent the normalized required power and the normalized outdoor temperature respectively.As shown in the figure, the mean and the standard deviation of the required power are higher than the outdoor temperature.Thus it could be concluded that the required power input is more influential than the outdoor temperature input.This result could be understood from the observation of the fuzzy rules of FLC-T shown in Table 4, where the changes of linguistic values (LOW, MED, HIGH) of the outdoor temperature do not change the output when the linguistic values of required power is MED or HIGH.The changes of the outdoor temperature affect the output, only when the value of required power is LOW.Therefore it is clearly shown that the influential effect of the required power is higher than the outdoor illumination.In Figure 14b, the red dot and the blue dot represent the normalized required power and the normalized outdoor illumination respectively.As shown in the figure, the mean and the standard deviation of the required power are higher than the illumination.Thus the required power input is more influential than the outdoor illumination input.Similar to the FLC-T discussed previously, the result could understood from the examination of fuzzy rules of FLC-I shown in Table 5. illustrated in Figure 14a,b respectively.In Figure 14a, the red dot and the blue dot represent the normalized required power and the normalized outdoor temperature respectively.As shown in the figure, the mean and the standard deviation of the required power are higher than the outdoor temperature.Thus it could be concluded that the required power input is more influential than the outdoor temperature input.This result could be understood from the observation of the fuzzy rules of FLC-T shown in Table 4, where the changes of linguistic values (LOW, MED, HIGH) of the outdoor temperature do not change the output when the linguistic values of required power is MED or HIGH.The changes of the outdoor temperature affect the output, only when the value of required power is LOW.Therefore it is clearly shown that the influential effect of the required power is higher than the outdoor illumination.In Figure 14b, the red dot and the blue dot represent the normalized required power and the normalized outdoor illumination respectively.As shown in the figure, the mean and the standard deviation of the required power are higher than the illumination.Thus the required power input is more influential than the outdoor illumination input.Similar to the FLC-T discussed previously, the result could understood from the examination of fuzzy rules of FLC-I shown in Table 5.The sensitivity indices calculated using the FAST method are shown in Figure 15, where Figure 15a shows the sensitivity indices of the FLC-T inputs, i.e., the normalized required power and the normalized outdoor temperature, while Figure 15b shows the sensitivity indices of the FLC-I inputs, i.e., the normalized required power and the normalized outdoor illumination.The sensitivity indices calculated using the FAST method are shown in Figure 15, where Figure 15a shows the sensitivity indices of the FLC-T inputs, i.e., the normalized required power and the normalized outdoor temperature, while Figure 15b shows the sensitivity indices of the FLC-I inputs, i.e., the normalized required power and the normalized outdoor illumination.Both figures show that the sensitivity index of the required power input is higher than the outdoor temperature input and the outdoor illumination input.These results conform to the previous EE analysis.The sensitivity analysis results show that it is reasonable to select the required power, the outdoor temperature, and the outdoor illumination as the inputs of the proposed FLCs, even though the last two inputs have a less influential effect compared to the required power input. Performance of Real-Time Implementation Since the objective of the proposed testbed is for testing the real-time implementation of the building energy management system, we conduct several experiments to verify our method in the term of the real-time implementation such as the execution time of the algorithm, the sampling time of the agents, and the efficiency of the optimization technique. The execution times of FLCs which are implemented on the Wemos modules are given in Table 11.As shown in the table, the execution time of FLC is about 10 to 12 ms.Thus to execute both FLC-T Both figures show that the sensitivity index of the required power input is higher than the outdoor temperature input and the outdoor illumination input.These results conform to the previous EE analysis.The sensitivity analysis results show that it is reasonable to select the required power, the outdoor temperature, and the outdoor illumination as the inputs of the proposed FLCs, even though the last two inputs have a less influential effect compared to the required power input. Performance of Real-Time Implementation Since the objective of the proposed testbed is for testing the real-time implementation of the building energy management system, we conduct several experiments to verify our method in the term of the real-time implementation such as the execution time of the algorithm, the sampling time of the agents, and the efficiency of the optimization technique. The execution times of FLCs which are implemented on the Wemos modules are given in Table 11.As shown in the table, the execution time of FLC is about 10 to 12 ms.Thus to execute both FLC-T and FLC-I on a Wemos module, it requires about 22 ms.The results verify that the proposed embedded agent using Wemos is suitable for a real-time implementation of the FLC algorithm.Since the sampling time of an agent is determined by the execution time of the algorithm and the transmission time of the communication protocol employed, we examine the sampling time of each agent as given in Table 11.Since the data communication between the load agents in the office room and the laboratory room with the simulators are performed directly, the sampling times of these load agents are quite fast, i.e., about 1.25 ms.Meanwhile, the sampling times of the agents with the Modbus protocol, i.e., the load agent in the classroom and the generator agent are 6.59 s and 2.63 s respectively.These results indicate that the sampling time is very affected by the transmission time of the Modbus protocol.Further, since the number of the Modbus slave devices in the classroom is higher than the generator agent, the sampling time is longer due to the fact that the master must poll the slaves. The sampling times of the load agent in the classroom and the load agent in the office room are illustrated in Figures 16 and 17, respectively.In the figures, the profiles of room temperature setpoints against time are shown, where the updated data are indicated with the marks on the graphs.Figure 16 shows that the room temperature setpoints in the classroom are updated in an average time of 6 s, while Figure 17 shows that the room temperature setpoints in the office room are updated in an average time of 1 s.The main contribution of our proposed system in the real-time implementation of the optimization technique using GA is shown in Table 11, where the execution time of GA on the Raspberry Pi module is 14.25 s (the number of population is 100 and the number of iteration is 30).While the sampling time of the central control agent is 16.60 s.The result implies that the communication task performed by the MQTT protocol only contributes a small portion of the time.Most of the time is consumed by the GA.It also suggests that the lightweight protocols such as the MQTT and the LWM2M protocols are the effective communication protocols between the agents in the MAS.Relying on the sampling time of the central control agent which is lower than one minute, we may conclude that our proposed testbed is suitable for implementing the BEMS, where the updating process is done in hourly basis even every minute. The main contribution of our proposed system in the real-time implementation of the optimization technique using GA is shown in Table 11, where the execution time of GA on the Raspberry Pi module is 14.25 s (the number of population is 100 and the number of iteration is 30).While the sampling time of the central control agent is 16.60 s.The result implies that the communication task performed by the MQTT protocol only contributes a small portion of the time.Most of the time is consumed by the GA.It also suggests that the lightweight protocols such as the MQTT and the LWM2M protocols are the effective communication protocols between the agents in the MAS.Relying on the sampling time of the central control agent which is lower than one minute, we may conclude that our proposed testbed is suitable for implementing the BEMS, where the updating process is done in hourly basis even every minute.The main contribution of our proposed system in the real-time implementation of the optimization technique using GA is shown in Table 11, where the execution time of GA on the Raspberry Pi module is 14.25 s (the number of population is 100 and the number of iteration is 30).While the sampling time of the central control agent is 16.60 s.The result implies that the communication task performed by the MQTT protocol only contributes a small portion of the time.Most of the time is consumed by the GA.It also suggests that the lightweight protocols such as the MQTT and the LWM2M protocols are the effective communication protocols between the agents in the MAS.Relying on the sampling time of the central control agent which is lower than one minute, we may conclude that our proposed testbed is suitable for implementing the BEMS, where the updating process is done in hourly basis even every minute. Effectiveness of Optimization Technique To verify the effectiveness of the proposed optimization technique, we evaluate the comfort index and the energy extracted from the grid.The objective is to maximize the comfort index while minimizing the energy extracted from the grid.The temperature comfort index (CIT) and the illumination comfort index (CII) are defined based on [64] where Temp opt , Temp min , Temp max is the optimized room temperature, the allowed minimum and maximum room temperatures respectively, Illum opt , Illum min , Illum max is the optimized room illumination, the allowed minimum and maximum room illuminations respectively.From Equations ( 20) and ( 21), the maximum comfort is achieved when the value of CIT or CII is 1, and the minimum comfort is achieved when the value is 0. In the experiments, we compare our proposed method that adjusts the temperature and the illumination setpoints to the existing method proposed by [2] (called as EXT), the fixed setpoint methods, i.e., the setpoint is set to the minimum comfort (called as Min-comfort), the setpoint is set to the middle comfort (called as Mid-comfort), and the setpoint is set to the maximum comfort (called as Max-comfort).The EXT [2] employs the FLC to adjust the temperature.The difference with our approach is described briefly in the following.Instead of using the required power and the outdoor temperature as the fuzzy inputs, the EXT uses the electricity price and the outdoor temperature.In this case, the electricity price has two linguistic values (PEAK, MD), whose membership functions are shown in Figure 18.As shown in the figure, the electricity price is expressed in respect to the time of use, which is adopted from [2].While the membership function of the outdoor temperature and the temperature setpoint are the same with our proposed method.The fuzzy rules are given in Table 12. illumination, the allowed minimum and maximum room illuminations respectively.From Equations ( 20) and ( 21), the maximum comfort is achieved when the value of CIT or CII is 1, and the minimum comfort is achieved when the value is 0. In the experiments, we compare our proposed method that adjusts the temperature and the illumination setpoints to the existing method proposed by [2] (called as EXT), the fixed setpoint methods, i.e., the setpoint is set to the minimum comfort (called as Min-comfort), the setpoint is set to the middle comfort (called as Mid-comfort), and the setpoint is set to the maximum comfort (called as Max-comfort).The EXT [2] employs the FLC to adjust the temperature.The difference with our approach is described briefly in the following.Instead of using the required power and the outdoor temperature as the fuzzy inputs, the EXT uses the electricity price and the outdoor temperature.In this case, the electricity price has two linguistic values (PEAK, MD), whose membership functions are shown in Figure 18.As shown in the figure, the electricity price is expressed in respect to the time of use, which is adopted from [2].While the membership function of the outdoor temperature and the temperature setpoint are the same with our proposed method.The fuzzy rules are given in Table 12.The room temperature setpoints for Min-comfort, Mid-comfort, and Max-comfort are set to 25 °C, 22.5 °C, and 20 °C respectively.The room illumination setpoints for Min-comfort, Mid-comfort, and Max-comfort are set to 300, 400 and 500 lx, respectively.The data of solar irradiation, the outdoor The room temperature setpoints for Min-comfort, Mid-comfort, and Max-comfort are set to 25 • C, 22.5 • C, and 20 • C respectively.The room illumination setpoints for Min-comfort, Mid-comfort, and Max-comfort are set to 300, 400 and 500 lx, respectively.The data of solar irradiation, the outdoor temperature, the outdoor illumination, and the electrical load from 7 h to 17 h given in Table 13 are used during comparisons.(FLC-T and FLC-I) have similar membership functions and the fuzzy rules. Figure 21 shows that the energy extracted from the grid of Min-comfort is always the lowest one, however since its comfort indices as shown in Figures 19 and 20 are also the lowest ones, we could not adopt this method.The same results are also achieved by the EXT, where as shown in the Figure 21, the energy extracted from the grid is lower than our proposed method, however the comfort index is also lower.index of or proposed method.This result could be understood from the fact that both FLC controller (FLC-T and FLC-I) have similar membership functions and the fuzzy rules. Figure 21 shows that the energy extracted from the grid of Min-comfort is always the lowest one, however since its comfort indices as shown in Figures 19 and 20 are also the lowest ones, we could not adopt this method.The same results are also achieved by the EXT, where as shown in the Figure 21, the energy extracted from the grid is lower than our proposed method, however the comfort index is also lower.It is worth noting that compared to Max-comfort and Mid-comfort, the energy profile extracted from the grid of our proposed method is lower than both methods.Therefore the effectiveness of our proposed in optimizing the comfort index and energy extracted from the grid is verified.Figure 21 shows that the energy extracted from the grid of Min-comfort is always the lowest one, however since its comfort indices as shown in Figures 19 and 20 are also the lowest ones, we could not adopt this method.The same results are also achieved by the EXT, where as shown in the Figure 21, the energy extracted from the grid is lower than our proposed method, however the comfort index is also lower. It is worth noting that compared to Max-comfort and Mid-comfort, the energy profile extracted from the grid of our proposed method is lower than both methods.Therefore the effectiveness of our proposed in optimizing the comfort index and energy extracted from the grid is verified. Conclusions An embedded platform for implementing the multi agent system (MAS) in the building energy management is proposed.The proposed MAS consists of the load agent, the generator agent, and the central control agent.The genetic algorithm (GA) is implemented on the central control agent to find the optimal power required by the load agents based on the availability of the PV-battery power and the requirement of the user comforts.The proposed objective functions require a few parameters, thus the data exchange between the agents could be performed efficiently.To provide a flexible implementation in the existing building infrastructure and to integrate with the state of the art IoT technology, our testbed employs the industrial Modbus protocol and the MQTT protocol, which are implemented on the low cost embedded platform.In the experiments, the execution time, the communication protocols and the sampling time of the embedded testbed are evaluated.The experiments results show that our proposed testbed is able to maximize the temperature and illumination comforts while minimizing the energy in a real-time manner.Further, the effectiveness of the optimization technique to optimize the energy cost and the user comfort of the building is verified. In future, the testbed system will be extended to cope with the complex building energy management by employing the complex models and advanced optimization algorithms.Further, the electrical system will be considered in the tested. Figure 1 . Figure 1.Architecture of the proposed system. Figure 1 . Figure 1.Architecture of the proposed system. Figure 2 . Figure 2. Input and output data of central control agent. Min. power consumption of laboratory room, Max.power consumption of laboratory room Central Control Agent (MOGA) Power required by laboratory room Power required by classroom Power required by office room Power supplied by Grid Min.power consumption of classroom, Max.power consumption of classroom Power produced by PV Power produced by Battery Min.power consumption of office room, Max.power consumption of office room Figure 2 . Figure 2. Input and output data of central control agent. Figure 3 . Figure 3. Input and output data of the load agent. Figure 3 . Figure 3. Input and output data of the load agent. Figure 3 . Figure 3. Input and output data of the load agent. Figure 6 . Figure 6.Input and output data of the generator agent. Figure 7 . Figure 7. Block diagram of the AC simulator. Figure 6 . Figure 6.Input and output data of the generator agent. Energies 2019 , 11 )Figure 6 . Figure 6.Input and output data of the generator agent. Figure 7 . Figure 7. Block diagram of the AC simulator. Figure 7 . Figure 7. Block diagram of the AC simulator. Figure 9 . Figure 9. Block diagram of the electrical load simulator. Figure 9 . Figure 9. Block diagram of the electrical load simulator. Figure 10 . Figure 10.Block diagram of the PV-battery simulator. Figure 12 . Figure 12.Profile of temperatures and power consumption of the AC simulator. Figure 12 . 29 Figure 13 . Figure 12.Profile of temperatures and power consumption of the AC simulator.Energies 2019, 12, x FOR PEER REVIEW 19 of 29 Figure 13 . Figure 13.Profile of illumination and power consumption of the lighting simulator. Figure 14 .Figure 14 . Figure 14.Mean and standard deviation of EEs of FLC inputs: (a) Normalized required power and outdoor temperature of FLC-T; (b) Normalized required power and outdoor illumination of FLC-I. Figure 15 . Figure 15.Sensitivity index of FLC inputs: (a) Normalized required power and outdoor temperature of FLC-T; (b) Normalized required power and outdoor illumination of FLC-I. Figure 15 . Figure 15.Sensitivity index of FLC inputs: (a) Normalized required power and outdoor temperature of FLC-T; (b) Normalized required power and outdoor illumination of FLC-I. Figure 16 . Figure 16.Sampling time of the load agent in the classroom. Figure 17 . Figure 17.Sampling time of the load agent in the office room. Figure 16 . Figure 16.Sampling time of the load agent in the classroom. Figure 16 . Figure 16.Sampling time of the load agent in the classroom. Figure 17 . Figure 17.Sampling time of the load agent in the office room. Figure 17 . Figure 17.Sampling time of the load agent in the office room. Figure 18 . Figure 18.Membership function of electricity price in the EXT. 11 Figure 18 . Figure 18.Membership function of electricity price in the EXT. Figure 19 . Figure 19.Temperature comfort index in the office room. Figure 20 .Figure 19 . Figure 20.Illumination comfort index in the office room. Figure 19 . Figure 19.Temperature comfort index in the office room. Figure 21 . Figure 21.Energy extracted from the grid. Table 1 . Typical agents of MAS in BEMS. Table 2 . Classification of MAS implementation in BEMS. * ND = Not defined clearly in the paper. Table 3 . Description of variables in the proposed MAS-based BEMS. Table 4 . Fuzzy rules of FLC-T. Table 5 . Fuzzy rules of FLC-I. Table 4 . Fuzzy rules of FLC-T. Table 5 . Fuzzy rules of FLC-I. Table 6 . Modbus data of the AC simulator. Table 6 . Modbus data of the AC simulator. Table 7 . Modbus data of lighting simulator. Table 7 . Modbus data of lighting simulator. Table 8 . Modbus data of the electrical load simulator. Table 8 . Modbus data of the electrical load simulator. Table 9 . Modbus data of the PV-battery simulator. Table 9 . Modbus data of the PV-battery simulator. Table 11 . Execution time and sampling time. Table 12 . Fuzzy rules of EXT. Table 12 . Fuzzy rules of EXT. Table 13 . Data inputs used in the experiments.	2019-10-03T09:12:08.399Z	2019-09-25T00:00:00.000	{ "year": 2019, "sha1": "516695b4f3ae1bbad50f9018dca13d9b7dcec5e6", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/1996-1073/12/19/3655/pdf?version=1569384532", "oa_status": "GOLD", "pdf_src": "ScienceParsePlus", "pdf_hash": "11532177c6a9e6acc9bbd02f42adc7189044182f", "s2fieldsofstudy": [ "Computer Science", "Engineering" ], "extfieldsofstudy": [ "Engineering" ] }
36058165	pes2o/s2orc	v3-fos-license	Removal of Fluoride from Groundwater by Carbonised Punica granatum Carbon ( “ CPGC ” ) Bio-Adsorbent This study applies the development and application of low cost, Punica granatum bio-adsorbent for the removal of fluoride in groundwater. The batch adsorption study was carried out to analyze the defluoridation by contact time variation, adsorbent dose, adsorbate concentration, adsorbent particle size and presence of co-anions at neutral pH. The analysis of the isotherm equilibrium data using the Langmuir and Freundlich equations by linear methods showed that the data fitted better with Freundlich model (R2 > 0.980). Prepared adsorbent showed enhanced removal of fluoride concentration by 78.1% at equilibrium contact time of 75 minutes. Carbonised Punica granatum Carbon (CPGC) seeds showed a high affinity for fluoride ions compared with other conventional adsorbents. Therefore, it can be considered as a potentially “good”, low-cost bio-adsorbent for de-fluoridation of water compared to other bio-adsorbent. Introduction Water is one of the most important elements for all forms of life and is indispensable to the maintenance of life on earth.Safe drinking water is the primary need of every human being.Pure water is scarce and is not easily available at all.Water may be contaminated by natural sources or industrial effluents.One such contaminant is fluoride.bent, it is significant to choose the suitable matrix materials used for loading of rare metals and consequently affect the adsorption behaviour for fluoride anion directly. The biomass produced in the agricultural industry is one of the most promising candidates for this application for its effectiveness, availability and abundance.In this aspect, orange juice residue has been used as the matrix material to load rare metal ions, showing quite excellent adsorption properties for anions including fluoride, arsenic and phosphate [36] [37].While for the wide application of this technique, more biomass should be sieved to develop highly cost-effective adsorbents in fluoride purification. In this study, a bio-adsorbent CPGC was developed for fluoride removal with a kind of easily available biowaste, Pomegranate seeds, which can be an alternative to the costly de-fluoridation products such as ion exchange resins and electro dialysis.In India, 810 × 10 3 metric ton Pomegranate fruit is consumed every year, and lots of seed waste is disposed, causing a severe problem in the community.So, for the environmental interest, this bio-waste could be a low-cost adsorbent and remove fluoride from groundwater.The aim of the present study is to evaluate the feasibility of using pomegranate seed waste for fluoride removal from groundwater and the effect of common experimental parameters, such as the initial pH value of the solution, equilibrium concentration of fluoride, contact time, adsorbent dose and coexisting anions. Adsorbent Preparation The Punica granatum seed (powdered sample), common name, Pomegranate, was purchased from market.Then the material was dried at 378 -383 K for 24 hours.It was washed with doubly distilled water to remove the free acid and dried at the same temperature for 3 hours.Later, the dried adsorbent was carbonised thermally in Muffle furnace between 1073 K to 1084 K temperature ranges.The resulting product was cooled to room temperature and sieved to the desired particle sizes, namely, <55, 55 -106, 106 -150, 150 -225 and 225 -305 micron.Finally, the product was stored in vacuum desiccators until required for treatment.The physiochemical properties of the adsorbent are listed in Table 1. Batch Adsorption Study The batch adsorption de-fluoridation study was conducted for the optimization of various experimental conditions like contact time, initial fluoride concentration, adsorbent dose, particle size and influence of co-ions with fixed dosage.The mixture was agitated in a thermostatic shaker at a speed of 250 rpm at room temperature.The reagents used in this present study are of analytical grade.A fluoride ion stock solution (100 mg/l) was prepared and other fluoride test solutions were prepared by subsequent dilution of the stock solution.Analytical grade sodium chloride, sodium nitrate, sodium sulphate and sodium phosphate were used to prepare 1.0 mol/L stock solutions, and mixed at an arbitrary volume ratio to adjust the concentration of the coexisting anions in the solution.Analytical grade sodium citrate and sodium nitrate were employed to prepare the total ion strength adjusting buffer (TISAB) solution for the pre-treatment of all sampling solutions before testing by using ion selective electrode of fluoride. All the experiments were carried out at room temperature.Fluoride ion concentration was measured with a specific ion selective electrode by use of total ionic strength adjustment buffer II (TISAB II) solution to maintain pH 5 -5.5 and to eliminate the interference effect of complexing ions [38].The pH of samples was measured by Orion ion selective equipment.All other water quality parameters were analyzed by using standard methods [39].Effect of different initial fluoride concentrations viz., 2, 4, 6, 8 and 10 mg/l were studied by keeping the mass of sorbent at 0.75 g and volume of solution at 100 ml in neutral pH. The fluoride concentration retained in the adsorbent phase, q e (mg/g), is calculated by Equation ( 1). ( ) where q e is the amount of fluoride adsorbed (mg/g); V is the volume of solution; c o and c e are the initial and residual concentration at equilibrium (mg/l), respectively, of fluoride and W is the weight (g) of the adsorbent. Effect of Contact Time and Initial Fluoride Concentration Contact time plays a very important role in adsorption dynamics.The effect of contact time on adsorption of fluoride onto CPGC is shown in Figure 1.Batch adsorption studies using the concentrations 2, 3, 4, 6, 8 and 10 mg/l of fluoride solution with 0.75 g of the adsorbent were carried out at 298 K as a function of time to evaluate the de-fluoridation and adsorption rate constants.The adsorption of fluoride increases with time and gradually attains equilibrium after 75 minutes.From Figure 1, the time to reach equilibrium conditions appears to be independent of initial fluoride concentrations.Therefore, 75 minutes was fixed as minimum contact time for the maximum de-fluoridation of the sorbent.The adsorption of fluoride decreased from 88% to 47% by increasing fluoride concentration from 2.0 to 10.0 mg/l.Further, it was observed that the removal curves are smooth and continuous indicating the possibility of the formation of monolayer coverage of fluoride ion at the interface of adsorbent. Effect of Particle Size The de-fluoridation experiments were conducted using CPGC with five different particle sizes viz.55, 55 -106, 106 -150, 150 -225 and 225 -303 μm.As the adsorption process is a surface phenomenon, the de-fluoridation efficiency of the sample with 55 μm registered high de-fluoridation efficiency due to larger surface area (Figure 2).The variation in the percentages of fluoride removal by the sample with different particle sizes was studied. Hence, the material with particle size of 55 μm has been chosen for further experiments.Higher percentage of adsorption by CPGC with smaller particle size is due to the availability of more specific surface area on the adsorbent surface. Effect of Adsorbent Dose One of the parameters that strongly affect the adsorption capacity is the concentration of the adsorbents.The effect of adsorbent dosage on the removal of fluoride from ground water was studied at neutral pH and fluoride concentration of 3 mg/l for 150 min.Figure 3 indicates that, increasing adsorbent dosage increases fluoride uptake, indicating that for a fluoride removal an optimum CPGC dosage of 0.75 g/L was required.The results further illustrates that the fluoride removal efficiency increases up to this optimum dosage beyond which fluoride uptake has no significant change with CPGC dosage.The increase in the adsorbent dosage provides greater surface area and hence increases the adsorption.However, by increasing the adsorbent dosage beyond the optimum dosage, there may be a slight improvement in fluoride removal efficiency but the adsorption capacity decreases. Effect of Interfering Co-Ions The effects of coexisting anions such as sulphate, nitrate, chloride and bicarbonate on fluoride adsorption by the CPGC adsorbent were examined and the results are given in Figure 4. Chloride and nitrate did not perceptibly interfere with fluoride removal even at a concentration of 500 mg/l, while sulphate began to show some adverse effects when the 2 4 SO − concentration increases.However, bicar- bonate showed great competitive adsorption with fluoride.The fluoride adsorption amount decreased quickly from 78.1 to 52% with the increase of bicarbonate concentration 0 -500 mg/l.This may be attributed to the competition of bicarbonate ions with the fluoride ions at the active site, on the surface of the sorbents.The selective nature of the fluoride by the sorbent depends on size, charge, polarizability, electro negativity difference, etc.The order of interference for fluoride removal was in the following order, Adsorption Isotherms The sorption isotherms express the specific relation between the concentration of sorbate and its degree to accumulation onto sorbent surface.The fluoride sorption capacity of CPGC at 298 K was evaluated using Langmuir and Freundlich isotherm models. The experimental data was applied in accordance with the linearized form of the Langmuir isotherm model.It can be represented as Equation ( 2): where q m is q e for a complete monolayer (mg/g); K a is the adsorption equilibrium constant (cm 3 /mg).To evaluate the adsorption capacity for a particular range of adsorbate concentration, the aforementioned equation can be used in the following linear Equation (3): 1 1 The constants q m and K a in Equation ( 2) can be determined from the slope and intercept of the linear plot/graph of c e /q e versus c e in Figure 5. Results indicate that the adsorbent CPGC has a high affinity for fluoride adsorption under these conditions.The present data fit well in Langmuir isotherm model with R 2 > 0.970.The average monolayer adsorption capacity (q m ) obtained for CPGC is 1.68 mg/g. Freundlich adsorption isotherm is based on adsorption on heterogeneous surface, and is the earliest known relationship describing the adsorption equilibrium and is given as Equation ( 4): where x is the amount of solute adsorbed (mg), m is the mass of adsorbent used (g), K f and 1/n are empirical constants, indicating the adsorption capacity and adsorption intensity, respectively.Above Equation ( 4) may be converted to a linear form (5) by taking logarithms: The values of K f and 1/n were obtained from the slope and intercept of the plot between log(q e ) and log(c e ).The Freundlich equation deals with physicochemical adsorption on heterogeneous surfaces.Linearized form of the Freundlich equation is demonstrated in Figure 6.Adsorption data fitted well in linearized form of model equation (R 2 > 0.980) which indicated the acceptability of the model. Conclusion Among various bio-waste materials, Punica granatum seeds were first time utilised in de-fluoridation of groundwater.In the present study, the fluoride is removed from aqueous solutions using Carbonised Punica granatum Carbon (CPGC) seeds as bio-adsorbent.It can be concluded that CPGC has good properties for the sorption of fluoride ions from aqueous solutions.The equilibrium time for removal of fluoride concentration is determined to be 75 min.The fluoride saturation capacity of CPGC is 1.68 mg F − /g at room temperature.The best fitting adsorption isotherm is Freundlich model (R 2 > 0.980), which indicates that fluoride bio-adsorption onto CPGC is characterized by chemisorptions on heterogeneous surfaces.The particle size is an important parameter that affects the sorption for fluoride on CPGC since the sorption of these ions increases as the particle size decreases.Finally, this low-cost material can be employed as an adsorbent for fluoride removal from groundwater, in particular in domestic systems where fluoride related problems exist. Figure 1 . Figure 1.Effect of contact time and fluoride concentration on fluoride removal. Figure 2 . Figure 2. Effect of particle size on fluoride removal. Figure 4 . Figure 4. Variation in adsorption capacity for different concentrations of co-ions. Table 1 . The physicochemical properties of CPGC adsorbent.	2017-10-30T11:47:09.137Z	2015-06-05T00:00:00.000	{ "year": 2015, "sha1": "86320ffd4b35caff8f2e44fd4afc3f60b1312d7e", "oa_license": "CCBY", "oa_url": "http://www.scirp.org/journal/PaperDownload.aspx?paperID=56946", "oa_status": "GOLD", "pdf_src": "ScienceParseMerged", "pdf_hash": "86320ffd4b35caff8f2e44fd4afc3f60b1312d7e", "s2fieldsofstudy": [ "Environmental Science", "Materials Science" ], "extfieldsofstudy": [ "Chemistry" ] }
11357141	pes2o/s2orc	v3-fos-license	Distinct Functions for the Drosophila piRNA Pathway in Genome Maintenance and Telomere Protection Transposons and other selfish DNA elements can be found in all phyla, and mobilization of these elements can compromise genome integrity. The piRNA (PIWI-interacting RNA) pathway silences transposons in the germline, but it is unclear if this pathway has additional functions during development. Here we show that mutations in the Drosophila piRNA pathway genes, armi, aub, ago3, and rhi, lead to extensive fragmentation of the zygotic genome during the cleavage stage of embryonic divisions. Additionally, aub and armi show defects in telomere resolution during meiosis and the cleavage divisions; and mutations in lig-IV, which disrupt non-homologous end joining, suppress these fusions. By contrast, lig-IV mutations enhance chromosome fragmentation. Chromatin immunoprecipitation studies show that aub and armi mutations disrupt telomere binding of HOAP, which is a component of the telomere protection complex, and reduce expression of a subpopulation of 19- to 22-nt telomere-specific piRNAs. Mutations in rhi and ago3, by contrast, do not block HOAP binding or production of these piRNAs. These findings uncover genetically separable functions for the Drosophila piRNA pathway. The aub, armi, rhi, and ago3 genes silence transposons and maintain chromosome integrity during cleavage-stage embryonic divisions. However, the aub and armi genes have an additional function in assembly of the telomere protection complex. Introduction Drosophila piRNAs have been implicated in transposon silencing and maintenance of genome integrity during female germline development. However, piRNA pathway mutations lead to complex developmental phenotypes [1,2,3,4], and piRNAs have been implicated in control of gene expression [5,6,7,8]. Furthermore, the majority of piRNAs in other systems, including mouse testes, are not derived from repeated elements [9,10,11,12,13]. The full extent of piRNA functions thus remains to be explored. Mutations in the majority of Drosophila piRNA pathway genes disrupt asymmetric localization of RNAs along the axes of the oocyte, and lead to maternal effect embryonic lethality [1,2,3,4]. The axis specification defects linked to several of piRNA pathway mutations are dramatically suppressed by a null mutation in mnk, which encodes a Checkpoint kinase 2 (Chk2) homolog required for DNA damage signaling, indicating that the loss of asymmetric RNA localization is downstream of DNA damage [1,2]. Oocyte patterning defects generally lead to embryonic lethality, but the mnk allele that suppresses the axis specification defects associated with piRNA mutations does not suppress embryonic lethality [1,2,3]. piRNAs thus have an essential function during embryogenesis that is independent of Chk2 activation and DNA damage signaling. To gain insight into potential new functions for the piRNA pathway, we have characterized the embryonic lethality associated with four piRNA pathway mutations. These studies reveal a novel function for a subset of piRNA genes in assembly of the telomere protection complex, and suggest that this process is directed by a subpopulation of 19-22 nt piRNAs. Results/Discussion The armi and aub genes encode a putative RNA helicase and a piRNA binding PIWI Argonaute protein, and recent studies suggest that they have distinct functions in piRNA biogenesis [2,8,14,15] Mutations in aub dramatically reduce piRNA species that overlap by 10 nt, which is characteristic of ping-pong amplification, while armi mutations reduce total piRNA production but enhance the ping-pong signature [15]. Mutations in aub and armi lead to maternal-effect embryonic lethality, however, suggesting that these genes share an essential function. To gain insight into the lethality associated with these mutations, we first analyzed DNA break accumulation during oogenesis. Germlinespecific DNA breaks normally form during early oogenesis, as meiosis is initiated [16]. In several piRNA mutants, however, DNA breaks persist, which could compromise the female pronucleus and thus lead to genetic instability in the early zygote [2,14]. DNA breaks trigger phosphorylation of histone H2Av, producing c-H2Av foci near the break sites [17]. In wild-type ovaries, c-H2Av foci begin to accumulate in region 2 of the germarium, as meiotic breaks are formed [16]. These foci are significantly reduced in stage 2 egg chambers, which have completed meiotic repair and budded from the germarium. Later in oogenesis, c-H2Av foci accumulate in the nurse cell nuclei, which undergo endoreduplication. However, these foci remain undetectable in the oocyte [16]. In ovaries mutant for aub or armi, c-H2Av foci appear in germarium region 2, but persist in nurse cells and the oocyte through stage 4. By stage 5, however, c-H2Av foci are undetectable in 50% of armi and aub mutant oocytes, and are significantly reduced in the remaining oocytes ( Figure S1 and data not shown). Both armi and aub mutations thus increase DNA damage during early oogenesis, but most of the damage in the oocyte appears to be repaired as oogenesis proceeds. As wild type oocytes mature and initiate meiotic spindle assembly, the major chromosomes form a single mass at the spindle equator and the non-exchange 4 th chromosomes move toward the poles [18,19]. In OregonR, we observed distinct 4 th chromosomes in 79% of stage 13 oocytes. In stage 13 aub and armi mutants, by contrast, distinct 4th chromosomes were observed in only 11% and 18% of stage 13 oocytes, respectively (Figure S2, Table S1). However, a single primary mass of chromatin was always observed. These observations are consistent with c-H2Av data suggesting that DNA breaks formed during early oogenesis are often repaired as the oocyte matures. In addition, both aub and armi mutations appear to inhibit separation of the small 4 th chromosomes, although it is also possible that this small chromosome is fragmented and thus difficult to detect cytologically. Drosophila oocytes are activated as they pass through the oviduct, which triggers completion of the meiotic divisions. The first meiotic division is completed in the oviduct, but meiosis II can be observed in freshly laid eggs and is characterized by four wellseparated meiotic products on tandem spindles ( Figure 1A). In aub and armi mutant embryos, the meiotic chromatin was either stretched across the paired meiotic spindles, or fragmented and spread over both spindles ( Figure 1A). No wild type meiotic figures were observed. Breaks thus appear to persist in some stage 14 oocytes, although this does not disrupt the karyosome organization during earlier stages. However, other oocytes appear to have intact chromosomes that fail to resolve during the meiotic divisions. Compromised zygotic genomic integrity in piRNA mutants Fertilization and pronuclear fusion initiate 13 rapid cleavage stage mitotic divisions [16]. These divisions are syncytial, but membranes surround the cortical nuclei to form cells following mitosis 13 [20]. 0 to 3-hr old cleavage stage aub and armi mutant embryos showed two distinct phenotypes. 60% of aub mutant embryos and 90% of armi mutant embryos contained dispersed chromatin fragments that were often associated with small spindlelike microtubule bundles ( Figure 1B, Table S2). The remaining embryos appeared to be progressing through cleavage divisions, and some cellularization and gastrulation stage embryos were observed. However, chromosome bridges/lagging chromosomes were present in 50% to 70% of the cleavage stage anaphase and early telophase figures ( Figure 1B and Figure 2C). Chromatin fragmentation could result from replication of broken chromosomes inherited from the female, or from postfertilization fragmentation of the zygotic genome. To directly assay zygotic genome integrity, mutant females were mated to wild type males and dual-label FISH was used to monitor physically separate regions of the Y chromosome. In male embryos derived from wild type females, the two Y chromosome probes always cosegregated through anaphase and telophase ( Figure 1C, 1D). Mutant embryos showing chromatin fragmentation, by contrast, contained chromatin clusters that did not label for either Y chromosome probe, or that labeled for only one of the two probes ( Figure 1C). In mutant embryos that proceeded through cleavage stage mitotic cycles, the majority of segregating chromatids retained both Y chromosome markers, indicating that chromosome continuity had been maintained. Chromatids with only one of two markers were observed, however, indicating that breaks had separated regions on a Y chromosome arm from the centromere ( Figure 1D). The axial patterning defects associated with piRNA mutations are suppressed by mutations in mnk [1,2], but mnk did not suppress either the chromatin fragmentation or segregation defects linked to aub and armi (Table S2, Figure S3). Mutations in aub and armi thus destabilize the genome of the zygote and disrupt chromosome resolution during the cleavage divisions through processes that are independent of DNA damage signaling. Mutations in the armi and aub genes disrupt piRNA production and transposon silencing, but have also been reported to inhibit homology dependent target cleavage by siRNAs [21,22]. In addition, null mutations in argonaute2 (ago2), which block siRNA based silencing, have been reported to disrupt mitosis during the syncytial blastoderm stage [23]. These observations raise the possibility that chromatin fragmentation and fusion in aub and armi mutants result from defects in the siRNA pathway. We therefore analyzed cleavage in embryos from females homozygous for null mutations in ago2 and dcr2, which block siRNA production and silencing [24]. Consistent with previous studies, we find that embryos from ago2 and dcr2 mutant females are viable [23,24]. However, we did not observe chromosome fragmentation or a statistically significant increase in anaphase bridge formation relative to wild type controls ( Figure S4, Figure 2C). The loquacious (loqs) gene encodes a Dicer-1 binding protein required for miRNA production [25], and we find that embryos from loqs mutant females also proceed through normal cleavage stage divisions ( Figure S4, Figure 2C). Chromosome segregation and maintenance of zygotic genome integrity during early embryogenesis thus appear to be independent of the siRNA and miRNA pathways, but require at least two components of the piRNA pathway. Telomere fusions in aub and armi embryos In S. pombe, mutations in ago1, dcr1 and rdp1 disrupt kinetochore assembly and thus lead to lagging mitotic chromosomes due to defects in centromere movement to the spindle poles [26]. To determine if Drosophila piRNA mutations disrupt kinetochore assembly, we performed dual label FISH for centromeric dodeca- Author Summary Transposons and other selfish genetic elements make up a significant fraction of all eukaryotic genomes, and the piRNA pathway appears to have a conserved function in transposon silencing and genome maintenance. However, other functions for this pathway have not been fully explored. Telomeres must be protected from recognition as DNA breaks by the repair machinery, which can covalently ligate unprotected chromosome ends and thus disrupt meiotic and mitotic chromosome segregation. We show that mutations in a subset of piRNA pathway genes disrupt meiotic and mitotic chromosome separation and that these segregation defects are suppressed by a mutation that blocks ligation of non-homologous DNA ends. These mutations also disrupt assembly of the telomere protection complex and reduce expression of a subpopulation of 19-to 22-nt telomere-specific RNA. We therefore propose that a subpopulation of short piRNAs direct assembly of the telomere protection complex. satellite sequences [27] and the telomere-specific transposon HeT-A. In aub and armi mutants, centromeric sequences segregated to the spindle poles in essentially every anaphase figure, but telomere specific sequences were consistently present at the chromatin bridges ( Figure 2A). These observations indicate that armi and aub are not required for kinetochore assembly, but are needed for telomere resolution. Telomeres are protected from recognition as DNA double strand breaks by the telomere-protection complex (TPC), and defects in telomere protection thus lead to covalent ligation of chromosome ends by the non-homologous end-joining (NHEJ) pathway [28,29]. DNA Ligase IV is required for NHEJ, and ligase IV mutations suppress fusions that result from covalent joining of unprotected chromosome ends [28,29]. To determine if chromosome fusions in aub and armi are due to NHEJ, we generated ligIV;aub and ligIV;armi double mutant females and analyzed chromosome segregation in the resulting embryos. In aub single mutant embryos, 50% of anaphase figures show bridges, but anaphase bridges are present in only 15% of ligIV;aub double mutants ( Figure 2B, 2C). By contrast, the fraction of embryos showing chromosome fragmentation increases in ligIV;aub double mutants (Table S2). Chromosome fragmentation also increased in ligIV;armi mutant embryos, and as a result morphologically normal anaphase figures could not be observed (Table S2). These findings strongly suggest that lagging chromosomes result from covalent ligation of chromosome ends by the NHEJ pathway, while chromatin fragmentation results from DNA breaks that are repaired by NHEJ. Mutations in armi and aub lead to significant over-expression of transposable elements [8,14,30], including DNA elements that are mobilized by a ''cut and paste'' mechanism that directly produces double strand breaks [31]. In addition, NHEJ pathway has been implicated in repair of gapped retroviral integration intermediates [32]. Chromosome fragmentation may therefore result from transposon over-expression and mobilization, which induces breaks that overwhelm the NHEJ pathway. Telomere fusions, by contrast, appear to result from defects in telomere protection, which lead to chromosome end recognition by the NHEJ pathway. Assembly of the telomere protection complex The Drosophila TPC includes HOAP and Modigliani (Moi), which may function only at chromosome ends, and HP1a and the MRN complex, which have additional roles in heterochromatic silencing and DNA repair [33,34,35,36]. To directly assay for TPC recruitment, we used chromatin immunoprecipitation (ChIP) to measure HP1a and HOAP binding to the telomere specific transposon HeT-A ( Figure 3B, 3C). In wild type ovaries, HOAP and HP1a bind to multiple regions of HeT-A ( Figure 3B, 3C). In armi and aub mutants, by contrast, HOAP and HP1a binding to the Het-A 59-UTR and ORF are significantly reduced ( Figure 3B, 3C). The 59end of Het-A is oriented toward the chromosome end, and is therefore likely to lie at the telomere. Ovarian tissue consists of germ cells with a surrounding layer of somatic cells, which complicates interpretation of these biochemical studies. However, ChIP on 0-3 hour old embryos from aub and mnk,aub mutant females revealed significant reduction in HOAP binding at the HeT-A 59-UTR ( Figure S5). The aub and armi genes thus appear to be required for TPC recruitment, consistent with ligation of chromosome ends in mutant embryos. To determine if other piRNA pathway mutations disrupt telomere protection, we analyzed the cleavage stage embryonic divisions in ago3 and rhi mutants. The ago3 locus encodes a PIWI clade protein that primarily binds sense strand piRNAs, and rhi encodes a rapidly evolving HP1 homologue required for production of precursor RNAs from a subset of piRNA clusters [14,30]. Essentially all of the rhi and ago3 mutant embryos showed chromatin fragmentation, as observed in the majority of aub and armi mutants ( Figure S6). We therefore biochemically assayed for TPC assembly in ovarian chromatin using ChIP for HOAP and HP1a. Surprisingly, neither ago3 nor rhi mutations disrupt HOAP or HP1a binding to Het-A, and rhi mutants show greater than wild type levels of HOAP binding to Het-A ( Figure 3B, 3C). By contrast, these rhi alleles reduce total piRNA production by 10 fold [14]. The ago3 mutations appear to be null, and the rhi mutations are strong hypomorphc alleles. Assembly of the TPC in the ago3 and rhi mutants is therefore unlikely to be mediated by residual protein. Instead, these findings strongly suggest that aub and armi have a function in telomere protection that is not shared by ago3 or rhi. In Drosophila, chromosome breaks can be converted to stable telomeres [37], called terminal deletions, which accumulate addi-tional copies of the telomeric elements HeT-A and TART. When terminal deletions are passaged in animals heterozygous for aub or the piRNA pathway gene spnE, the number of terminal TART repeats increase [38]. The defects in TPC assembly in aub and armi could therefore be triggered by increased HeT-A and TART copy number, which could titrate TPC components. We therefore assayed telomeric transposon copy number in aub and armi mutants, which show defects in TPC assembly, and in rhi and ago3 mutants, which do not. We also assayed telomeric transposon copy number and mitotic chromosome segregation in a wild-type variant, Gaiano, that has been reported to carry additional HeT-A repeats [39]. Consistent with previous reports, we find that Gaiano has 10 to 15 fold more HeT-A copies than OregonR controls ( Figure 3D). Despite the increase in telomere length, this stock is viable and fertile, and we did not observe telomere fusions or lagging chromosomes during the cleavage stage embryonic divisions ( Figure S6). In addition, we found that aub mutants that show defects in TPC assembly do not accumulate additional copies of HeT-A or TART, while rhi and ago3 mutants that are wild type for TPC binding show an increase in telomere-specific transposon copy number ( Figure 3D). Assembly of the TPC is therefore independent of telomere specific transposon copy number ( Figure S6). Aub and Armi are required for production of a subpopulation of 19-22 nt piRNAs piRNAs are proposed to direct PIWI clade proteins to targets through sequence specific interactions. Our observations raised the possibility that armi and aub promote production of piRNAs that direct the telomere protection complex to transposons that make up chromsome ends. We therefore analyzed published small RNA deep sequencing data [14,15,30] for species derived from a fourth chromosome cluster, defined by a high density of uniquely mapping piRNAs, containing multiple repeats of the telomeric transposons [40]. Our bioinformatic analysis showed that 70-80% of telomere specific piRNAs match this cluster ( Figure 4, Table S3). Figure 4 shows length histograms for small RNAs from wt, rhi, ago3, aub and armi mutant ovaries that map to this cluster. Data are normalized to sequencing depth, and small RNAs mapping to the plus genomic strand are represented in blue and RNAs mapping to the minus strand are in red. Significantly, aub and armi mutations lead to a preferential loss of shorter piRNAs mapping to the minus genomic strand ( Figure 4B, 4C). Loss of these shorter RNAs highlights the peak at 21 nt, which is retained in all of the mutants and likely represent endogenous siRNAs ( Figure 4A, black arrow). The telomeric elements (HeT-A and TART) are almost exclusively on the minus genomic strand in this cluster, and the RNAs that are lost in aub and armi thus correspond to the sense strand of the target elements. Ovaries mutant for ago3 and rhi, by contrast, retain these shorter sense strand RNAs. We quantified the relative abundance of typical 23-29nt long piRNAs and the shorter 19-22nt species, excluding the 21nt endo-siRNA peak. All four mutations significantly reduce 23 to 29 nt piRNAs, although rhi mutants retain approximately 50% of wild type minus strand species. Loss of these piRNAs is consistent with over-expression of transposons matching this cluster in all four mutants ( Figure S8). By contrast, the shorter minus strand RNAs are reduced by 3 to 10 fold in armi and aub, but are expressed at 80% to 95% of wild type levels in ago3 and rhi ( Figure 4B, 4C). In addition, short piRNA species from the telomeric cluster coimmunoprecipitate with Piwi protein [15,30], which localizes to the nucleus and is a likely effector of chromatin functions for the piRNA pathway ( Figure S7). Binding of this subpopulation of piRNAs by Piwi is retained in ago3 mutants, which assemble the TPC, but significantly reduced in armi mutants, which block assembly of the TPC ( Figure S7). Taken together, these observations suggest that the piRNA pathway has two genetically distinct functions during oogenesis and early embryogenesis. The pathway prevents DNA damage during oogenesis and maintains the integrity of the zygotic genome during the embryonic cleavage divisions, which likely reflects the established role for piRNAs in transposon silencing [2,8,14,30]. This function requires aub, armi, rhi and ago3, which are also required for wild type piRNA production. In addition, our studies reveal a novel function for the piRNA genes aub and armi in telomere protection, whch may be mediated by a novel class of short RNAs that bind to Piwi. Consistent with this hypothesis, it has been reported that germline clones of piwi null alleles do not significantly disrupt oogenesis, but lead to maternal effect embryonic lethality and severe chromosome segregation defects during the cleavage divisions [41]. A subpopulation of Piwi-bound piRNAs may therefore direct assembly of the TPC. Fly stocks Flies were reared at 25uC on standard corn meal medium. OregonR and w1118 were used as controls. Stocks carrying the following alleles were obtained from the Bloomington Stock Center: ago2 51B , ago2 Df , aub HN2 , aub QC42 , dcr2 L811fsX , mnk P6 , ligIV 5 , rhi 02086 and rhi KG00910 . ago2 51B is an imprecise P-element induced deletion of the first two exons of ago2 locus. aub HN2 and aub QC42 are both EMS-induced point mutations [42,43]. dcr2 L811fsX is an EMSinduced loss-of-function allele described in [24]. rhi 02086 and rhi KG00910 are both P-element insertion alleles, which act as strong hypomorphs [44]. Both armi 1 and armi 72.1 alleles are strong hypomorphic alleles which produce armi transcript at low levels Binding of the telomere protection complex proteins HOAP and HP1 to HeT-A. Chromatin Immunoprecipitation (ChIP) was used to recover bound DNA, and the percent of input chromatin precipitated was determined by qPCR. Fold change in binding relative to wild type is shown, and was calculated by dividing mutant by wild type (wt) values. D. Genomic copy number for HeT-A and TART. Copy number was determined by qPCR, using the single copy Rp49 gene as an internal standard. Gaiano is a wild-type stock previously shown to carry additional telomeric transposon repeats. doi:10.1371/journal.pgen.1001246.g003 [4]. mnk P6 ,aub HN2 and mnk P6 ,aub QC42 [2] recombinants were generated using standard genetic procedures. The loqs f00791 and loqs KO alleles were from Bloomington and Dennis McKearin [25], respectively. Stocks carrying ago3 4931 and ago3 3658 , which are lossof-function alleles with premature stop codons [30], were obtained from the Zamore lab (University of Massachusetts Medical School). Immunostaining and fluorescence in situ hybridization 0-30-min-old or 0-3-hr-old embryos were fixed in methanol and immunostained for a-tubulin (Dm1a, Sigma Chemical Co., 1:300) and 0.2 mM TOTO-3 (Molecular Probes) using standard procedures [45]. For staining of egg chambers, the ovaries were dissected in Robb's medium and fixed in 4% formaldehyde as described [2]. c-H2Av antibody was generously provided by Kim McKim (Rutgers) and was used at 1:500 dilution. The dodecasatellite probe for the fluorescent in situ hybridization was made by 39 end labeling using terminal deoxynucleotidyl transferase (Roche), followed by direct fluorophore conjugation using ARES DNA labeling kit as described by the manufacturer (Molecular Probes). The dodeca satellite sequence from the pBK6E218 plasmid was amplified using T3 and T7 primers [27]. The telomeric probe was made by indirect substitution of DIG-dUTP using the PCR DIG probe synthesis kit (Roche). The sequence was amplified from genomic DNA using the following primers-telF-59-GACAATGCACGACAGAGGAA-39 and telR-59-GTCTT-TTTGGGTTTGCGGTA-39. The Y-chromosome satellites (AA-TAC)n and (AATAAAC)n were purchased as oligos with direct conjugation of FAM and Cy-3 fluorophores at the 39end (IDT). Hybridization was performed as described previously [46]. Fluorescently labeled samples were imaged using a Leica TCS-SP inverted scanning confocal microscope or a Nikon TE-2000E2 inverted microscope and captured using Metamorph software (Universal Imaging). All images were processed using Image J (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 1997-2006) and Adobe Photoshop. Chromatin bridges quantification To quantify chromatin bridges, the ratio of anaphase/telophase (A/T) bridges to total A/T figures was calculated for 10 to 30 embryos. The mean bridge frequency was determined by designating each embryo as an independent experiment, and the standard error was determined using an Anova test. Two-tailed ttests were also used to compare specific data sets, using a = 0.05. Pvalues are noted on the graphs. Chromatin Immunoprecipitation and quantitative PCR (qPCR) Whole ovaries were dissected from 2-5-day old flies and fixed using 1.8% formaldehyde for 10 minutes at room temperature. For ChIP using embryos, 0-3 hr old embryos were collected and fixed using 1.8% formaldehyde for 20 minutes at room temperature. The ChIP assay was performed as per manufacturer's instructions (Invitrogen) and as previously described with some modifications [14]. Immunoprecipitation was done using HOAP polyclonal serum previously described [14] or the monoclonal HP1 antibody (Developmental Studies Hybridoma Bank, IA). The Figure 4. piRNAs linked to a 4 th chromosome cluster containing telomeric transposon fragments. A. Length histograms showing plus genomic strand (blue) and minus genomic strand (red) mapping piRNAs in wt, armi, aub, rhi and ago3 mutants. The relative abundance is normalized to sequencing depth and is plotted on the y-axis. Note that sense strand of the transposon fragments in this cluster are on the minus genomic strand, and that the scales differ. Preferential loss of shorter piRNAs from aub and armi leads to a prominent endo-siRNA peak at 21 nt (marked by a black arrow). B. Abundance of longer (23-29 nt) plus strand (blue) and minus strand (red) piRNAs in the indicated mutants relative to their respective wildtype controls. All four mutations reduce plus strand piRNAs, which are anti-sense to the telomeric transposons. C. 19-22 nt genomic plus and minus strand piRNAs in the indicated mutants. All four mutations reduce plus strand RNAs. However, minus strand species are retained at near wild type levels in both rhi and ago3 mutants. For panels B and C, bars show normalized reads in mutants divided by normalized reads in wild-type controls. doi:10.1371/journal.pgen.1001246.g004 purified DNA was subjected to qPCR using Applied Biosystems 7500 system, and data was analyzed by calculating the % of immunoprecipitated DNA compared to the input DNA sample. All ChIPs were performed at least twice and the data presented is an average of two different biological replicates with technical triplicates for each of them. The data was plotted with error bars representing standard deviations for individual samples. The difference between primer efficiencies was calculated by preparing standard curves and was taken into consideration while calculating % IP values. The primer sequences are available upon request. Sequence extraction and annotation For each sequence read, the first occurrence of the 6-mer perfectly matching the 59-end of the 39-linker was identified. Sequences without a match were discarded. The extracted inserts for sequences that contained the 39-linker were then mapped to the female Drosophila melanogaster genome (Release R5.5, excluding chromosome YHet). Inserts that matched fully to a genomic sequence were collected using Bowtie (Langmead et al., 2009) and the corresponding genomic coordinates were determined for downstream functional analysis. Sequences corresponding to pre-miRNAs or non-coding RNAs (ncRNAs) were identified and removed. For analyis of the telomeric cluster, small RNA length distributions were determined for reads that mapping to chr4:1280000-1350999, normalizing for sequencing depth (genome mapping reads excluding ncRNAs).	2016-05-04T20:20:58.661Z	2010-12-01T00:00:00.000	{ "year": 2010, "sha1": "689094074c7306e2700f6a2bde4b8732d307a5c5", "oa_license": "CCBY", "oa_url": "https://journals.plos.org/plosgenetics/article/file?id=10.1371/journal.pgen.1001246&type=printable", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "689094074c7306e2700f6a2bde4b8732d307a5c5", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Medicine", "Biology" ] }
225735015	pes2o/s2orc	v3-fos-license	Resilient Schedule Coordination for a Bus Transit Corridor Providing convenient transit services at reasonable cost is important for transit agencies. Timed transfers that schedule vehicles from various routes to arrive at some transfer stations simultaneously (or nearly so) can signiﬁcantly reduce wait times in transit networks, while stochastic passenger ﬂows and complex operating environments may reduce this improvement. Although transit priority methods have been applied in some high-density cities, operating delays may cause priority failures. This paper proposes a resilient schedule coordination method for a bus transit corridor, which analyzes link travel time, passenger loading delay, and priority signal intersection delay. It maximizes resilience based on realistic passenger ﬂow volume, whether or not transit priority is provided. The data accuracy and result validity are improved with automatically collected data from multiple bus routes in a corridor. The Yan’an Road transit corridor in Shanghai is used as a case study. The results show that the proposed method can increase the system resilience by balancing operation cost and passenger-based cost. It also provides a guideline for realistic bus schedule coordination. Introduction Providing convenient transit services for passengers at reasonable total cost is one of the main purposes of transit agencies. Bus routes operate under high pressure in China because of large passenger flow and complex road conditions, so a resilient bus system is needed. As urban areas expand, transit networks become more complex. A rail transit network may serve as a backbone network, while bus routes connect and feed the rail transit network, serve as shuttle systems, and provide door-to-door transit services. In metropolitan areas, transfers among transit routes are needed because fixed-route buses cannot economically provide direct service among all origins and destinations. e potential value of schedule coordination is that user waiting time may be reduced at transfer stations if vehicles from different routes can arrive at transfer stations simultaneously (or nearly so). e passengers can then transfer quickly among vehicles that stop briefly near one another. A schedule coordination system can greatly reduce passenger wait times at transfer stations but cannot completely eliminate them in a system with probabilistic running times and delays. Metropolitan areas have complex traffic conditions as well as stochastic transit demand, which may force some vehicles off their schedules and greatly increase the difficulty of achieving coordinated timed transfers. Slack time, which is a buffer parameter, should be included and optimized in schedules in order to reduce the probability of missed connections for passengers at transfer stations. Additional slack time increases operation cost but also increases transfer reliability by countering some travel time randomness [1,2]. For each bus route, it is important to optimize the headway, which influences the vehicle operating cost and passenger waiting time. erefore, a method is proposed here for jointly optimizing bus headways and slack times in an integrated bus system with multiple transfer stations in a transit corridor. Schedule coordination has been well researched in different environments, while analysis and optimization of coordination performance in changing transit environments are rarely researched. Resilience reflects how a transit system is influenced by changes in its environment and how it recovers from such change. is paper proposes a method for schedule coordination considering transit system resilience. is method increases system resilience while balancing operator and user costs. is optimization is based on real-world environments, involving passenger flow fluctuations and travel times based on traffic conditions. Schedule Coordination. Bus schedule coordination has been researched over decades. Many models have been developed for optimizing bus systems while considering transfer passengers. Among them, Rapp and Gehner [1] first developed a four-stage interactive computerized system for transfer optimization, with minimum total transfer waiting time as its objective function. en, service quality has been considered by Andréasson [3] and Abkowitz et al. [4]. Since schedule coordination is difficult to solve analytically, a computerized tool has been developed for solving it as early as 1992 [5]. In 2001, Daganzo [6] generated a synchronized timetable for a given network by maximizing the number of simultaneous bus arrivals at the transfer nodes of the network. Since then, many extensions have been presented for solving this problem more realistically [7][8][9][10]. Relevant studies have explored whether timed transfers are appropriate for large networks with decentralized transfers [11] and how slack times can be optimized at transfer points with simple deterministic models [12] or simulation models [4]. Since passenger demand and vehicle travel times are stochastic, researchers developed probabilistic models for this problem [11]. Being limited by data sources and computational performance, research in this period mainly focused on simplifying and modeling schedule coordination in given numerical cases. In recent years, researchers have researched this problem with more detailed and realistic methods. Ting and Schonfeld [13] proposed a schedule coordination method for a multiple hub transit network, which is more challenging than that for single hub networks. As noted in [14], the bus transit network planning problem can be presented as a sequence of four main phases, in which bus timetable preparation is the second step. Some works focus on jointly optimizing this step with vehicle scheduling [15][16][17]. Wu et al. presented a comprehensive review of multiobjective resynchronizing of a bus timetable, involving model and solution [18]. With real-time information, transit agencies can now predict travel time with high accuracy, thus enabling reliable and dynamic timetable optimization to be combined with bus coordination [19,20]. Data driven modeling, as well as sufficient computational ability, makes schedule coordination more realistic and readier for application. A rail transit network typically has a backbone role in an urban public transport system, while bus routes are usually used to connect rail transit stations with the passengers' travel origins and destinations. Multimode transit schedule coordination is well researched [21,22]. A transport corridor is a generally linear area defined and served by one or more modes of transportation such as highways, railroads, or public transit, which share a common course. Transfers occur mainly at transfer stations where multiple routes converge. Hence, studies have been conducted to characterize different kinds of transfers and then optimize them [23][24][25]. Algorithms for solving this problem have been widely researched. Ibarra-Rojas and Rios-Solis have proved that the synchronized timetabling problem is NP hard. ey have proposed a multistart iterated local search algorithm and a mixed integer programming method [9]. However, heuristic methods are more usually used to quickly produce timetables. Ibarra-Rojas et al. extended the heuristics from their previous work to solve a multiperiod case [26]. Other methods, including a genetic algorithm [27,28], mixed integer programming [9], and tabu search [17], have also been used for solving this problem. Although previous studies considered travel times when coordinating schedules, these studies usually assumed stable travel times. However, different components including the links, the stations, and even the transit priority operations may be influenced by different factors and have different impact on travel time estimation [29,30]. For example, link travel times may be influenced by traffic, while dwell times may mainly be affected by passenger flows. With the development of advanced public transportation technologies, such as automatic vehicle location (AVL) and automatic fare collection (AFC) systems, more realistic situations with detailed travel times should be considered for schedule coordination. Resilience. Resilience in transportation research is divided into two parts: elasticity analysis [31,32] from the economic aspect and system resilience or vulnerability [33][34][35] from the physical aspect. In 1992, Wakabayashi and Iida proposed the concept of overall resilience of the system and introduced system resilience to traffic network analysis [36]. After 2000, researchers analyzed the resilience of transport system for two main cases: the recovery ability under foreseeable events (e.g., large-scale passenger flows and changes of road environment) [37,38] or unforeseeable events (e.g., hurricanes, snowstorms, and earthquakes) [39,40]. Compared with foreseeable system changes, unforeseeable changes are more serious and yield slower recoveries. In this field, researchers consider the change intensity and recovery ability of different traffic modes, such as rail transit compared with taxi. In addition, the study of combined resilience and vulnerability is also a hot topic. For example, the recovery cost of the system can be analyzed through the critical values [41,42]. In transport system resilience, the main research objective is the system's restorability, which contains transportation capacity and transportation time. Methodology e proposed method analyzes a transit corridor containing a multimode bus system connecting a rail transit station. Such cases are common in real-world operations since bus routes are usually used to connect rail transit stations with the passengers' travel origins and destinations. e following assumptions are made in this study: (1) e network of transit routes is predetermined and buses stop at every station along their routes (2) It is assumed to be independent of transit service quality, deterministic, and uniformly distributed over time during the specified time period (3) Preplanned transfer coordination is assumed under a no-hold policy (4) e capacity of each bus station is unlimited For coordinated operation, we search for optimized headways that are integer multiples of the base cycle in order to increase the possibility of immediate connections at transfer stations. To do so, schedules for all the routes have to be optimized jointly. In the next sections total system costs with timed transfers are formulated. en the station delay and signal priority intersection delay are checked to ensure that these optimized headways and slack times for each bus routes are reliable. Both uncoordinated and coordinated operations include nontransfer and transfer costs. Nontransfer costs include vehicle operating cost and passenger waiting cost. Transfer costs include slack-time cost, missed-connection cost, and dispatching-delay cost. Analytic solutions for uncoordinated operation are obtained. A heuristic algorithm for coordinated operation is also presented. All the results for a real-world case are verified by comparisons with the previous study of Ting and Schonfeld [13]. Symbols used in this paper are shown in Table 1. Predetermined parameters have certain baselines, which come from "TCRP 165: Transit Capacity and Quality of Service Manual" [43] and field research by the Shanghai Science and Technology Committee (unpublished reports): Uncoordinated Operation. is paper aims to optimize bus headway in a multimode transit system. e rail transit is a part of input of bus passenger flow volume. As we can get passengers' location by both smart card data and bus's GPS data, the passenger volume can be acquired as follows: (1) Passengers transferring from rail transit to bus can be identified from 2 consecutive smart card swiping records (rail transit to bus) in one time period (from 6:00 am to 12:00 pm) (2) Passengers transferring from one bus line to another can be identified as 2 consecutive smart card swiping records (bus line 1 to bus line 2) in 1 time period (from 6:00 am to 12:00 pm) (3) Other passengers can be identified as smart card swiping record In uncoordinated operation, the objective is to minimize the total system cost by optimizing the headway independently for each route. However, many factors, such as road delay, intersection delay, and station delay, can affect bus travel time [29,30]. With real-time information, transit agencies can now predict travel time with high accuracy [29,30,44]. e round-trip time, T k , of route k is the sum of travel times on all the links of route k. e travel time can be divided into three parts: link travel time, dwell time at stations (T 0 + T b ), and delay at signal priority intersections. e travel time can be expressed as In reality, since some bus routes have long headways, the travel time data are insufficient for analysis. Multiple routes' data can be gathered to obtain E(t r ). Figures 1 and 2 show a transit corridor with three bus routes in Shanghai. While the planned headway of route 936 is 30 minutes, it only operates 30 times a day, so link travel time data are insufficient. However, when we combine these three routes, we collect over 300 bus round trips per day. e detailed method for combining travel times of each route in the same corridor can be found in [45]. e vehicle size used here and the linear function of vehicle operating cost proposed by Jansson [46] are as follows: In (2), the demand Q k is multiplied by a maximum load factor L k to allow for more-than-expected passengers/bus. e operating cost of route k is the product of the needed fleet size T k /h k and the unit operating cost. e total operating cost is According to the stochastic process for passenger arrivals at stops (randomly and uniformly over time), the waiting time may be estimated as that by Welding [47] or Osuna and Newell [48]: e waiting cost is a product of waiting time w k , unit waiting cost μ, and passenger value Q k : When bus routes operate independently, the transfer waiting time is equal to w k , so C f can be formulated as Journal of Advanced Transportation Total boarding delay cost (RMB/min) - e lowest total cost (RMB/min) in time period -C sig Delay cost at signal priority intersection (RMB/min) -D mk Number of passengers already on board at transfer center m on route k (passengers/min) - Transfer demand from other routes to route k at transfer center m (passengers/min) - Probability density function of arrival time on route k g jk Greatest common divisor of β j and β k - Frequency of missing signal priority (buses/hour) - When the passenger flows per bus increase, bus dwell times at stops also increase. e stopping delay can be expressed as follows: Although the bus wait time at intersections is included in E(t r ), there should be no delay at intersections with signal priority. Priorities including bus lane along with signal priority have been applied on particular bus routes to improve their operation efficiency and reliability. However, since priorities are only given in a certain period during a signal circle, when delays exceed this certain period, buses may miss the signal priority. If buses operate on exclusive lanes, these delays are mainly caused by boarding delay at stations. us, signal priority missing cost is introduced as follows: During the optimization process, μ 3 is set extremely high to avoid missing signal priority. To sum up, the total cost is In this paper, the goal of the optimization process is to maximize resilience in the transfer system. e resilience here is defined as the ability to resist and recover from passenger flow fluctuation. In current typical studies, when analyzing system resilience, we first define (10) as the system service efficiency function. Based on the system efficiency function, the resilience function can be defined by the system efficiency rate (C T /C T0 ). e system resilience function can be expressed as Jiangsu road metro station Transit corridor Figure 1: Layout of the transit corridor in Shanghai. Journal of Advanced Transportation e optimization process is to get the maximum R. Coordinated Operation. Slack-time cost includes passenger cost and vehicle cost. Specifically, it can be divided into three parts: e first term is the slack-time delay cost to passengers already on board. e second term is the waiting cost for passengers transferring to route k. e last term is the increased vehicle operating cost due to the slack time at each transfer station on route k. e slack-time cost can be expressed as e transfer waiting time should consider the difference in headways and slack times between two routes. In real cases, the passenger flows of different routes, and hence the optimized headways of those routes, may differ greatly. erefore, this paper uses the Integer-Ratio Headways, introduced in [13]. In this case, the vehicle operating cost, passenger waiting cost, and in-vehicle cost are the same as for the uncoordinated operation. In [13] passenger waiting times are analyzed based on the condition that the headways as well as scheduled travel times between transfer stations are the same or integer multiples of the same cycle time. e waiting time is us, the transfer waiting times between connecting routes j and k are as follows: Assuming reasonably that passengers may miss at most one bus, the missed-connection cost and delay-cost formulations are simplified. Since buses from both routes meet at the transfer station once every h j × h k /(g jk × y) minutes, the average transfer demand is q jk × g jk × y/h k for each connecting bus. e missed-connection cost is e dispatching-delay cost is e optimization procedure is specified as follows: We first optimize the headway for each bus route separately. We assume T b � 0 and T sig � 0. Since these two parameters are 0, the total cost C T in our model will be the same as in [13]. e optimal headway of route k can be obtained by setting the first derivative of the total cost with respect to headway h k to be zero and solving for h k . e first derivative is . (17) us, the optimal h k is e second derivative of total cost with respect to h k is Since each component in (19) is always positive, the second derivative is nonnegative, and thus h * k globally minimizes the total system cost; then, the resilience is maximized. e maximal headway (policy headway) permitted between two adjacent buses in route k, h max k � S k l k /Q k , must be checked for each route. As in [13], the base cycle is optimized as a round fraction of 60 min (i.e., 60 min divided by an integer). For optimizing coordinated operations, the headways of different bus routes at the same transfer station should be optimized for multiples of the base cycle y. Based on that, slack time is set to minimize the expected total costs of bus operations and passenger waiting times, including those due to missed connections, which are affected by slack times. Compared with the heuristic algorithm provided in [13], the optimizing goal and computational basis are changed here from system cost (C T ) to system resilience (R). Besides, more parameters are checked. We check h * k to find if T b > 0 or T sig > 0. Since T i precludes signal priority, T sig should be 0. We rank T b in ascending order, reduce h * k to reduce T b , update T k , and calculate h * k . is procedure is repeated until N k � 0 and no significant further improvement is obtained. Background of is Case. is paper focuses on a bus corridor which connects a rail transit station and includes multiple bus routes. It contains a rail transit station (Jiangsu Road Rail Transit Station, which is among the busiest in Shanghai) and a bus corridor (Yan'an Road transit corridor, which is the earliest established and largest transit corridor in Shanghai). We formulate a system resilience function and find the combinations of headways and slack times which maximize that function. ere are two bus network configurations in this transit corridor, as shown in Figures 2 and 3. e pre-2017 network, in which there were three bus routes operating concurrently, is shown in Figure 2. After 2017, route 71 serves as the main bus route in this corridor. It has been provided with exclusive bus lane and transit signal priority. First, analytical solutions for optimal headways in the uncoordinated transit network are found. en, a heuristic algorithm is developed to jointly find the headways and slack times that maximize the system resilience for the coordinated transit network. Yan'an Road bus lane starts at Zhongshan Road and ends at Gubei Road, with a total length of 10 km. It opened in about 2000 as Shanghai's first bus lane. e section considered here extends from the West Yan'an Road-Kaixuan Road (KX) bus stop to the Mid Yan'an Road-Shimen Road No. 1 (SM) bus stop. Its characteristics are illustrated in Figures 1-3. e data acquisition process uses an automated collection system and related equipment based on AVL, to gather GPS and smart card data. e bus routes have been changed in early 2017. us, our case study uses two data series: February to July in 2015 and February to July in 2018. e raw data includes the following data types: Smart card data: card id, fare collection machine id, time point Vehicle-route data: vehicle ID, route ID, route name Bus stop data: stop ID, stop name, stop longitude, stop latitude Route-stop data: route ID, route name, operating direction, stop number GPS data: route ID, vehicle ID, time point, longitude, latitude, speed, direction angle e baseline parameter values, adopted for the present work from "TCRP 165: Transit Capacity and Quality of Service Manual" [43] and field research by the Shanghai Science and Technology Committee (unpublished reports), are B k � 4.0 RMB/bus-min, μ 1 � 1.5 RMB/passenger-min, μ 2 � 5.0 RMB/passenger-min, μ 3 � 10,000 RMB/passengermin, and v � 1.0 RMB/passenger-min. In real networks, the travel times on each link between two hubs may differ considerably by period and travel direction. In this case, the travel direction is from suburban to urban area, and the time period is 6:00 am to 12:00 pm. In both conditions, we calculate the total cost by five methods: e first one is planned operation, which means we use the headways provided by the transit agency. In the second method, called uncoordinated operation, the headways are optimized independently for the different routes. In the coordinated operation, the headways and slack time of different routes are optimized jointly, using the method in [13], based on average passenger volume (method 3) and maximum passenger volume (method 4). e method proposed in this paper is presented as the fifth method, called the resilience optimized operation. In this method, the headways and slack times of different routes are optimized jointly, considering boarding delay and missed signal priority delay. Initial (Pre-2017) Bus Routes. is corridor included three bus routes: 71, 127, and 936, as shown in Figure 1. e Journal of Advanced Transportation configuration of these bus routes is shown in Figure 2. Station ids a2 to a7 mean stations on route 71, station id b means stations on route 127, and station id c means stations on route 936. Note that when i � 1 to 7, a i , b i , and c i are the same station on different routes. However, a1, b1, c1, a8, b8, and c8 are different stations. e blank points c4, b5, and c6 mean that there are no stations on these routes, and thus buses pass through without stopping. e comparison of different operation methods is shown in Table 2. Table 2 shows that all four optimizing methods increase the system resilience and reduce the total cost compared to the planned operation (method 1). e method proposed in this paper can increase the system resilience more than the other methods and does not increase the total cost (304.499 RMB compared with 306.623 RMB and 303.976 RMB). It performed well in real cases with real passenger volumes. Coordinated operation is preferable to uncoordinated operation. Its operation cost is higher than that of uncoordinated operation, but the waiting cost and transfer cost decrease more, so the total cost decreases, as shown in Table 2. After Changing Bus Routes. Bus route 71 is the main route in this corridor. It operates on an exclusive lane and has signal priority. At stations 4 and 10, there are two feeder bus routes: route 1250 (marked as route a) at station 4 and route 974 (marked as route b). e configuration of these three bus routes is shown in Figure 3. e comparative results for different operation methods after bus route restructuring are shown in Table 3. Table 3 shows that all the four optimizing methods increase the system resilience and reduce the total cost compared to the planned operation. Coordinated operation is preferable to uncoordinated operation. Its operation cost is higher than that of uncoordinated operation, but the waiting cost and transfer cost decrease more, so the total cost decreases. e table also shows that bus route 71 misses signal priority 5 times per hour when it operates as planned. All the optimized operations reduce the missing times, and Detailed Comparison. To understand how these results arise, we take the results of initial bus routes to compare these five methods in detail within the whole time period. e comparison is based on the results in February to July 2018. e minute-based evaluation graph is shown below. From Figure 4, we can understand the following: (1) e efficiency rate (C T /C T0 ) decreases when morning peak period starts and increases when it ends. All these 5 methods can make the system recover from passenger volume increasing, since the efficiency rates of all these 5 methods increase to 1 finally. (2) e planned method performs the worst. When passenger volume increases, the efficiency rate decreases and reaches a very low level. (3) e uncoordinated method performs better than the planned method but worse than the coordinated methods. (4) When we use the methods introduced by Ting and Schonfeld [13], the results differ based on different passenger volume. When we use average passenger volume, the efficiency decreases greatly since the headway is too long. At the maximum passenger volume, the operation cost is high even when passenger volume is not very high (before 6:30 am and after 9:30 am), so the efficiency rate recovers slowly. (5) e proposed method performs best since it uses reasonable headways with realistic passenger volume. According to "Transit Capacity and Quality of Service Manual" (3 rd edition) [43], the service quality can be divided into five levels. In this case, we can use this standard to divide the efficiency rate into five levels, which are shown in Table 4. en, we compare these 5 methods based on the results in Figure 4 and the results are shown in Table 5. From Table 5, we can find that the proposed method not only resists the efficiency rate decrease but also has the shortest time for recovering to level A, so it has the highest resilience. Besides, among these methods, coordinated operation performs better than uncoordinated operation. Planned operation performs the worst. Coordinated operation avoids level D, and the proposed method avoids level C. Conclusions and Recommendations is paper proposed an extension of the schedule coordination method by Ting and Schonfeld [13] for jointly optimizing headways and slack times in coordinated schedules for public transportation routes within the aspect of resilience. is method is applied in a realistic case, with passenger flow fluctuations as well as travel time variability based on operating conditions. e main contributions are as follows. Firstly, this paper applies and extends a schedule coordination model within a real multihub environment (stations 2-7 in Figure 2 and stations 4, 6, and 10 in Figure 3), considering passenger flow fluctuations and whether transit priority is provided, within the new resilience angle. is advance improves this method's practicality. Secondly, the case study presented here shows that the coordinated schedule obtained with the proposed method can ensure high system resilience based on the verification with realistic passenger volumes, which means it performs well in resisting and recovering from passenger volume fluctuations and traffic variations. It balances the operation cost and passenger cost. e proposed method improves the resilience of the schedule. irdly, this paper focuses on a transit corridor, combining data from several bus routes for analysis. It improves data volume and accuracy. is paper allows transit agencies to optimize coordinated schedules considering passenger flow fluctuations and transit priority. Resilience is important in analyzing and optimizing transit schedules. e value of the resilient schedule coordination method proposed in this paper is demonstrated for balancing operator and user costs in real world, considering (6) 37 (4) 0 (0) 0 (0) 0 (0) Level E (min) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Note. e variance is shown in the brackets. passenger flow fluctuations and traffic variations. Further research may not only consider more parameters in different environments, but also explore more efficient algorithms for solving this problem. Besides, this method can be further extended from corridors to urban transit networks, intercity trains, buses, and airlines. Data Availability e GPS and smart card data used to support the findings of this study are restricted by the Shanghai Science and Technology Committee. Requests for data will be considered by the corresponding author for researchers who meet the criteria for access to confidential data. Conflicts of Interest e authors declare that they have no conflicts of interest. Authors' Contributions Study conception and design were conducted by Xiongfei Lai, Jing Teng, and Paul Schonfeld. Data were collected by Xiongfei Lai and Jing Teng. Analysis and interpretation of results were performed by Xiongfei Lai. Manuscript draft was prepared by Xiongfei Lai Jing Teng, Paul Schonfeld, and Lu Ling. All authors reviewed the results and approved the final version of the manuscript.	2020-06-18T09:03:27.782Z	2020-06-15T00:00:00.000	{ "year": 2020, "sha1": "43edb058303ece8dba4cd93b65c9f0cd2d066d2a", "oa_license": "CCBY", "oa_url": "https://downloads.hindawi.com/journals/jat/2020/5398298.pdf", "oa_status": "GOLD", "pdf_src": "Adhoc", "pdf_hash": "d0a15c80384c917e55aa30dfcf30c884735fddd0", "s2fieldsofstudy": [ "Business" ], "extfieldsofstudy": [ "Computer Science" ] }
266769288	pes2o/s2orc	v3-fos-license	Long-Term Outcomes of Ultrasound-Guided Thread Carpal Tunnel Release and Its Clinical Effectiveness in Severe Carpal Tunnel Syndrome: A Retrospective Cohort Study Ultrasound-guided thread carpal tunnel release (TCTR) was proposed as an effective and safe surgical technique with faster recovery and fewer complications. This study was conducted to confirm the long-term outcomes after TCTR and verify its clinical effectiveness in severe carpal tunnel syndrome (CTS) for more insights into TCTR procedure. A total of 168 TCTR procedures were performed in 152 individual patients by two physiatrists during 36-month period. In an assessment of 82 hands, surgical outcomes of 2 years after TCTR could be obtained, and the grade 6 CTS group of 21 hands, classified as extremely severe grade by Bland’s classification, was compared with other severity groups (grade 1–5). The Boston Carpal Tunnel Syndrome Questionnaire (BCTQ) was used to assess surgical outcomes. No adverse events occurred in all cases including the case of severe CTS and anatomical variants. TCTR showed significant improvement in BCTQ scale within 1–2 weeks, which continued up to 2 years with no recurrence (p < 0.01). Although slower and more progressive than the other severity group, there was also significant improvement relative to the BCTQ scale around 4 weeks after procedure in the grade 6 CTS group (p < 0.05). With the familiarity of ultrasound, ultrasound-guided TCTR is an effective and reliable surgical treatment for CTS in long-term outcomes and in severe CTS. Introduction Carpal tunnel syndrome (CTS) is the most frequent of the compressive neuropathies in the upper extremity and defined by compression and/or traction of the median nerve at wrist level.Its prevalence is between 4 and 5% among the general population, particularly affecting individuals between 40 and 60 years of age [1]. The treatments for non-deficit forms of CTS are medication, a night-time immobilization brace, and corticoid infiltration.However, in the case of resistance to conservative treatment or deficit forms, surgical treatment is indicated to achieve a reduction in intratunnel pressure by sectioning the transverse carpal ligament (TCL) [2]. Open carpal tunnel release (OCTR) is most frequently performed surgical procedure with relatively favorable outcomes [3].However, because an incision of 3-4 cm is made, cutaneous nerve injury, hypertrophic scar with tenderness, pillar pain, and a delay in return to work are known to occasionally occur [3,4].Endoscopic carpal tunnel release (ECTR) was introduced to reduce these complications in the late 1980s and resulted in a lower incidence of scar-related complications and earlier return to work than OCTR, but the risk of median nerve injury by insertion of an endoscope into carpal tunnel, a steep learning curve and its significant setup time are drawbacks [4,5]. With the development of interventional procedures using high-frequency ultrasound, ultrasound-guided percutaneous carpal tunnel release (USCTR) using various dividing instruments was proposed to overcome these limitations of OCTR and ECTR [6].Among the USCTR, the thread carpal tunnel release (TCTR) was first proposed by Danqing Guo and his colleagues [7], which can transect the TCL without skin incision, leaving only two needle punctures [8].Compared to other methods of USCTR, TCTR has the advantages of not using a sharp dividing instrument, requiring no repetitive cutting motions, and less iatrogenic injury by using the hydrodissection technique [9]. The clinical effectiveness and safety of TCTR, as well as its faster recovery and fewer complications compared to OCTR and ECTR, were reported by our previous study and other groups [8][9][10][11][12][13], and TCTR was expected to become the more popular surgical procedure for the treatment of CTS gradually.Therefore, it is necessary to investigate what extent TCTR can be applied for the surgical treatment of CTS in various clinical setting.However, there is a relative paucity of data available with reference to the long-term results of TCTR and its clinical effectiveness in severe CTS. The purpose of this study was to determine the scope of clinical application of TCTR procedure by confirming the long-term outcomes after TCTR over a period of 2 years and verifying its clinical effectiveness in severe CTS. Participants A total of 184 hands in 168 patients (>18 years old) with symptoms of CTS were enrolled in this study from 1 March 2020 to 28 February 2023.Clinical symptoms that allowed the suspicion of CST Symptoms were hand numbness, tingling sensation, pain or weakness within median nerve territory and positive signs with provocation tests, such as Tinel at wrist and Phalen.Ultrasonographic and electrophysiologic evaluation were conducted in all patients for confirming the diagnosis and classifying the severity of CTS.For more detailed analysis, Bland's more subdivided classification was used [14].All enrolled patients failed to conservative treatment for more than 3 months. Equipment All surgical procedures were performed under local anesthesia without tourniquet by two experienced physiatrists in the clinical procedure room using an LOGIQ S7 ultrasound machine (GE Healthcare, Seoul, Republic of Korea) fitted with a ML6-15 (8 MHz) linear array transducer.Smartwire CTS (0.27 mm in diameter, Smartwire Inc., Seoul, Republic of Korea) was used as cutting thread and it was developed for the percutaneous dissecting thread technique with higher cutting force, better visibility on ultrasound, and easier handling by additional coating of thin titanium nitride [13,15]. Preoperative Evaluation Preoperative ultrasound evaluation was performed before surgical procedure in all participants.The presence of intra-tunnel space occupying lesion, such as ganglion cyst or intra-tunnel aberrant muscle, such as proximal origin of lumbricals was evaluated first.Then, the relevant anatomical structures were evaluated including the course of median nerve, third common digital nerve (TCDN), RMB, superficial palmar arch (SupPA), and distal end of TCL, which called "duck's beak" due to its shape [16].Finally, it was confirmed whether there were some anatomical variations that could affect the surgical procedure, such as hypertrophic abductor pollicis brevis (APB), ulnar side branching of RMB, bifid median nerve with or without persistent median artery, Berrettini communicating branch (BCB), variant of superficial palmar arch (SupPA), and aberrant hypothenar muscle, if present. Surgical Procedure First, the entry and exit points were determined and marked on the skin.The entry point was placed at the middle of palm, where the 3rd ray and a horizontal line from the apex of the interdigital fold between the thumb and index finger crosses perpendicularly.The exit point was placed just medial to the palmaris longus tendon, 1-2 cm proximal to the distal wrist crease.After sterile preparation and draping, 1-2 mL of lidocaine without epinephrine was injected subcutaneously at the entry and exit points with 27G needle.Under the ultrasound guidance, another 27G 2-inch needle with 5 mL of normal saline was inserted at the entry point and advanced over the SupPA and under the distal edge of TCL through the superficial and deep palmar aponeurosis (PA) to open up the distal carpal tunnel space with hydrodissection then removed. Following the pathway made by previous step, a pre-bent 18G Tuohy needle (20-degree angle 1.5 cm distal to the tip with bevel up and 20-25-degree angle 2.0 cm proximal to the tip) with 5 mL of normal saline was inserted at the entry point and advanced through the dorsal side of the TCL with additional hydrodissection.After the needle tip exited at the determined exit point, a cutting thread was passed through the needle.Leaving the thread at the dorsal side of the TCL, the needle was removed. With continuous ultrasound guidance, another pre-bent 18G Tuohy needle (10-degree angle 1.0 cm distal to the tip with bevel up) with 5 mL of normal saline was inserted at the same entry point and advanced through palmar side of TCL.After the needle tip exited at the same determined exit point, the cutting thread left at the dorsal side of the TCL was passed through the needle distally then looped around the TCL by removing the needle. Using the axial and sagittal sonographic view, the absence of RMB, TCDN, BCB, and SupPA within the loop of cutting thread was confirmed.Then, the TCL was dissected by moving the cutting thread reciprocally.After the removal of the thread, the patient's dissected TCL was confirmed through ultrasound examination and RMB was checked immediately by asking thumb opposition. After hemostasis by gentle compression, compressive dressing is applied with commercial wrist brace. Postoperative Instruction All patients were allowed to commence the simple daily activities as tolerated immediately after procedure and instructed to remove the dressing by themselves and wash their hands in 3 days.Lifting heavy objects or clenching hands was prohibited until 2 weeks after surgery. Participant's occupation was classified into 3 categories: white-collar worker, bluecollar worker with light, repetitive hand motions, and blue-collar worker with heavy, repetitive hand motions, and return to work was permitted in 3 days, 2 weeks, and 3-4 weeks, respectively, as tolerated.However, there were no restrictions on any activities or return to work, if no pain. Materials The clinical effectiveness of TCTR was assessed by Boston Carpal Tunnel Syndrome Questionnaire (BCTQ), which is a validated patient-centered measure for quantifying the symptom severity and disability [13].BCTQ scores range between 1 and 5, with 1 representing the no pain or no difficulty and 5 representing the most severe symptom or functional limitation.The BCTQ scores of pre-TCTR and 1 week after TCTR were measured in the clinic, and the BCTQ scores of 2 weeks, 4 weeks, 2 months, 3 months, 6 months, 1 year, and 2 years after TCTR were measured via google survey link or phone interview in most cases. The follow-up period to determine the long-term prognosis after TCTR was set 2 years, because the mean time to revision surgery was 1.23 years after OCTR or ECTR [17]. Because of considerable differences among the post operative outcomes according to the severity, especially in grade 6 CTS [14], Bland's classification was used for severity classification instead of Steven's relatively simple one [18].The effectiveness of TCTR in grade 6 CTS was evaluated separately from other grades, and the proportion of "unchanged" plus "worse" after TCTR was measured additionally in that grade. Statistical Analysis Medical records collected by two physiatrists were anonymized by separated data collector and then sent to the data analyzer.Statistical analysis was carried out using SPSS statistics version 22 (IBM, Armonk, NY, USA).The normality of the distribution was assessed by the Shapiro-Wilk test.The paired t-test and Wilcoxon signed-rank test were performed to analyze pain reduction and functional gain over time in each group.The independent t-test was performed to compare the grade 6 CTS group with other severity group (grade 1 to 5) at each outcome-measured period.Statistically significant level was set to p-value of <0.05. Long-Term Outcomes A total 168 hands in 152 individual patients were included in this study and longterm outcomes over a period of 2 years could be evaluated in 82 hands.Their pre-TCTR BCTQ severity score was 3.21 (SD ± 0.82) and showed a significant improvement of 1.65 (SD ± 0.55) in 1 week (p < 0.01).This improvement continued progressively until 4 weeks (BCTQ severity score = 1.41,SD ± 0.55) and plateaued after that.At 1 year and 2 years after TCTR, BCTQ severity score showed 1.05, SD ± 0.07 and 1.03, SD ± 0.05, respectively, and was well maintained without worsening up to 2 years (Table 1).Regarding hand function, the pre-TCTR BCTQ function score was 2.42 (SD ± 0.84) and showed a significant improvement of 1.76 (SD ± 0.56) in 2 weeks (p < 0.01).Additional improvements were shown until 3 months (BCTQ function score = 1.32,SD ± 0.34) and plateaued thereafter as the severity remained stable.At 1 year and 2 years after TCTR, BCTQ function score showed 1.08, SD ± 0.11 and 1.04, SD ± 0.08, respectively, and was well maintained without worsening up to 2 years (Table 1). Clinical Effectiveness in Severe Carpal Tunnel Syndrome Twenty-one hands of grade 6 CTS were analyzed separately to confirm the clinical effectiveness of TCTR in severe CTS. In contrast to the other severity group, where a notable improvement in the BCTQ severity score occurred within 1 week and plateaued by 4 weeks post-TCTR, the grade 6 CTS group exhibited comparatively less improvement during the first 1 to 4 weeks.The plateau for this group was reached around 3 months after TCTR, as illustrated in Figure 2A Despite a slower and less distinct recovery compared to severity, hand function showed an overall similar pattern to severity and significant improvements in BCTQ function score were shown after TCTR. Pre-TCTR BCTQ function scores were 2.77 (SD ± 0.63) in the grade 6 CTS group and 2.42 (SD ± 0.87) in other severity group.In contrast to BCTQ severity score, there was significant difference in pre-TCTR BCTQ function score between two groups (p = 0.029).Both groups showed significant improvements in BCTQ function score until 6 months as 1.37 (SD ± 0.28) and 1.14 (SD ± 0.19), respectively (p < 0.05 in the grade 6 CTS group, p < 0.01 in other severity group) (Table 2). In contrast to the other severity group, where a notable improvement in the BCTQ severity score occurred within 1 week and plateaued by 4 weeks post-TCTR, the grade 6 CTS group exhibited comparatively less improvement during the first 1 to 4 weeks.The plateau for this group was reached around 3 months after TCTR, as illustrated in Figure 2A Despite a slower and less distinct recovery compared to severity, hand function showed an overall similar pattern to severity and significant improvements in BCTQ function score were shown after TCTR. Pre-TCTR BCTQ function scores were 2.77 (SD ± 0.63) in the grade 6 CTS group and 2.42 (SD ± 0.87) in other severity group.In contrast to BCTQ severity score, there was significant difference in pre-TCTR BCTQ function score between two groups (p = 0.029).Both groups showed significant improvements in BCTQ function score until 6 months as 1.37 (SD ± 0.28) and 1.14 (SD ± 0.19), respectively (p < 0.05 in the grade 6 CTS group, p < 0.01 in other severity group) (Table 2). In the other severity group, a significant improvement in BCTQ function score was shown within 2 weeks and plateaued in 3 months after TCTR, but a much slower and progressively improving trend was observed throughout 6 months after TCTR in the grade 6 CTS group (Figure 2B). The proportion of "unchanged" plus "worse" after TCTR in the grade 6 CTS group was 9.5% (2 in 21 hands).In the other severity group, a significant improvement in BCTQ function score was shown within 2 weeks and plateaued in 3 months after TCTR, but a much slower and progressively improving trend was observed throughout 6 months after TCTR in the grade 6 CTS group (Figure 2B). The proportion of "unchanged" plus "worse" after TCTR in the grade 6 CTS group was 9.5% (2 in 21 hands). Adverse Events and Others No adverse events were reported, including nerve injury, vascular damage, or infection even in the case of anatomical variants, such as bifid median nerve or ulnar side branching of RMB.The surgical time from the first local anesthesia to the resection of TCL was within 20 min and more than 5 min of compression for hemostasis was not necessary in all cases.All patients could commence their activities of daily living and return to work according to the instructed schedules as follows: within 3 days in white-collar workers, 2 weeks in blue-collar workers with light, repetitive hand motions, and 3-4 weeks in blue-collar workers with heavy, repetitive hand motions after the procedure, except one case. One patient suffered painful wrist swelling after the procedure and iatrogenic partial resection of accessory abductor digiti minimi (ADM) muscle was identified on a follow-up ultrasound examination.However, his symptoms subsided spontaneously without any invasive interventions, and he could resume the daily activities in 2 weeks.He was a video camera man with heavy, repetitive hand motions and returned to work in 5 weeks after TCTR without restrictions. No patient was necessary to perform revision surgery except for one case.Nearly complete relief of symptoms was shown immediately after TCTR in that case, but relapse of symptoms occurred in 5 months after the procedure.Due to the patient's preference, revision TCTR was performed again, and the patient had complete symptom relief without recurrence. Discussion Ultrasound-guided TCTR was proposed by Danquing Guo and colleagues first.They verified the clinical effectiveness and safety of TCTR and improved the technique of procedure through their cadaveric and clinical studies [7][8][9].Other groups also reported faster clinical improvement in various parameters and a shorter return to work compared to OCTR and ECTR with fewer complications [10][11][12].With these advantages, TCTR seems to play an important role for the surgical treatment of CTS in the future.However, there was relatively little evidence of long-term outcomes after TCTR and its clinical effectiveness in severe CTS.In this clinical perspective, the objective of this study was to confirm the long-term outcomes of a 2-year period after TCTR and verify its clinical effectiveness in severe CTS for more insights about the scope of its clinical application. Current Surgical Technique The modified TCTR technique suggested by Danquing Guo and colleagues [8], which was also considered standardized technique in other groups [10][11][12][13], was used in this study.However, our technique differed from Guo's modified TCTR technique with two minimal modifications.First, because the boundary between PA and TCL was unclear on ultrasound in some cases, especially at the proximal part of TCL, PA was included in the looped thread and not preserved in this study to avoid incomplete resection of TCL.However, there was no significant difference from other clinical studies of TCTR with PA preservation in pillar pain and recovery time to work [8,12,13].Second, during the second Tuohy needle passage, which was palmar to TCL, needle-tip manipulation with bevel turning was used to locate the thread along the fusiform shape of proximal part of TCL (Figure 3A) and avoid unnecessary resection of aberrant hypothenar muscle, especially in the muscular type of accessory ADM [19].After we experienced one case of self-limited painful wrist swelling caused by iatrogenic partial resection of accessory ADM, this modification was used to reduce post-operative pain and time to recovery. Long-Term Outcomes Long-term outcomes of the 2-year period could be evaluated in 82 hands.Because the mean time to revision surgery was 1.23 years after OCTR or ECTR [17], long-term follow up of 2 years was a reliable period for the evaluation of recurrence after TCTR. As per previous clinical studies [8][9][10][11][12][13], the recovery process of TCTR was also much faster than that of OCTR or ECTR in this study.There were significant improvements in 1 or 2 weeks, and they continued progressively about 4 weeks and plateaued after that in both severity and function.Until 2 years after TCTR, these improvements were well maintained without recurrence of symptoms. Clinical Effectiveness in Severe CTS One of the purposes of this study is to determine whether TCTR can be performed effectively in severe CTS.Although the clinical effectiveness of TCTR in severe CTS was reported in some previous clinical studies [10][11][12][13], the classification proposed by Stevens, which divides the CTS simply into 3 grades as mild, moderate, and severe, was used in all of them.This classification may be useful to determine the proper treatment options according to the individual severity, but it cannot reflect degree of median nerve damage Long-Term Outcomes Long-term outcomes of the 2-year period could be evaluated in 82 hands.Because the mean time to revision surgery was 1.23 years after OCTR or ECTR [17], long-term follow up of 2 years was a reliable period for the evaluation of recurrence after TCTR. As per previous clinical studies [8][9][10][11][12][13], the recovery process of TCTR was also much faster than that of OCTR or ECTR in this study.There were significant improvements in 1 or 2 weeks, and they continued progressively about 4 weeks and plateaued after that in both severity and function.Until 2 years after TCTR, these improvements were well maintained without recurrence of symptoms. Clinical Effectiveness in Severe CTS One of the purposes of this study is to determine whether TCTR can be performed effectively in severe CTS.Although the clinical effectiveness of TCTR in severe CTS was reported in some previous clinical studies [10][11][12][13], the classification proposed by Stevens, which divides the CTS simply into 3 grades as mild, moderate, and severe, was used in all of them.This classification may be useful to determine the proper treatment options according to the individual severity, but it cannot reflect degree of median nerve damage in CTS accurately. Bland and his colleagues categorized the severity of CTS into 6 grades and evaluated the overall subjective outcomes after OCTR or ECTR using five-point rating scale: "cured", "much better", "better", "unchanged" and "worse" [20].According to the more detailed classification suggested by Bland, the severe grade proposed by Stevens was subdivided into severe (grade 4), very severe (grade 5), and extremely severe (grade 6) [14].Interestingly, the surgical outcome in grade 6 CTS was much worse than that in grade 4 or grade 5.The proportion of "unchanged" plus "worse" after OCTR or ECTR in grade 6 was 31.7%, but only 12.2% in grade 4 and 13.7% in grade 5 [14].Thus, Stevens' classification was not suitable to evaluate the exact outcomes following carpal tunnel release in severe CTS.To verify whether TCTR can be performed effectively in severe CTS, it would be the better way to separately evaluate the outcome in grade 6 CTS alone, excluding grade 4 and 5, which have relatively favorable surgical outcomes.If the surgical outcomes of TCTR are favorable even in grade 6 CTS, TCTR can be proposed as an effective surgical treatment for CTS regardless of its severity. Twenty-one hands of grade 6 CTS were included in the current study.Unlike the other severity group, which showed significant improvement within 1-2 weeks after TCTR, the BCTQ severity score took 2-4 weeks to improve and BCTQ function score improved gradually over 6 months in the grade 6 CTS group.Although there were transient exacerbations of symptoms in some cases, most patients of the grade 6 CTS group showed overall favorable outcomes in both severity and function within 6 months.The transient aggravation of symptoms was presented 1-2 weeks after TCTR, not immediately after procedure, then subsided in 1 to 2 months spontaneously.Before the procedure, practitioners should warn about the possibility of transient worsening of symptoms after TCTR and assurance of its benign course, especially to the patients of grade 6 CTS.This slow recovery and transient exacerbation of symptoms in that grade may be due to time-consuming nerve regeneration and resulting hypersensitivity.The increase in intratunnel pressure during the TCTR procedure also may be the potential cause of this transient exacerbation. Although the number of grade 6 CTS included in this study was much smaller than that in Bland's, as the two studies consisted of 21 hands vs. 483 hands, respectively [14], the proportion of "unchanged" plus "worse" after TCTR in the grade 6 CTS group was 9.5% (2 in 21 hands) without any "worse" cases, which was much smaller than 31.7%, as Bland reported.The two "unchanged" patients' symptoms were not too severe to interfere their daily living, and they did not want an additional surgical procedure to be performed.Because their data were collected at 6 months and 1 year after TCTR, respectively, it seems to be relatively insufficient time period to confirm their overall recovery. Although the recovery process was relatively slow and progressive, TCTR was effective and showed favorable outcomes even in grade 6 CTS.Thus, it was verified that TCTR can be performed effectively in most CTS regardless of its severity. Considerations for Anatomical Variations Major and digital nerve injuries were very rare in OCTR and ECTR [21] and also are not reported in the clinical studies of TCTR [8][9][10][11][12][13] including this study.However, due to the high variability of RMB with regard to its course in relation to TCL [22], RMB may be susceptible to iatrogenic injury during TCTR, especially in the type of ulnar side branching.However, because the incidence of ulnar side branching of RMB was very low, ranging from 0.9 to 3.6% [22], and its reliable visualization on high-resolution ultrasound [23], iatrogenic injury of RMB can be avoided through the thorough ultrasound evaluation of median nerve before and during TCTR.In this study, 2 cases (1.2%) of ulnar side branching of RMB were identified and performed TCTR without any complications (Figure 3B). Another considerable variation of RMB is the transligamentous type with a pooled prevalence rate of 11.3% [22].Because of the possibility of compression within the reticular fibers, isolated RMB neuropathy can occur in transligamentous type [24].Because the pooled prevalence of transligamentous course was 23.4% in patients with hypertrophic APB, as compared to 1.7% without it, practitioners should consider the potential RMB variants in patient with hypertrophic APB.Although isolated RMB neuropathy was either rare or possibly underdiagnosed [23] and was not encountered in current study, OCTR seem to be indicated instead of TCTR in isolated motor deficiency because the exploration of RMB may be needed [2,25]. The digital nerves most likely to be damaged during TCTR are the TCDN of the median nerve and the BCB between the median and ulnar nerve, which is present in around 80% [26].However, because of their clear visualization on high-frequency ultrasound and the hydrodissection technique, iatrogenic injury could be prevented during TCTR in this study.This holds true, irrespective of the presence or classification of the nerves, aligning with findings from other clinical studies [8][9][10][11][12]. The bifid median nerve, with a prevalence of 9-13% [27], can make the TCTR procedure difficult by narrowing the safety zone, which is the space between the median nerve and ulnar artery.We identified 11 cases (6.5%) with a bifid median nerve, which were not excluded but instead included in our study.These cases underwent TCTR without encountering any adverse events.Despite instances where a portion of the thread was positioned palmar to the ulnar-sided bifid median nerve, minimal movements along the long axis of the median nerve did not result in any damage.This was attributed to the smooth surface of the thread. Although SupPA had 18.7% of incomplete variants [28], which was also encountered in our study, initial hydrodissection was performed successfully through just under the distal part of TCL in all cases. Reoperation after TCTR Another consideration is the possibility of reoperation after TCTR.The reported incidence of revision surgery was 1.4-4.4% in OCTR and 2.8-6.5% in ECTR [17,21].Although the cumulated number of cases that underwent TCTR was much smaller than that of OCTR and ECTR, the pooled reoperation rate of TCTR was about 1.4% (7 in 497 hands) including one case of revision TCTR in current study [8][9][10][11][12][13].Because the focal swelling of median nerve with an hourglass deformation was a prominent feature on follow-up ultrasound evaluation in that case, the incomplete release of TCL was thought to be a more reliable cause of recurrence rather than adhesion or scar tissue formation.Most cases of reoperation after TCTR were related to the incomplete division of distal TCL in early stage of learning curve.However, after the introduction of the currently standardized proximal to distal cut technique [11], or an additional step for the confirmation of complete TCL resection with a blunt, rigid cannula [12], there were no further cases requiring the revision surgery. Another Considerations Because the whole process of TCTR was performed under ultrasound guidance, the sufficient skill of handling and interpreting the ultrasound is essential.Contraindications of TCTR should include not only the indications of surgical exploration, but also the failure of identification of relevant anatomy and thread position on ultrasound.A significant learning curve is also required for successful outcomes [12].Schrier et al. suggested that 25 and 50 cases of supervision are needed for the practitioner with and without previous ultrasound experience, respectively [29].In the case of motor form only [25], intra-tunnel space occupying lesion, and acute CTS like median artery thrombosis, OCTR with exploration should be indicated rather than TCTR [1]. Depending on the medical system of each country, the use of ultrasound and disposable dissecting thread may increase the medical cost of TCTR procedure.However, because it does not require general or brachial anesthesia and hospitalization for postoperative pain control, TCTR can offset this increase in direct surgical cost.Furthermore, the patient's early return to work with faster recovery after TCTR has the potential to reduce the overall socioeconomic burden of carpal tunnel release [9]. Limitations of the Study The limitations of this study are that it was retrospective and used a relatively small amount of long-term follow up and grade 6 CTS.Well-designed, controlled randomized studies with direct comparison of TCTR to OCTR and ECTR for longer time periods using more patients are needed in the future. Conclusions Ultrasound-guided TCTR showed reliable and favorable outcomes in the long-term follow up.It was not only effective even in the CTS of extremely severe grades but also safe in CTS with anatomical variants.If the practitioner is familiar with ultrasound-guided procedures, it can be performed effectively and safely in most CTS regardless of its severity or presence of anatomical variants. Author Contributions: Conceptualization, I.J.K. and J.M.K.; methodology, I.J.K. and J.M.K.; investigation, I.J.K. and J.M.K.; writing-original draft preparation, I.J.K. and J.M.K.; writing-review and editing, I.J.K. and J.M.K.; supervision, J.M.K.All authors have read and agreed to the published version of the manuscript. Informed Consent Statement: The need for patient consent was waived by ethics committee due to the research involves no more than minimal risk to the subjects. Figure 2 . Figure 2. Post-TCTR time-dependent changes of patients in grade 6 CTS group and other severity group.(A) BCTQ severity scale and (B) BCTQ function scale.TCTR, thread carpal tunnel release; BCTQ, Boston Carpal Tunnel Syndrome Questionnaire.* Denotes significant difference between groups at each follow-up time by independent t-test, p < 0.05. Figure 2 . Figure 2. Post-TCTR time-dependent changes of patients in grade 6 CTS group and other severity group.(A) BCTQ severity scale and (B) BCTQ function scale.TCTR, thread carpal tunnel release; BCTQ, Boston Carpal Tunnel Syndrome Questionnaire.* Denotes significant difference between groups at each follow-up time by independent t-test, p < 0.05. Figure 3 . Figure 3. Realtime ultrasound images of thread placement during TCTR procedure.(A) Green arrows: thread placement along the fusiform proximal TCL (longitudinal view).(B) Yellow arrow: median nerve; red arrow: ulnar side branching of RMB; white arrows: thread placement medial to the ulnar side branching of RMB (axial view).TCTR, thread carpal tunnel release; TCL, transverse carpal ligament; RMB, recurrent motor branch. Figure 3 . Figure 3. Realtime ultrasound images of thread placement during TCTR procedure.(A) Green arrows: thread placement along the fusiform proximal TCL (longitudinal view).(B) Yellow arrow: median nerve; red arrow: ulnar side branching of RMB; white arrows: thread placement medial to the ulnar side branching of RMB (axial view).TCTR, thread carpal tunnel release; TCL, transverse carpal ligament; RMB, recurrent motor branch. Funding: This research received no external funding.Institutional Review Board Statement: This study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Incheon St. Mary's Hospital (Research number OC23RADI0107; date of approval 8 September 2023). 3Paired t-test results in each follow-up interval. Table 2 . Outcomes following TCTR according to the severity.	2024-01-06T16:23:14.315Z	2024-01-01T00:00:00.000	{ "year": 2024, "sha1": "64e418ab15df0c802a8b29790a4358f81d925092", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2077-0383/13/1/262/pdf?version=1704188445", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "13c66669972d6ce024a750d5ae0e478c882c1dd3", "s2fieldsofstudy": [ "Medicine", "Engineering" ], "extfieldsofstudy": [ "Medicine" ] }
26976718	pes2o/s2orc	v3-fos-license	Identification and characterization of RNA-dependent RNA polymerase activity in recombinant Japanese encephalitis virus NS5 protein Summary The complete nonstructural NS5 gene of Japanese encephalitis virus (JEV) was amplified and cloned into an expression vector. The NS5 protein was expressed in Escherichia coli and purified by His-tag based affinity chromatography. This recombinant NS5 protein exhibited RNA-dependent RNA polymerase (RdRp) activity in vitro in the absence of other viral or cellular factors. The RNA polymerase activity was dependent on divalent cations, and Mn2+ was found to be 20 times more effective than Mg2+ in coordinating the catalytic reaction of RdRp, while Ca2+ inhibited enzyme activity. The optimal reaction conditions for the in vitro RdRp reaction were established. Characterization of the RdRp reaction products demonstrated that the JEV NS5 protein can initiate RNA synthesis through a de novo initiation mechanism in our in vitro reaction system. Comparing the efficiency of different RNA templates, we found that JEV NS5 protein was more efficient in using negative-strand RNA templates, indicating that the JEV NS5 protein is involved in regulating the ratio of positive- to negative-strand RNA. Four amino acid sequence motifs crucial for RdRp activity were also identified using site-directed mutagenesis analysis. All substitutions of the conserved residues within these motifs led to a complete inactivation or severe loss of enzyme activity. Introduction Japanese encephalitis virus (JEV), a member of the family Flaviviridae, is transmitted by Culex mosquitoes and causes serious viral encephalitis in humans. The virus is found across a vast geographic area that includes China, India, Japan and virtually all of South-East Asia. Approximately 3 million people live in areas that are JEV endemic. As many as 50,000 cases of JEV infection and about 15,000 associated deaths are reported every year. A high proportion of survivors develop serious neuorologic and psychiatric sequelae [15,26]. The complete nucleotide sequence of the JEV genome has been determined, and the structural features of the genome are similar to those of other flavivirus genomes [5,15,21]. The genome of JEV contains a single positive-strained RNA molecule, which has a type I cap at its 5 0 end and lacks a poly(A) tail at its 3 0 end. It contains a single open reading frame, which is translated into a large polyprotein that is subsequently processed by both host and viral protease into three structural (C, prM, E) and seven nonstructural proteins (NS1 to NS5) in the order 5 0 NC-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-NC3 0 . The NS5 protein, with a molecular weight of 103 kD, is the largest and most highly conserved of the viral proteins [5]. Comparing the deduced amino acid sequence of the JEV NS5 protein with the polymerases of several positive-strand animal and plant RNA viruses, several short homologous regions are found that probably represent common functional domains of RNA-dependent RNA polymerases (RdRp), including the highly conserved G-D-D polymerase motif [14,18]. This suggests that the NS5 protein is the RdRp of JEV. JEV-infected C6=36 cell extracts have been found to have polymerase activity, and this enzymatic activity was shown to be present in the perinuclear endoplasmic reticulum membranes, where both the NS3 and NS5 proteins are located. Rabbit antiserum to NS3 or NS5 was able to inhibit the polymerase activity in cell fractions, confirming that NS3 and NS5 are involved in RNA replication [7,22]. Uchil and Satchidanandam also characterized the RNA synthesis of JEV using virus-infected porcine kidney cell extracts [25]. These studies did not exclude the contribution of other viral or host proteins present in the infected cell extracts. Although it has been demonstrated for other flaviviruses such as dengue virus, Kunjin virus and hepatitis C virus that recombinant NS5 protein has RdRp activity [1,4,8,9,11,23], there has been thus far no such report for JEV. In this study, we expressed the complete JEV NS5 protein in E. coli and demonstrated that recombinant NS5 protein has RdRp activity in the absence of additional viral or host cell factors. The chemical and molecular characterization of Japanese encephalitis virus NS5 protein was thoroughly analyzed. The establishment of an in vitro RdRp assay system using recombinant JEV NS5 protein could provide a basis for further studies to identify factors involved in viral replication and offer a useful tool for the selection of effective antiviral drugs. Virus and cell culture The JE virus strain used in this study was JaOH0566, which was isolated from the brain of a JE patient in 1966 in Osaka, Japan, using suckling mice. After isolation, the virus was passaged more than 10 times in C6=36 cells. For virus preparation, C6=36 cells were used. They were cultured in minimum essential medium with non-essential amino acids (MEM; ICN Biomedicals Inc., USA) supplemented with 10% fetal bovine serum (FBS) at 28 C. The maintenance medium was MEM with 2% FBS. Construction of recombinant plasmids The JEV NS5 gene was amplified by RT-PCR. RNA extraction and RT-PCR were performed as described previously [28]. PCR amplification was carried out using the primers 5 0 AGGGGATCCCCTGGGGGCAGGA 3 0 and 5 0 -TTAA GTCGACCTAGATGACCCTG-3 0 (generated restriction sites are underlined). The 2.8-kb PCR-amplified DNA fragment was digested with BamHI and SalI and cloned into the corresponding restriction site of pQE30 (Qiagen, Hilden, Germany). The presence and the identity of the insert in the recombinant plasmid were confirmed by DNA sequencing; the deduced amino acid sequence of the insert was the same as JaOH0566 (GenBank accession no. AY508813). The expression construct, encompassing amino acids (aa) 1-905, the full length of JEV NS5 protein with a vector-derived His-tag (histidine hexmer) at the N-terminus, was obtained. Site-directed mutagenesis of JEV NS5 protein Site-directed amino acid substitutions were introduced by using paired complementary oligonucleotides for the desired point mutations to generate specific mutations in the JEV NS5 protein using a QuickChange site-directed mutagenesis kit (Stratagene, USA) according to the manufacturer's instructions. Revertant mutants for all the mutants produced were constructed using the same method but with oligonucleotides to reverse the previously introduced changes. Both mutations and revertant mutations were confirmed using the dideoxy sequencing method. The mutant plasmids were introduced by transformation into E. coli XL1-Blue cells, and the mutant NS5 protein were expressed and purified in the same way as wild-type JEV NS5 protein. The six resultant mutant proteins were designated NS5mutA, NS5mutB, NS5mutC, NS5mutC2, NS5mutCn and NS5 mut D. Expression and purification of the recombinant NS5 protein To express the recombinant NS5 protein, the recombinant plasmid containing the JEV NS5 sequence was used to transform E. coli strain XL1-Blue. The transformed E. coli cells were then cultured at 30 C in Luria-Bertani medium containing 100 mg=ml of Ampicillin. The recombinant proteins were 1860 F. Yu et al. induced by the addition of 0.2 mM isopropyl b-D-thiogalactoside (IPTG) when the OD600 of the culture reached 0.5. After IPTG induction at 30 C for 3 h, the cells were harvested by centrifugation, washed in phosphate-buffered saline solution (PBS), resuspended in 20 mM Tris-HCl pH 7.5, 500 mM NaCl, and frozen at À80 C. After freezing and thawing 3 times, the cell suspension was sonicated in 1-sec. pulses at 1-sec. intervals for 2 min. and centrifuged at 30,000 g for 15 min. at 4 C. The supernatants were then applied to a Talon TM IMAC resin column (Clontech, USA). After washing with a buffer containing 20 mM Tris-HCl, 500 mM NaCl, and 10 mM imidazole, the purified protein was then eluted with 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 250 mM imidazole. The proteins were aliquoted and stored in 10% glycerol at À80 C until use. The protein concentrations were determined using a Bio-Rad protein assay reagent kit (Bio-Rad, USA), and protein purity was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analysis The Western blot procedure was performed as described by Towbin et al. [24]. A semi-dry protein transfer method was used to immobilize the recombinant protein to a polyvinylidene difluoride (PVDF) membrane (Immobilon, Millipore, USA). Briefly, protein separated in a 10% polyacrylamide gel was transferred to a PVDF membrane using a semi-dry electroblotter (Sartorius, Germany). The membrane was initially blocked overnight at 4 C with Blockace (Yokijirushi, Japan), then reacted with Blockace-diluted mouse anti-histidine (1:1000 dilution Amersham Biosciences, USA) or JEVinfected mouse immune serum(1:400) for 1 h at 37 C, then incubated with goat anti-mouse IgG peroxidase conjugate (1:1000 dilution Biosource, CA, USA) for 1h at 37 C. Finally, the reaction was visualized by dymethyl amino benzidine (DAB) staining. RNA template synthesis RNA templates used for the RdRp reactions were in vitro transcribed from PCR products. PCR primers were designed in which the sense or antisense strand contained a T3 promoter sequence. Approximately 1-kilobase (kb) PCR products were amplified for JEV 5 0 end, JEV 3 0 end, dengue-2 virus (strain 16681) 5 0 end and dengue-2 virus 3 0 end from the respective cDNA clones. The PCR products were gel purified using a gel extraction kit (Qiagen, Germany) and used for RNA transcription using in vitro transcription kits (Ampliscribe, USA). The transcribed RNA were quantitated spectrometrically and stored at À80 C until use. When the RNA template was to be labelled, transcription was performed in presence of 10 mCi of [a-32 P] UTP. Six RNA products were produced by in vitro transcription and used as templates for in vitro RdRp assays: 1-kb JEV 3 0 (À)RNA; 1-kb JEV 3 0 (þ) RNA; 1-kb Den-2 virus 3 0 (À) RNA;1-kb Den-2 virus 3 0 (þ) RNA; 1-kb HCV 3 0 (þ)RNA and one 1.3-kb non-specific RNA produced using the control DNA supplied with the transcription kit. RNA-dependent RNA polymerase(RdRp) assay This assay was based on the incorporation of radiolabelled nucleotides into a newly synthesized RNA molecule [10]. The RdRp activity assay was performed at 30 C in 50-ml reaction volumes containing 3 mg of purified NS5 protein, 50 mM HEPES, pH 7.3, 100 mM UTP, 0.5 mM ATP, 0.5 mM CTP, 0.5 mM GTP, 4 mM DTT, 3 mM MnCl 2 unless specifically indicated, 6 mM ZnCl 2 , 5 mg of in vitro transcribed RNA template, 60 units of HPRI and 5-10 mCi of [a-32 P] UTP (3000Ci=mM, Amersham, USA). The reaction mixture was incubated for 4 h. A volume of 5 ml was removed hourly and spotted onto a DE81 Whatman filter (Whatman, USA), washed twice with 500 mM Na 2 HPO 4 , pH 7.5, and finally with 100% ethanol. The filter was air-dried, solubilized in 1 ml of scintillation fluid (Amersham, USA), and radioactivity was measured using a scintillation counter (Amersham, USA). To check the effect of different divalent ions, similar reactions were performed using different solutions containing MnCl 2 , MgCl 2 or CaCl 2 . After a 4-h incubation, 5 ml of the reaction mixture was removed and spotted onto a DE81 filter membrane (Whatman, USA), which was washed, and radioactivity was measured. The effect of CaCl 2 was further studied by increasing CaCl 2 concentration in the presence of 3 mM MnCl 2 . When analyzed by electrophoresis, the RNA products were extracted with phenol=chloroform and precipitated with ethanol. The RNA pellet was air-dried, resuspended in loading buffer (95% formamide, 0.5 mM EDTA, 0.025% bromophenol blue, 0.025% xylene cyanol), heated for 5 min at 95 C and loaded onto a 8M-urea-denatured 3.5% polyacrylamide gel in Tris=borate=EDTA (TBE) buffer. Electrophoresis was carried out at a constant 20 mA until the bromophenol blue marker migrated to the bottom of the gel. The gel was then dried and autoradiographical data analysed using a Bio-Rad image developing system. Ribonuclease A digestion of RdRp reaction product RNase A digestion was performed at 37 C for 30 min in 20-ml volumes of 20 mM Tris-HCl, pH 7.5, and 5 mM EDTA in the absence or presence of 0.3 M NaCl containing the RNA product of the RdRp reaction assay. Reaction products were analyzed by electrophoresis and autoradiography. Sodium periodate treatment of RNA transcripts The 3 0 -hydroxyl group of the RNA template was blocked by sodium periodate treatment as described by Ackerman et al. [1,27]. Briefly, 10 mg of the RNA transcripts was dissolved in 200 ml 50 mM NaOAc, pH 5.0. After the addition of 50 ml NaIO 4 (100 mM), the reaction was incubated for 1 h at 22 C, Characterization of RdRp activity of JEV NS5 protein extracted with PCA and precipitated with propanol. The pellets were washed with 70% EtOH, dissolved in water and quantified spectrophotometrically. Expression and purification of JEV NS5 protein The JEV JaOH0566 strain NS5 coding region was amplified using reverse transcription polymerase chain reaction (RT-PCR) and cloned into expression vector pQE30. The constructed expression plasmid spanned the entire 905 amino acids of NS5. The recombinant protein was expressed in E. coli and purified using Talon metal affinity resin (Clontech USA) under natural conditions. Analysis of purified recombinant NS5 protein by SDS-PAGE and Coomassie blue staining revealed a single protein band of 103 KD, as predicted (Fig. 1A). The identity of the recombinant NS5 protein was further confirmed by Western blot with mouse antiserum to both histidine and Japanese encephalitis virus (Fig. 1B, C). Demonstration of RdRp activity in NS5 Polymerase activity of the purified recombinant NS5 protein was demonstrated by measuring the level of incorporation of [ 32 P]-UMP into newly synthesized RNA strands using RNA templates. The RdRp assay was carried out as described under ''Materials and Methods'' except that the initial RdRp reactions were performed as detailed in Materials and methods. Briefly, 5 ml was removed hourly from the reaction volume and spotted onto a DE81 Whatman filter and washed with 0.5 M Na 2 HPO 4 , pH7.5. Radioactivity was then measured by scintillation counting. B The 32 P-labeled RNA polymerase products were analyzed on an 8M-urea-denatured 3.5% PAGE gel. T 32 P-labeled RNA template obtained by in vitro transcription; 2 RdRp product with template omitted; 3 with protein omitted; 4 1 kb JEV 3 0 (À)RNA as template. C The 32 P-labeled RNA polymerase products using non-specific RNA templates were analyzed on a 8M-urea-denatured 3.5% PAGE gel. 1 1-kb HCV 3 0 (þ) RNA as template; 2 1.3-kb non-specific RNA as template assays were conducted in the presence of actinomycin D (50 mg=ml) to inhibit any possible contaminating bacterial DNA-dependent RNA polymerase. There was no difference in polymerase activity recorded in the presence or absence of actinomycin D (data not shown). Therefore it was excluded in subsequent assays. As shown in Fig. 2A, when using 1-kb JEV3 0 (À) RNA as the RNA template, the incorporation of [ 32 P]-UMP into newly synthesized RNA increased linearly over a period of 4 h at 30 C, while in reactions without RNA template there was no detectable incorporation, indicating that the recombinant NS5 protein was dependent on RNA, as expected for an RdRp. The 32 P-labelled RdRp products were also visualized on an 8.0 M-urea-denatured 3.5% SDS-PAGE gel, as shown in Fig. 2B. There was no synthesized RNA band in reactions without RNA template or NS5 protein (Fig. 2B, Lanes 2, 3). When using 1-kb JEV3 0 (À) RNA as the RNA template, the main polymerase product was the same size as the RNA template used. In addition, the recombinant NS5 protein also synthesized several smaller polymerase products. Similar to other RNA polymerases, these smaller RNA species may represent partially or incompletely synthesized RNA products [23]. To determine whether JEV NS5 could use nonspecific RNA templates, the enzyme were incubated with 1-kb-long HCV 3 0 (þ) RNA and a 1.3-kb-long non-specific RNA transcribed from the control DNA supplied in the transcription kit (Ampliscribe, Fig. 3. Characterization of RNA polymerase of JEV. The RdRp reaction was assayed in the standard reaction mixture with 3 mg purified JEV NS5 and 5 mg JEV 3 0 (À) RNA template. A Optimization of the concentration of MnCl 2 or MgCl 2 . B Effect of CaCl 2 . C Determination of optimal reaction temperature. D Optimization of reaction pH USA). In both cases, mainly template-sized products were obtained (Fig. 2C). Characterization of the RNA polymerase activity of JEV NS5 protein To characterize the RNA polymerase activity of purified JEV NS5 protein, its RdRp activity was examined under various assay conditions (Fig. 3). JEV NS5 purified with buffers lacking divalent metals was used to examine the effect of an increasing concentration of exogenously provided divalent metal ions. In the absence of exogenously provided divalent metal ions, no RNA synthesis was observed, indicating that the RNA polymerase activity was dependent on divalent cations. Manganese ions were 20 times more effective than magnesium in coordinating the catalytic reaction of RdRp at optimal concentration (3 mM) (Fig. 3A). The coordinating effect of Ca 2þ ions was tested in 3, 6 and 12 mM solutions, and in all cases, no RNA synthesis was found (data not shown). The effect of Ca 2þ on the RdRp reaction in the presence of 3 mM Mn 2þ was also checked. Increasing concentrations of Ca 2þ decreased the RNA synthesis by JEV NS5, indicating that Ca 2þ inhibits the RNA polymerase activity of JEV NS5 (Fig. 3B). The RdRp reaction was carried out at different temperatures, establishing that the optimal temperature for the reaction is 30 C (Fig. 3C). The reaction was also tested at different pH levels; the optimal pH of JEV NS5 polymerase ranged from 7.5 to 8.0 (Fig. 3D). Characterization of polymerase assay products RdRp products generated by JEV NS5 in our reaction system were mainly the same size as the input template RNAs (Fig. 2B and C). To check whether the template-length products were produced through de novo initiation of the template RNA, 1-kb JEV 3 0 minus strand and plus strand RNA was pre-treated with sodium periodate. RdRp reactions were performed using templates with a blocked 3 0 hydroxyl group. As shown in Fig. 4, the sodium-periodatetreated RNA can also serve as a template for RdRp, and the reaction products were almost the same as the untreated RNA, indicating that the product in this reaction system is not produced through a self-priming copy-back mechanism using the template RNA's 3 0 end. We sought to determine whether the RdRp product was in fact the plus strand formed by de novo initiation at the 3 0 end of the RNA template. The RdRp reaction product obtained using 1-kb JEV 3 0 (À) RNA as template was treated with RNase A(10 mg=ml) under conditions of high salt(0.3 M NaCl) or low salt(0.01 M NaCl) concentrations at 37 C for 30 min. The resultant RNase A digestions showed that under high-salt conditions, which facilitate degradation of single-stranded but not double-stranded RNA species, the labelled product remained undigested (Fig. 5. Lane 3). However, under low-salt conditions, which allow digestion of all RNA species, no labelled product was detected ( Fig. 5. Lane 2). These results indicate that the products of JEV NS5 protein RdRp reactions were double-stranded RNA molecules, verifying that the radioactive RNA visualized on the gel is not simply a terminally radiolabeled input template RNA, as these would be degraded by RNase A. These results, taken together, indicate that the purified NS5 protein initiates primer-independent de novo RNA synthesis. Activity of JEV NS5 RdRp with different RNA templates In flavivirus genomes, two conserved sequences within the 3 0 -untranslated region (3 0 -UTR) as well as stem-loop structures within the 3 0 -and 5 0 -UTRs are thought to be important for viral RNA replication. Hence, assuming that the two terminal regions of the JEV genome contain domains that are important for the regulation of RNA replication, we compared the efficiency of RNA synthesis with templates of the plus-or the minus-strand JEV virus RNAs. Approximately 1-kilobase(kb) RNAs corresponding to the regions of the 3 0 end of the plus-or the minus-strand JEV genome were transcribed from PCR products in vitro and used as RNA template. As shown in Fig. 6A, both the plus-strand RNA and the minus-strand RNA were used as templates, but the incorporation of [ 32 P]-UMP was much lower when using positive-strand RNA as template than when using negative-strand RNA -an almost 10-fold difference in efficiency. A similar result was observed when using around 1 kilobase of 3 0 end plus-and minus-strand RNA from the genome of another flavivirus( dengue 2 virus), which gave a difference in yield of about 5-fold. When the reaction products were analyzed on an 8M urea-denatured 3.5% PAGE gel, the synthesized RNA bands were much stronger when Fig. 6. Comparison of RNA synthesis by JEV NS5 using JEV and dengue-2 virus 3 0 end plus-and minus-strand RNA templates. Around 1 kb plus or minus strands of JEV or dengue-2 virus 3 0 end RNA were used in equal amounts (5 mg) as templates for standard RdRp reaction as mentioned in Materials and methods. The reaction products were analyzed after 4 h of reaction at 30 C and checked by scintillation counting (A). B The reaction products (5 ml) were denatured and run on a 8M urea, 3.5% PAGE gel. T 32 P-labeled 1-kb JEV 3 0 (À)RNA template obtained by in vitro transcription. 1 Without RNA template. 2 1-kb JEV 3 0 (À)RNA as template. 3 1-kb JEV 3 0 (þ) RNA as template. 4 1-kb Den-2 virus 3 0 (À) RNA as template. 5 1-kb Den-2 virus 3 0 (þ) RNA as template using the minus-strand RNA as template (Fig. 6B, Lanes 2 and 4) than when using the plus-strand RNA template (Fig. 6B, Lanes 3 and 5). Mutation analysis of the RNA polymerase of JEV NS5 protein By comparing the sequences of various reverse transcriptases and various viral RdRps, Poch et al. identified four sequence motifs for RdRps [18]. By sequence alignments of JEV NS5 with other viral RdRps, the same four sequence motifs could be identified. To identify the importance of these motifs for the JEV NS5 RdRp activity, single amino acid substitutions were introduced into the JEV NS5 gene by site-directed mutagenesis. The exact substitution were: Asp536 to Ala in Motif A, Gly605 to Ala in Motif B, Asp668 to Ala, Asp669 to Ala and Asp669 to Asn in Motif C, Lys691 to Ala in Motif D (illustrated in Fig. 7A). The mutant proteins were expressed in E. coli and purified as described for the parental JEV NS5. The mutations were confirmed by DNA sequencing. For all proteins, the expression level and purity were comparable to those of the wild type (data not shown). To measure the RdRp activities of all of the mutant NS5 proteins, standard reactions were performed with JEV 3 0 -end 1-kb (À) RNA as template, and 3 mg of each purified NS5 protein mutant was used for each reaction. Of the 6 mutants in the 4 motifs we produced, 5 substitutions led to complete inactivation of the enzyme. Only one mutant, NS5 mutCn, the Asp-to-Asn substitution at position 669 in Motif C showed a reduction in activity to about 5% relative to the parental NS5 (Fig. 7B). Revertant mutation of each mutant was performed in the same way, and polymerase activity was recovered in all the revertants (data not shown). These results also confirm that the RNA polymerase activity is due to the wild JEV NS5 protein rather than contaminating proteins derived from bacterial cell lysates. Discussion We successfully expressed JEV NS5 protein in E. coli and purified the recombinant protein to near homogeneity. The purified recombinant protein was enzymatically active, and we were able to demonstrate polymerase activity in the absence of either viral or host cofactors. This indicates that the product of the viral NS5 gene is an RdRp, which is involved in the replication of the virus. This is the B RNA synthesis by mutated JEV NS5 proteins. The mutant proteins were expressed and purified in the same way as wildtype NS5. Standard RdRp reactions were conducted using these mutant NS5 proteins and 1-kb JEV 3 0 (À) RNA as template. The reaction products were extracted and analyzed on an 8M-urea-denatured 3.5% PAGE gel. T labeled 1-kb JEV 3 0 (À) RNA template obtained by in vitro transcription; 1 RdRp products obtained using NS5mutA; 2 RdRp products obtained using NS5mutB; 3 RdRp products obtained using NS5mutC; 4 RdRp products obtained using NS5mutC2; 5 RdRp products obtained using NS5mut Cn; 6 RdRp products obtained using NS5 mut D; w RdRp products obtained using wild-type NS5 1866 F. Yu et al. first direct confirmation of JEV's NS5 as a viral RdRp. As with other enzymes of this class, the RdRp activity of JEV was found to be dependent on divalent ions, Mn 2þ or Mg 2þ . We found that Mn 2þ is much more effective than Mg 2þ in the RdRp reactions, similar to reports for HCV NS5B [8,16]. Taken together, this would suggest that Mn 2þ is more favoured by flaviviral RdRps. Ca 2þ inhibits polymerase activity. The optimal reaction conditions for in vitro RdRp assay were determined. Like the report for Polio virus RdRp, the optimum reaction temperature is 30 C [10]. A possible reason for a low optimal reaction temperature may be the fact that the rate of UMP incorporation at lower temperature was linear over a longer time period. This in vitro RdRp reaction system would be a good system for studying the viral and host factors involved in viral replication and also for assessing antiviral drugs targeting viral replication. RNA viruses initiate RNA synthesis mainly by two mechanisms: a primer extension mechanism and a de novo initiation mechanism [12,13]. In this report, RNA synthesis occurred without adding primers; the RdRp products were mainly the same size as the input template when analysed on 8M-ureadenatured PAGE gel, no products twice the size of template were found. Sodium periodate treatment of the template, which blocked the 3 0 end of the template to prevent ''copy-back'' synthesis [1,27], does not otherwise change the RdRP reaction products (Fig. 4), and RNase A treatment did not digest the synthesised RNA under high-salt conditions (Fig. 5). All of these results indicates that JEV NS5 RdRp initiate RNA synthesis on RNA templates by a de novo mechanism. Ranjith-Kumar et al. reported that Mn 2þ cations significantly increased the de novo initiation products of HCV RdRp [19]. Ackermann et al. reported that at low initiation temperature, more de novo initiation products were produced [1]. In our reaction system, we use Mn 2þ instead of Mg 2þ , and the reaction temperature we use is low. This may partially explain why in our reaction system, no primer extension products were found. This may also be related to the RNA template used and the RdRp characteristics of each virus. In RdRp of dengue 1 virus and Kunjin virus, only template-sized products were produced [11,23]. The NS5-mediated reaction was found not to be limited to JEV RNAs. RNA synthesis was also observed with HCV and unrelated input RNA templates. This is a common phenomenon among RNA polymerases [4,23]. This suggests that NS5 is necessary but not solely sufficient to direct the replication of the JEV genome as other viral and=or cellular factors must by necessity be involved in this process in order to restrict the RNA synthesis exclusively to viral templates. During the plus-strand RNA virus replication cycle, the genomic RNA serves as a template for synthesis of negative-strand RNA, and the negativestrand RNA serves as a template for synthesis of further positive-strand genomic RNA. The synthesis of positive-strand RNA versus negative-strand RNA must be regulated because a ratio of positiveto negative-strand RNA of 10:1 to 100:1 has been observed in virus-infected cells [2,3,5,6,20]. When we compared the efficiency of RNA synthesis of JEV NS5 protein using JEV and dengue-2 virus minus-strand RNAs templates with that of the corresponding plus-strand templates, a 5-10 fold efficiency difference was observed (Fig. 6). JEV NS5 protein used the minus-strand RNA to synthesize the plus-strand product much more efficiently than it uses the plus-strand template to synthesize the minus RNA in vitro. This correlates with findings in virus-infected cells in which 10-100 times more plus-strand RNA was synthesized than minus-strand RNA. Our results indicate that the RNA ratio in infected cells is, at least partially, regulated by the viral RdRp. In a recent report, Filomatori et al. suggest that the 5 0 UTR of dengue 2 virus genome functions as a promoter for RdRp activity [9]. Comparing RNA molecules carrying the 5 0 -terminal or the 3 0 -terminal sequences of the dengue 2 virus genome RNA, they observed a remarkable preference (100 fold) for the template carrying the 5 0 -terminal sequences (5 0 DV RNA) over that of the RNA template containing the 3 0 end sequences [9]. Similar to their report, we observed a much lower RNA synthesis efficiency using the 3 0 -end plus-strand RNA template with the NS5 protein, which may be due to the lack of a 5 0 end promoter sequence in the template. Numerous studies have shown that nucleic acid polymerases have fundamental structural and mech-anistic similarities, which are reflected by four distinct sequence motifs, Motif A, Motif B, Motif C and Motif D [14,17,18]. Using linear sequence alignments of JEV NS5 with other RdRps as a basis for site-directed mutagenesis experiments, we also identified four amino acid motifs which appear to play a vital role in the polymerase activity for JEV. Substitutions of almost all these 4 motifs led to a complete inactivation of the enzyme, except for the Asp-to-Asn substitution at position 669 in Motif C, which markedly reduced the activity to about 5% that of parental NS5. This GDN motif was the RdRp motif for some of the negative-strand RNA viruses in the alignment comparison, and this higher tolerance of the second Asp residue of the GDD motif for substitutions has also been reported for other RNA polymerases [17,18]. These mutatagenesis results also confirm that the RNA polymerase activity is due to the wild JEV NS5 protein rather than contaminating proteins derived from the bacterial cell lysates.	2017-08-02T20:46:02.884Z	2007-06-18T00:00:00.000	{ "year": 2007, "sha1": "3460b5eca649b253db4b80593a5cf90afe5511bf", "oa_license": "implied-oa", "oa_url": "https://europepmc.org/articles/pmc7086876?pdf=render", "oa_status": "GREEN", "pdf_src": "PubMedCentral", "pdf_hash": "3460b5eca649b253db4b80593a5cf90afe5511bf", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
15905011	pes2o/s2orc	v3-fos-license	Setting oral health goals that include oral health-related quality of life measures : a study carried out among adolescents in Thailand The aim of this study was to assess the association between oral diseases and condition-specific oral health-related quality of life (CS-OHRQoL) as a basis for proposing OHRQoL-based goals for the population of 15-year-olds in Thailand. Oral examinations and OHRQoL interviews were conducted with 871 15-year-olds as part of the Sixth Thailand National Oral Health Survey. The severity of oral impacts was categorized using “intensity”. Associations between oral diseases and CS-OHRQoL were analyzed using chi-square and logistic regression. Thirty-nine percent of 15year-olds experienced moderate/higher levels oral impacts on quality of life. Compared to those individuals with no tooth decay, adolescents with one or four or more decaying teeth were three and seven times more likely to experience moderate/ higher impacts, respectively. Adolescents with extensive gingivitis in 3 or more mouth sextants were twice as likely to experience moderate/higher CS-impacts. Based on these findings, it is proposed that goals should focus on untreated decaying teeth and extensive gingivitis. Oral health goals for 15-year-olds should include specific OHRQoL measures. Dental Caries; Adolescent; Gingivitis; Oral Health; Quality of Life Introduction The major goal of the dental profession is to improve peoples’ quality of life 1. Therefore, quality of life measures must be included in setting oral health goals for the profession. Incorporating quality of life measures into national oral health goals is vital because oral health is not the absence of oral diseases, but the physical, psychological and social well-being in relation to oral status 2. The World Dental Federation (FDI)/ World Health Organization (WHO)/International Association for Dental Research (IADR) collaboration published guidelines recommending the development of broader oral health goals rather than only using oral disease goals 3. The guidelines stressed the importance of oral health-related quality of life (OHRQoL). They stated that the goals of oral health services were “to minimize the impact of diseases of oral and craniofacial origin on health and psychosocial development” 3 (p. 286). They encouraged local actions to adopt such a broader approach to goal setting and oral health service planning. The FDI/ WHO/IADR guidelines also provided examples of goals for reductions in specific oral diseases that are known to cause impacts on quality of life. A number of OHRQoL indexes have been developed and applied to compliment clinical indices 4. Although some countries have included OHRQoL measures in their national oral health surveys 5,6,7,8,9 and some studies have proposed ARTIGO ARTICLE Krisdapong S et al. 1882 Cad. Saúde Pública, Rio de Janeiro, 28(10):1881-1892, out, 2012 specific applications of OHRQoL to oral health service planning 10,11, no country had ever comprehensively included OHRQoL measures in setting oral health goals for the population. Previous oral health goals for the Thai population were disease-based and typical of those set by many countries. The oral health goals for adolescents in the year 2000 stated that not less than 75% of 18-year-olds should retain all 28 permanent teeth, and all 18-year-olds should have at least two sextants, on average, with healthy periodontal tissues (Community Periodontal Index; CPI = 0) 12. In 2007, the Thai government published its oral health goals for 2020 in accordance with the FDI/WHO/IADR guidelines and a theoretical framework for linking clinical status and quality of life was developed. The ultimate goal of the Thai Oral Health Service is: “people of all ages should have oral health that enables good quality of life and social well-being” 13 and clinical goals were set based on the assumption that a reduction in oral diseases would lead to better quality of life. Clinical data from previous National Oral Health Surveys 14,15 was used to provide a baseline and only one oral health goal was set for adolescents: not more than 60% of 17 to 19-yearolds should have periodontal disease (CPI = 1 or above) 13. Although evidence suggests a positive association between oral status and OHRQoL, the defined clinical goal for Thai adolescents was theoretical and not supported by scientific evidence of an association. Relatively few studies exist on the association between oral disease and OHRQoL in adolescent populations and their findings are inconclusive. Dental caries and progressive forms of periodontal disease were significantly associated with adolescents’ OHRQoL 16,17,18, while findings regarding general periodontal disease, missing teeth and dental fluorosis were equivocal 16,17,19,20. Moreover, most of the studies used generic OHRQoL measures that have poorer discriminative ability than condition-specific (CS) measures 21,22. As the latest Thai oral health survey included condition-specific OHRQoL measures and Thai oral health goals for 2020 explicitly mention OHRQoL as the ultimate goal of the oral health service, it was considered important to assess the relation between clinical and OHRQoL measures using a CS-OHRQoL measure developed in Thailand. Findings from the Thai oral health survey regarding overall OHRQoL in 15-year-olds suggested that, although oral impacts in this age group were very common (83%), the prevalence of severe and very severe impacts was only 8%. In addition, oral ulcers and toothache were the main perceived causes of overall oral impacts 23. In addition to the information from the above study, data from Thailand is used to illustrate the broader principle of setting oral health goals as recommended by the FDI/WHO/IADR guidelines. The objectives of this study were therefore to assess the association between specific oral diseases and CS-OHRQoL and classify adolescents in terms of level of risk of adverse effects on quality of life. Finally, based on these findings, the study aimed to propose OHRQoL-based oral health goals for this age group in Thailand. Setting oral health goals that include oral health-related quality of life measures: a study carried out among adolescents in Thailand Incorporação da qualidade de vida relacionada à saúde bucal em metas de saúde bucal: estudo conduzido em adolescentes tailandeses Introduction The major goal of the dental profession is to improve peoples' quality of life 1 .Therefore, quality of life measures must be included in setting oral health goals for the profession.Incorporating quality of life measures into national oral health goals is vital because oral health is not the absence of oral diseases, but the physical, psychological and social well-being in relation to oral status 2 .The World Dental Federation (FDI)/ World Health Organization (WHO)/International Association for Dental Research (IADR) collaboration published guidelines recommending the development of broader oral health goals rather than only using oral disease goals 3 .The guidelines stressed the importance of oral health-related quality of life (OHRQoL).They stated that the goals of oral health services were "to minimize the impact of diseases of oral and craniofacial origin on health and psychosocial development" 3 (p. 286).They encouraged local actions to adopt such a broader approach to goal setting and oral health service planning.The FDI/ WHO/IADR guidelines also provided examples of goals for reductions in specific oral diseases that are known to cause impacts on quality of life.A number of OHRQoL indexes have been developed and applied to compliment clinical indices 4 .Although some countries have included OHRQoL measures in their national oral health surveys 5,6,7,8,9 and some studies have proposed Cad.Saúde Pública, Rio de Janeiro, 28 (10):1881-1892, out, 2012 specific applications of OHRQoL to oral health service planning 10,11 , no country had ever comprehensively included OHRQoL measures in setting oral health goals for the population. Previous oral health goals for the Thai population were disease-based and typical of those set by many countries.The oral health goals for adolescents in the year 2000 stated that not less than 75% of 18-year-olds should retain all 28 permanent teeth, and all 18-year-olds should have at least two sextants, on average, with healthy periodontal tissues (Community Periodontal Index; CPI = 0) 12 .In 2007, the Thai government published its oral health goals for 2020 in accordance with the FDI/WHO/IADR guidelines and a theoretical framework for linking clinical status and quality of life was developed.The ultimate goal of the Thai Oral Health Service is: "people of all ages should have oral health that enables good quality of life and social well-being" 13 and clinical goals were set based on the assumption that a reduction in oral diseases would lead to better quality of life.Clinical data from previous National Oral Health Surveys 14,15 was used to provide a baseline and only one oral health goal was set for adolescents: not more than 60% of 17 to 19-yearolds should have periodontal disease (CPI = 1 or above) 13 .Although evidence suggests a positive association between oral status and OHRQoL, the defined clinical goal for Thai adolescents was theoretical and not supported by scientific evidence of an association. Relatively few studies exist on the association between oral disease and OHRQoL in adolescent populations and their findings are inconclusive.Dental caries and progressive forms of periodontal disease were significantly associated with adolescents' OHRQoL 16,17,18 , while findings regarding general periodontal disease, missing teeth and dental fluorosis were equivocal 16,17,19,20 .Moreover, most of the studies used generic OHRQoL measures that have poorer discriminative ability than condition-specific (CS) measures 21,22 .As the latest Thai oral health survey included condition-specific OHRQoL measures and Thai oral health goals for 2020 explicitly mention OHRQoL as the ultimate goal of the oral health service, it was considered important to assess the relation between clinical and OHRQoL measures using a CS-OHRQoL measure developed in Thailand.Findings from the Thai oral health survey regarding overall OHRQoL in 15-year-olds suggested that, although oral impacts in this age group were very common (83%), the prevalence of severe and very severe impacts was only 8%.In addition, oral ulcers and toothache were the main perceived causes of overall oral impacts 23 .In addition to the information from the above study, data from Thailand is used to illustrate the broader principle of setting oral health goals as recommended by the FDI/WHO/IADR guidelines.The objectives of this study were therefore to assess the association between specific oral diseases and CS-OHRQoL and classify adolescents in terms of level of risk of adverse effects on quality of life.Finally, based on these findings, the study aimed to propose OHRQoL-based oral health goals for this age group in Thailand. National Oral Health and OHRQoL Survey The Sixth Thailand National Oral Health Survey was conducted in 2007 15 .The total sample of 15-year-olds was 1,742.Full details of sampling procedures have been described elsewhere 15,23 .Two out of the four provinces/sub-districts selected by the national survey were randomly selected for this study resulting in a sample size of 871 individuals that represents half the national survey.All adolescents in the randomly selected provinces/sub-districts were recruited as study participants.The protocol was approved by the Ethics Committee of Chulalongkorn University. Adolescents were orally examined by trained and calibrated community dentists using WHO criteria 24 for the diagnosis of caries and fluorosis.The CPI 25 was used to assess periodontal conditions of six mouth sextants: upper right, upper anterior, upper left, lower left, lower anterior and lower right.Components of the CPI were scored separately in the following manner: gingival bleeding without calculus (score one), calculus without gingival bleeding (score two), shallow pocket (score three), deep pocket (score four) and calculus with bleeding (score five).To assess OHRQoL, the adolescents were interviewed by trained and calibrated interviewers using the Thai version of the Oral Impacts on Daily Performances (OIDP) 26 .This index assesses oral impacts during the past six months in relation to eight daily performances: (1) eating, (2) speaking, (3) cleaning teeth, (4) emotional stability, (5) relaxing/sleeping, (6) smiling without feeling embarrassment, (7) studying, and (8) social contact.For each performance, a frequency and severity score, both ranging from zero to five, were recorded.When an impact was detected, the adolescent was asked to report the oral conditions that were perceived to be the main causes.Ten percent of adolescents were re-examined and re-interviewed.The Intraclass Correlation Coefficients (ICC) was 0.926 for the oral examinations and 0.862 for OHRQoL interviews.In addition, socio-demographic information regarding sex, region, place of residence (municipal/rural) and school type (private/public) was recorded. Data analysis Stata 10.0 (Stata Corp., College Station, USA) was used for data analysis.To assess dental caries and fluorosis the DMFT score and its components (DT, MT and FT) were calculated.Periodontal disease was analyzed in terms of overall periodontal disease (codes one to five) and using the CPI components (gingivitis without calculus = score one, calculus without gingivitis = score two, periodontal pocket = score three to four and calculus with gingivitis = score five).In addition, an analysis of gingivitis without or with calculus (score of one or five), and calculus without or with gingivitis (score of two or five) was performed.OHRQoL was analyzed in relation to overall oral impacts and CS-impacts.CS-impacts were impacts caused by specific oral conditions, calculated by taking into account the oral conditions that adolescents perceived as the main causes of impacts.Eight groups of CS-impacts were calculated: (1) dental caries: main perceived causes of oral impacts were toothache, sensitive teeth, hole in a tooth, broken filling, toothache after filling, (2) periodontal disease: main perceived causes were inflamed gums, gum pain, tartar, bad breath, (3) edentulous area: main perceived cause was tooth space due to extracted permanent tooth, (4) oral lesions: main perceived causes were oral ulcer or other oral lesions such as herpes, dry/cracked lips, (5) discoloration: main perceived cause was tooth/teeth colour, (6) malocclusion: main perceived cause was tooth/teeth position, (7) traumatic dental injuries: main perceived cause was a ffractured tooth, and (8) natural process: main perceived causes were tooth space due to unerupted permanent tooth, exfoliating primary tooth and erupting permanent tooth. The "intensity" of impacts was used instead of impact score to reflect the severity of oral impacts.The OIDP score is the sum of eight performance scores, where each performance score is derived by multiplying the frequency score with the severity score.Thus, a score reflects both extent (the number of performances with impacts) and intensity (the severity of impacts on each performance) 27 .The concept of intensity focuses on the most severe impact on any performance regardless of the number of performances that are affected.For each performance, frequency and severity scores were recorded ranging from zero to five.There are therefore 15 possible performance scores (0, 1, 2, 3, 4, 5, 6, 8, 9, 10, 12, 15, 16, 20, 25).Intensity was defined as the high-est performance score out of the eight performances, classified into six levels: none, very little, little, moderate, severe and very severe, where the highest performance scores were 0, 1-2, 3-5, 6-12, 15-16 and 20-25 respectively.Moderate or higher levels of impacts were defined by the highest performance score of six or over.The classification of intensity levels has been fully described elsewhere 23 .These measures were used instead of impact scores since scores are not a clear representation of the severity of impacts 28 . The association between oral diseases and OHRQoL was investigated using chi-square for trend and logistic regression.Oral diseases were analyzed as independent categorical variables predicting the presence or absence of CS-impacts.Socio-demographic variables and other possibly confounding oral diseases were controlled for multiple logistic regression.Multicollinearity existed for the three socio-demographic variables: region, place of residence (municipal/ rural) and school type (private/public).Bangkok was considered municipal, while the other four regions consisted of municipal and rural areas.All private schools were in municipal areas while public schools were in both municipal and rural areas, therefore creating a variable of "regionplace of residence" consisting of nine categories (e.g.Bangkok, central-municipal, central-rural, north-municipal, north-rural) that was used instead of the individual variables region, place of residence and school type.The stepwise technique was used and the goodness-of-fit test of the models using the log-likelihood and pseudo r2 revealed that the best fit models contained sex and region-place of residence as socio-demographic variables. Panel of dental public health professionals To propose OHRQoL-based oral health goals, dental public health professionals, academics and dentists employed by the Dental Health Division of the Department of Health of the Ministry of Public Health were asked to give their opinions and comments on the findings of this study.In addition to the SMART concept for goal setting (specific, measurable, attainable, realistic and time-bound) 29 , compatibility with current strategies and plans for oral health services were also considered. Oral status and OHRQoL A total of 811 15-year-olds (93.1% response rate) completed the OHRQoL interviews and had oral examinations, of which 48% were male, 43% resided in municipal areas and 89% were attending public schools (Table 1).Caries prevalence was 68.6%.The mean DMFT score was 2.4 (±2.7):DT = 1.2 (±1.9),MT = 0.1 (±0.4) and FT = 1.1 (±1.9).Prevalence of periodontal disease was 81.5% with an average of 2.9 (±2.1) diseased sextants per child.Most adolescents (53.1%) had gingivitis without calculus, while 46.1% and 39.2% had calculus without and with gingivitis respectively.None of the adolescents had periodontal pockets and only 1% had dental fluorosis (Table 1).Eighty-three percent of 15-year-olds reported experiencing oral impacts on quality of life during the past six months, 39% of which at a moderate/ higher level of intensity.CS-impacts attributed to dental caries were the most prevalent impacts (40.7%), followed by CS-impacts attributed to oral lesions (36.4%) and periodontal disease (29.6%) (Table 1). Associations between oral diseases and moderate/higher level of oral impacts on quality of life Following discussions with dental public health professionals, academics and dentists from the Ministry of Public Health, it was decided that only oral impacts of moderate to very severe levels of intensity should be considered when setting oral health goals, since the majority of impacts reported were of very little or little level of intensity.With a moderate threshold, CS-impacts attributed to dental caries, periodontal disease, tooth discoloration and edentulous area were 18.4%, 11.3%, 3.1% and 0.1%, respectively.The association between moderate/higher CS-impacts and dental caries, periodontal disease and fluorosis with actual oral diseases was investigated.The percentage of adolescents experiencing moderate/higher CS-impacts attributed to caries increased significantly with increasing number of decayed teeth (p < 0.001) (Table 2).Significant differences in moderate/higher CS-impacts attributed to caries were also observed among adolescents with and without missing teeth due to caries (p < 0.05) but not for filled teeth.Moderate/higher CS-impacts attributed to periodontal disease were significantly associated to increasing numbers of sextants with periodontal disease (p < 0.01), gingivitis (p < 0.001), gingivitis without calculus (p < 0.01) and calculus with gingivitis (p < 0.01), whereas having more sextants with calculus or calculus without gingivitis was not significantly associated to moderate/higher CS-impacts attributed to periodontal disease.Moderate/higher CS-impacts attributed to tooth discoloration were not significantly associated with dental fluorosis (Table 2).Multivariate logistic regression was performed to ascertain the association between oral diseases and moderate/higher CS-impacts (Table 3).After adjusting for sex and regionplace of residence, the likelihood of experiencing moderate/higher CS-impacts attributed to caries increased with the number of decayed teeth.When compared to adolescents with no decayed teeth, adolescents with one decayed tooth were 3.3 times (95%CI: 1.90-5.5)more likely to experience moderate/higher impacts.This likelihood increased to 6.5 (95%CI: 2.9-15.0)and 7.6 (95%CI: 3.8-15.0)times in adolescents with four and five or more decayed teeth respectively.The association between missing teeth and moderate/higher CS-impacts attributed to caries, after adjusting for socio-demographic variables and decayed teeth, was not significant.These findings reveal the confounding role of decayed teeth.For moderate/higher CS-impacts attributed to periodontal disease, adjusted analyses revealed a non-significant association with periodontal disease and calculus with gingivitis.Gingivitis and gingivitis without calculus occurring in one to two sextants did not significantly increase the likelihood of experiencing moderate/ higher CS-impacts.However, when present in three or more sextants, the likelihood of experiencing these impacts increased almost twofold (Table 3). Classification of the risk of adverse effects on quality of life and proposals for OHRQoLbased oral health goals in Thailand Based on an increased likelihood of experiencing moderate/higher impacts found by this study, four levels of risk of adverse effects on quality of life due to oral diseases were identified: Small Risk, Great Risk, Greatest Risk and No Risk (Figure 1). "Small Risk" refers to adolescents with gingivitis in three or more sextants.Currently, 35.4% of 15-year-olds are in this category and are approximately two times more likely to experience moderate/higher oral impacts on quality of life. "Great Risk" refers to adolescents with one to three untreated decayed teeth.Currently, 37.5% of 15-year-olds are in this category and are three to four times more likely to experience moderate/higher oral impacts on quality of life. "Greatest Risk" refers to adolescents with four or more untreated decayed teeth.Currently, 11% of 15-year-olds are in this category and are up to seven times more likely to experience moderate/ higher oral impacts on quality of life. "No Risk" refers to adolescents with no untreated tooth decay and with gingivitis in less than three sextants, regardless of the number of sextants with calculus.Currently, 35.6% of 15year-olds are in this category and do not experience moderate/higher oral impacts on quality of life. The above results were discussed by a panel of dental public health professionals who, based on these findings agreed that goals should focus on reducing the number of untreated decayed teeth and extensive gingivitis in order to improve the quality of life of Thai adolescents.A panel of dental public health professionals proposed a reduction in the percentage of adolescents subject to Small, Great and Greatest Risk.In addition to existing oral health goals for 15-year-olds, the following OHRQoL-based goals are recommended: (1) "no more than 25% of 15-year-olds should have untreated decayed teeth and none should have more than three untreated decayed teeth", and (2) "no more than 25% of 15-year-olds should have extensive gingivitis occurring in three or more sextants". Discussion The integration of OHRQoL with clinical measures provides a theoretical framework for oral health goal-setting for 2020 where oral disease is linked to the ultimate goal of quality of life 13 .However, the OHRQoL-based oral health goals proposed by this study differ from the official oral health goals for adolescents in Thailand.Dental caries was considered the most important oral problem by this study because the likelihood of experiencing moderate/higher impacts on quality of life is greatest in the case of untreated tooth decay.An adolescent with one to three untreated decaying teeth was three to four times more likely to experience moderate/higher oral impacts, while an adolescent with four or more untreated decaying teeth is seven times more likely.This study categorized these groups (one to three and four or more untreated decaying teeth) as Great Risk and Greatest Risk, respectively, in terms of adverse effects on quality of life, and therefore urges oral health services to reduce the percentage of adolescents belonging to these groups, particularly the Greatest Risk group.Our findings suggest that early treatment is needed for adolescents with untreated dental caries and that oral health promotion is necessary to further reduce the number of adolescents with dental caries.Further investigations are necessary to identify the geographic concentration of the Great and Greatest Risk groups and the underlying social causes of dental caries in vulnerable populations that need to be addressed.Population-based interventions targeting all adolescents in deprived areas are recommended 30,31,32 . The difference between the published oral health goals in Thailand and those proposed by this study reflects the different concepts of oral health and oral disease.Although oral diseases may have adverse affects on quality of life, the impacts of some levels of oral diseases can be insignificant.Studies on overall periodontal disease found no significant association between adolescents with and without disease 19,20 .Although generally consistent with the findings of the current study, it was shown that acute necrotizing ulcerative gingivitis and periodontal attachment loss were significantly associated with poor OHRQoL 18 .In the present study, although exten- * p < 0.001; p < 0.05; * p < 0.01.All adjusted analyses were controlled for sex and region-place of residence.Adjusted analyses for untreated decay were additionally controlled for missing teeth, adjusted analyses for missing teeth were additionally controlled for untreated decay, adjusted analyses for gingivitis without calculus (code 1) were additionally controlled for calculus with gingivitis (code 5), and adjusted analyses for calculus with gingivitis (code 5) were additionally controlled for gingivitis without calculus (code 1). Proportions of 15-year-old Thai children at different levels of risk of adverse effects on quality of life due to oral diseases (N = 881). sive gingivitis was significantly associated with OHRQoL, no association was found between calculus and OHRQoL, even in more severe cases.These findings are similar to those from Tanzania that show that there is no significant positive association between severe calculus and overall oral impacts.It is also interesting to note that the same study showed a positive association with high levels of dental plaque 17 .In contrast, Mbawalla et al. 17 , applying different clinical measures to those used by this study, found that CS-impacts attributed to periodontal disease were not significantly associated with high levels of plaque or calculus.Therefore, further studies using CS-OHRQoL measures are recommended, particularly in relation to periodontal disease, in order to ascertain the degree or extent and form of disease and define the focus of dental public health interventions since attempts to reduce high disease prevalence with limited resources is impractical.Although the findings of this study show that overall periodontal disease and calculus in adolescents were not associated with oral impacts and gingivitis and calculus did not invariably progress to periodontitis 33 , longitudinal studies are recommended to investigate the long-term impacts of such forms of periodontal disease.This study's findings regarding untreated tooth decay corroborate the findings of all previous studies and confirmed that adolescents with untreated tooth decay were significantly more likely to have poor OHRQoL 16,17,19,20 . A limitation of the present study was the lack of clinical data on oral lesions and malocclusion since these factors were not included in Thai National Oral Health Survey 15,23 .Due to the absence of such clinical data, it was not possible to perform an analysis of the relationship between OHRQoL and oral conditions.Therefore, it was not possible to assess the importance of clinical oral lesions and malocclusion in terms of their effect on significant oral impacts on adolescents' quality of life.To accurately set OHRQoL-based oral health goals for adolescents, it is suggested that oral examination of these conditions should be included in national oral health surveys. This study has important implications for oral health service planning.Using a diseasebased approach results in high levels of normative oral health needs and unrealistic oral health goals 10,11 .The OHRQoL approach follows the FDI/WHO/IADR guidelines 3 , provides evidence of the type and extent of oral diseases in the country that should be considered as real dental public health problems, and therefore leads to more appropriate and realistic oral health goals Table 1 Socio-demographic characteristics, oral health status and oral impacts on quality of life of 15-year-old Thai adolescents (N = 811). Table 1 ( continued) Socio-demographic characteristics, oral health status and oral impacts on quality of life of 15-year-old Thai adolescents (N = 811). * Data on fl uorosis available for only 314 children. Table 2 Percentage of 15-year-old Thai adolescents with moderate to very severe condition-specifi c impacts (CS-impacts), by different levels of oral diseases (N = 811). Table 3 Logistic regression of moderate to very severe condition-specifi c impacts (CS-impacts) on oral diseases in 811 15-year-old Thai adolescents. 34e findings of this study are not a universal representation of the association between oral diseases and OHRQoL since individuals' perceptions of oral health vary from one social context to another34.Moreover, the classification of levels of risk of the adverse effects on quality of life is relative to a population's percep-tions and therefore may vary between countries.Nevertheless, all countries are recommended to integrate the conceptual ideas of the FDI/WHO/ IADR guidelines into their oral health service planning to assure that attempts to reduce oral diseases lead to improvements in the quality of life of the population.ResumoO objetivo deste estudo foi avaliar a associação entre doença bucal e a condição específica de qualidade de vida associada à saúde bucal (CS-OHRQoL), como base para propor OHRQoL metas para adolescentes tailandeses.Exame clínico bucal e entrevista foram realizados em 871 adolescentes na faixa etária de 15 anos, como parte da 6 a Pesquisa Nacional Tailandesa de Saúde Bucal.A severidade do impacto bucal foi categorizada usando-se a "intensidade".A associação entre doença bucal e CS-OHRQoL foi investigada usando-se o teste qui-quadrado e regressão lógica.Trinta e nove por cento da amostra reportaram impactos bucais de grau moderado/elevado.A probabilidade de reportar um impacto bucal de grau moderado/elevado dos adolescentes com um dente cariado e aqueles com 4 ou mais foi 3 e 7 vezes maior, respectivamente, quando comparada à dos adolescentes sem dentes cariados.A presença de gengivite severa em 3 ou mais sextantes dobrou a probabilidade de ocorrência do CS-impacto de grau moderado/elevado.Metas de saúde bucal para adolescentes devem incluir instrumentos específicos de OHRQoL.Krisdapong contributed to study conception, the literature review, research design, data collection, input, validation, analysis and interpretation, drafting of the manuscript and final approval of submitted manuscript.P. Prasertsom contributed to research design, data collection and input, critical revision of the manuscript for important intellectual content and final approval of the submitted manuscript.K. Rattanarangsima contributed to research design, data collection and input, critical revision of the manuscript for important intellectual content and final approval of submitted manuscript.S. Adulyanon contributed to study conception, research design, critical revision of the manuscript for important intellectual content and final approval of the submitted manuscript.A. Sheiham contributed to study conception, interpretation of data, critical revision of the manuscript for important intellectual content and final approval of the submitted manuscript.	2017-04-08T14:38:20.820Z	2012-10-01T00:00:00.000	{ "year": 2012, "sha1": "e01e865f456086b8ac90074a60c21b57ac4f45cc", "oa_license": "CCBY", "oa_url": "https://www.scielo.br/j/csp/a/krdWwPQkshRyqqYrvk6ncfC/?format=pdf&lang=en", "oa_status": "GOLD", "pdf_src": "ScienceParseMerged", "pdf_hash": "e01e865f456086b8ac90074a60c21b57ac4f45cc", "s2fieldsofstudy": [ "Medicine", "Psychology" ], "extfieldsofstudy": [ "Medicine" ] }
232113788	pes2o/s2orc	v3-fos-license	Preparation for Adulthood: Shifting Responsibility for Management of Daily Tasks From Parents to Their Children Importance: Limited research has described the timing of acquisition of the broad range of skills required for the transition to adulthood. Objective: To describe the timing of the shift of responsibility for daily tasks from parent to child. Design: This study used an existing data set of parent responses to 49 items in the Responsibility domain of the Pediatric Evaluation of Disability Inventory Computer Adaptive Tests. Participants: A U.S. nationally representative sample of 2,205 typically developing children and youth ages 0 to 20 yr. Outcomes and Measures: Descriptive analyses focused on two ages: (1) starting age (when >50% of parents reported their child was taking at least some responsibility for a task) and (2) full responsibility age (when >50% of parents reported their child was taking full responsibility for the task). Results: The process of shifting responsibility for daily life tasks from parent to child typically occurred over a long period. Many task items had an interval of 5 yr from starting age to full responsibility age; the longest interval was 15 yr. Youth began assuming responsibility for more complex tasks and tasks that involved more risk at ages 10 to 15. Conclusions and Relevance: Results can serve as a reference for the timing of the transition to greater self-management of daily life tasks across childhood and adolescence. Timing of responsibility shifts may reflect a combination of development of underlying capacities and social transitions. Executive functioning may be especially relevant for management of the more complex tasks required in daily life in adulthood. What This Article Adds: The transfer of responsibility for managing tasks of daily life from parents to children often extends over a period of many years. Clinicians may find the results helpful when discussing the future with parents of young people with disabilities and other chronic conditions and the tasks that their children must learn to manage for independent living as an adult. adapted to the current context. The shift of responsibility for daily tasks from the caregiver to the young person is complex and is likely shaped by many factors, including development of the young person's underlying performance capacities (e.g., motor coordination, memory, executive functions), culturally specific transitions (e.g., timing of school entry), and cultural and family values and practices (e.g., whether the young person is expected to attend college away from home). Despite the universality of the phenomenon, limited research has described the timing, sequence, and variation in patterns of acquisition of the broad range of skills that are required for managing daily life tasks in adulthood. Most of the existing literature on responsibility development focuses more on the development of internal cognitive processes involved in taking on responsibility than on changes in task performance as a topic in its own right. For example, several lines of inquiry have focused on the construct of autonomy, which involves the extent to which young people of different ages have decision-making power over their engagement in selected daily activities. Wray-Lake and colleagues (2010,2016) reported developmental data that illustrate how parents may grant autonomy for low-risk decisions, such as choosing one's clothes, at a younger age than for higher risk activities, such as money management, for which the consequences of making an error are more significant. Literature in several fields has examined facets of the general construct of responsibility from different perspectives, such as self-reliance or self-sufficiency (Ochs & Izquierdo, 2009), identity as a "responsible person" (Bjerke, 2011;Wood et al., 2009), and variations in the personality trait of conscientiousness (Bogg & Roberts, 2004). Other studies have examined the association between parenting style and various adolescent outcomes such as risk taking, communication around decision making, and self-reliance (Grolnick et al., 2014;Huebner & Howell, 2003). Although these studies have contributed important knowledge regarding development of young persons' identity or growth in capacity to evaluate the consequences of their decisions, research has not addressed observable differences in daily occupational performance. One line of research that does focus on observable differences in performance has investigated taking responsibility for household tasks or chores (Goodnow, 1988;Larson, 2004;White & Brinkerhoff, 1981). Dunn (2004) developed a parent-report measure that describes a child's current participation in specific household tasks and the extent of caregiver support typically provided when they participate. Studies using this measure have examined similarities and differences in the participation of children with and without attention deficit hyperactivity disorder (ADHD), Down syndrome, and cerebral palsy and with families of different socioeconomic circumstances (Amaral et al., 2014;Drummond et al., 2015;Dunn et al., 2009;Dunn & Gardner, 2013). Other research on the management of daily tasks has involved studies of young people with health conditions and disabilities. This attention may have been stimulated, in part, by data showing the relatively poor employment and independent living outcomes for young adults with disabilities (Newman et al., 2011). Disability or illness in childhood presents unique challenges to learning and applying skills needed in daily life. In addition to the challenges posed by particular impairments, characteristics of the condition may also introduce unique demands that require specialized skills to manage, such as monitoring and managing blood sugar levels for a young person with diabetes (Babler & Strickland, 2015) or managing alternative methods of bowel and bladder care for a young person living with spina bifida (Tarazi et al., 2007). Moreover, recent literature has documented an association between executive function (EF) and management of daily life tasks (Rosenberg, 2015). EF problems are associated with disorders including ADHD and learning disabilities, autism spectrum disorder, and spina bifida, and studies have shown an association between EF limitations and limitations in daily functioning in people with these disorders (Gardiner & Iarocci, 2018;Jacobson et al., 2013;Sharfi & Rosenblum, 2016). Families of children with EF challenges may find that the informal approach of guided participation is insufficient to effectively teach these skills, and the families may need additional support to ensure the young person is prepared for successful adulthood. To date, there is no clear description of the developmental pattern as children and adolescents transition toward taking more responsibility across a broad range of daily life tasks expected in adulthood. This information would provide a useful guide for families and service providers about when these transitions typically happen, which tasks youth take over earlier, and which tasks require parent involvement for a longer time. In addition, a comprehensive description of developmental patterns may suggest productive areas for further investigation to understand factors that contribute to individual or group variations in the timing and outcome of the transition process. The current study addresses this need by examining two transition points in the shift of responsibility for a wide range of daily life tasks in a representative sample of children and adolescents in the United States. The specific research questions were as follows: 1. When do parents begin to engage their child by giving them some responsibility for a daily life task? 2. At what age do parents generally hand over management of the tasks completely to the youth? Method This study used existing data originally collected to examine the psychometric properties of a revised measure of function for children with disabilities, the Pediatric Evaluation of Disability Inventory Computer Adaptive Tests (PEDI-CAT; Haley et al., 2012). The data are parent reports from a nationally representative sample of 2,205 typically developing children and youth in the United States ages 0-20 yr. Data and Participants The data were collected via the internet by an online survey company (YouGov, London, England) between May 2009 and August 2009. The company has an online panel of members (n = 115,000) who have regularly participated in online surveys. The survey company contacted only panel members with one or more children younger than age 21 yr with addresses within the contiguous United States. Details regarding data collection were reported in a prior article (Haley et al., 2011). Parents were asked to answer a series of screening questions (e.g., Was the child receiving early intervention services? Did the child have any limitation in personal care activities, routine needs, play, or recreation?) to determine their eligibility and placement in either the normative or the disability sample. The analysis in this article focuses on the normative sample, which included 2,205 parents reporting on their typically developing child or adolescent. YouGov used a quota sampling method based on child's age to ensure that sufficient cases were collected in each of the age strata (100 cases for each age year under age 21). The company monitored sample composition to ensure that there were equal proportions of male and female children and youth and that the overall sample was representative of the U.S. population. In general, the distribution of race and ethnicity of the final sample matched the profile of the 2000 U.S. Census (U.S. Census Bureau, 2001), except that this sample had less Asian representation (1.1% vs. 3.6%). Table 1 provides demographic information for the 2,205 children and youth. To limit respondent burden during data collection, a set of four overlapping, shorter PEDI-CAT forms was created for each age group (ages 0-7, 8-14, and 15-21); a total of 12 forms were used. Parents of children in each age group were randomly assigned to one of the four forms in that age group. Using this multiform design, the number of valid responses for each item ranged from 915 (41.5%) to 1,535 (69.6%) of the 2,205 children and youth, with approximately 25% of the participants (n = 573) completing all items in the Responsibility domain. There were no unplanned missing data. For more details regarding this multiform design, see the manual of the PEDI-CAT (Haley et al., 2012). Instrument The Responsibility domain is one of four domains in the PEDI-CAT (Haley et al., 2012). This component of the instrument examines parents' perspective on the extent to which a young person is taking responsibility for managing life tasks. The Responsibility domain includes 51 items that describe a range of important daily tasks that enable independent living (e.g., taking care of daily needs, health management, staying safe, organization and planning). An example question is "How much responsibility does your child take for the following activities?" The item "Getting ready in the morning on time" includes the tasks of "Getting up; Getting dressed; Grooming and hygiene activities; Eating breakfast; Completing on time" (Haley et al., 2012, p. 53). Respondents rate the extent to which responsibility for each task is being assumed by the parent and child using a 5point scale in which 1 = Adult/caregiver has full responsibility; the child does not take any responsibility; 2 = Adult/caregiver has most responsibility and child takes a little responsibility; 3 = Adult/caregiver and child share responsibility about equally; 4 = Child has most responsibility with a little direction, supervision, or guidance from an adult/caregiver; and 5 = Child takes full responsibility without any direction, supervision, or guidance from an adult/caregiver (Haley et al., 2012, p. 58). Table 2 provides more examples. The test-retest reliability and discriminant validity of the Responsibility domain have been supported (Haley et al., 2012). The raw score on each item in the Responsibility domain was used for analysis. Two items, managing menstrual cycle and voting, were excluded from the analysis because they were not applicable to a large proportion of the respondents because of gender or age. Data Analysis The data for the original PEDI-CAT sample included the child's age in whole years. For this analysis, we focused on the timing of two transition points, identified using the frequency table for each item. Starting age for a task was defined as the first age group (year) in which more than 50% of parents reported their child was taking at least some responsibility (a rating of 2 or above). This age indicates the point at which, on average, transfer of responsibility from parent to child has begun to occur. Full responsibility age was defined as the age at which more than 50% of parents first reported that their child was taking full responsibility for the task (a rating of 5). This age indicates the point at which, on average, the responsibility shift is complete. Results Figures 1 and 2 graphically present the results, showing the two transition ages for each Responsibility domain item. The items are ordered by the starting age; for items toward the bottom of the figures, the responsibility shift began at a younger age, and for those toward the top, the shift began at an older age. Items with an earlier starting age (see Figure 1) cover a wide range of areas: basic self-care (e.g., managing bowel and bladder through the day), safety (e.g., eating safely), preparation for school life (e.g., getting ready in the morning), chores (e.g., putting items or objects away after use, maintaining cleanliness and upkeep of living space), and selfregulation (e.g., coping with stress, worry, or anger). The majority of parents started to transfer some responsibility in these areas when their child was around 2 or 3 yr old. Items with a later starting age (see Figure 2) also reflect a wide range of areas: money management (e.g., paying bills), health (e.g., managing health appointments), and some complex life tasks (e.g., resolving errors in personal business, locating needed services or supports). The majority of parents reported transferring some responsibility in these areas when their child was between ages 13 and 15. Items for which parents reported that their child had full responsibility at an earlier age (see Figure 1) include managing bowel and bladder through the day, managing bowel and bladder through the night, eating safely, and testing and adjusting water temperature. The majority of parents reported that their child was fully in charge in these areas when between ages 5 and 9. Items with a later full responsibility age (see Figure 2) include completing legal and/or other personal paperwork, locating needed services or supports, resolving errors in personal business, and organizing important papers and information. These four items also had a late starting age. For most items in the Responsibility domain, the transfer of responsibility occurred across a long time interval. The item with the longest interval is maintaining cleanliness and upkeep of living space; the starting age was 3 yr, whereas the full responsibility age was 18. Other items with a long gap between ages are coping with stress, worry, or anger; putting items and objects away after use; and getting ready in the morning. Discussion This cross-sectional study describes the timing of the shift in responsibility for managing daily tasks from parents to children in a typically developing sample in the United States. Overall, we found that there was a long period between the starting and full responsibility ages for most tasks represented by the items in the Responsibility domain of the PEDI-CAT. For many of these items, the gap was 5 yr or longer, and the longest gap was 15 yr. In other words, the shift of responsibility from parent to child occurred gradually in most areas of daily life. This finding is consistent with Young and colleagues' (2008) report in which parents described the experience of preparing children for adulthood as a natural and gradual process. Rating scale a □ Adult/caregiver has full responsibility; the child does not take any responsibility. □ Adult/caregiver has most responsibility and child takes a little responsibility. □ Adult/caregiver and child share responsibility about equally. □ Child has most responsibility with a little direction, supervision, or guidance from an adult/caregiver. □ Child takes full responsibility without any direction, supervision, or guidance from an adult/caregiver. a In the data analysis, we assigned a score to each rating option. The option with the child taking full responsibility was assigned a score of 5, and the option with the adult or caregiver taking full responsibility was assigned a score of 1. As discussed earlier, the timing of the shift of responsibility across the different tasks likely reflects a complex set of factors. For example, Wray-Lake and colleagues (2010), in a study of developmental patterns of decision-making autonomy, found that money management was the area in which autonomy increased most slowly. Similarly, in the current study, the age of full responsibility was much later for items such as paying bills and managing daily expenses. In addition, Wray-Lake et al. found that for chores, decision-making autonomy remained low across childhood and through adolescence. In our study, although parents started to shift responsibility to their children at an early age for some chores, such as putting items away after use and maintaining cleanliness and upkeep of living space, there was a long gap of 13-15 yr between the starting and full responsibility ages for these items. Similarly, the area of health was identified by Wray-Lake et al. as one in which autonomy remained low across middle childhood to late adolescence, and we found that youth assumed full responsibility for health-related tasks at older ages (e.g., age 18 for managing health appointments). The timing of the shift of responsibility for many tasks may be related to the development of children's EF. A previous study found that children's EF was associated with the level of assistance needed in daily life situations (Rosenberg, 2015). In the current study, we found that many of the items had a starting age of around 5-8 yr and a full responsibility age around 15-19 yr. In the development of EF, children show significant gains in working memory in the 5-8 yr age range (De Luca & Leventer, 2008). As children's working memory develops, they are more able to learn and recall the steps required to complete a daily life task. When children start to remember the procedure or the directions for a task, parents may begin to involve children more in the task or may lessen supervision. Although planning and problem solving continue to mature during young adulthood, adolescents show significant gains in strategic planning and problem solving around ages 15-19 (De Luca & Leventer, 2008). This growth may help explain why youth assumed full responsibility for many items in the Responsibility domain in this age period. Many of the items with a later transition age require the young person to plan actions and identify feasible methods to solve problems, which reflect EF skills (e.g., prioritizing multiple goals, resolving errors in personal business). The timing of responsibility shifts may also be related to important milestones in young people's lives-for example, entering school or leaving home for college or work. The shift we found around ages 5-8 yr on many items is also the period when formal schooling starts in the United States. Parents may become more focused on preparing their children for school life and teaching them to be responsible for school-related tasks. There also are many changes in children's social context during this period. A child's world broadens when entering school. Exposure to a new social environment and new performance demands within the school environment may give children opportunities to learn new skills. In turn, these skills enable children to begin to take responsibility for some daily tasks. Many tasks had a full responsibility age between 17 and 19 yr. This is the period when many young people in the United States leave home for college or work. As young people move away from home, their parents may no longer be geographically close enough to provide regular supervision or guidance for most tasks. Items with the greatest gap between the two transition ages were in areas in which research has shown that parents and adolescents have frequent conflicts: maintaining cleanliness and upkeep of living space, putting objects away after use, and getting ready in the morning. Riesch et al.'s (2000) survey of 163 parent and young adolescent dyads found that getting up in the morning, putting clothes away, helping out around the house, and cleaning up one's bedroom were ranked among the top 10 topics that parents and adolescents had the most frequent disagreements about. Parents may have certain expectations for how these tasks should be performed, whereas children may want the freedom to decide how they handle these tasks. Parents and children may argue about these tasks for many years, but eventually a balance may be reached when parents adjust their expectations, children meet their parents' expectations, or a combination of both. This interpretation echoes a theme in the literature on autonomy development suggesting that parents' expectations and children's expectations gradually converge between early and late adolescence (Zimmer-Gembeck & Collins, 2006). Several features of the study design and secondary data analysis must be considered when interpreting these findings. First, the data were collected in the United States, and thus the results may not be generalizable to other countries. Different countries may have different regulations regarding the age at which young people may complete legal documents, work, obtain a driver's license, or access certain financial services (e.g., debit or credit card). These regulations are likely to influence the timing of transfer of responsibility for these tasks. In addition, culture may influence parents' beliefs about appropriate child-rearing practices and shape the ways in which parents engage their children in daily life, such as whether children should have chores (Drummond et al., 2015;Rogoff, 2003). Given the likelihood of differences across countries and cultures, cross-cultural comparison studies would be valuable to help service providers better understand how the timing of responsibility shifts differs between cultures and what factors are associated with those differences. Second, although the overall sample of this study represented the racial and ethnic proportions of the 2000 U.S. Census, further research is needed with minority (e.g., Hispanic Americans, Asian Americans) and other subgroups (e.g., young people growing up in rural areas) to determine whether the timing of transitions varies and, if so, in which task areas. Third, this study used a cross-sectional design to describe the timing of the transfer of responsibility from parents to their children; therefore, it is not possible to conclude that the reported timetable reflects only the effects of age. A crosssectional study is an efficient way to expand knowledge regarding this everyday phenomenon in a population across a broad age range. However, future research with a longitudinal design that follows families for many years would be valuable to help service providers better understand the process through which parents transfer responsibility to their children and the factors associated with variations in this process. Moreover, studies of young people with disabilities would provide valuable information about how variations in disability and environmental factors affect the timing of transfer of responsibility to provide further guidance for service providers working with families and youth as they transition to adulthood. Implications for Occupational Therapy Practice The findings of this study have the following implications for occupational therapy practice: n The timing of the shift of responsibility for important daily tasks from parent to child varies significantly by task. n Data on the typical timing of transitions in responsibility can provide a useful starting point for conversations with parents about their expectations and priorities for their children and the ways they are assisting them to manage daily tasks. n Clinicians who work with children with disabilities may reference these overall patterns to guide programming throughout childhood and adolescence to prepare young people for adult tasks. n Clinicians should consider families' cultural beliefs, expectations, and unique circumstances when discussing the timing of their children's responsibility for daily tasks. Conclusion The results of this study indicate that the transfer of responsibility for managing tasks of daily life from parents to their children often extends over a period of many years. The findings may serve as a useful starting point for conversations with parents about their expectations for their children and the steps they are taking to assist their children to manage important tasks on their own. Clinicians may find the results helpful when discussing the future with parents of young people with disabilities and other chronic conditions and the tasks that their children must learn to manage for independent living as an adult. Readers are cautioned that the timetable described in this article reflects the overall patterns of the population sampled and that the timetable for an individual child and family may vary according to their unique circumstances.	2021-03-05T06:22:59.344Z	2021-02-02T00:00:00.000	{ "year": 2021, "sha1": "3fad229f2afe29260b9b0bd3a48a7ac9d9d54cc3", "oa_license": null, "oa_url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7929602", "oa_status": "GREEN", "pdf_src": "PubMedCentral", "pdf_hash": "acd0a287e93f061168a804378c8dd2c237e9bb58", "s2fieldsofstudy": [ "Psychology" ], "extfieldsofstudy": [ "Medicine" ] }
26963959	pes2o/s2orc	v3-fos-license	A Role for Androgens in Epithelial Proliferation and Formation of Glands in the Mouse Uterus The endometrium consists of stromal and epithelial compartments (luminal and glandular) with distinct functions in the regulation of uterine homeostasis. Ovarian sex steroids, namely 17 (cid:2) estradiol (E 2 ) and progesterone (P 4 ), play essential roles in modulating uterine cell proliferation, stromal-epithelial cross-talk and differentiation in preparation for pregnancy. The impact of an-drogensonuterinefunctionremainspoorlyunderstood.Thecurrentstudyinvestigatedtheimpact of the non-aromatizable androgen dihydrotestosterone (DHT) on mouse endometrial function. Ovariectomized female mice were given a single subcutaneous injection (short treatment) or 7 daily injections (long treatment) of vehicle alone (5% ethanol, 0.4% methylcellulose) or vehicle with the addition of 0.2 mg DHT (n (cid:2) 8/group) and a single injection of Bromodeoxyuridine (BrdU) 2 hours prior to tissue recovery. Prl8, Prlr ) as well as a striking impact on the number of endometrial glands. This study has revealed a novel role for androgens in regulating uterine function with an impact on the glandular compartment of the endometrium. This previously unrecognised role for androgens has implications for our understanding of the role of androgens in regulation of endometrial function and fertility in women. ment group). Statistical analysis performed using GraphPad Prism 6.0. Student’s t test was used for comparison between VC and DHT groups, with statistical significance for P (cid:5) .05. T he endometrium is a hormone-dependent multicellular tissue that consists of stromal and epithelial compartments. The stromal compartment contains mesenchymal fibroblasts, a well-developed vasculature and immune cells, while the epithelium is divided into a luminal and a glandular component, each with a distinct identity and role in reproductive physiology (1). Both compartments respond to hormonal cues in a coordinated spatial and temporal manner characterized by extensive cross-talk to regulate the development and homeostasis of the tissue (2)(3)(4). While the impacts of estrogens on endometrial function have been extensively studied, androgens have only recently emerged as key players in the regulation of endometrial function. Analysis of circulating steroid hormone levels utilizing sensitive liquid chromatography-tandem mass spectrometry has revealed significant concentrations of androgens present throughout the menstrual cycle in women exceeding those of circulating estrogens (5). In-vestigation of circulating androgen levels in cycling female mice reported detectable concentrations of the circulating testosterone metabolite 5␣-dihydrotestosterone (DHT; 2.8 Ϯ 0.4 pg/ml) during all phases of the estrous cycle, with concentrations peaking during proestrus and metestrus (6). In aging female mice, there is a significant increase of circulating DHT, with a mean value of 12.8 Ϯ 0.6 pg/ml. To our knowledge there are no studies in the literature regarding the circulating levels of DHT during mouse pregnancy, however, there are several reports that androgen levels increase during pregnancy in women (reviewed in (7)). In women androgens appear to be 'Goldilocks' factors with both deficiency and excess contributing to pregnancy-and fertility-related disorders such as polycystic ovarian syndrome (8,9), endometriosis (10) and recurrent pregnancy loss (11). The effects of androgens are mediated via the androgen receptor (AR), a ligand-activated transcription factor that binds testosterone (T) and its metabolite 5␣-dihydrotestosterone (DHT) with high affinity and specificity. In women, expression of AR in the endometrium is regulated both temporally and spatially during the normal menstrual cycle and detailed analysis of full-thickness uterine tissue sections identified endometrial stromal fibroblasts as a key targets for androgen action (12). Administration of antiprogestins in women and nonhuman primates induces upregulation of AR in the uterine epithelium, which may mediate the antiproliferative effects of these compounds (13). In the uteri of mice, quantitative in situ hybridization detected uniform labeling of Ar mRNA in all compartments, including the epithelium (14). A recent study reported nuclear stromal AR staining in the mouse uterus but AR was not detected in the luminal or glandular epithelium (15). A study using a transgenic mouse model of an androgen response element (ARE) construct fused to a luciferase reporter revealed strong AR activity in the uterus, with the antiandrogen bicalutamide inhibiting luciferase activity (16). Female global AR knockout (ARKO) mice are subfertile, with a neuroendocrine and ovarian phenotype accompanied by defects in uterine development (17). Studies in female-to-male transsexuals have reported a negative impact of androgens on endometrial proliferation in response to long-term administration of testosterone, resulting in endometrial atrophy (18,19). Treatment of primary human endometrial stromal cells with DHT also results in a significant reduction in both proliferation and apoptosis (12). Androgen-estrogen cross-talk has been implicated in the initiation and progression of endometrial cancers, with androgens acting in an antagonistic manner to the proliferative effects of estrogens, restricting endometrial carcinogenesis (reviewed in (20)). Weihua et al reported that the proliferative effects of E 2 in the rat uterus were blocked by either the antiestrogen tamoxifen or the antiandrogen flutamide (21), suggesting a functional overlap between androgen and estrogen action in this rodent species. Microarray analysis of uteri recovered from ovariectomized mice after a single injection of DHT revealed time-dependent changes in the expression of genes involved in the regulation of cell division and organ development between 12 and 24 hours post injection (22). Sex steroids have been shown to exert different effects in cell proliferation and apoptosis resulting from either acute or chronic stimulation (reviewed in (23,24)). In the 1970s, Lee and colleagues recorded three phases of epithelial cell proliferation within the mouse uterus in response to continuous oral treatment with estrogen, characterized by pronounced uterine epithelial cell proliferation on days 2 and 3, basal proliferation on days 4 and 5 and a second wave of proliferation on days 19 and 20 (25,26). Similar effects have been reported in prostate cancer cells in response to androgen treatment, characterized by an early proliferative response followed by a refractory period to androgen-induced proliferation (27). Taking into account this biphasic effect of androgens in controlling cell proliferation of target tissues, a short-term (24 hrs) and a long-term (7 days) treatment paradigm was selected to elucidate the impact of DHT on endometrial growth in a mouse model. Experiments were performed in ovariectomized mice to avoid the confounding impacts of endogenous ovarian hormones. In the current study, we show that treatment with DHT has compartment-specific impacts on the immunoexpression of AR, cellular proliferation and expression of cellcycle regulators and epithelial growth factors resulting in an increase in the number of uterine glands. These striking findings identify a novel role for androgens in the regulation of endometrial homeostasis. Animals and Treatments Female C57BL/6J mice were purchased from Charles Rivers Laboratories and allowed to acclimate for a week with ad libitum access to food and water. Mice were maintained in accordance with UK legal requirements under licensed approval from the UK Home Office. Eight-to ten-week old female mice were ovariectomized (ovx) by dorsal bilateral ovariectomy and allowed to recover for 7 days prior to treatment to deplete endogenous sexsteroid hormones. Surgery was performed under isoflurane anesthesia followed by a postoperative analgesic, buprenorphine (0.1 mg/kg) for pain management. Mice were randomly assigned into four treatment groups (n ϭ 8/group, 32 in total). To explore the short-term and long-term impacts of androgens mice received either one (24 hrs group) or seven (7 days group) daily subcu-taneous injections of DHT (0.2 mg/mouse) or vehicle control (5% ethanol-0.4% methylcellulose). Two hours prior to being sacrificed, mice received an intra-peritoneal injection of 0.1 ml BrdU (25 mg/ml in PBS). To measure the serum concentration of DHT after treatments, blood was collected via cardiac puncture and exsanguination from mice treated with VC (5% ethanol-0.4% methylcellulose) or DHT (0.2 mg/mouse) for 24 hrs (n ϭ 4/group). Serum DHT concentrations were assayed using a commercially available mouse DHT ELISA kit (Amsbio, BlueGene; Cat. Number E03D0001 -Assay sensitivity: 1 pg/ml). Samples were assayed in duplicate and a standard curve was generated using a four parameter logistic curve fit (4-PL); sample DHT concentrations were interpolated from the standard curve. For analysis of tissues, one uterine horn was fixed in 4% neutral buffered formalin (NBF) for paraffin embedding, sectioning and histology while the other horn was kept in RNA Save at -80°C. RNA Extraction and Reverse Transcription Total RNA was extracted from homogenized mouse uterine tissue (20 mg/sample) using TriReagent and chloroform followed by RNeasy Mini Kit (Qiagen, UK) column isolation and elution as per the manufacturer's instructions. RNA concentration and quality was measured using a Nanodrop® ND-1000 spectrophotometer (Nanodrop Technologies, USA) and the samples were standardized to 100ng/l in RNAse-free water. Reverse transcription was performed using SuperScript VILO cDNA Synthesis Kit and Master Mix (Invitrogen, UK) as per the manufacturer's instructions using a thermal cycler programmed at: 25°C for 10 minutes, 42°C for 60 minutes and 52°C for 5 minutes. Two negative controls (no enzyme control and no RNA control) and one positive control (mouse RNA positive control) were included for each set of RNA samples. Quantitative RT-PCR Analysis Quantitative PCR was performed using SuperMix with Premixed ROX dye (Invitrogen, UK), primer sets designed using the Roche Universal Probe Library Assay Design Centre (sequences in Table 1) purchased from Eurofins (MWG Operon) and probes from the Roche Universal Probe Library Mouse Set (Roche Applied Science, UK). Samples were assayed in duplicate and run on an ABI 7900 HT Fast Real Time PCR machine using the following cycling conditions: 95°C for 10 minutes then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Primer amplification efficiency was validated and analysis was performed using the relative standard curve method. Data were normalized to 18S and fold-change is expressed as the ratio of expression of each gene of interest in the treated groups vs the average of the VC groups (one for the 24 hrs and one for the 7 days time point respectively). Statistical analysis was performed using GraphPad Prism 6.0. Data are presented as mean-/ϩSEM. Student's t test was used for comparison between VC and DHT groups. Criterion for significance was P Ͻ .05. Primer pair and probe information is provided in Supplemental Table 1. Immunohistochemistry and Immunofluorescence Samples were fixed in 4% NBF overnight at room temperature, transferred in 70% ethanol and the tissues were processed for paraffin wax embedding. Transverse sections of 5 m thickness were cut using a microtome (Leica RM2135) and the slides were incubated overnight at 55°C. For histology, the tissues were dewaxed, rehydrated and antigen retrieval was performed using citrate (0.01M citrate, pH 6.0) in a decloaking chamber followed by several blocking steps with serum from the species the secondary antibody was raised in, to avoid nonspecific binding and hydrogen peroxide was used to block endogenous peroxidase activity. Sections were incubated with the primary antibody diluted in serum blocking solution overnight at 4°C. Negative controls were included, in which the primary antibody was omitted to identify any nonspecific binding by the secondary antibody. The following day, detection was performed by using either a conjugated secondary antibody specific for the primary antibody or a polymer-based detection system (ImmPress, Vector Labs, UK). For chromogenic immunohistochemistry, DAB (Vector, UK) was used as a chromogen, while for immunofluorescence the Tyramide Signal Amplification system (Perkin Elmer, UK) was used as per the manufacturer's instructions. Between incubations, washes were performed with TBS-Tween. Hematoxylin was used as a counterstain for immunohistochemistry and DAPI as a counterstain for immunofluorescence. Antibody information is provided in Table 1. Image Acquisition and Stereology For chromogenic immunohistochemistry images were acquired with a Provis AX70 microscope (Olympus Optical, London, UK) fitted with a Canon DS6031 camera (Canon Europe, Amsterdam) while fluorescent images were acquired with a LSM 710 confocal microscope (Carl Zeiss, UK) and processed with the ZEN software (Carl Zeiss). Tissue surface area was measured using a Zeiss AXIO Imager A1 (Carl Zeiss, UK) while quantification of BrdU-positive epithelial cells and of the number of glands was performed by blinded manual counting by two independent investigators. For the calculation of cell density, sections were counterstained with DAPI and acquired images were analyzed using the image analysis software Fiji (28). In all cases, a minimum of two nonserial sections were used (n ϭ 4 -8/treat- Trophic effects of androgens in the mouse uterus In order to elucidate the actions of DHT in mouse uterine physiology, ovx mice were treated with either vehicle or DHT under a short-or long-term treatment regime (24 hrs and 7 days respectively). ELISA analysis of serum from mice injected with vehicle control or DHT 24 hrs previously had a mean DHT concentration of 180 pg/ml in the DHT group, while DHT concentrations in the control group were below the level of detection (Ͻ1 pg/ml) (Supplemental Figure 1). These results appear consistent with those of Zhang et al who previously demonstrated that ovx C57BL/6 female mice injected subcutaneously with 0.1 mg/mouse DHT, exhibit a peak of circulating DHT 1 hour postinjection (ϳ14 ng/ml) which rapidly declines over the following 24 hrs (29). The short-term treatment with DHT (24 hrs) induced a significant increase in uterine weight compared to vehicle which was further increased when treatment was extended to 7 days ( Figure 1A, P Ͻ .001 at 7 days). The effect of DHT on uterine weight was accompanied by significant increase of total uterine surface area ( Figure 1B), endometrial area ( Figure 1C) and myometrial area ( Figure 1D) at both time points. Analysis of cell density suggested the change in uterine size was due to an increase in cell numbers accompanied by a significant reduction in cell density at both time points ( Figure 1E, F). Treatment with DHT induces epithelial AR expression in the mouse uterus Positive immunoexpression of AR was detected in the stromal compartment at both 24 hrs and 7 days in both vehicle and DHT-treated mice. DHT treatment induced expression of AR in the luminal and glandular epithelium at 24 hrs ( Figure 2B) that was absent in vehicle groups at both time points (Figure 2A, C). In mice treated with DHT for 7 days, AR was detected in the stroma and the glandular epithelium but not the luminal epithelium ( Figure 2D). Real-time PCR showed a significant decrease of Ar mRNA after 7 days of DHT treatment compared to vehicle ( Figure 2E). Investigation of the expression pattern of ER␣ revealed no difference between vehicle and DHT treatedgroups at both time points (Supplemental Figure 2). Treatment with DHT induces epithelial proliferation in the mouse uterus Immunofluorescence was performed with an antibody targeting the proliferation marker MKi67 that detects all cells in interphase or undergoing mitosis. Staining identified extensive proliferation of luminal and glandular epithelial cells after treatment with DHT at 24 hrs compared to vehicle ( Figure 3A DHT regulates genes necessary for proliferation, cell-cycle regulation and stromal-epithelial crosstalk in the mouse uterus Real-time PCR analysis identified dynamic changes in the expression of several genes associated with cell-cycle regulation and stromal-epithelial cross-talk following treatment with DHT. Wee1 expression was significantly upregulated by DHT after 24 hrs, followed by a significant downregulation at 7 days ( Figure 5A). Ccnd1 expression was significantly increased by DHT at 24 hrs followed by a significant decrease after 7 days ( Figure 5B). Rb1 expression was significantly downregulated by DHT at both time points ( Figure 5C). Wnt signaling, involved in stromal-epithelial cross-talk in the uterus, was significantly affected by treatment with DHT. Wnt4 expression was significantly downregulated after treatment with DHT for 24 hrs and 7 days ( Figure 5D). Wnt5a mRNA concentration was significantly increased by DHT at both time points (Figure 5E), while press.endocrine.org/journal/endo 5 Wnt7a expression was downregulated by DHT only at 7 days ( Figure 5F). Cdh1 which is expressed in the uterine epithelium was significantly upregulated by DHT at both 24 hrs and 7 days ( Figure 5G), while expression of the adherens junction component vinculin (Vcl) involved in intercellular adhesion and signaling displayed a significant upregulation by DHT at 24 hrs, with no significant change observed between vehicle and DHT at 7 days ( Figure 5H). Expression of the putatively androgen-regulated epithelial growth factor Igf1 was significantly upregulated by DHT at both time points compared to vehicle treatments, reaching an average 10-fold increase at 7 days ( Figure 5I). Prl8, whose role has been reported in gland formation in the mouse uterus, was significantly upregulated more than 20-fold at both time points ( Figure 5J), with a similar pattern observed for it receptor (Prlr, Figure 5J). Furthermore, expression of the epithelial growth factors Fgf7 ( Figure 7L) and Hgf ( Figure 7M) was significantly altered after treatment with DHT. The transcription factor Foxa2, which is exclusively expressed in uterine glands exhibited a trend for an increase after DHT treatment at 24 hrs (P ϭ .14) but the changes at both time points didn't reach statistical significance ( Figure 5K). A role for androgens in regulating gland formation in the mouse uterus Histological examination of uterine sections at 7 days suggested more glands with DHT treatment. FOXA2 is a transcription factor expressed exclusively in the glands in the mouse uterus and FOXA2 immunohistochemistry was performed to quantify the number of glands between the treatment groups ( Figure 6A, B). A significant increase in the total number of glands was observed with DHT compared to vehicle treatment verifying the qualitative observations ( Figure 6C). To compare the effect of DHT with that with E 2 in glandular expansion, ovx mice treated with an E 2 pellet for a week were used as a comparator. Staining for FOXA2 and quantification of glands demonstrated fewer glands in the E 2 group compared to the DHT-treated group accompanied by a weaker staining pattern (Supplemental Figure 3A, D). Real-time PCR identified a significant reduction in Foxa2 expression levels between E 2 and DHT-treated groups (Supplemental Figure 3). Discussion In this study, we have demonstrated that androgens induce epithelial proliferation resulting in an increased numbers of endometrial glands in the adult mouse uterus. Uterine glands are essential for uterine receptivity and implantation and secrete factors which are necessary for the survival and development of the conceptus. Our results suggest that homeostatic balance of androgens and the local steroidogenic environment could be important physiological drivers of gland formation during endometrial remodelling in early pregnancy. Animal models in which uterine gland development is disrupted, exhibit compromised fertility and altered expression of decidualization factors (30,31), highlighting the potential for prospective therapeutic targeting of androgen signaling in the uterus for fertilityassociated disorders (32). Although a trophic effect of androgens in the rodent uterus has previously been reported, we identify for the first time the molecular mechanisms involved and selective effects of androgens on distinct tissue compartments within the uterus. Notably, we describe for the first time that DHT promotes gland formation in the adult mouse uterus through directly stimulating glandular epithelial proliferation and by driving expression of regulators of cell cycle and stromal-epithelial interactions. The novel data generated in the current study provide new insights into the role of androgens in the regulation of endometrial function. In the uterus, sex steroid action occurs via direct steroid hormone receptor signaling in target cells and indirect paracrine effects on neighboring cellular compartments. Such examples of intercellular paracrine signaling include the stromal-derived secretion of IGF1 and WNT5␣ binding on epithelial receptors controlling proliferation and gland formation respectively. Thus, regulation of the complex stromal-epithelial interactions resulting from androgen action can be investigated using in vivo model systems, rendering rodents invaluable models. To investigate the impact of androgen treatment in the steroid-depleted mouse uterus, we treated ovx mice with DHT at a supraphysiological dose (0.2 mg/mouse) previously shown to elicit uterine responses (29,33). Although the DHT concentrations generated in our experimental conditions are above the physiological levels detected in cycling female mice (6), this dose is consistent with those used in several previous studies investigating the direct action of DHT in the rodent uterus (22,34) and adipose tissue (29,35). In this study, DHT induced a time-dependent increase in uterine weight in agreement with previous reports in rats treated with DHT or the synthetic androgen mibolerone, where a dose-and time-dependent increase in uterine weight after androgen treatment was detected in that species (34). In the present study, increases in total uterine area, endometrial area and myometrial area accompanied by a significant decrease in cell density, possibly due to water imbibition, were detected, demonstrating the trophic effects of DHT in both uterine compartments in the mouse. Although trophic effects are also induced by estrogens in the rodent uterus, in the current study uterine morphology after DHT treatment lacked the characteristic distended phenotype, edema and thinness of the endometrial layer induced by E 2 (36,37) consistent with a unique effect of androgens in regulating uterine physiology that is distinct from that of estrogens. Early studies using in vitro cell-free systems and in vivo rodent models have demonstrated interaction between testosterone and DHT with ER, leading to nuclear translocation of ER, while cotreatment with antiestrogens but not antiandrogens blocked this effect (38,39). However, a later study demonstrated that the in vivo doses of DHT required to elicit ER cytoplasmic depletion and nuclear translocation in the rat uterus are very high (5-10 mg), suggesting that the uterine trophic effects of DHT observed in our study are unlikely to be mediated by ER (40). In addition, ovariectomized female rats cotreated with DHT and the AR antagonist bicalutamide exhibited a complete suppression of DHT-induced expression of Igf1, a growth factor whose upregulation is both ER␣-and ARcontrolled (34). This finding together with the fact that putative ER␣ target-genes are not induced after treatment with DHT in the mouse uterus (34) suggest more of a functional interaction between AR and ER␣ than a physical one. Notably, in a study describing the development of a uterine glandular epithelial specific ARKO mouse model (ugeARKO), uterine parameters (uterine weight and surface area) of adult ugeARKO female mice were within the normal range but androgen-induced uterine growth in ovx mice was impaired (41) consistent with the results in the present study which describe androgenic induction of uterine growth via epithelial AR. Since androgens promote growth of the rodent uterus, we investigated expression of proliferation markers in the tissue to elucidate compartment-specific effects. Assessment by immunofluorecence of the proliferation marker MKi67 which detects all actively proliferating cells (cells not in G 0 of the cell-cycle) revealed extensive proliferation within the uterine epithelium that was associated press.endocrine.org/journal/endo with a significant increase in concentrations of Mki67 mRNA after 24 hours that was also accompanied by nuclear translocation of cyclin D1 in the epithelium (data not shown). The proliferative effect of DHT was confirmed by assessing incorporation of BrdU, which revealed extensive proliferation in the epithelial compartment but not in the stroma after treatment with DHT. Notably, increased proliferation was only detected after 24 hrs of treatment consistent with DHT inducing rapid changes in the tissue. Our findings are further complemented by Terada et al who reported that treatment of ovx E 2 -primed mice with DHT significantly decreased the apoptotic index of the uterine epithelium, thus arguing for an inhibitory effect of DHT on cell death on the mouse uterine epithelium (42). Furthermore, the effects of DHT partially mimic those seen after treatment with E 2 where maximal epithelial proliferation rate is observed after 21 hrs of E 2 treatment followed by a decline in proliferation and increased apoptosis after repeated E 2 injections (43). Treatment of ovx mice with DHT resulted in a striking increase in AR immunoreactivity in the uterine stroma accompanied by induced expression of AR in glandular and luminal epithelial cells. Previous studies have demonstrated that while androgens decrease AR transcription, they positively modulate translation efficiency and stability of the AR protein, thus increasing AR protein levels (44 -47). These findings are in agreement with our data where we detected a significant decrease of Ar mRNA following treatment with DHT for 7 days accompanied by an increase of AR protein levels. As already mentioned, previous studies have reported that androgenic effects in the mouse uterus can be mediated via ER␣ signaling (21,34), with extensive cross-talk between the two receptors (40,48). However no change was observed in the expression pattern of ER␣ after DHT treatment, suggesting that if any functional interaction occurs between AR and ER␣ following DHT treatment it is not due to altered ER␣ levels. Taking together the uterine epithelial proliferation following DHT treatment with the known role of androgens in controlling cell-cycle progression in the prostate (49) and the mouse uterus (22), transcript expression of cell-cycle regulators was analyzed by real-time PCR. The multifunctional cell-cycle regulator Wee1 controls chromatin integrity and entry into mitosis by regulating the transcription of histones and forming active complexes with factors that control cell-cycle progression (reviewed in (50)). DHT induced upregulation of Wee1 at 24 hrs, consis- tent with the actively proliferating epithelial compartment at that time point, while its expression after 7 days of DHT treatment was significantly reduced compared to vehicle control suggesting a negative feedback mechanism restoring tissue proliferation to basal levels. The same expression pattern was observed for Ccnd1 (cyclin D1), whose levels and nuclear localization have been shown to be regulated by E 2 in mouse uterine epithelial cells (51). Expression of the cell-cycle inhibitor and tumor suppressor Rb1 was significantly downregulated by DHT at both time points consistent with increased proliferation and growth of the uterus detected in response to DHT. Since the changes seen in uterine size reflect changes in all cellular compartments (including the stroma) further studies are required to investigate whether changes in stromal proliferation occur in response to DHT across other time points post androgen administration. The results of the current study extend previous transcriptional analyses of samples generated from DHT-treated isolated human endometrial stromal cells which demonstrate AR-dependent repression of cell cycle regulators (52) and DHT-dependent decreases in both proliferation and apoptosis (12). Taken together these results suggest androgens may impact on the stromal compartment to inhibit stromal cell apoptosis, rather than promoting proliferation, in addition to the direct effects on the uterine epithelial proliferation described in the current study. Wnt signaling is a major pathway involved in stromalepithelial interactions and organ patterning during neonatal and postnatal uterine development. Wnt4 and Wnt5␣ are expressed by stromal fibroblasts and Wnt7␣ by epithelial cells with paracrine and autocrine signaling controlling cell fate and differentiation of the uterus (53). We observed significant changes in the expression pattern of these factors, with Wnt4 and Wnt7a being significantly downregulated and Wnt5a significantly upregulated after DHT treatment. Studies with mice have demonstrated that all three Wnts are required for gland formation in the uterus and knockout mice for these Wnts have compromised fertility (54 -56). In addition, administration of either progesterone or the synthetic estrogen diethylstilbestrol (DES) during an early postnatal time window inhibits endometrial formation of glands (adenogenesis) in the mouse uterus (3,57). Similarly, cadherin 1 (Cdh1) has been to shown to be vital for endometrial differentiation of the uterine epithelium, while loss of Cdh1 in the uterus leads to absence of glands, a disorganized luminal epithelium and upregulation of Wnt4a with concomitant downregulation of Wnt5a in the neonatal mouse uterus (58). In our study, treatment with DHT induced a significant upregulation of Cdh1 at both 24 hrs and 7 days accompanied by downregulation of Wnt4␣ and upregulation of Wnt5␣, suggesting that androgens in the mouse uterus positively regulate formation of glands. press.endocrine.org/journal/endo 9 Trophic effects in tissues are often accompanied by tissue remodelling and that led us to investigate the expres-sion of vinculin (Vcl), an adherens junction component involved in cell-cell adhesion, whose expression is andro- gen-regulated in the prostate (59). Vcl was significantly upregulated after 24 hrs of DHT treatment and by 7 days its expression was the same as the vehicle, indicating an initial phase of alteration in cell adhesion properties followed by restoration to basal levels. In addition, this is the first evidence of androgenic regulation of Vcl in the uterus. To corroborate the increased epithelial proliferation after treatment with DHT, expression of epithelial growth factors was assessed. IGF1 is predominantly produced by mouse endometrial stromal cells following E 2 treatment and signals to the epithelium to proliferate (51) and previous studies have reported that the Igf1 promoter contains androgen response elements (60). In the current study, a significant increase in the expression of the epithelial growth factor Igf1 was detected after 7 days of DHT treatment. Notably, Nantermet et al reported that DHT did not induce uterine hypertrophy in mice lacking a functional estrogen receptor (estrogen receptor ␣ knockout mice; ERKO), suggesting that ER␣ is required to mediate DHT responses (34). However, Klotz et al have previously reported that ER␣ expression is necessary to mediate the proliferative effect of IGF1 in the mouse uterus (61). Thus, taken together with the data in the current study these results are consistent with DHT also mediating indirect effects on proliferation in the uterus through stromal-epithelial cross-talk following induction of the IGF signaling pathway. The stromal-derived epithelial growth factors FGF7 and HGF both mediate stromal-epithelial interactions in the uterus by promoting epithelial proliferation with important roles in epithelial morphogenesis (62). While progesterone administration in the neonatal mouse uterus inhibits uterine gland formation accompanied by a significant reduction in the expression of Fgf7 and a significant increase in the expression of Hgf (62), in our study, treatment with DHT induced the opposite effect by significantly increasing Fgf7 mRNA levels and decreasing Hgf mRNA levels. The most striking result observed was the impact of DHT on uterine glands. In control uteri, AR expression was not detected in glandular epithelial cells, however DHT treatment induced AR expression and increased glandular epithelial proliferation. Quantification of FOXA2-positive glands demonstrated more uterine glands in the DHT-treated mouse uterus, an effect which was unique to DHT, as treatment with E 2 did not produce this effect. FOXA2 is expressed exclusively in the uterine glandular epithelium of mice and Foxa2 uterine knockout mice fail to develop glands (30). In the current study, DHT induced changes in expression of a number of factors that promote adenogenesis in adult mice. Although expression of Foxa2 mRNA in whole tissue homogenates was not significantly different between control and DHT treated mice, treatment with E 2 significantly decreased Foxa2 mRNA concentrations highlighting the importance of androgen-estrogen balance in the regulation of this cellular compartment in the uterus. Taken together these data are consistent with DHT regulating gland formation in the adult mouse uterus by inducing a transcriptional program that results in de novo adenogenesis. In the present study, treatment of mice with DHT revealed androgenspecific effects on endometrial architecture characterized by a rapid increase in proliferation of epithelial cells lining the glands and significant DHT-dependent changes in expression of genes associated with cell-cycle regulation and stromal-epithelial cross-talk. These results suggest that any endocrine pathology that resulted in significant deviation from normal intra-tissue concentrations of androgens could have a long term impact on the abundance of glandu- press.endocrine.org/journal/endo lar epithelial cells, with consequent impacts on glandular secretions and the epithelial-stromal cell cross-talk required to support establishment of pregnancy in women.	2018-04-03T00:50:29.967Z	2016-03-10T00:00:00.000	{ "year": 2016, "sha1": "5008c02bbf206587e13d289342e92dd09451bf87", "oa_license": "CCBY", "oa_url": "https://academic.oup.com/endo/article-pdf/157/5/2116/20218301/endo2116.pdf", "oa_status": "HYBRID", "pdf_src": "ScienceParsePlus", "pdf_hash": "677191d85a11ca59bbc839bb80c89629667002a2", "s2fieldsofstudy": [ "Biology", "Medicine" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
54468390	pes2o/s2orc	v3-fos-license	Nurses’ knowledge on pressure injury prevention: a systematic review and meta-analysis based on the Pressure Ulcer Knowledge Assessment Tool Introduction Inadequate knowledge on pressure injury (PI) can have a detrimental effect on preventive care strategies. The aim of this study was to assess the overall knowledge of nurses on PI prevention based on their scores on the Pressure Ulcer Knowledge Assessment Tool (PUKAT) and its subscales in different settings. Methods In this systematic review and meta-analysis, databases including Web of Science, Science Direct, Google Scholar, PubMed, and Scopus were searched, using the following keywords: Pressure Ulcer, Pressure injury, Bedsore, Pressure Sore, Decubitus Ulcer, knowledge, and their possible combinations. Based on heterogeneity between the studies, the data were analyzed using a random effects model. All of the analyses were performed using STATA v.12 software. Results In all three groups (nurses, assistant nurses, and nursing students), the lowest knowledge scores were for prevention measures to reduce the amount of pressure/shear. Nurses’ knowledge (55.4%, 95% CI: 42.4–68.4) was higher than that of nursing students (52.7%, 95% CI: 3–49.56) and assistant nurses (42.2%, 95% CI: 16.4–68). Conclusion The overall knowledge of nurses on PI prevention was lower than the recommended level (60%). Regular training courses and review of PI prevention guidelines can be useful in updating the knowledge of nurses, especially assistant nurses and nursing students on PI prevention. Introduction Pressure injury (PI) is a painful, costly, but potentially preventable problem that is common in older people and patients with limited mobility. 1 The cost of the treatment of PI is 2.5 times than its prevention. 2 PI increases the length of stay in the hospital from 4 to 30 days, decreases quality of life, and increases pain, morbidity, and mortality. [3][4][5] Limited use of knowledge is a common problem in clinical practice. Nurses are not completely aware of up-to-date care protocols and may not have enough knowledge on the current evidence-based practices. Sometimes, nurses' activities are not based on knowledge, but rather on intuition, experience, or habit. 6 Control and prevention of PI requires interdisciplinary collaboration. In order to keep the integrity of patients' skin and prevent the complications of PI, nurses need to receive support and advice from other health professionals. 7 9 Prevention of PI begins by identifying high-risk individuals, systematical examination of skin, using bed and chair support surfaces, changing posture, mobility, and nutritional support. 10 Low knowledge on PI prevention negatively affects preventive care strategies. 11 The review of literature suggests that nurses' knowledge on PI prevention is limited and that this lack of knowledge can negatively influence their performance. 12 There are various tools for evaluating nurses' knowledge on PI prevention that often lack adequate validation, so their results cannot be generalized. [13][14][15][16] The Pressure Ulcer Knowledge Assessment Tool (PUKAT) is a 26-item questionnaire, designed by Beeckman et al to assess nurses' knowledge on pressure injury in six areas of etiology and development (six items), classification and observation (five items), risk assessment (two items), nutrition (one item), reduction in the amount of pressure/shear (seven items), and reduction in the duration of pressure/shear (five items). A score of 16 and higher (out of 26) indicates acceptable level of knowledge and proficiency on PI (60% of the total score). 17 The PUKAT has been used in different countries, including Australia, Mexico, China, Italy, Sweden, Ireland, and Belgium, to assess nurses' knowledge on PI prevention. 11,[18][19][20][21][22][23][24] PI is an index of nursing care quality, and management of PI is one of the main nursing tasks, which is influenced by nurses' knowledge on this issue. Different studies have reported different results about nurses' knowledge on PI prevention. The results indicated that nurses' level of knowledge on PI prevention ranged from 28% to 74%. Considering the importance of improving nurses' knowledge on PI prevention, we should first have an insight on their current level of knowledge; this systematic review and meta-analysis was conducted with the aim of evaluating nurses' overall knowledge on PI prevention. search strategy Nurses' knowledge on PI prevention was evaluated based on their scores on the PUKAT, reported in articles conducted from 2010 to March 2018. 17 The year 2010 was selected because the PUKAT was published in that year, and ever since it has been cited in various research studies. In terms of language, the articles published in English and Spanish were included in the analysis. The search was conducted in Web of Science, Science Direct, Google Scholar, PubMed, and Scopus using the following keywords: Pressure Injury, Pressure Ulcer, Bedsore, Pressure Sore, Decubitus Ulcer, and Knowledge, and their possible combinations. The reference lists of the articles were also reviewed to improve the coverage. selection of studies and data extraction First, all the articles that had used the keywords in their titles were selected based on inclusion and exclusion criteria. The inclusion criteria were the use of the PUKAT for measuring nurses' knowledge on PI prevention and reporting the required data. Lack of access to the article's full text and use of other tools to gather data were the exclusion criteria. Using the abovementioned criteria, the titles and abstracts of the articles were independently reviewed by two researchers, and the related materials were extracted. In the next step, the full texts of the articles providing useful information were analyzed. The methodological quality of the articles was analyzed using the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE). This checklist contains 22 items assessing six different sections of research articles, including title and abstract, introduction, method, results, discussion, and sponsorship. 25 Disagreements between the two researchers were resolved by the correspondent author who was experienced in meta-analysis. The data from the selected articles were recorded in an Excel chart, including the name of the first author, year of publication, country of study, total sample size, target group (nurses, assistant nurses, and nursing students), total scores on the PUKAT, and scores on the six dimensions of the instrument. statistical analysis Because in the selected studies, total scores on the PUKAT and scores on its six dimensions were provided as percentages, the scores were estimated using the binomial distribution. The variance of each study was calculated using the binomial distribution formula. A weighted mean was used to combine the percentages of the scores in each study, so that each study was weighted in proportion to its variance. Because of the percentage difference in total PI prevention knowledge scores and dimension scores between different studies, and due to the significance of heterogeneity indices, the random effects model was used to combine the studies and estimate the percentage of total and dimension scores. The I 2 index and Cochran's Q test were used to examine the heterogeneity between the studies (I 2 statistics below 25% indicated low heterogeneity, between 25% and 50% moderate heterogeneity, and over 75% high heterogeneity). For the Cochran's Q test, the P-value was set at <0. 1 615 nurses' knowledge on pressure injury prevention was used for a comprehensive demonstration of the selected studies in terms of effect size and 95% CI. The selected studies were classified by continents of origin (Europe, Asia, Australia, and America) and target groups (nurses, assistant nurses, and nursing students). In addition, the percentage of overall knowledge on PI prevention and knowledge on the six dimensions of the PUKAT were calculated using the subgroup analysis and the random effects model. The relationship between year of publication and sample size with the percentage of total knowledge on PI prevention was also evaluated using a meta-analysis. Finally, the funnel plot based on the Egger's regression test was used to examine the role of each study in the final result of sensitivity analysis and to investigate the effect of small studies and potential publication error. All analyses were performed using the STATA v.12 software. Results In this systematic review and meta-analysis, all the studies on nurses' knowledge on the prevention of PI were reviewed based on the PRISMA statement. In the initial search, 692 studies were identified, of which 684 were excluded based on the inclusion and exclusion criteria ( Figure 1). Finally, eight studies with 11 groups and a total sample size of 4,766 were analyzed. In terms of methodological quality, four studies had moderate quality [19][20][21]23 and four had excellent quality. 11,18,22 Also, four studies were from Europe (Sweden, Ireland, Belgium, and Italy), 11,[22][23][24] two studies from Asia (China and Turkey), 20,21 one from Australia, 18 and one from Mexico. 19 The studies had been conducted between 2012 and 2018. The highest and the lowest knowledge on PI prevention scores were in the Lui et al's study in China (among nurses) and Demarré et al's study in Belgium (among assistant nurses), respectively. 21,24 More details are reported in Table 1. Nurses' total score on PI prevention is presented in Figure 2.The percentage of total PI prevention knowledge was 53.1% (95% CI: 47.5-58.8), which was acceptable, but not desirable ( Figure 2). Results based on continent showed that the highest percentage of PI prevention knowledge based on the six dimensions of the PUKAT was for the studies conducted in Asia. The lowest scores on all dimensions, except on risk management (D3), were for the studies conducted in Europe. The lowest scores on risk management (D3) were for the studies conducted in the other continents (North America and Australia). (2012) Gill et al (2013) Gunningberg et al (2015) Gunningberg et al (2015) Gunningberg et al (2015) Simonetti et al (2015) Tulek et al (2016) Lui et al (2016) Hernandez et al (2017) Usher et al (2018) Overall Sensitivity analysis indicated that none of the studies alone had a significant effect on estimating the percentage of total knowledge. In addition, the publication bias was not significant (P=689; Figure 4). The results of meta-regression analysis showed that there was no relationship between sample size and nurses' total knowledge on PI prevention (P=0.922). Although nurses' knowledge increased by year of publi- Discussion In the present meta-analysis, a total of eight studies with a total sample size of 4,766 were analyzed in order to assess nurses' knowledge on PI prevention. The results indicated that the total score of nurses on PI prevention was lower than the cutoff point of the PUKAT (more than 60% of the total score). 17 The lowest score of nurses, assistant nurses, and nursing students was on preventive measures to reduce the amount of pressure/shear. In addition, knowledge on PI prevention was higher for nurses than assistant nurses and nursing students. The study results indicated that nurses' total knowledge on PI prevention was 53.1%. This shows that nurses still do [26][27][28] In other words, nurses' limited knowledge on PI prevention is not influenced by geographical regions. The findings showed that nurses' total knowledge on PI prevention was higher than that of nursing students and assistant nurses. The knowledge score of nursing students was also higher than that of assistant nurses. Factors that may have contributed to this result were nurses' having more clinical experience, more opportunities to visit patients with various levels of pressure ulcer, and receiving training courses in the hospital useful in updating their knowledge on PI prevention. Contrary to our expectation, nursing students' knowledge was lower than that of nurses; this can be attributed to their limited clinical experiences. In the study by Aydin and Karadag (2010), receiving training and previous experiences with patients was associated with nurses' knowledge on PI prevention. 26 The highest and the lowest PI prevention knowledge scores were on nutrition and preventive measures to reduce the amount of pressure/shear. In fact, for all the three groups of nurses, assistant nurses, and nursing students, the lowest scores were on preventive measures to reduce the amount of pressure/shear. This dimension includes nurses' knowledge on repositioning, positions for reducing the risk of ulcer, timing of repositioning in patients lying on the viscoelastic foam, disadvantages of water mattresses, and the common location of pressure ulcer. In the study by Hulsenboom et al, less than half of the nurses knew that putting heels on ring-shaped cushions or water-filled gloves could reduce the pressure applied to the heel. 29 In Schoeps et al's study (2017), less than half of the nurses followed the strategies for PI prevention, such as repositioning. 30 In a study by Källman and Suserud (2009), pressure relieving was the third commonly used strategy for PI prevention after regular repositioning and the use of pressure reducing mattresses. 15 The results of this study indicated that between 2012 and 2018, nurses' knowledge on PI prevention remained unchanged. The results of the study by Hulsenboom et al showed an increase in nurses' knowledge on PI prevention between 1991 and 2003. 29 In a study by Mwebaza et al, one-third of nurses admitted that they did not assess patients' bodies for pressure ulcer. 31 In the Lawrence et al's study, 93% of nurses were still unaware that massage was not recommended for the prevention of PI. 5 PI is a multifactorial problem that, despite being preventable in theory, may occur in care set-tings with the highest quality. 8 Appropriate nursing care is an essential component of PI prevention. Therefore, nurses' level of knowledge on this issue is vital in the prevention of PI in the patients, based on evidence-based practice. 20 The PUKAT was developed 8 years ago, and ever since it has been used in different studies. One of the strength of the present meta-analysis was that it was the first study assessing and reporting nurses' knowledge on PI prevention based on the PUKAT. By drawing on the findings of this meta-analysis, researchers can easily compare nurses' knowledge on PI prevention with the expected level, and get some insight on the nurses' current level of knowledge on this issue. Among the limitation of the meta-analysis was that some articles had not reported the necessary information, or had used modified versions of the PUKAT (adding some items to the original items); therefore, they could not be used in the analysis. Another limitation was related to the translation of the original scale that had some semantic difference with the original form. Only in psychometric studies, the translated instrument is sent to the developer to be examined and verified in terms of translation, and these studies are not concerned with the quality of translation. Among the eight analyzed articles, only two were psychometric studies that had been conducted in China and Turkey. Among the six studies, five were in English and one in Spanish. Considering that the original questionnaire and the other studies were all in English, the concern of proper translation was only with the Mexican study that was in English. Review of the studies conducted on nurses' knowledge on PI prevention showed that nurses' knowledge on this issue was lower than the expected level. Nurses' limited knowledge on PI prevention can both reduce the quality of nursing care and increase the risk of PI in the patients. Regular training courses and review of PI prevention guidelines can be useful in improving nurses' knowledge, help the application of this knowledge in clinical practice, and ultimately reduce the incidence of PI.	2018-12-16T18:46:00.964Z	2018-11-01T00:00:00.000	{ "year": 2018, "sha1": "f7389ae126302a36eace2e286f7f52bd38ee0133", "oa_license": "CCBYNC", "oa_url": "https://www.dovepress.com/getfile.php?fileID=46444", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "f1b3e63294f85776f83243be2f1b908c043a47de", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
236319690	pes2o/s2orc	v3-fos-license	Maternal Bochdalek Hernia during Pregnancy: A Systematic Review of Case Reports Background: Since the first report of a diaphragmatic hernia from Ambroise Paré’s necropsy in 1610, the Bochdalek hernia (BH) of the congenital diaphragmatic hernia (CDH) has been the most common types with high morbidity and mortality in the neonatal period. Due to the nature of the disease, CDH associated with pregnancy is too infrequent to warrant reporting in the literature. Mortality of obstruction or strangulation is mostly due to failure to diagnose symptoms early. Data sources and study selection: A systematic literature search of maternal BH during pregnancy was conducted using the electronic databases (PubMed and EMBASE) from January 1941 to December 2020. Because of the rarity of the disease, this review included all primary studies, including case reports or case series that reported at least one case of maternal BH in pregnant. Searches, paper selection, and data extraction were conducted in duplicate. The analysis was performed narratively regardless of the control groups’ presence due to their rarity. Results: The search retrieved 3450 papers, 94 of which were deemed eligible and led to a total of 43 cases. Results of treatment showed 16 cases in delayed delivery after hernia surgery, 10 cases in simultaneous delivery with hernia surgery, 3 cases in non-surgical treatment, and 14 cases in hernia surgery after delivery. Of 16 cases with delayed delivery after hernia surgery, 13 (81%) cases had emergency surgery and three (19%) cases had surgery after expectant management. Meanwhile, 10 cases underwent simultaneous delivery with hernia surgery, 6 cases (60%) had emergent surgery, and 4 cases (40%) had delayed hernia surgery after expectant management. 3 cases underwent non-surgical treatment. In this review, the maternal death rate and fetal/neonatal loss rate from maternal BH was 5% (2/43) and 16% (7/43), respectively. The preterm birth rate has been reported in 35% (15/43) of maternal BH, resulting from maternal deaths in 13% (2/15) of cases and 6 fetal loss in 40% (6/15) of cases; 44% (19/43) of cases demonstrated signs of bowel obstruction, ischemia, or perforation of strangulated viscera in the operative field, resulting from maternal deaths in 11% (2/19) of cases and fetal-neonatal loss in 21% (4/19) of cases. Conclusion: Early diagnosis and surgical intervention are imperative, as a gangrenous or non-viable bowel resection significantly increases mortality. Therefore, multidisciplinary care should be required in maternal BH during pregnancies that undergo surgically repair, and individualized care allow for optimal results for the mother and fetus. Introduction A diaphragmatic hernia is the protrusion of abdominal organs into the thorax. Since three cases of diaphragmatic hernia, including one traumatic origin, were first reported Sources To find articles published in all languages from January 1941 to December 2020, we searched the PubMed (all fields) and the EMBASE databases concerning maternal diaphragmatic hernia during pregnancy using the following search strategies: ("hernia, diaphragmatic" (Medical Subject Headings (MeSH Terms)) ("hernia" AND "diaphragmatic") OR "diaphragmatic hernia" OR ("diaphragmatic" AND "hernia")) AND ("pregnancy" (MeSH Terms) OR "pregnancy" OR "pregnancies") for PubMed, and ("diaphragm"/exp OR diaphragm) AND ("hernia"/exp OR hernia) AND ("pregnancy"/exp OR pregnancy) for EMBASE. We also hand-searched for additional studies through the references of related articles. The search ended in 2020. Study Selection We performed a two-step study selection to verify eligibility and inclusion criteria. In the first step, two reviewers (Sang Hun Lee and Jin Young Choi) independently screened the titles and abstracts of all studies for the management of maternal BH during pregnancy. After the screening, the two reviewers then independently assessed the eligibility of fulltext articles. Any discrepancy was resolved through mutual discussion, and a citation was included in our study if both agreed. We included all primary studies, including case reports and case series, reporting any type of maternal BH (traumatic, congenital, hiatal, or unknown), herniated organ (stomach, small intestine, large intestine, spleen, omentum, pancreas, kidney, or appendix), symptom (acute/sub-acute abdominal/pelvic pain, nausea, vomiting, dyspnea, and pain (shoulder, epigastric, chest, or back)), treatment of herniated organs (surgery or conservative treatment), management according to trimester (conservative treatment, operation after delivery, or simultaneous delivery), or adverse maternal and fetal/neonatal outcome. We excluded case reports or case series reporting maternal diaphragmatic hernia in pregnant patients presenting with traumatic, hiatal, and unknown types. The evidence was summarized and classified using a standard sorting method in Tables S1 and S2. We did not conduct meta-analyses due to the rarity of cases, but the analyses of case reports were summarized narratively regardless of the presence of control groups. Results From January 1941 to December 2020, the search retrieved 3450 papers, 94 of which were deemed eligible, and 42 articles were reviewed. Therefore, we studied 43 cases based on 42 articles that satisfied the inclusion criteria. Figure 1 presents a PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) flowchart with a summary of the search results. Tables 1 and 2 summarizes the clinical features, treatment modalities, and adverse maternal and fetal/neonatal outcomes of all published cases related to maternal BH during pregnancy. The clinical feature of maternal BH is shown in Table 3. Of the 43 cases, the mean age of presentation was 28.5 years, and the parity was 19 primigravida (43%), 16 multiparous (36%), and nine unknown (21%). When the clinical picture accompanying the symptoms was diagnosed, the gestational ages of the diaphragmatic hernias were 28 in the antenatal period--one in the first trimester, 13 in the second trimester, and 14 in the third trimester (65%), as well as 15 in the postpartum period (35%). However, four patients were asymptomatic before pregnancy. Maternal BH demonstrated maternal death in 5% of cases (2/43) and fetal/neonatal loss in 16% (7/43-five stillborns and two neonatal deaths). The preterm birth rate was 35% (15/43) for maternal BH, resulting from maternal deaths in 13% (2/15) of cases and fetal/neonatal loss in 40% (6/15) of cases. 44% (19/43) of cases demonstrated signs of bowel obstruction, ischemia, or perforation of strangulated viscera in the operative field, resulting in maternal deaths in 11% (2/19) of cases and fetal/neonatal loss in 21% (4/19) of cases. Table 4 summarizes the relation between treatment for maternal BH during pregnancy and the time interval from hernia diagnosis to hernia surgery according to trimesters and adverse maternal and fetal/neonatal outcomes. Our data showed 79% (11/14) in 1st and 2nd vs. 36% (5/14) in the 3rd trimester for delayed delivery after surgical repair with diagnosing a hernia and 14% (2/14) in 1st and 2nd vs. 50% (7/14) in 3rd trimester for simultaneously delivery with surgical repair. Of 11 cases in the 1st and 2nd trimester with delayed delivery after surgical repair with diagnosing hernia, 11 immediately surgery were undergone. Of 8 cases in the 3rd trimester with delayed delivery after surgical repair with diagnosing hernia, 5 immediate surgery and 3 surgical repair with expectant management were undergone. Bochdalek hernia was incidentally diagnosed in a chest X-ray obtained for work five years ago. BH, Bochdalek hernia; NA, not available; VD, vaginal delivery; C/S, cesarean section. Embryology and Pathogenesis In the 18th and 19th centuries, the renowned anatomists and pathologists Giovanni Battista Morgagni and Alexander Bochdalek dedicated themselves to elucidating the development of anatomy. The anteromedial defect of diaphragmatic hernia was named Morgani in 1765, while a congenital postero-lateral diaphragmatic defect was named Bochdalek in 1848. The diaphragm is a thin skeletal muscle located at the base of the thorax between the abdomen and the thorax, consisting of a thin central aponeurosis and a peripheral muscle. The muscular diaphragm begins developing at the 3rd week of embryologic development and is fully formed at the 12th week. The normal diaphragm development is formed by organogenesis from (1) the transverse septum, formed during the third embryonic week that eventually forms the central tendon, (2) the right and left pleuroperitoneal membranes closed with the dorsal mesentery of the esophagus medially, the transverse septum laterally and caudally, and body wall posteriorly at approximately the 6th week of the embryonic period. (3) the dorsal mesentery of the esophagus forming the crura of the diaphragm, (4) inter and outer body wall muscles splitting the pleural cavities and their costodiaphragmatic fold during the 9th and 12th embryonic weeks. CDH account for most of the developmental abnormality of the diaphragm. Developmental defects in the fetus's diaphragm in utero may range from weak-to-partial to complete. The pathogenesis of CDH still is not well known and usually exhibits sporadic patterns; however, teratogenic and genetic factors can affect it. According to embryonic theory, a CDH may result from a delay or variation in the organogenesis timetable between diaphragmatic components and abdominal viscera. By the end of the 6th week of the embryonic period, the primordial diaphragm originated from four diaphragmatic components is produced. Failure of fusion of composite structure related to the primordial diaphragm is the most common causal factor of CDH. CDH is characterized by the presence of abdominal viscera in the thoracic cavity. During the 10th week, the intestines return to the abdomen. If some component of the primordial diaphragm is still open and has the failure of closure when the intestine returns the abdomen from the umbilical cord at this period. The herniation arises as to the protrusion of the abdominal viscera into the thorax. The CDH has a prevalence of 0.8-5/10,000 in live births. The incidence in the prenatal period may be much higher due to the effects of fetuses that die in utero without a pathological postmortem. This occurs equally for both males and females. The Bochdalek hernia(postero-lateral hernia) represents 70-75% of all CDH, while the Morgagni hernia (anterior hernia) 23-28%, central hernias 2-7%, and the other types (congenital epigastric hernia, hiatal hernia, and eventration of the diaphragm) [46][47][48]. A Bochdalek hernia is caused by the defective development of the pleuroperitoneal membrane in the posterolateral portion of the diaphragm. The location is 85% on the left and 13% on the right, with 2% of cases presenting bilaterally [49,50]. In an adult, the defect is 78% on the left, 20% on the right, and 2% bilateral [51]. Our data also showed left-side dominance for infants and adults. The pleuroperitoneal fold (PPF) is two pyramidal-shaped mesodermal structures between the abdominal and thoracic cavities and forms the dorsal-lateral portions of the primordial diaphragm [52]. Recently, more studies have suggested that diaphragmatic defects occur earlier in the developmental period of PPF than in the period associated with fusion. A "mesenchymal hit" hypothesis has been suggested to explain the effects in the PPF of muscle connective tissue fibroblasts and somatic genetic mutations associated with CDH. In experimental animal models, an impaired PPF of cellular etiology and genetic mutations of transcription factors such as GATA-4 and its coregulator FOG-2 appear to be potent causes for abnormality and CDHs [52,53]. Etiology Carl Hedblom [2] systematically classified diaphragmatic hernias into the following four etiologies: congenital, caused by an imperfect developmental defect of the diaphragm; traumatic, the result of various causes (e.g., knife wound, crushing injury, fall, collision, or fractured rib); acquired, caused by gradual irritation of the anatomical region with the least resistance such as the esophageal ring; and indeterminate. A hernia may be classified into the congenital type due to developmental disabilities in the embryonic period, acquired type due to gradual irritational pressure or sudden trauma, and mixed type. A Bochdalek hernia in an adult can stem from increased intra-abdominal pressure, such as pregnancy, chronic constipation, severe coughing, binge eating, fits of laughter, upside-down hanging, or diving. In 173 reviewed cases, Brown et al. [51] reported that precipitating factors or triggering events accounted for 25% of BH cases and that pregnancy was the most common cause, accounting for 34% of cases with one or more triggering factors or 8% overall. Our data showed that pregnancy-associated maternal BHs occur regardless of maternal age or parity (primigravida 43%, multiparous 36%, and unknown 21%). Although labor pain is the major precipitating factor manifesting symptoms, 28/43 cases (65%) presented in the antenatal period (one in the first trimester, 13 in the second trimester, and 14 in the third trimester) and postpartum (15; 35%). Symptoms occurred more frequently in the antenatal period. In this review, the second trimester (30%) had as significant a proportion as the third trimester or postpartum. One study reported a rare case for the first trimester [10]. However, four cases were primigravida with no symptoms before pregnancies: Two cases of maternal BH surgically repair in the neonate period [20,28], and two cases of maternal BH incidentally recognized through chest X-rays five years and several years earlier, respectively [31,33]. Clinical Presentation The clinical symptoms of patients with diaphragmatic hernias depend primarily on the degree of protrusion of the herniated organs into the chest. Therefore, most symptoms are related to the abdomen and chest and range from no symptoms to life-threatening complication. However, initial symptoms such as upper-abdominal pain, nausea, and vomiting are often misdiagnosed as mild and non-specific symptoms during the antenatal or postnatal period. In the case of pregnant women, it is difficult to diagnose maternal BH due to a particular unique disease entity and various symptoms related to pregnancy. Therefore, maternal BH should be suspected when dyspepsia, postprandial vomiting, epigastric pain, and hematemesis do not disappear in the last stages of pregnancy. Strangulation Strangulation occurs when a hollow organ, the vessel etc. has become tightly constricted, such that the flow of blood or air is blocked. The following finding suggests the diagnosis of strangulated BH: (1) signs of acute gastro-intestinal obstruction, (2) cardiac shift to the right or the left side, (3) bloody fluid on aspiration of the thoracic cavity, (4) radiographic finding of diaphragm higher than the contralateral diaphragm. Strangulation has been recognized as severe diaphragmatic hernia complications. Strangulation of diaphragmatic hernia complicating the puerperium is very rare, especially obstruction of or gangrene in the herniated viscera due to strangulated diaphragmatic hernia which is a life-threatening emergency accompanying fetal-neonatal and maternal death. Hedblom, in 1931, revealed that mortality doubled due to intestinal obstruction during a diaphragmatic hernia. The mortality rate was 53.1% in 126 cases with obstruction and 23.8% in 252 cases without obstruction [2]. Pearson et al., in 1953, analyzed four cases of their own and 70 cases from the literature between 1798 and 1952. The mortality rate was 52.7% in 74 cases, with an operative mortality of 32.6% and non-operative mortality of 100% [59]. Diagnosis In pregnant women with BH, accurate diagnosis is particularly important because treatment decisions based on the diagnosis are related to life-threatening maternal and fetal events if surgical treatment is not performed at an optimal time. A BH should be suspected if the following chest radiographic features appear: displacement of the heart across the mediastinum to the opposite side of herniated viscera, air bubbles above the diaphragm level, very high position to the contralateral side, and an opacified hemithorax, which is evidence of fluid in the chest cavity. However, it can be difficult to diagnose BH only with chest radiographic examination. Thus we should be considered that this diagnosis cannot be completely ruled out this diagnosis even if previous chest radiographs are a normal finding. Ultrasonography has been recognized as the screening modality in obstetric imaging due to the advantage of easy accessibility and real-time scanning. Ultrasonography features include a fragmented diaphragm, inability to identify the liver, spleen, kidney, the superior mesenteric and portal vessels within the normal position in the abdomen, displacement of the heart across the mediastinum, and the identification of bowel and liver in the chest. Direct visualization and detailed evaluation of the herniated viscera are crucial for accurate diagnosis and treatment. Ultrasonography is insufficient for an adequate diagnosis. Therefore, computed tomography (CT) and MRI are useful for evaluating detailed information in pregnant patients. The exposure effects of radiation on the developing conceptus or perinatal period are related to spontaneous abortion and fetal growth restriction in the first trimester and slightly increased childhood leukemia and childhood cancer risk in the second trimester or more. In general, the use of CT during pregnancy should be avoided. The National Council on Radiation Protection and Measurements has provided guidelines on radiation doses to ensure that a diagnostic CT conducted during preg-nancy is safe for the fetal well-being. These guidelines consider ≤5 rads (0.5 Gy) to be negligible compared to the other risks of pregnancy. Fetal radiation exposure from an abdominal/pelvis CT is 2.5 rad (25 mGy). CT can evaluate the maternal abdomen by performing a relatively high pitch and relatively thick slice (7-10 mm) to minimize any risk [22,28,30,31,34,36,40,41,43,45,60], but imaging should be performed only if the benefits of diagnosis exceed the theoretical risk of fetal exposure. However, the important advantage of MRI is its demonstration of a better depiction of soft-tissue contrast. MRI can be useful for evaluating abdominal pain in the pregnant patient, such as adnexal torsion, appendicitis, uterine rupture, pelvic vein thrombosis, biliary disease, and small bowel obstruction that complicate pregnancy and using MRI as the first-line modality without USG can be against the recommendation. In strangulated herniated viscera with obstruction or ischemia, a high signal on T1W, T2W, and T1W fat-saturated sequences indicates ischemia infarction. DWI can also be used as the imaging modality and is the best for describing early ischemia. Some authors have postulated that T2W contrast information of the strangulated stroma may be useful for predicting the severity of ischemic infarction. Identification of the strangulated herniated viscera with obstruction or ischemia is important because it is necessary to determine the conversion through an abdominal approach in patients considering thoracic repair. We suggest that MRI be used as the first-line modality in pregnant women with a BH considering surgery. Guidelines for Treatment during Pregnancy In particular, a diaphragmatic hernia during pregnancy should be treated according to the clinical picture of the gestation age in which the disease is diagnosed. In an asymptomatic pregnant woman with a previously recognized CDH, the choice between conservative or surgical treatment is controversial. Given the reason for selection bias due to the disease's rarity and that most symptomatic patients had been multiparous women who experienced normal childbirth, an argument was opposed to routine repair for an asymptomatic pregnant patient with a previously recognized CDH, even though pregnancy can trigger worsening symptoms of an unrecognized hernia, Most of the authors reviewed by Brown agreed with the surgical indication for Bochdalek hernias in adults, suggesting that it also includes those diagnosed incidentally in radiological images. For maternal BH during pregnancy, most authors, including Kurzel et al. [52] claim that defects in the first or second trimester should be repaired under elective conditions after administering antenatal corticosteroids before labor, regardless of the presence or absence of symptoms. In the third trimester, asymptomatic patients should undergo elective cesarean section and hernia repair simultaneously after close observation until fetal maturity. The reasons for the routine surgical repair for the maternal BH complicating pregnancy are as follows: (i) these hernia defects may easily manifest symptoms during labor or the early postpartum period, but can manifest symptoms in the 2nd or 3rd trimester without labor; (ii) delays in treating abdominal viscera's incarceration will lead to the greater visceral displacement into the thorax by the enlarging uterus; (iii) in the later stages of pregnancy, these viscera have lost the "right of domain," and operative closure may be extremely difficult. In symptomatic pregnant women with a strangulated maternal BH, or at any time during observation of visceral strangulation and obstruction, emergency surgery should be performed regardless of gestational age and fetal maturity. Further delay may result in ischemia, gangrene, or perforation. However, some authors, including Genc and Fleyfel [21,61] suggest that gastric decompression of the nasogastric tube might improve the bowel obstruction symptoms caused by herniation. It is crucial to consider the time until surgery if antenatal corticosteroids to enhance fetal lung maturity are to be administered before transfer to a tertiary center. However, such an improvement under close monitoring of fetal and maternal vital signs is allowed for a delayed surgery. Surgical Management Since fetal outcomes are directly related to maternal conditions, optimal surgical intervention in pregnant women is as important as diagnosing without delay maternal and fetal well-being. Although treatment for pregnant women should be the same as that for non-pregnant women, the surgeon should operate in close relationships with obstetricians for cardiotocographic fetal monitoring before or during an operation. It is still debatable whether the abdominal or thoracic approach and laparoscopy or laparotomy are best. Because surgeon need to find an excellent way to access the diaphragm reducing the modality during surgery. Proponents of the abdominal approach think it offers a better chance of evaluating all abdominal organs and of treating a strangulated or perforated bowels or a volvulus. If malrotation of herniated organs is identified, repair through an abdominal approach should be implemented rather than thoracic repair. In an emergency abdominal surgery of a maternal BH, a laparotomy provides good organ access and a good view of the surgical field, and permits efficient repair of the defect. This review demonstrated that surgery for patients with bowel obstruction, ischemia, or perforated strangulated viscera resulted in a maternal mortality rate of 11% because of sepsis and multisystem organ failure, while fetal loss was 21%. Maternal BH with strangulated viscera should be performed before the onset of perforation or bowel necrosis. Abdominal skin incisions are often determined by associated conditions and can be midline, paramedian, or subcostal. The surgeon should plan the incision based on the operative exposure desired to complete the procedure safely. Types of abdominal skin incisions are often determined by the associated conditions and have included midline, paramedian, and subcostal incisions. The surgeon should plan the incision based on the operative exposure desired to complete the procedure safely. There is no evidence yet that which skin incision method is preferred or better. Securing visibility during surgery is the most important. During surgery, a subcostal abdominal incision is a preferred approach in posterolateral BH. This incision had the advantage of being extended across the midline with or without a vertical upper midline extension to achieve wide exposure. This incision is a preferred approach in posterolateral BH. Regardless of the surgical approach, there are four important steps to repairing maternal BH: reducing the hernia contents; lysing the adhesion; reconstructing the herniated viscera after complete excision of the hernia sac from the posterior pleural cavity; and repairing the hernia defect. Primary closure repair for the herniated defect is the best treatment. Closure with excessive tension must be avoided to prevent hernia recurrence. If a tension-free closure due to large defects is not possible, the surgical technique of flaps was inevitably performed in the past. Due to tremendous advances in prosthetic material over recent decades, the surgeon can use prosthetic material that has the advantages of reducing operation time and a tension-free repair [15,[26][27][28]30,32,33,36,38]. Kurzel, in 1988, reported the first hernia repair using synthetic polypropylene mesh in maternal BH during pregnancy. Prosthetic material for hernia repair consists of (a) synthetic mesh with different polymers, (b) biological mesh with the regenerative extracellular matrix, (c) composite mesh with two different surfaces, and (d) recently, drug-loaded mesh. Despite the development of novel meshes through new technologies, recent mesh still remains a challenge to overcome postoperative complications and hernia recurrence. There was the first hernia repair of maternal BH during pregnancy through the thoracoscopic approach by Julien in 2011 [32] and laparoscopic approach by Brusciano in 2003 [60]. Maternal BH with strangulated viscera should be performed before perforation or bowel necrosis occur. With the advancement of modern surgical techniques, if the hernia defect size is small and asymptomatic, it can be achieved primarily with minimally invasive surgery. Some authors argue that the emergence of minimally invasive surgical techniques such as thoracoscopy via the thoracic approach (i.e., video-assisted thoracoscopic surgery, VATS) had decreased postoperative pain, short hospitalization compared to thoracotomy and laparotomy. For maternal BH cases with a stable condition during pregnancy that does not require laparotomy, if there are findings such as abnormal chest radiograph, related abdominal injury, or a right-sided defect, VATS can be recommended. VATS can quickly and easily repair the diaphragm defect after adhesiolysis of the strangulated herniated visceral organ from the pleural cavity. There are running or interrupted sutures in the method of repair. The diaphragm repair should be performed using the interrupted suturing technique of polypropylene instead of the continuous running suturing technique. Maternal Expectant Management after Primary Surgical Intervention The other important point is expectant treatment until pregnancy termination after primary emergent or elective surgical intervention. It is important to prevent preterm labor or preterm birth during maternal expectant management until delivery after surgery. Studies have demonstrated that preterm labor by non-obstetric elective surgical intervention accounts for 5% of cases. According to trimester, our results of treatment showed 16 cases underwent delayed delivery after surgical repair with diagnosing hernia. Of 11 cases (69%) in the 1st and 2nd trimester, 10 surgeries were immediately performed, and 1 surgical repair after expectant management was attempted. The duration of expectant management until delivery after hernia repair surgery in this period ranged from 5 days to 13 weeks. In five cases (31%) in the 3rd trimester, three immediate surgery and two surgical repairs with expectant management were performed. The duration of follow-up until delivery after hernia repair surgery ranged from 3 days to 10 weeks. Ten cases underwent simultaneous delivery with surgical repair. Of two cases (22%) in the 1st and 2nd trimesters, one surgery was immediately performed, and one surgical repair after expectant management was attempted. In seven cases (82%) in the 3rd trimester, four immediate surgery and three surgical repairs with expectant management were performed. In one case in the postpartum, one surgery was immediately performed. The duration of follow-up until delivery after hernia repair surgery ranged from 7 days to 3 weeks. Tocolytics (i.e., terbutaline, magnesium) used for postoperative preterm labor is generally well controlled in the patient with elective surgical intervention. However, preterm delivery may be uncontrolled in some patients with emergent surgical intervention. Gestational age at treatment and severity of the underlying disease is the most predictive indicator of patients at risk for preterm delivery. The risk of preterm delivery is higher in the later trimester and for severe BH. During expectant management, it is important to achieve appropriate nutritional support. Because malnutrition for this period can be associated with maternal and fetal morbidity, including fetal growth restriction, maternal weight loss, and electrolyte imbalances, therefore, specialized nutritional support can be delivered enterally, usually via nasogastric tube feeding, feeding jejunostomy, or parenteral feeding via intravenous vessels with peripheral or central venous access Strengths and Limitations This study's main strength is that both maternal and fetal outcomes related to maternal BH were documented. Until now, there had been limited attempts to systematically review the outcome data during pregnancy. There has been no randomized study due to the rarity of the condition. Therefore, a meta-analysis, including the multiplicity of confounding factors influencing the outcome, could not be performed. Under such a circumstance, an evidence-based survey by case series or case report was adopted as the only way to conduct a systematic review, which identified all eligible studies through detailed data extraction. First, this review was affected by selection bias because of the limited data available for maternal BH, as well as publication bias. Second, all patients under review were classified as having maternal BH according to the inclusion criteria. However, it may be difficult to determine whether the hernia could be classified as a preexisting congenital type due to developmental disabilities in the embryonic period, an acquired type due to gradual irritational pressure, or sudden trauma, or even a mixed type. Conclusions Early diagnosis and definitive surgical intervention are imperative, as gangrenous or a non-viable bowel resection significantly increase mortality. Therefore, multidisciplinary care should be required for maternal BH cases who undergo surgical repair during pregnancy, and the mother and fetus should receive individualized care for optimal results.	2021-07-26T05:34:34.630Z	2021-07-01T00:00:00.000	{ "year": 2021, "sha1": "1664571d61fea0601a9fc4e1ae5139a340e09a3a", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2075-4418/11/7/1261/pdf", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "1664571d61fea0601a9fc4e1ae5139a340e09a3a", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
231812295	pes2o/s2orc	v3-fos-license	Prevalence of Metastatic Lateral Lymph Nodes in Asian Patients with Lateral Lymph Node Dissection for Rectal Cancer: A Meta-analysis Importance Rectal cancers occupy the eighth position worldwide for new cases and deaths for both men and women. These cancers have a high tendency to form metastases in the mesorectum but also in the lateral lymph nodes. The therapeutic approach for the involved lateral lymph nodes remains controversial. Objective We performed a systematic review and meta-analysis to assess the prevalence of metastatic lateral lymph nodes in patients with lateral lymph node dissection (LLND) for rectal cancer, which seems to be a fundamental and necessary criterion to discuss any possible indications for LLND. Methods Data sources–study selection–data extraction and synthesis–main outcome and measures. We searched MEDLINE, EMBASE and COCHRANE from November 1, 2018, to November 19, 2018, for studies reporting the presence of metastatic lateral lymph nodes (iliac, obturator and middle sacral nodes) among patients undergoing rectal surgery with LLND. Pooled prevalence values were obtained by random effects models, and the robustness was tested by leave-one-out sensitivity analyses. Heterogeneity was assessed using the Q-test, quantified based on the I2 value and explored by subgroup analyses. Results Our final analysis included 31 studies from Asian countries, comprising 7599 patients. The pooled prevalence of metastatic lateral lymph nodes was 17.3% (95% CI: 14.6–20.5). The inter-study variability (heterogeneity) was high (I2 = 89%). The pooled prevalence was, however, robust and varied between 16.6% and 17.9% according to leave-one-out sensitivity analysis. The pooled prevalence of metastatic lymph nodes was not significantly different when pooling only studies including patients who received neoadjuvant treatment or those without neoadjuvant treatment (p = 0.44). Meta-regression showed that the pooled prevalence was associated with the sample size of studies (p < 0.05), as the prevalence decreased when the sample size increased. Conclusion The pooled prevalence of metastatic lateral lymph nodes was 17.3% among patients who underwent rectal surgery with LLND in Asian countries. Further studies are necessary to determine whether this finding could impact the therapeutic strategy (total mesorectal excision with LLND versus total mesorectal excision with neoadjuvant radiochemotherapy). Supplementary Information The online version contains supplementary material available at(10.1007/s00268-021-05956-1) Introduction Rectal cancer metastasizes to the perirectal lymph nodes contained within the mesorectum and along the iliac arteries [1]. Assessment of lymph node involvement is a strong predictor of recurrence-free survival and overall survival in patients with rectal cancer [2]. Currently, total mesorectal excision constitutes the gold standard for removing perirectal lymph nodes [3]. However, the therapeutic strategy regarding lymph nodes, notably those located along the iliac arteries, may include neoadjuvant radiochemotherapy, as performed in Western countries [3], or lateral lymph node dissection (LLND), as performed in Japan when the lower border of the tumour is located under the reflection of the peritoneum and when it has passed the muscularis propria [4]. However, to date, only two randomized controlled trials (RCTs) [5,6] have compared the outcomes of the two therapeutic strategies in terms of survival, looking at local and distant recurrences and complications. Furthermore, the choice of the best strategy, either LLND or radiochemotherapy or the combination of the two treatments, is difficult to identify, as the prevalence of metastatic lateral lymph nodes in patients with rectal cancer is poorly documented. Indeed, before comparing two therapeutic strategies and their outcomes, it is necessary and more relevant to know if it is legitimate to propose them as a treatment; in other words, if they would even correctly target metastatic lateral lymph nodes (LLNs). Therefore, our objective was to perform a systematic review and meta-analysis reporting on the prevalence of metastatic lateral lymph nodes in patients with rectal cancer with the hypothesis that this meta-analysis will elucidate the best therapeutic strategy in the current absence of reliable assessment of tumour aggressiveness. Materials and methods The present methodology is in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist (Table S1). Literature search and study selection A literature search was conducted in MEDLINE, EMBASE and COCHRANE from inception (DATE??) until November 19, 2018. Keyword combinations are reported in Table S2. Additional records were identified by manually searching the reference lists of the included publications. To be included, studies had to be written in English or French and to report the prevalence of metastatic lateral lymph nodes among patients with rectal cancer who underwent surgery with LLND. LLND was defined by the dissection of nodes included in areas such as the common iliac (IC), internal iliac (II), external iliac (EI), obturator (O), middle sacral (MS) and aorta bifurcation (Ao). We excluded case series, conference abstracts, letters to the editor and secondary analyses of previously published papers. Data extraction Two independent reviewers (NC and JM) independently selected articles for inclusion and extracted the data according to a pre-established data collection form. Discrepancies were resolved by reaching a consensus with the senior authors (NCB and FR). The following data were extracted: first author; publication year; country where the study took place; study period, after and before 2010 due to modifications of the Japanese Guidelines for LLND; study design; number of patients who underwent LLND, prophylactic: dissection and removal of nodes that seem invaded (enlarged) based on pre-and peri-operative examinations, versus therapeutic/curative: removal of all nodes in the ''lateral lymph area''; sex; number of patients with metastatic lateral lymph nodes; number of patients who underwent pre-operative radio-and/or chemotherapy; type of neoadjuvant treatment in those patients; and oncological stages of included patients. Statistical analysis Models with random effects (DerSimonian and Laird's approach [7]) were used to combine the prevalence of metastatic lateral lymph nodes across the studies. A logit transformation was applied to prevalence before statistical pooling, and the pooled logit of prevalence was then transformed back. Heterogeneity was assessed by using the I 2 statistic, and a leave-one-out sensitivity analysis was conducted to check the robustness of the pooled prevalence. Potential sources of heterogeneity were investigated by comparing the pooled prevalence between subgroups of studies. A sensitivity analysis was also conducted excluding stage 4 patients from the denominator and from the numerator of the prevalence because stage 4 patients were 4 Division of Clinical Epidemiology, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211 Geneva 14, Switzerland assumed to have metastatic lateral lymph nodes (model with random effects). In addition, a meta-regression analysis was conducted to assess the relationship between the sample size of studies and the prevalence with the restricted maximum likelihood method. [8] All analyses were performed with the Meta and Metafor packages for R version 3.3.1 (R Foundation for Statistical Computing, Vienna, Austria). Literature search and study characteristics Two hundred and sixty-five publications were identified in MEDLINE, EMBASE and COCHRANE. Three publications were identified from other sources. Fifteen duplicates were removed. Of the 250 publications that were identified as eligible, 155 were excluded after title/abstract screening, and 64 were excluded after full-text screening (44 publications did not report the number of patients with metastatic lateral lymph nodes and 20 publications did not distinguish patients with metastatic lateral lymph nodes from those with metastatic lymph nodes from other areas). Ultimately, 31 publications [5,6, were included in the quantitative synthesis, all coming from Japan except one from China ( Fig. 1, Table 1). To determine whether neoadjuvant treatment could alter the prevalence of metastatic lateral lymph nodes, we performed subgroup analyses by separately pooling studies reporting patients who received neoadjuvant treatment from others (Fig. S2). The difference in pooled prevalence of metastatic lateral lymph nodes between studies with versus without neoadjuvant treatment was 16.9% (95% CI: 14.1-20.2), but the inter-study variability (heterogeneity) was high important, with I 2 = 88%. Then, we explored whether the heterogeneity could be explained by changes in the therapeutic strategies across the years. The pooled prevalence of metastatic lateral lymph nodes between studies published before and after 2010 was 17.3% (95% CI: 14.6-20.4), but the inter-study variability (heterogeneity) was noteworthy, with I 2 = 89% (Fig. S3). Furthermore, we excluded patients with TNM stage IV (in six studies). After exclusion of these patients, the pooled prevalence of metastatic lateral lymph nodes was 15.8% (95% CI 13.1 to 18.9) (Fig. S4). Then, we separately pooled studies according to the type of LLND performed (systematic versus curative). The difference in the pooled prevalence of metastatic lateral lymph nodes between studies with systematic or curative dissection was not statistically significant (p = 0.7111) (Fig. S5). However, the pooled prevalence of metastatic lateral lymph nodes appeared to be associated with the sample size of studies (p = 0.005): the prevalence decreased when the sample size increased (Fig. S6). Meta-regression showed that, on average, a study with a sample size that was 10 times larger than that of some other study had an estimated odds ratio for prevalence that was reduced by approximately 50% (Fig. S7). Discussion We performed a systematic review and meta-analysis estimating the prevalence of metastatic lateral lymph nodes in patients with rectal cancer. By pooling 31 studies of Asian origin (30 from Japan and one from China) totalling 7599 patients, we found a prevalence of metastatic lateral lymph nodes of 17.3%, thereby underlining the importance of further examination of the treatment of these node areas to avoid cancer recurrence. Six studies included patients with TNM stage IV rectal cancer. According to the most recent Japanese guidelines, metastatic disease is not the most important stage to indicate LLND [4]. However, within this stage, some patients may present both primary tumour and resectable distant metastases, thus proceeding to the possibility of undergoing LLND. As a result, to understand the prevalence of metastatic lymph nodes within the most common indications, we excluded patients with TNM stage IV disease by considering them to have metastatic lateral lymph nodes. After exclusion, the pooled prevalence of metastatic lateral lymph nodes remained stable (15.8%). In the present systematic review and meta-analysis, we were not able to show any improvement in neoadjuvant treatment in terms of lateral lymph node metastasis. However, it should be noted that the power of this subgroup analysis might be insufficient. Additionally, Asian protocols differed from Western protocols, and only a small proportion of patients among studies with neoadjuvant treatment benefited from that modality. Therefore, it is not possible to draw any conclusions about the efficacy of neoadjuvant treatment in terms of the prevalence of metastatic lateral lymph nodes based on our results. Despite this, it is worth noting that neoadjuvant treatments Fig. 1 Flow chart showing the selection of publications for quantitative review. Forest plot of the prevalence of metastatic lateral lymph nodes in patients who underwent lateral lymph node dissection. Each horizontal bar summarizes a study. The bars represent 95% confidence intervals. The grey squares represent each of the studies' weights in the meta-analysis. The diamond in the lower part of the graph depicts the pooled estimate along with 95% confidence intervals. Events = number of patients with metastatic lateral lymph nodes, total = number of patients who underwent lateral lymph node dissection for rectal cancer between the studies were quite similar using 5-fluorouracil, oxaliplatin and irinotecan (Table S3). Some centres performed systematic LLND, whereas others performed LLND in patients with enlarged lateral lymph nodes diagnosed by pre-operative imaging. We pooled these studies separately and found no difference in terms of metastatic lateral lymph nodes. After 2010, as Japanese guidelines changed and recommended limiting LLND for all cT3-T4 tumours with a lower edge below the peritoneal reflection, we reported a subgroup analysis of the pooled prevalence of metastatic LLN according to the publication date. Due to the high heterogeneity, we cannot interpret this result as significant. Furthermore, we note that meta-regression showed that the pooled prevalence of metastatic lymph nodes was associated with the sample size of studies, as the prevalence of metastatic lymph nodes decreased when the sample size increased. We think that this might be explained by more selective indications for LLND in studies including fewer patients with LLND. Thus, even if the number of studies between curative and systematic LLND intention was not the same, the tendency of the sample size of studies with curative LLND was lower. This indirectly shows that smaller studies were more likely to have a selected population. As a consequence, studies with larger sample sizes (in other words, studies with prophylactic LLND) were more likely to represent the ''real'' proportion of patients with rectal cancer with LLN metastasis. Our meta-analysis is the first to examine the prevalence of metastatic lateral lymph nodes, which seems to be an essential prerequisite before considering any treatment. Indeed, recent literature [40][41][42] directly questions the oncological and functional results of LLND without even first investigating whether it is necessary. Advantages of the study The strengths of our systematic review and meta-analysis are as follows: 1) the inclusion of a large number of studies reporting the prevalence of metastatic lateral lymph nodes based on histopathological analysis and to clarify, for the first time to our knowledge, its subsequent effects in patients operated on for rectal cancer; and 2) the highlighting of the necessity of systematic treatment of lateral lymph nodes due to the high prevalence (17.3%) of LLN metastasis. Indeed, despite the heterogeneity of the studies, which could have impacted the aim of our work, we were able to make some sound calculations due to a specific statistical analysis methodology. Limitations of the study The limitations of the present study are as follows: 1) the high heterogeneity of the calculated pooled prevalence of metastatic lateral lymph nodes, resulting from the quality and heterogeneity of the included publications; and 2) the final analysis included 31 papers, most of which were from Japan and were retrospective studies. Therefore, some patients might have overlapped because these papers were published from some limited leading hospitals. Moreover, it is worth noting that by focussing on patients who have benefited from LLND, a surgical procedure that is most common in Asian countries, selection has been carried out. As a result, it is expected that the selected studies tend to oversample patients with indications for LLND, with these patients being more likely to have advanced disease. Thus, a bias could have been introduced. The percentage of 17.3% of metastatic LLNs could have been overestimated. This element can explain the counter-intuitive (but nonsignificant) results of a higher prevalence of metastasis in the prophylactic LLND group (17.3%) versus the curative group (15.9%). Conclusion In conclusion, this systematic review and meta-analysis of Asian studies indicates that the prevalence of metastatic lateral lymph nodes in patients with low rectal cancer who underwent surgery is 17.3%. This prevalence is of clinical importance, as it may result in cancer recurrence and calls for the systematic treatment of these lymph node areas. Future randomized controlled trials should determine which therapeutic strategy, whether neoadjuvant radiochemotherapy versus systematic or imaging-guided lateral lymph node dissection, offers the best improvement in overall and recurrence-free survival. Author contributions JM conceived and designed the study. JM and NC acquired the data. CC analysed the data. NC, JM, CC, AB, NCB and FR interpreted the data. NC, JM, CC, AB, JRY, NCB and FR contributed to the writing of the manuscript and to its critical revision. NC, JM, CC, AB, JRY, NCB and FR approved the final version of the manuscript. Compliance with ethical standards Conflict of interest The authors declare that they have no conflicts of interest. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/.	2021-02-05T15:17:24.197Z	2021-02-04T00:00:00.000	{ "year": 2021, "sha1": "cfa4269916cb61d46ce2cea326e95a163e6e1566", "oa_license": "CCBY", "oa_url": "https://link.springer.com/content/pdf/10.1007/s00268-021-05956-1.pdf", "oa_status": "HYBRID", "pdf_src": "PubMedCentral", "pdf_hash": "cfa4269916cb61d46ce2cea326e95a163e6e1566", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
11197431	pes2o/s2orc	v3-fos-license	D-instantons and matter hypermultiplet We calculate the D-instanton corrections (with all D-instanton numbers) to the quantum moduli space metric of a single matter hypermultiplet with toric isometry, in the effective N=2 supergravity arising in type-IIA superstrings compactified on a Calabi-Yau (CY) threefold of Hodge number h_{2,1}=1. The non-perturbative quaternionic hypermultiplet metric is derived by resolution of a complex orbifold singularity, thus generalizing the known (Ooguri-Vafa) solution in flat spacetime to N=2 supergravity. Introduction D-instanton quantum corrections to the moduli space metric of a single matter hypermultiplet for the CY-compactified type IIA superstrings near a conifold singularity were investigated by Ooguri and Vafa [1]. They found an unique solution consistent with N=2 rigid supersymmetry and toric isometry. The solution [1] was interpreted as the infinite D-instanton sum coming from multiple wrappings of the Euclidean D-branes around the vanishing cycle [1]. The Ooguri-Vafa solution is given by the hyper-Kähler metric in the limit of flat 4d spacetime, i.e. when N=2 supergravity decouples, by taking the Planck mass to infinity. Including N=2 supergravity may lead to new physical phenomena at strong coupling [2], so that it is important to generalize the Ooguri-Vafa solution to the case of local N=2 supersymmetry. One immediate consequence of the local N=2 supersymmetry is that the hypermultiplet moduli space metric is no longer hyper-Kähler but quaternionic [3]. In particular, the standard (Gibbons-Hawking) hyper-Kähler Ansatz [4] (it was one of the key technical tools in ref. [1]) cannot be applied to quaternionic metrics. Hence, the quaternionic generalization of the Ooguri-Vafa solution is a non-trivial problem. In our earlier papers [5] we solved a similar problem in the case of the universal hypermultiplet that has a dilaton amongst its bosonic field components. The D-instanton corrections to the universal hypermultiplet moduli space metric were derived in ref. [5] by requiring, in addition to N=2 local supersymmetry and toric isometry, the SL(2, Z) duality invariance that is not the case for a single matter hypermultiplet. The moduli space metric of the matter hypermultiplet is periodic (or T-selfdual) with respect to one of its variables [1] but it is not S-selfdual, so that another solution is needed. The quaternionic constraints in the case of a single hypermultiplet are given by the (integrable) Einstein-Weyl system of non-linear partial differential equations. The key to the explicit construction of ref. [5] is the remarkable fact that any Einstein-Weyl metric with toric isometry is governed by the real pre-potential that is an eigenfunction of the Laplace-Beltrami operator ∆ H in the hyperbolic plane H with the eigenvalue 3/4 [6]. The four-dimensional hyper-Kähler metrics (including the Ooguri-Vafa metric [1]) with a triholomorphic isometry are known to be governed by harmonic functions in flat three dimensions (except some isolated points) [4]. In this Letter we also use yet another mathematical fact that the eigenfunctions of ∆ H correspond to the homogeneous harmonic functions [7]. The solution [1] is formally obtained by applying T-duality to the 'basic' Green function in three dimensions. By using the Calderbank-Singer correspondence [7] we can lift the Ooguri-Vafa solution [1] to a curved spacetime of N=2 supergravity by applying T-duality to the corresponding basic eigenfunction of ∆ H . In the next sect. 2 we review the hyper-Kähler solution [1] that is the pre-requisite to our derivation of the corresponding quaternionic solution in sect. 3. Ooguri-Vafa solution The Ooguri-Vafa (OV) solution [1] describes the D-instanton corrected moduli space metric of a single matter hypermultiplet in N=2 four-dimensional superstrings compactified on a Calabi-Yau threefold of Hodge number h 2,1 = 1, when both N=2 supergravity and the universal hypermultiplet are switched off, and five-brane instantons are suppressed. The corresponding low-energy effective action is given by the fourdimensional N=2 supersymmetric non-linear sigma-model that has the Ooguri-Vafa metric in its target space. Given any four-dimensional hyper-Kähler metric with a tri-holomorphic isometry ∂ t , it can always be written down in the standard (Gibbons-Hawking) form [4], that is governed by linear equations, and The one-formΘ = Θ 1 dx + Θ 2 dy + Θ 3 dz is fixed by the 'monopole equation' (3) in terms of the real scalar potential V (x, y, z). Equation (2) means that the function V is harmonic, with possible isolated singularities, in three Euclidean dimensions R 3 . The singularities are associated with the positions of D-instantons. Given extra U(1) isometry, after being rewritten in the cylindrical coordinates (ρ = √ x 2 + y 2 , θ = arctan(y/x), η = z), the hyper-Kähler potential V (ρ, θ, η) becomes independent upon θ. Equation (1) was used by Ooguri and Vafa [1] in their analysis of the matter hypermultiplet moduli space near a conifold singularity. The conifold singularity arises in the limit of the vanishing period, where the CY holomorphic 3-form Ω is integrated over a non-trivial 3-cycle C of CY. The powerful singularity theory [11] can then be applied to study the universal behaviour of the hypermultiplet moduli space near the conifold limit. In the context of the CY compactification of type IIA superstrings, the coordinate ρ represents the 'size' of the CY cycle C or, equivalently, the action of the D-instanton originating from the Euclidean D2-brane wrapped about the cycle C. The physical interpretation of the η coordinate is just the expectation value of another (RR-type) hypermultiplet scalar. The cycle C can be replaced by a sphere S 3 for our purposes, since the D2-branes only probe the overall size of C, as in ref. [1]. The pre-potential V is periodic in the RR-coordinate η since the D-brane charges are quantized [2]. This periodicity should also be valid in curved spacetime. We normalize the corresponding period to be 1, as in ref. [1]. The Euclidean D2-branes wrapped m times around the sphere S 3 couple to the RR expectation value on S 3 and thus should produce the additive contributions to the pre-potential V , with the factor of exp(2πimη) each. In the classical hyper-Kähler limit, when both N=2 supergravity and all Dinstanton contributions are suppressed, the pre-potential V (ρ, η) of a single matter hypermultiplet cannot depend upon η since there is no perturbative superstring state with a non-vanishing RR charge. Accordingly, the classical pre-potential V (ρ) can only be the Green function of the two-dimensional Laplace operator, i.e. whose normalization is in agreement with ref. [1]. The calculation of ref. [1] to determine the exact D-instanton contributions to the hyper-Kähler potential V is based on the idea [2] that the D-instantons should resolve the singularity of the classical hypermultiplet moduli space metric at ρ = 0. A similar situation arises in the standard (Seiberg-Witten) theory of a quantized N=2 vector multiplet (see, e.g., ref. [9] for a review). Equation (2) formally defines the electrostatic potential V of electric charges of unit charge in the Euclidean upper half-plane (ρ, η), ρ > 0, which are distributed along the axis ρ = 0 in each point η = n ∈ Z, while there are no two charges at the same point [1]. A solution to eq. (2) obeying all these conditions is unique, After the Poisson resummation eq. (6) takes the singularity resolution form indeed [1], where the modified Bessel function K 0 of the 3rd kind has been introduced, valid for all Re z > 0 and Re s > 0, while µ is a constant. By inserting the standard asymptotical expansion of the Bessel function K 0 near ρ = ∞ into eq. (7) one finds [1] A dependence upon the string coupling constant g string is easily reintroduced into eq. (9) by a substitution ρ → ρ/g string . The factors of exp (−2πmρ/g string ) in eq. (9) are just the expected multi-D-instanton contributions [1]. D-instantons in N=2 supergravity Any four-dimensional quaternionic manifold has the Einstein-Weyl geometry of negative scalar curvature [3], where W abcd is the Weyl tensor, R ab is the Ricci tensor of the metric g ab , and the constant Λ > 0 is proportional to the gravitational coupling constant. It is the theorem [6] that any four-dimensional Einstein-Weyl metric (of nonvanishing scalar curvature) with two linearly independent Killing vectors can be written down in the form in some local coordinates (ρ, η, θ, ψ) inside an open region of the half-space ρ > 0, where ∂ θ and ∂ ψ are the two Killing vectors, the one-formsα andβ are given bŷ while the whole metric (12) is governed by the real function (= hypermultiplet prepotential) F (ρ, η) obeying a linear differential equation, Equation (14) is thus a consequence of 4d, local N=2 supersymmetry and toric isometry. It is highly non-trivial that the linear master equation (14) governs all U(1) × U(1)-symmetric solutions to the highly non-linear Einstein-Weyl system (11). Equation (14) means that the quaternionic pre-potential F is a local eigenfunction (of the eigenvalue 3/4) of the two-dimensional SL(2, R) Laplace-Beltrami operator on the hyperbolic plane H equipped with the metric The 'basic' η-independent solutions to eq. (14) are given by Their linear combination gives some perturbative contributions to the hypermultiplet pre-potential [5]. The only η-dependent 'basic' solution to eq. (14) is given by [6,7] F 0 (ρ, η) = ρ + η 2 ρ . The basic eigenfunction (17b) is simply related to the harmonic (outside the origin) function V 0 (see the end of sect. 2), This is an example of the general correspondence between the harmonic functions V of homogeneity α in R 3 and the ∆ H -eigenfunctions F in H of eigenvalue α(α − 1) [7]. In the perturbative region at large ρ the sum in eq. (22) represents the nonperturbative contributions with all D-instanton numbers, when using the asymptotical expansion of the K 1 -function at x → ∞, in full similarity to ref. [1]. The first two terms in eq. (22) are perturbative contributions that are to be η-independent. Hence, the functionf (η) is actually a constant, f = C. Our final result for the D-instanton-corrected pre-potential of the matter hypermultiplet moduli space metric is thus given by The logarithmic factor in the second term of eq. (25) is apparently to be interpreted as the N=2 superstring (one-loop) renormalization effect. I am grateful to the referee of my paper [5] in Nucl. Phys. B, whose remarks led me to this investigation.	2014-10-01T00:00:00.000Z	2003-02-01T00:00:00.000	{ "year": 2003, "sha1": "eb6af03d08a4cc3dc15231868f29149836aa5900", "oa_license": "CCBY", "oa_url": "https://doi.org/10.1016/s0370-2693(03)00244-2", "oa_status": "HYBRID", "pdf_src": "Arxiv", "pdf_hash": "eb6af03d08a4cc3dc15231868f29149836aa5900", "s2fieldsofstudy": [ "Physics", "Mathematics" ], "extfieldsofstudy": [ "Physics" ] }
221696185	pes2o/s2orc	v3-fos-license	Mizaj as an Index in Persian Traditional Medicine Index Could Associate with Sensitivity to the Radiation Background: The sensitivity to the radiation among human population depends on various parameters. This variation could lead to dissimilar outcome of radiotherapy in similar situations. Mizaj is a well-known term in Persian medicine that present an individualized medicine viewpoint. All of the people will be categorized in cold, moderate, and warm Mizaj. In this study, we aimed to evaluate the possible association between Mizaj and radiosensitivity by comet assay. Materials and Methods: Peripheral blood sample of 30 healthy volunteers (10 cold, 11 moderate and nine warm Mizaj) were taken and divided into two identical parts. The first part was exposed to 4 Gy x-rays, and the second part was regarded as the sham control. Then, DNA damages of samples were evaluated by the neutral comet assay. Results: The results showed that the mean percentage of damaged cells, in all of the irradiated groups including A (warm), B (moderate) and C (cold) was significantly higher than the controls (P<0.001). Moreover, DNA damage rate in the irradiated warm Mizaj group was higher than both cold and moderate irradiated groups, but the difference between moderate and cold irradiated groups was not statistically significant. Conclusion: The results are indicating that warm Mizaj persons could be more radiosensitive than other groups, which their importance in radiotherapy individualization should be evaluated in more extensive studies. Introduction R adiosensitivity is relative vulnerability to the dangerous effects of ionizing radiation. The response to radiation depends on many factors such as radiation type, energy, and dose rate. Also. The biological and chemical properties of cells or tissues could affect the response [1] then, similar irradiation condition may induce different reactions to radiation in different patients [2]. Genetic is one of the most important biological factors which could affect the radiation sensitivity [3]. The results of studies have shown that the genes are involved in DNA repairs, such as ATM and NBS have an essential role in the biological response to radiation. It is shown that mutations in the ataxia-telangiectasia gene could alter the response to radiation [4]. Also, a study of radiosensitivity in head and neck cancers have reported that differential expression of ku80 gene can be a predictive factor for radiosensitivity [5]. The correlation between some SNPs and cellular response to radiation has investigated too [6,7]. Differences in genome sequencing and gene expression cause various phenotypes, and these differences can potentially affect the risk of diseases and responses to therapies or drugs [8]. This difference is the basic theory of personalized medicine, a relatively new medical approach that was based on differences among peoples. The concept of Mizaj is considered as a principle in many traditional medicine cultures. In traditional Persian medicine (PM), an individualized viewpoint is introduced by the term of Mizaj. The root of Mizaj (hot and cold natures), is from ancient Greece (Hippocrates, a Greek physician, 460-375). The most critical activity in this area in the east was carried out by Avicenna (Persian physician, 937-1037), who tried to collect and organize all the ancient theories and mixed them with the medical approaches of the time and wrote the book "Canon of medicine". The maintenance of the equilibrium between the fundamental body elements was the most important principle of the book. Ten criteria focused on physical, physiological, and mental status are propose for Mizaj assessment in PM [9]. Some studies showed that some disorders are related to Mizaj. The type of Mizaj can be responsible for the risk of some diseases [10]. Also, Mizaj can alter the response to treatment, e.g., it was reported that some drugs are more efficient for cold Mizaj persons, another study described an association between blood groups and types of Mizaj [11]. Consequently, as the theory of Mizaj in PM is a sort of individualized viewpoints in medicine, it can theoretically cause different radiosensitivity in persons. Comet assay is a cytogenetic method that directly detects DNA damage such as single-strand break (SSB) and double-strand break (DSB) and also repair. The assay is relatively cheap, rapid, and easy to work. It does not need cell culture, and the results could be obtained within hours after sampling [12]. The assay is previously used for the study of radiosensitivity in human cells [13]. The possible association between Mizaj and radiosensitivity was not studied yet; then, the present study aimed to compare radiosensitivity in warm and cold Mizaj persons by the neutral comet assay. Objective and Study Design In this case-control study, we have evaluated the association between radiation-induced comet formation rate (as a technique for evaluating of radiosensitivity) and Mizaj in peripheral blood lymphocytes, based on PM theories. The study duration was 26 weeks started from 28 April up to 7 December 2016, and it was approved by Ethical Committee of Babol University of Medical Sciences, Babol, Iran. Study Population and Recruitment Strategy Individuals who fulfilled the following criteria entered into the study: Twenty to 40 years old men or women, general good health, without any known disease and continues the use of drugs. Handedness and blood group had a relationship with radiosensitivity, so we selected right-hander and O blood group individuals to control the effects of these factors. The exclusion criteria were the history of radiation therapy, radiation workers, smoking, alcohol use, and pregnancy. Screening Process Potential participants were subjected to visit GMJ.2020;9:e1705 www.gmj.ir 3 by physicians to assess if they met the eligibility criteria. Fifty-two participants completed a screening questionnaire. The procedure of the study was explained, and the written informed consents were completed, then they attended the study (Figure-1). 1.3.Blood sampling and irradiation Two-milliliter blood samples of the volunteers were taken under the sterile condition and collected in micro-tubes containing sodium heparin anticoagulant. The samples were divided into two equal values (1 ml) and kept in similar conditions. One part of each sample was exposed to 4 Gy (200 cGy per min, SSD=100 cm) of x-rays from Linac (ELEKTA Compact 6MV). The second part, regarded as the control. Neutral Comet Assay After irradiations, the DNA damages were evaluated by the neutral comet assay. Briefly, the samples were centrifuged at 2500 rpm for 5 min, and the remainder (10µL) was mixed with 140 µL of 0.75% low melting point agarose (Fermentaz, Poland) dissolved in distilled water. Then, 50 µL of the suspension was applied to each window slides coated with a layer of 1% normal melting point agarose (Fermentaz, Poland). The slides were covered and solidified in 5 min. Then, the slides were submersed within 30 minutes at 4 ºc in lysis solution, which consist of (2. Microscopic Analysis The slides were analyzed with a fluorescent microscope (E800, Nikon, Japan) with Wavelength (WL) 590 nm and magnification power 200. The slides were analyzed by visual detection and classified into 4 grade (N1-N4) base on the length of the comet tail that shows the severity of DNA damage [14]. The total Statistical Analysis The data were analyzed by SPSS 14 software (SPSS for Windows, SPSS, Chicago, USA), and the normality was checked by the Kolmogorov-Smirnov test. The analyses were performed by paired t-test between the control and exposed groups and ANOVA with significance accepted at the 5% level were used to compare the DNA damage between three exposed groups of Mizaj. The demographic characteristics were described by descriptive statistics, including mean ± standard deviations, frequency, and percentage. The error bars were applied by 95% confidence interval. Results After considering the inclusion and exclusion criteria, 30 out of 50 volunteers entered into the study. The Mizaj assessment process is shown in Figure-1. The Demographic characteristics, including age, height, weight, and body mass index (BMI) of the included population were shown in Table-1. The Mean frequency of damaged cells in blood samples exposed to 4 Gy irradiation were shown in Figure-1. The results showed that warm Mizaj persons have the highest radiosensitivity (P<0.001) but the difference between moderate (177.73±27.13) and cold Mizaj (170.40±19.66) groups was not significant (P=0.84). According to Figure-2, the mean percentage of damaged cells, in the irradiated samples for all of the groups including warm, moderate, and cold Mizaj was significantly higher than the controls (P<0.001). The increase of damages after exposure to 4 Gy x-rays for all of the three groups was significant. A photomicrograph of our comet assay results were shown in Figure-3. Discussion The idea of the difference in radiosensitivity among individual's Mizaj characters is proposed. 30 entered individuals were categorized in 3 Mizaj of warm, moderate, cold, and the rate of DNA damages were compared after x-ray irradiation. We have applied an expert panel to determine the Mizaj. The Mizaj assessment was performed in two separate sessions to authenticate the result to enhance its validity. Disagreement in two sessions was excluded from the study. The most interesting finding of our study indicated that the number of DNA damage in the warm Mizaj group is significantly higher than the cold Mizaj individuals (P-value<0.001). Based on the traditional PM, the Mizaj varies with age, and we selected healthy young volunteers to enroll in the study. In this study, warm groups had more instability in DNA than moderate and cold Mizaj. Although there was a difference between radiosensitivity of two groups of cold and moderate Mizaj persons, it was not statistically significant. This result may be due to the Mizaj assessment protocol that results in more nearness between moderate and cold Mizaj groups than the similarities between moderate and warm Mizaj ones. In this study, radiation exposure induced significant DNA damage in agreement with studies of Koksal et al. [15]. The radiation-induced DNA damage is a fact, and we were not surprised by this outcome. A clinical study of radiosensitivity in breast cancer patients has shown the different reactions of patients to the same dose and reported that some patients displayed severe reactions to radiation therapy [2]. According to the present study, warm Mizaj persons shown higher radiosensitivity but the difference between moderate Mizaj and cold Mizaj groups was not significant. Based on Traditional medicine, Mizaj is an inherent trait that is identified with physical, physiological, and psychological differences among people and as a result, different criteria have been proposed for Mizaj assessment based on PM. Shahabi et al. reported that warm nature individuals have a more sympathetic activity. They also cited that their Th2 immune system responses are more often [16]. Another study has declared high frequency of allergic reactions exist in warm Mizaj persons [17]. Investigation of Cheng A et al. displayed that the efficiency of anti-inflammatory drugs in patients with a cold pattern is higher than those with a warm pattern [18]. A previous study performed by Rashid Bikha et al. indicated that some disorders such as diabetes and hypertension are more common in persons with warm Mizaj [10]. Also, it is suggested that warm Mizaj persons are more prone to disorders such as acute fever, stroke, dehydration. The study performed by Jongmans W et al. described an association between genes such as CABLES1, WWOX and IFI27 and warm Mizaj persons with RA [18]. Thus, Mizaj, as an individualized medicine indicator, can be used for predicting the radiosensitivity. Based on PM theories, warmness can cause instability, so it supposed that warm individuals have more movement, with high temperament, various and rapid reactions to external stimulators. Although these reactions were not surveyed yet and in the case of approval, it could be generalized to the level of cells, and our findings can confirm this theory. It means that warmness of individuals widespread in their cells and shows the same reaction that we suppose to see in general reaction of a person. Lake of a reliable and valid standard questionnaire of Mizaj is a limitation of our study. We tried to eliminate this defect with two session assessment of Mizaj based on an expert panel opinion that is the best available gold standard. The low sample size is another limitation of our study, but the results have been significantly based on the interim analysis. More studies can be suggested with dif-ferent tests of radiosensitivity and with higher sample size. As it is proposed in previous studies, some genes such as ATM and NBS, have roles in DNA repair, surveying the correlation between the existence of these genes and Mizaj of individuals, can be the matter of future studies. Conclusion In conclusion, the results are indicating that warm Mizaj persons could be more radiosensitive than other groups, which their importance in radiotherapy individualization should be evaluated in extensive studies.	2020-08-20T10:12:42.963Z	2020-08-19T00:00:00.000	{ "year": 2020, "sha1": "113a67b28d12436623b1a782fab378f04824bad2", "oa_license": "CCBY", "oa_url": "https://doi.org/10.31661/gmj.v9i0.1705", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "bf5ae2fefb90d7f8fdf9c537375c453a3bcbe5bc", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
256825967	pes2o/s2orc	v3-fos-license	Unravelling homologous recombination repair deficiency and therapeutic opportunities in soft tissue and bone sarcoma Abstract Defects in homologous recombination repair (HRR) in tumors correlate with poor prognosis and metastases development. Determining HRR deficiency (HRD) is of major clinical relevance as it is associated with therapeutic vulnerabilities and remains poorly investigated in sarcoma. Here, we show that specific sarcoma entities exhibit high levels of genomic instability signatures and molecular alterations in HRR genes, while harboring a complex pattern of chromosomal instability. Furthermore, sarcomas carrying HRDness traits exhibit a distinct SARC‐HRD transcriptional signature that predicts PARP inhibitor sensitivity in patient‐derived sarcoma cells. Concomitantly, HRDhigh sarcoma cells lack RAD51 nuclear foci formation upon DNA damage, further evidencing defects in HRR. We further identify the WEE1 kinase as a therapeutic vulnerability for sarcomas with HRDness and demonstrate the clinical benefit of combining DNA damaging agents and inhibitors of DNA repair pathways ex vivo and in the clinic. In summary, we provide a personalized oncological approach to treat sarcoma patients successfully. Introduction Genomic instability is a hallmark of many cancers. Aberrant proliferation associated with cancer cells promotes the accumulation of genomic alterations and mutations in genes regulating cell division and tumor suppression (Negrini et al, 2010;Nguyen et al, 2020). Most cells rely on the homologous recombination repair (HRR) pathway for repairing DNA double-strand breaks (DSB) with high fidelity (Li & Heyer, 2008). Inactivation of genes belonging to the HRR pathway leads to additional genomic alterations, correlates with poor prognosis, and is associated with metastases development (Lord & Ashworth, 2016;Turajlic & Swanton, 2016;Bakhoum et al, 2018). The most widespread genetic biomarker of HRR deficiency (HRD) in the clinic is germline or somatic BRCA1/2 mutation status. However, this approach overlooks partial or complete deletions of the BRCA1/2 loci or their epigenetic silencing (Farmer et al, 2005;Nguyen et al, 2020). The prevalence of HRD extends beyond BRCA1/2 inactivation and includes other genes of the HRR pathway such as ATM, ATR, CHK1, CHK2, PALB2, RAD51, and the FANC protein family (McCabe et al, 2006). The term BRCAness has been employed to define defects in the HRR pathway without germline BRCA1/2 mutations, and by name is associated with breast cancer susceptibility (Lord & Ashworth, 2016). In contrast, HRDness encompasses tumor-agnostic HRR pathway deficiencies and will be used throughout this study. Accurate detection of HRD is of clinical relevance as it is indicative of sensitivity to targeted therapy with poly ADP-ribose polymerase inhibitors (PARPi) as well as to DNA damaging agents (McCabe et al, 2006;Nguyen et al, 2020). PARPi trap PARP1/DNA nucleoprotein complexes and stall the advancement of the replication fork. Cancer cells that cannot adequately repair DSB DNA damage via the high-fidelity HRR pathway alternatively use more error-prone DNA repair mechanisms. Treatment with PARPi ultimately induces synthetic lethality in cancer cells with HRD, sparing normal tissue. PARPi can further enhance DNA damage and treatment eventually results in chromosomal instability (CIN), which is a form of genomic instability common in most cancers. CIN refers to the high rate of structural and numerical abnormalities in cancer cells compared with normal cells (Negrini et al, 2010;Sonnenblick et al, 2015). HRD is frequently observed in ovarian and breast cancer, followed by prostate and pancreatic cancer (Nguyen et al, 2020). Recent studies have identified traits of HRDness in soft tissue and bone sarcoma (Kovac et al, 2015;Seligson et al, 2019;Li et al, 2020). Sarcomas are rare mesenchymal cancers, accounting for approximately 1% of all malignancies (Cancer Genome Atlas Research Network, 2017b). Sarcoma comprise a very heterogeneous group of more than 100 distinct histological subtypes originating anywhere in the soft tissue of the body and in bone. To date, there are few treatment options for most sarcoma entities, which often translates into dismal prognosis. Standard of care for soft tissue sarcoma (STS) is usually radiotherapy and resection, while distinct soft tissue and mainly bone sarcoma undergo neoadjuvant chemotherapy, surgery and adjuvant chemotherapy (Meyer & Seetharam, 2019). Locally advanced or metastatic STS often show chemo resistance and metastatic disease is related with a poor prognosis. First-line therapy for advanced disease includes doxorubicin as single agent or in combination with ifosfamide. Median progression-free survival (PFS) in first-line therapy for unresectable STS ranges between 4.5 and 6 months. Second-line treatment is increasingly tailored toward distinct histological subtypes and encompasses the use of chemotherapeutic agents such as trabectedin, eribulin, gemcitabine and taxanes, and the multitargeted receptor tyrosine kinase inhibitor pazopanib (Linch et al, 2014;Gomez & Tsagozis, 2020). Median PFS for most second-line therapies remains below 5 months (Meyer & Seetharam, 2019), thus highlighting the unmet medical need to identify new therapeutic venues for STS treatment. The rarity and the molecular heterogeneity of sarcoma have prevented a detailed study of these mesenchymal cancers, which partially explains the lack of targeted and immunotherapy-based treatment approaches for the distinct sarcoma entities. Only few sarcomas exist with a relevant alteration that can be targeted, the most common examples being activating KIT mutations, NTRK or ALK fusions. Complex karyotype sarcomas lack well-understood genomic driver alterations and commonly harbor copy number alterations and loss of tumor suppressor genes such as TP53, RB1, NF1, and CDKN2A, which are challenging targets . Our understanding of defects in DNA repair mechanisms in sarcoma remains at an early stage. Recent preclinical studies in bone and STS models have shown enhanced PARPi sensitivity when combined with the DNA damaging agent trabectedin (Ordonez et al, 2017;Pignochino et al, 2017) or the alkylating agent temozolomide (Oza et al, 2020). In addition, PARP1, RAD51, and MCM4 expression have been suggested as promising predictive biomarkers for PARPi sensitivity, HRD phenotype and/or genomic instability (Pignochino et Here, we broaden the understanding of HRDness in STS and bone sarcoma by performing a comprehensive molecular analysis of HRR deficiency biomarkers and signatures in several independent cohorts of sarcoma using a multi-omics and cross-platform approach. We show that distinct sarcoma entities exhibit elevated HRD scores, harbor numerous alterations in common HRR pathway genes, high levels of CIN and are characterized by mutational signatures associated with HRD. Furthermore, HRD high sarcoma entities show a distinct SARC-HRD transcriptional signature that predicts PARPi sensitivity. We further validate our in silico findings using functional data from our own established and molecularly characterized patient-derived ex vivo sarcoma models. We show a lack of RAD51 nuclear foci formation in HRD high sarcoma cells suggesting defective HRR upon DNA damage together with a functional dependency toward PARPi and DNA damaging agents. Both agents in combination exert a synergistic effect in HRD high sarcoma models and show a therapeutic response in a leiomyosarcoma patient, thus evidencing the clinical efficacy and feasibility of this approach. Finally, we show therapeutic benefit of targeting other DNA repair pathways, which advocates for a more general mechanism for treating HRDness. A subset of sarcoma entities exhibit HRDness characteristics Cancers with defects in HRR exhibit characteristic chromosomal changes reflecting the use of alternative and more error-prone DNA repair pathways (Chopra et al, 2020). To investigate whether STS and bone sarcoma subtypes exhibit such HRDness features, we calculated the genomic instability signatures loss of heterozygosity (LOH; Abkevich et al, 2012), telomeric allelic imbalance (TAI; Birkbak et al, 2012) and large-scale transitions (LST; Popova et al, 2012) in the TCGA-SARC cohort consisting of 247 cases (Data ref: Cancer Genome Atlas Research Network, 2017a) and the TARGET-OS cohort of 69 cases (TARGET Osteosarcoma Project, 2009). The HRD score is the sum of all three genomic instability signatures and predicts PARPi sensitivity (Marquard et al, 2015;Hodgson et al, 2018;Smeby et al, 2020). HRD scores for high-grade serous ovarian cancer (HGSOC), triple-negative breast cancer (TNBC), and colorectal cancer (CRC) from the TCGA cohorts were previously reported and were used here for comparison (Cancer Genome Atlas Network, 2012a;Data ref: Cancer Genome Atlas Research Network, 2011bData ref: Cancer Genome Atlas Network, 2012b;Marquard et al, 2015;Takaya et al, 2020). Our analysis confirmed the reported HRD scores for HGSOC, TNBC, and CRC and showed striking differences in the genomic instability signatures and HRD scores in the different STS and bone sarcoma entities (Fig 1A-D). Undifferentiated pleomorphic sarcoma (UPS), osteosarcoma (OS), myxofibrosarcoma (MFS) and uterine leiomyosarcoma (ULMS) exhibited the highest LOH, LST, and TAI values followed by malignant peripheral nerve sheath tumor (MPNST), extra-uterine leiomyosarcoma (LMS) and dedifferentiated liposarcoma (DDLPS). While the sarcoma entities with high levels of genomic instability signatures are known to carry complex karyotypes including complex rearrangements in their genomes, the pathogenesis of synovial sarcoma (SS) is associated with a unique chromosomal translocation that results in the expression of the fusion gene SS18-SSX, and mutations in the CTNNB1 or APC genes are the most common cause of desmoid tumors (DT; Cancer Genome Atlas Research Network, 2017b; Kim et al, 2018;Knijnenburg et al, 2018), most likely explaining the observed differences in the signatures. We next investigated the somatic mutational landscape of genes that play a role in core reactions of HRR, as well as genes that are associated with the pathway and might confer PARPi sensitivity (Appendix Table S1; Cancer Genome Atlas Research Network, 2011a). Despite the fact that only 4% of sarcoma carry mutations in BRCA1, the best characterized HRR pathway gene, we found several other alterations, most in the form of amplifications and losses, in essential and associated members of the HRR pathway, particularly in UPS, MFS, LMS, ULMS, DDLPS and MPNST ( Fig 1E). The Fanconi anemia genes FANCB and FANCA exhibited recurrent amplifications and losses in 37 and 20% sarcoma cases, respectively. In striking contrast to BRCA1, BRCA2 alterations were found in 22% sarcoma. MDM2 amplifications were found in DDLPS cases and accounted for 21% of all cases. 21% sarcoma cases showed mostly missense mutations and losses of the tumor suppressor PTEN, and 17% were characterized predominantly by amplifications of the cell cycle checkpoint gene RAD1. Consistent with our previous analysis, SS and DT harbor fewer alterations in the reported HRR genes (Fig 1E). The total number of alterations in HRR genes per sarcoma in the TCGA-SARC cohort showed a bimodal distribution, which was not observed for any of the genomic instability signatures (compare Fig EV1E with Fig EV1A-D). Applying a finite mixture model, which is a probabilistic model for representing the presence of subpopulations within an overall population, we classified each sample status as high or low for HRR-CIN. As the HRD score is one of the most clinically used biomarkers to define HRDness, we plotted a receiver operating characteristic (ROC) curve with HRD score data considering the predicted HRR-CIN status. We next applied the Youden index to summarize the ROC curve and infer the optimum HRD score cut-off point as the value with maximum potential effectiveness of a given biomarker. The optimal cut-off value for the HRD score of soft tissue sarcoma was set at 32. Employing the same method to infer LOH considering the HRD score status, we obtained a cut-off at 10. The HRD score cut-off was implemented in the TCGA-SARC cohort to stratify each STS case (Fig EV1F and G A subset of sarcoma entities exhibit high levels of chromosomal instability Most solid tumors have a form of genomic instability termed CIN that is commonly associated with cancer recurrence and multi-drug resistance (Negrini et al, 2010;Bakhoum et al, 2018). CIN refers to the increased rate by which chromosome structure and number changes over time in cancer cells in comparison with normal cells, including gain or loss of whole chromosomes or large chromosomal fragments (Negrini et al, 2010). To perform a differential characterization of CIN levels in HRD high and HRD low STS and bone sarcoma cases, we computed several features of CIN in the TCGA-SARC and TARGET-OS cohorts (Fig 2). HGSOC and TNBC with BRCA1 or BRCA2 loss are characterized by high levels of CIN due to defects in HRR and were used as positive controls for comparison. In contrast, most colorectal cancer (CRC) cases are HR-proficient (Kuo et al, 2009;Data ref: Cancer Genome Atlas Research Network, 2011aData ref: Cancer Genome Atlas Research Network, 2012b;Telli et al, 2016;Chen et al, 2020) and were also included in the analysis as a negative control. Soft tissue and bone sarcoma cohorts showed high levels of aneuploidies mainly due to chromosomal duplications in HRD high UPS, MFS, OS, ULMS, MPNST, LMS and DDLPS, but not in SS and DT cases (Fig 2A and E). Consistent with the HRD score, the known genomic complexity of the high CIN sarcoma entities compared to the low CIN likely reflects the observed differences. We next computed the number of gains (Fig 2B and F) and losses (Fig 2C and G) within each chromosome as well as the sum of both (total CIN; Fig 2D and H). While complex karyotype sarcomas were characterized by frequent copy number alterations (CNA) mainly due to high levels of amplifications, fusion-driven sarcomas such as SS and the mutation-driven DT displayed considerably fewer CNAs. Higher CIN levels were found in sarcoma entities bearing high HRD scores (Fig 2A-H). Chromosome 12 of DDLPS harbored the highest level of amplifications and CIN (Fig 2B, D, F, and H) irrespective of the HRD score status, confirming previous reports showing highly recurrent focal amplifications at 12q13-15 known to contain several genes involved in its pathogenesis such as MDM2 and CDK4 (Knijnenburg et al, 2018). While well-differentiated LPS (WDLPS) is also characterized by amplification in the same chromosomic region, only the dedifferentiated counterpart acquires more genomic alterations (Coindre et al, 2010). When assessing CIN at the level of each chromosome cytoband, we observed significant differences based on the HRD score status. Cytobands in which 52 core or associated genes of the HRR pathway are located, among other genes, showed significantly higher CIN levels in HRD high compared to HRD low sarcoma cases ( Table S2). Higher CIN levels were also identified in patients with high HRD scores after radio-and chemotherapy or both combined ( Fig 2N). We observed a high correlation among the different genomic instability signatures as well as between the genomic instability signatures and CIN ( Fig 2O). Taken Statistical significance in (I-L) and (N) was determined using Mann-Whitney U-test and showed statistical differences in (N): Chemo-& radiotherapy, P = 0.008; Chemotherapy, P = 0.047; and Radiotherapy, P = 0.0001; P < 0.05; P < 0.01; **P < 0.001. Exact P-values for (I-L) can be found in Appendix Table S3. Source data are available online for this figure. together, we identified high levels of aneuploidies, a complex pattern of amplifications and deletions and a high proportion of HRD high cases with multiple alterations in HRR genes across sarcoma entities. Altogether, these results provide a rationale for employing targeted treatments against DNA damage and repair pathways for sarcoma with HRDness traits. Mutational signatures suggestive of HRDness in distinct sarcoma entities To gain better insights into the mutational processes specific to sarcoma, we next investigated the most relevant signatures based on single base substitutions (SBS) across sarcoma entities ( Fig 2P). The mutational signature SBS5 showed the highest percentage contribution across STS and was reported to correlate with the age of individuals (Alexandrov et al, 2013). SBS3 was the second most prevalent mutational signatures in UPS, MFS and MPNST have been linked with defective HR-based DNA damage repair, which manifests predominantly as small deletions and insertions and genome rearrangements due to abnormal DSB repair (Nik-Zainal et al, 2012). SBS3 is strongly associated with BRCA1/2 mutations and correlates with platinum therapy response and therapeutic approaches that exploit HRD. Since tumors with unstable genomes and HRR defects have been shown to respond to agents that further enhance DNA damage, these data also support the implementation of such therapeutic strategies in a personalized manner for sarcoma patients exhibiting HRDness features. In contrast, the fusion-driven SS exhibited a lower contribution of the SBS3 signature, in line with our previous results that show low CIN and genomic instability signatures as well as few alterations in the HRR pathway in this entity (Figs 1 and 2A-H). Additional sarcoma cohorts show a similar pattern of HRDness and chromosomal instability in distinct sarcoma entities To further validate our finding that MFS exhibited characteristics of HRDness, we characterized a cohort of five treatment na€ ıve MFS patients by whole-genome sequencing, including one low-grade and four high-grade tumors compared with paired normal samples, and investigated genomic instability signatures and fraction of genome altered within this cohort. The average HRD score for the high-grade MFS cases (MFS2-MFS5) was 59.3 AE 11.8, and the low-grade MFS1 presented an HRD score of 5 ( Fig 3A). In addition, HRD high MFS2-MFS5 were characterized by a high degree of their genome exhibiting chromosomal gains and losses ( Fig 3B). To gain further insight into the mutational landscape in MFS, we investigated copy number variations among 70 genes involved in HRR regulation (Appendix Table S1). The HRR pathway genes ATR, BCL2L1, BRCA1, BRCA2, CHEK1, FANCA, FANCL, GEN1, HUS1, PTEN, RAD51, RAD52, RAD54L, RPA1, TEX15, TP53BP1, and XRCC2 showed copy number alterations (gains, amplifications, losses, and deletions) in all HRD high MFS. The only altered HRR gene in the HRD low MFS1 was FANCL ( Fig 3C). We next performed in-depth analysis of chromosomal instability features and identified a complex pattern of amplifications and deletions in regions of most chromosomes, exclusively in the four cases with higher HRD scores (Fig 3D-F). We expanded our investigation of genomic instability in STS with an additional cohort of 21 cases of angiosarcoma, a malignancy that affects blood and lymphatic vessels, that were profiled using wholegenome sequencing. One third of the angiosarcoma cases were HRD high (Fig 3G), were associated with a high percentage of gains and losses in their genome (Fig 3H), and characterized by numerous copy number alterations in HRR genes. EME1, FANCA, RAD51, TP53BP1, RAD51C, RPA1, and XRCC2 exhibited alterations in more than half the cases of the angiosarcoma cohort ( Fig 3I). Elevated levels of gains, losses, and total CIN were observed specially in HRD high angiosarcoma cases ( We additionally characterized 282 STS and bone sarcoma patients by clinical genomic profiling of a panel of 409 genes, for which all types of genomic alterations in cancer can be detected, including base pair substitutions, insertions, deletions, CNAs and rearrangements. Assessment of the genomic instability signature LOH confirmed UPS, OS, MFS, LMS, ULMS and DDLPS as sarcoma entities with high levels of this biomarker. Furthermore, we identified a subset of angiosarcoma and Ewing sarcoma that also exhibited high LOH values ( Fig 3M). Targeted panel sequencing detected mutations and structural alterations in 19 HRR essential and associated genes out of 22 included in the panel. 56.6% of all analyzed sarcoma samples carried alterations in HRR pathway genes; BRCA2, FANCA, and ATM were found among the top altered genes in 11, 10, and 8% of all cases, respectively ( Fig 3N). Tumor mutational burden (TMB), defined as the total number of somatic mutations per coding area of a tumor genome, can predict response to checkpoint blockade in certain cancers (Galuppini et al, 2019). Typically, high TMB occurs in cancer types developed as a consequence of exposure to carcinogens like tobacco or mutagens such as ultraviolet (UV) light in melanoma (Kang et al, 2020). Within our soft tissue and bone sarcoma cohort, dermal sarcoma and a subset of angiosarcoma found in sun-exposed locations exhibited high TMB, consistent with a UV-induced mutational pattern ( Fig 3M). Taken together, the genomic analysis of several independent cohorts revealed a similar pattern of genomic instability signatures, alterations in HRR genes and CIN features across a broad spectrum of soft tissue and bone sarcoma. Notably, sarcomas with high HRD scores consistently exhibited molecular alterations in genes of the HRR pathway. HRD high sarcomas show a distinct SARC-HRD gene signature and enrichment in DNA repair and cell cycle control pathways To explore the transcriptional landscape of sarcoma with HRDness traits, we performed a differential gene expression analysis in HRD high cases compared with HRD low of the TCGA-SARC cohort. Our analysis showed an upregulation of 10 genes in HRD high sarcoma: BRCA1, BRCA2, BLM, EME1, FANCB, FANCD2, FANCI, RAD51, RAD54L and XRCC2 (Figs 4A and C, and EV3), which we named SARC-HRD signature, as well as enrichment of genes in the HRR pathway ( Fig 4B). MDM2 and DMC1 were significantly downregulated in HRD high sarcoma, but showed a sarcoma histotypespecific expression. While MDM2 was upregulated in all DDLPS regardless of the HRD status, it appeared significantly downregulated only in UPS (Figs 4A and C, and EV3A). DMC1 was upregulated in an LMS cluster and unchanged in all other sarcoma entities (Figs 4C and EV3). We next performed a gene set enrichment analysis (GSEA) in HRD high cases compared with HRD low cases, which showed an enrichment in DNA repair pathways including the HRR pathway, among others, across all sarcoma entities except for ULMS ( A Volcano plot showing enrichment of HRR genes in HRD high compared with HRD low sarcoma cases. B Enrichment score of HRR genes in HRD high compared with HRD low sarcoma cases. Normalized enrichment P-value = 2.5e10 À10 . C Heatmap with hierarchical clustering of differentially expressed genes in HRD high compared with HRD low sarcoma cases. D, E GSEA showing hallmark (D) and KEGG (E) pathways enriched in HRD high compared with HRD low sarcoma cases. Data information: Datasets from TCGA-SARC (n = 247) were used; n indicates biological replicates. Source data are available online for this figure. 8 of 21 The Authors p53 pathway and interferon immune responses among the common downregulated gene sets ( Fig EV3). Altogether, we showed an upregulation of 10 HRR genes creating a specific SARC-HRD gene signature, which was further corroborated at the pathway level. Interestingly, the upregulation of the HRR pathway was accompanied by a general enrichment of DNA damage repair pathways, such as mismatch repair and nucleotide excision repair in HRD high sarcoma ( Fig 4E). Genomic and transcriptomic characterization of patient-derived ex vivo sarcoma cell models To investigate novel therapeutic strategies for sarcoma with HRDness traits, we first established genomically and transcriptomically characterized patient-derived ex vivo sarcoma cell models. All cell models faithfully recapitulated the molecular alterations from their corresponding tumor of origin (Fig 5A and B;Bangerter et al, 2022;Chen et al, 2022;Pauli et al, 2022). We molecularly characterized four human MFS cell models, namely NMFH1, OH931, PM197, USZ-21_MFS2, and one UPS cell model (USZ-21_UPS1) by whole-genome sequencing. NMFH-1 showed loss of TP53; OH931, PM197 and USZ-21_MFS2 exhibited loss of CDKN2A and CDKN2B. In addition, USZ-21_MFS2 carried mutations in ATR, MDM2 and TP53, and loss of MLH1. USZ-21_UPS1 showed loss of ATRX and RB1. Genomic profiling also revealed high levels of genomic instability signatures (HRD scores ranging from 62 to 73), a high degree of genomic gains and losses and multiple molecular alterations in HRR pathway genes (Figs 5A-C and EV4A-C). We also established five HRD low models, a BCOR-rearranged sarcoma (USZ-20_REA1), an unclassified low-grade sarcoma (USZ-21_LG1), a fusion-driven CIC-DUX sarcoma (USZ-21_CIC1), a solitary fibrous tumor (USZ-20_SFT1) with an NAB2-STAT6 fusion and an extraskeletal myxoid chondrosarcoma (USZ-22_EMC2) harboring a TAF15-NR4A3 fusion. The HRD scores for these sarcoma cell models scores were 0, 13, 4, 0, and 2, respectively ( Fig 5A). The fraction of altered genome and total number of molecular alterations in HRR genes were lower in HRD low models when compared with HRD high (Fig 5B and C). In addition, we performed differential gene expression analysis comparing the transcriptome of the five genomically characterized patient-derived HRD high MFS and UPS cell models with five HRD low sarcoma cell models. Our analysis identified an upregulation of the SARC-HRD gene signature, enrichment for HRR genes and DNA repair pathways in HRD high sarcoma cells (Figs 5D and EV4D-G). The expression of all genes belonging to the SARC-HRD signature was significantly upregulated and the expression of MDM2 significantly decreased in patient-derived HRD high sarcoma cells compared with HRD low (Figs 5D and EV4D), thus validating the SARC-HRD gene signature in an independent sarcoma cohort. HRD high sarcomas show sensitivity to PARP inhibition PARP1 and PARP2 play critical roles in single-strand DNA break repair and in maintenance of genomic stability mainly through the base excision repair (BER) pathway. PARPi block the BER pathway and, in cells with HRD, cells rely on the error-prone nonhomologous end joining (NHEJ) pathway for DNA repair (McCabe et al, 2006). As a result, cells accumulate massive genetic damage, which ultimately leads to cell death. In vitro sensitivity to DNA double-strand break-inducing drugs, such as platinum salts, is also a feature of HRD cells. We have identified sarcoma entities with HRDness characteristics, thus supporting the use of PARPi and platinum salts for treating sarcoma patients with an HRD high status. We subjected 10 patient-derived ex vivo sarcoma cell models to treatment with two PARPi, olaparib and niraparib, as well as to the standard chemotherapy agents doxorubicin, an anthracycline antibiotic that blocks topoisomerase 2, and the alkylating agents oxaliplatin and trabectedin (Fig 5E-L). To evaluate cellular responses to PARP inhibition, we treated cells for 8 and 12 days with six doses of PARPi and performed ATP measurements (CellTiter-Glo) as a surrogate for metabolically active and thus viable cells. We employed the ovarian cancer cell line UWB1.289 that presents a pathogenic frameshift mutation in BRCA1 and an HRD score of 67 as a control to evaluate the drug response of the HRD high sarcoma models. Our five patient-derived HRD high MFS and UPS cell models show sensitivity to both PARPi, with IC 50 in a comparable range as the IC 50 in BRCA1-mutated ovarian carcinoma cells. In contrast, HRD low ▸ Figure 5. HRD high sarcomas show sensitivity to PARP and WEE1 inhibition and synergy with chemotherapy drugs. A Quantification of LOH, LST, TAI and HRD score in patient-derived sarcoma cell models. B Fraction of genome with gains and losses in patient-derived sarcoma cell models. C Oncoprint depicting the molecular alterations in HRR genes in patient-derived sarcoma cell models and the total number of alterations. D Heatmap with hierarchical clustering of the SARC-HRD gene signature in HRD high compared with HRD low sarcoma cell models. Data was mean-centre and scaled. E-G Ex vivo treatment of HRD high (NMFH-1, OH931, PM197, USZ-21_MFS2 and USZ-21_UPS1) sarcoma models compared with HRD low (USZ-20_REA1, USZ-21_LG1, USZ-22_EMC2, USZ-20_SFT1 and USZ-21_CIC1) sarcoma models for 4 days with six doses of the chemotherapy agents oxaliplatin, doxorubicin, and trabectedin. sarcoma models exhibited significantly lower sensitivity to olaparib and niraparib treatment compared with the HRD high models (Figs 5I,J,and L,and EV5A,B,D,F,and G). This data shows that MFS and UPS cell models characterized by high HRD scores are susceptible to PARP inhibition in a dose-response manner. We found no major differences between HRD high and HRD low sarcoma models in their response to oxaliplatin, doxorubicin, and trabectedin in monotherapy (Figs 5E-H and EV5E). We next assessed whether a combinatorial modality of trabectedin and olaparib resulted in synergism in HRD high sarcoma. After 3 days combinatorial drug therapy, we observed a synergistic effect of the combined drugs in HRD high sarcoma cells as well as in BRCA1-mutated ovarian carcinoma cells, but not in two HRD low sarcoma cells (compare Fig 5M-O with Fig EV5H and I). We employed SynergyFinder to compute the four drug synergy scores zero interaction potency (ZIP), Loewe, Bliss and highest single agent (HSA), which evidenced the synergistic effect between olaparib and trabectedin ( Fig 5P). These data highlight the therapeutic potential of trabectedin and olaparib combination in sarcoma with HRDness, which is currently explored in clinical trials (NCT04076579). Inhibition of WEE1 is a new therapeutic strategy for HRD high sarcoma We hypothesized that our patient-derived UPS and MFS cell models might show a generalized susceptibility to other agents targeting DNA damage and repair pathways. To identify additional drug susceptibilities in HRD high sarcoma based on their genomic instability traits, we focused on the WEE1 protein, which belongs to the serine/threonine family of protein kinases. WEE1 acts by inhibiting CDK1 and CDK2 and thus promotes temporary cell cycle arrest and DNA damage repair. WEE1 exhibits a high number of CNA in sarcoma and its inhibition has been linked to increased genomic instability Martin et al, 2011). In ovarian and endometrial cancers, WEE1 inhibition renders TP53-deficient cells sensitive to radiation and DNA-damaging agents such as PARPi and chemotherapeutic agents (Meng et al, 2018). To investigate the effect of WEE1 inhibition in HRD high sarcoma, we performed a sixpoint dose-response curve with the WEE1 inhibitor adavosertib in our ex vivo patient-derived sarcoma models. HRD high , but not HRD low , sarcoma cells showed adavosertib sensitivity with IC 50 at the nanomolar range after 8 and 12 days treatment (Figs 5K and L, and EV5C, D, F, and G). The response of the HRD high sarcoma models to WEE1 inhibition was comparable with the BRCA1mutated ovarian carcinoma cells. We next assessed whether doxorubicin increased the sensitivity to adavosertib in HRD high sarcoma cells. We observed a synergistic effect of adavosertib together with 0.1 lM doxorubicin in HRD high UPS and MFS but not in HRD low sarcoma (compare Fig 5Q-T with Fig EV5J and K). Altogether, these data show that targeting DNA damage response and DNA repair pathways are powerful therapeutic strategies for sarcoma with HRDness traits. Absence of RAD51 nuclear foci in patient-derived HRD high sarcoma cells upon trabectedin and olaparib-induced damage To functionally assess HRR ex vivo, we investigated biomarkers of HRR in our patient-derived sarcoma models and their corresponding originating tumor tissue (Fig 6). The DNA repair protein RAD51 is a central player in the core mechanism of HRR, catalyzing the recognition of homology and strand exchange between homologous DNA partners to join a processed DNA break and the repair template. The presence of RAD51 nuclear foci is considered a functional biomarker of HRR and can be predictive of PARPi resistance (Li & Heyer, 2008;Castroviejo-Bermejo et al, 2018;Cruz et al, 2018). We evaluated the formation of RAD51 nuclear foci using immunofluorescence in ex vivo MFS and UPS sarcoma cell models upon trabectedin and olaparib-induced DNA damage. We visualized the extent of DNA damage by staining for the phosphorylated histone variant H2A.X forming cH2A.X, which marks DNA DSB (Mah et al, 2010). High levels of cH2A.X are also associated with high sensitivity to agents that further enhance DNA damage (Huang & Zhou, 2020). 10 nM ▸ Figure 6. Absence of RAD51 nuclear foci in patient-derived HRD high sarcoma cells upon trabectedin and olaparib-induced DNA damage. trabectedin and 100 nM olaparib in monotherapy increased cH2A.X nuclear expression, and the combination regimen further enhanced it in all our tested cell models (Fig 6A and C; Appendix Fig S1A). In contrast, whereas we observed a 2-fold increase in RAD51 nuclear intensity as well as formation of nuclear foci in the HRD low USZ-21_LG1, neither formation of RAD51 nuclear foci nor increased RAD51 nuclear intensity were observed in the MFS and ovarian carcinoma cell models (Fig 6B and D; Appendix Fig S1B). The slight increase (0.4-fold) in RAD51 nuclear expression in UPS cells treated with the combinatorial modality did not translate into formation of nuclear foci (Fig 6B and D; Appendix Fig S1B), which is a key step for its function (Haaf et al, 1999). Moreover, RAD51 expression and nuclear foci were reduced in HRD high UPS and MFS tissue compared with both HRD low sarcoma patient samples (Fig 6E and F). Our combined results demonstrate defective HRR in sarcoma cell models with increased genomic instability levels and PARPi sensitivity. Trabectedin and olaparib combination treatment show clinical benefit in a leiomyosarcoma patient A 47-year-old female patient presented with multiple lesions in lungs and liver in April 2020, but the location of the primary tumor was unknown. Pathological review confirmed metastatic LMS, most likely of uterine origin. The patient immediately underwent a systemic therapy with pegylated liposomal doxorubicin (PLD). Due to the widespread metastatic disease, broad molecular profiling was performed, which evidenced a loss of exons 8-11 and a rearrangement in intron 7 within the TP53 gene. In addition, targeted panel sequencing also revealed a stable microsatellite status, TMB of 7 Mut/Mb and an LOH score of 26%, which was considered high in the hospital-based molecular tumor board (Fig 6G). Treatment with PLD had to be terminated because the patient suffered from a myocardial infarction and underwent cardiopulmonary resuscitation, due to an anthracycline-induced cardiomyopathy. From October 2020, the patient was treated off-label with a combination of trabectedin and olaparib and remained in a lasting radiological partial remission for one and a half years (Fig 6G and H). Genomic profiling of a stomach metastasis in late 2022 revealed an HRD score of 83 and 22% of structural genomic alterations (Fig 6I). Genomic gains in the HRR genes HUS1, RAD1 and RAD52 as well as losses in PCNA, RPA2 and XRCC2, and absence of nuclear RAD51 expression in the metastatic tissue further supported a defect in HRR mechanisms ( Fig 6J and K). This case highlights the potential benefits of extending the therapeutic indications for these drugs alone or in combination. Altogether, our data provide the groundwork for clinical testing of DNA damaging agents and DNA repair targeting drugs in new sarcoma indications. Discussion To date, very few targeted options exist to successfully treat sarcoma, thus highlighting the need to investigate specific disease mechanisms for the distinct sarcoma entities to guide development of new personalized therapies. Here, we performed a comprehensive and systematic analysis of genomic instability biomarkers in sarcoma and cross-validated our results using multiple independent sarcoma cohorts. We identified high levels of CIN and other genomic instability signatures in sarcoma with complex karyotypes. Determining the genomic instability scars LOH, LST and TAI as well as the HRD score has proven clinically relevant as it can predict sensitivity to PARPi, the gold-standard approach to treat HRD tumors. Commercial clinical tests such as the MyriadMyChoiceâCDx assay and the FoundationOneâCDX assay are widely used to detect HRD at the genomic level. The MyriadMyChoiceâCDx assay uses an HRD cut-off value of 42 to predict response to platinum-containing neoadjuvant chemotherapy in patients with TNBC (Telli et al, 2016) and recurrent ovarian cancers treated with niraparib (Mirza et al, 2016;Gonzalez-Martin et al, 2019). The FoundationOneâCDX assay uses a cut-off value of LOH > 16% to predict response to PARPi in ovarian cancers (Coleman et al, 2017). Currently, no clinically validated diagnostic test exists for determining HRDness in soft tissue or bone sarcoma and the designated threshold for the different genomic signatures in ovarian and breast cancer is not necessarily applicable to other tumor entities, as has been reported for pancreatic cancer (Zhuang et al, 2021). By assessing the CIN levels in HRR genes, the only signature that showed a bimodal distribution pattern, we could infer an optimal cut-off value for the HRD score in STS applying the Youden index. We defined an HRD score higher or equal to 32 to classify the HRD status of each sarcoma case. This cut-off correlates with high PARPi sensitivity in patient-derived ex vivo sarcoma models. An independent study defined a cut-off of 35 based on sarcoma patient survival curves (Li et al, 2020). Nevertheless, a clinical trial addressing PARPi sensitivity is required for validating the clinical utility of this biomarker for soft tissue and bone sarcoma. An alternative mechanism of HRDness was reported in Ewing sarcoma. Interestingly, this fusion-driven bone sarcoma lacks genomic instability but shows PARPi sensitivity (Gorthi & Bishop, 2018). The proposed underlying mechanism is an exacerbated transcription that is considered to cause a widespread accumulation of R-loops, which in turn prevents BRCA1 relocation to sites of damage and HRR . Moreover, complex chromosomal rearrangements resembling chromothripsis were observed in LMS, OS and LPS (Chudasama et al, 2018;Cortes-Ciriano et al, 2020). Chromothripsis is another form of genomic instability observed across many tumor types in particular those with TP53 loss. It is characterized by a catastrophic chromosomal event that causes clustered genomic rearrangements in a few cell divisions (Maciejowski et al, 2015;Cortes-Ciriano et al, 2020). Our mutational analysis evidenced that most STS patients carry alterations in genes involved in the HRR pathway and mutational signatures based on single-base substitutions common in HRdeficient cancer. BRCA1/2 mutations are well-known causes of HRD, 4 and 22% of sarcoma patients carry mutations in BRCA1 and BRCA2, respectively. Notably, reversion mutations in BRCA2 mediating PARPi resistance have been reported in ovarian, breast, pancreatic and prostate cancer, highlighting the benefit of implementing molecular profiling to pre-emptively detect them in clinical settings. Moreover, two members of the Fanconi anemia core complex, FANCA and FANCB, the TP53 regulator MDM2, the tumor suppressor PTEN, cell cycle checkpoint regulators RAD1 and CHEK1, DNA damage and replication proteins ATM, RPA1 and H2AFX completed the list of the 10 top altered HRR genes in the TCGA-SARC cohort. Our genomic analyses are in line with recently published work (Gounder et al, 2022;Nacev et al, 2022) that described molecular patterns and genomic instability signatures in distinct sarcoma histotypes using a large cohort of sarcoma patients. In contrast to their findings, we identified a significant number of gene alterations in an extended HRR gene set, thus contributing to elucidate potential molecular mechanisms for HRDness in distinct sarcoma histotypes. Although molecular alterations in HRR genes besides BRCA1/2 are less established surrogate markers for HRD, mutations in ATM and PALB2 but not in other HRR genes were shown to be associated with sensitivity to PARPi in prostate carcinoma in a phase 3 clinical trial (Hussain et al, 2020). In addition, not only BRCA1/2-mutant patients but also BRCA1/2 wild type exhibiting high LOH were shown to benefit from PARPi therapy (Coleman et al, 2017). How individual genetic aberrations or the overall changes within the HRR molecular landscape in sarcoma contribute to creating HRDness or predict platinum and PARPi sensitivity is yet to be shown. In addition to our comprehensive genomic analyses, we also identified a distinct SARC-HRD gene expression signature that was able to predict PARPi and WEE1i sensitivity in ex vivo sarcoma cell models. The SARC-HRD gene signature includes 10 HRR genes whose expression was significantly increased across all HRD high sarcoma cases from the TCGA-SARC cohort. The activation of the HRR pathway might be due to HR-deficient cells trying to compensate at the transcriptional level for the dysfunction in the pathway. Similarly, upregulation of HRR genes was previously reported for osteosarcoma (Barenboim et al, 2021). We could validate a significant increase in the 10 genes of the SARC-HRD signature in our patientderived sarcoma models. Thus, the SARC-HRD signature shows promise and warrants further corroboration and clinical validation. In contrast to the other soft tissue sarcoma entities, the transcriptomic profiling analysis of HRD high ULMS showed an enrichment neither in HRR nor in other DNA repair pathways. ULMS has been shown to separate from other sarcomas into a distinct molecular cluster (Cancer Genome Atlas Research Network, 2017b), likely explaining the observed differences. Interferon responses were commonly downregulated in most HRD high sarcoma histotypes. Notably, PARPi treatment was shown to induce the innate immune interferon response via the cGAS-STING pathway (Chopra et al, 2020;Kim et al, 2020;Bruand et al, 2021), which underlines a potential additional mechanism of action of this drug. RAD51 is a key player for restarting stalled replication forks via HRR, thus reducing DNA damage and protecting cells from apoptosis. We demonstrated a functional impairment in HRR in patientderived sarcoma cell models harboring multiple alterations in HRR genes and high HRD scores. HRD high UPS and MFS cells lacked RAD51 nuclear expression and foci formation upon DNA damage elicited with a combinatorial modality of PARPi and chemotherapy. In addition, we showed that HRD high UPS and MFS patient tissue have reduced RAD51 nuclear expression compared with HRD low low grade and a BCOR-rearranged sarcoma patient tissue. Formation of RAD51 nuclear foci was proposed as a predictive biomarker for platinum and PARPi response in pre-clinical studies (Castroviejo-Bermejo et al, 2018;Pellegrino et al, 2022). Our work also supports the use of RAD51 as a potential and easily affordable biomarker for HRDness, particularly in diagnostic settings where NGS is not readily available. Interestingly, RAD51 overexpression contributes to genomic instability and increased RAD51 levels in BRCA-deficient cells are a described mechanism of resistance to DNA damaging treatment (Richardson et al, 2004;Hannay et al, 2007;Liu et al, 2017). Additional candidate biomarkers whose clinical utility warrants further clinical validation are PARP1 (Pignochino et al, 2017), MCM4 (Liu et al, 2021) and high levels of pH2AX and MAP17 (Perez et al, 2020). By using patient-derived ex vivo sarcoma cell models, we showed that PARPi was effective against HR-deficient sarcoma cells that lacked BRCA mutations but exhibited (i) elevated levels of CIN in core and associated genes of the HRR pathway, and (ii) high genomic instability scores. Our HRD high sarcoma cell models also showed high sensitivity to WEE1 inhibition, a tyrosine kinase involved in G 2 checkpoint signaling. Either combination of the PARPi olaparib with the alkylating agent trabectedin or the WEE1i adavosertib with the anti-tumor antibiotic doxorubicin elicited a high degree of drug synergy in HRD high sarcoma cells, demonstrating the efficacy of two different therapeutic approaches for treating HR-deficient sarcoma cells. The efficacy of both olaparib and trabectedin combination therapy as well as adavosertib in combination with chemotherapeutic agents are currently under clinical investigation in either metastatic or advanced sarcoma (NCT04076579), recurrent ovarian cancer (NCT02101775) or relapsed or refractory solid tumors in pediatric patients (NCT02095132). We have already demonstrated that an off-label use of such drug combinations is beneficial in an LMS patient, providing additional support for such clinical trials. Interestingly, we observed high levels of CIN and high HRD scores in patients that had previously undergone radio-, chemotherapy or both, which can exacerbate genomic instability (Hendry, 2001). Whether PARPi is a viable therapeutic option for treatmentacquired HRDness is yet to be clinically proven. Measuring the degree of genomic instability in a treatment na€ ıve tumor may prove relevant to guide personalized treatment. Beyond PARP inhibition, other targeted agents have been explored in HR-deficient tumors, such as against topoisomerase II and c-Abl (Siddiqui et al, 2021), PLK1 andCHEK (Yoshida et al, 2022), mTOR alone or in combination with DNA repair inhibitors (Mo et al, 2016;El Botty et al, 2018), among others. In summary, we provide a comprehensive genomic, transcriptomic and functional investigation of HRDness in sarcoma. The use of an HRR-CIN score or the newly identified SARC-HRD gene expression signature might enhance the identification of patients that benefit from DNA damage and DNA repair-based therapies and therefore warrants further investigation to personalize treatment for sarcoma patients. Future research could benefit from exploring specific mechanisms of PARPi resistance in sarcoma and focus on the identification of treatment combination regimens to prevent and overcome development of resistance. Data collection Data license was acquired for TCGA data portal (https://portal.gdc. cancer.gov) and VCF files, allele-specific copy number, gene-level copy number and RNA-sequencing raw counts were downloaded. (2009)) were included in the analysis. Targeted next-generation sequencing data obtained with the FDA-approved, broad molecular diagnostic test FoundationO-neâHEME assay (Foundation Medicine Inc.) for the following cases: 41 angiosarcoma, 5 BCOR-rearranged sarcoma, 14 chondrosarcoma, 4 CIC-DUX4 sarcoma, 3 clear cell sarcoma, 4 DDLPS, 36 dermal sarcoma, 4 epithelioid sarcoma, 20 Ewing sarcoma, 4 intimal sarcoma, 46 LMS, 12 MFS, 13 myxoid liposarcoma, 4 NTRKfused sarcoma, 12 OS, 9 SS, 16 ULMS and 35 UPS. The assay sequences DNA of the complete exons of 409 cancer-related genes for the detection of short variants (single nucleotide variants, insertions and deletions), copy number alterations as well as microsatellite status and tumor mutational burden (TMB), selected introns and promotor regions of 31 genes involved in rearrangements, in addition to RNA sequencing of 265 genes. Differently to LOH scores reported by scarHRD (Sztupinszki et al, 2018), LOH scores determined by the FoundationOneâHEME assay are measured as genome-wide percentage of LOH events. Tumor tissue DNA was profiled without paired normal control DNA, which may lead to unintentional inclusion of germline variants. Short nucleotide polymorphism (SNP) analysis detects sample purity and possible contaminations, thus allowing for detection and exclusion of any cross-contaminated samples. An OS cohort and a mixed cohort of UPS and LMS profiled using Affymetrix Genome-wide Human SNP 6.0 arrays were downloaded from the Gene Expression Omnibus database repository with accession numbers GSE33153 and GSE154591, respectively (Data ref: Kuijjer et al, 2012;Data ref: Lesluyes et al, 2019). In addition, the ULMS cohort with accession number GSE119043 profiled using the Oncoscan array was also downloaded (Data ref: Gultekin et al, 2021). After filtering low-quality samples, 30 OS, 34 ULMS, 30 LMS and 20 UPS were analyzed. Whole-genome sequencing (WGS) DNA was extracted with the Maxwellâ 16 DNA/RNA Purification Kits (Promega) from fresh frozen native tumor tissue or FFPE together with matched normal tissue. DNA quantification was performed via Nanodrop (ThermoFisher Scientific) and Qubit ds DNA BR Assay Kit (ThermoFisher Scientific) or Qubit 3.0 Fluorometer (ThermoFisher Scientific). 200 ng of dsDNA was fragmented intõ 200 bp fragments by sonication (Covaris system) prior to purification (AMPure XP Beads; Agencourt). Library construction was performed using NEBNext kits (NEB, E6040S). DNA extracted from four high-grade and one low-grade MFS tissues together with matched normal samples were sequenced using the Illumina HiSeq X instrument as described by the manufacturer. Primary tumor tissue derived from UPS, MFS, BCOR-rearranged sarcoma, unclassified low-grade sarcoma, fusion-driven CIC-DUX sarcoma, solitary fibrous tumor and extraskeletal myxoid chondrosarcoma patients, their respective patient-derived cell models (USZ-21_UPS1, USZ-21_MFS2, USZ-20_REA1, USZ-21_LG1, USZ-21_CIC1, USZ-20_SFT1 and USZ-22_EMC2), as well as an angiosarcoma cohort consisting of 21 cases together with matched normal samples were sequenced using Illumina NovaSeq 6000 instrument following the manufacturer's instructions. Three MFS cell models (NMFH-1, OH931 and PM197) and ovarian carcinoma cells (UWB1.289) were also subjected to whole-genome sequencing for genomic instability signature and copy-number variation analysis. Cells, tumor and normal sample sequencing was performed with 60 or 30-fold coverage depth and analyzed with the Dragen Bio-IT platform v3.9.5 (Illumina). Segmentation and allele-specific copy number (ASCN) calling Raw data (.CEL) from SNP arrays (Oncoscan and SNP6.0) were processed using the R package EaCoN (https://github.com/ gustaveroussy/EaCoN). BAF and LRR data were used as input in ASCAT copy number R package (https://github.com/cancerit/ ascatNgs), which infers a sample's ASCN profiles. Gamma values were selected for each sample based on the goodness of fit curve plots. BAM files from tumor and normal pairs were input in ascatNgs to generate allele-specific copy number data from WGS. The reference SNP positions for both array and WGS data were recommended by ASCAT. Genomic instability signatures LOH, LST, TAI and HRD score HRD score was computed using the R package scarHRD (Sztupinszki et al, 2018). This score is the unweighted, linear sum of genetic alterations embracing loss-of-heterozygosity (LOH), largescale transitions (LST) and telomeric allelic imbalance (TAI). LOH implies to the loss of genomic regions larger than 15 Mb without covering whole chromosomes. LST includes chromosomal breaks between adjacent regions of at least 10 Mb, with an inter breakage distance no larger than 3 Mb. TAI is the number of unequal contributions of allele sequences in the telomeric regions of chromosomes. Genomic instability Chromosomal instability (CIN) from TCGA data was computed on the allele specific copy number segments by adding the number of gains (CNV > 2) and the number of losses (CNV < 2) per sample and chromosome. The same threshold method was applied at the level of specific cytobands in order to compute their genomic instability. For array and WGS data, gains and losses were computed overall on the segments based on the L2R values. Segments with a positive L2R greater than 0.1 were considered as gains, whilst negative segments less than À0.1 were considered as losses. To identify high level amplifications and homozygous deletions, copy number variations (CNV) with log2R > 0.7 (high level gain) and < À0.7 (deep deletions) were implemented as used by convention in cBioPortal (Cerami et al, 2012). Total genomic instability was computed as the fraction of the whole genome that presented gains and losses. The same was implemented at the chromosome level, where the genomic instability was computed as the fraction of every chromosome that presented genomic aberrations of gains or losses. Chromosomal aneuploidy events were analyzed using the CNV for each segment. Events with a loss or gain greater than 90% found within a chromosome were considered as aneuploidy. HRR-CIN score and HRD score cut-off The HRR-CIN score was calculated from the copy number profiles by computing the CIN specifically in genes belonging to the HRR pathway. A finite mixture model to assess bimodal data was used and thereafter a cut-off value that separates the two peaks in the bimodal distribution was implemented as described in the R package cut-off (https://github.com/choisy/cutoff). The confidence interval from the cut-off was computed by Monte Carlo simulations and resulted in an HRR-CIN cut-off value of 37. Sample population was stratified into HRR-CIN high and HRR-CIN low . In order to infer a cut-off for the HRD score, we plotted a receiver operating characteristic (ROC) curve with HRD score data. An ROC curve illustrates the diagnostic ability of a binary classifier as the classification thresholds are varied. ROC curves show the trade-off between sensitivity (x axis: true positive rate) and specificity (y axis: false positive rate, 1-specificity). While a random classifier gives curves along the diagonal, classifiers that fives curves closer to the top-left corner indicate better performance. Subsequently, the Youden index was applied using the R package cutpointr as it summarizes the ROC curve and shows the maximum potential effectiveness of a biomarker, which in this case resulted in an optimal value of 32 for the HRD score. The same method applied to LOH data from the TCGA-SARC cohort and resulted in an optimal cut-off value of 10 for LOH. Oncoprints and mutational signatures Oncoprints including genes from the HRR pathway (Appendix Table S1; Cancer Genome Atlas Research Network, 2011a) were generated using maftools (Mayakonda et al, 2018) from MAF (mutation annotation format) files downloaded from TCGA. Additionally, copy number amplifications and copy number deletions were computed as described above and added to the oncoprints. For the analysis of WGS, VCF files were filtered to include somatic variants and variants with a quality equal to PASS. Filtered VCF files were annotated by VEP (https://github.com/Ensembl/ensembl-vep) and converted to MAF using vcf2maf tool (https://github.com/mskcc/ vcf2maf). From samples sequenced with the FoundationOne â HEME assay, somatic mutations of known significance, reported amplifications and deletions as well as reported tumor mutational burden and percentage LOH were included. Of note, not all HRR pathway genes are covered in this assay. RNA-sequencing RNA from patient-derived sarcoma cell models was extracted using the Maxwell RSC simplyRNA Tissue kit (Promega AS1340). The extracted RNA was quantified with a Nanodrop (ThermoFisher Scientific) and the High Sensitivity RNA ScreenTape Assay for TapeStation systems (Agilent 5067). RNA-sequencing libraries were prepared using the Illumina Stranded mRNA sample preparation protocol following the manufacturer's instructions. Each library was sequenced in paired-end mode using the NovaSeq 6000 platform and resulting reads were mapped to the human transcriptome. Differential gene expression between HRD high and HRD low sarcoma samples from the TCGA cohort was investigated by downstream processing of raw counts using the R package DESeq2 (Love et al, 2014). In-house RNA-sequencing data were processed using the R package tximportData to import the quantification files generated by Salmon into DESeq2. Genes with a two-fold change and a P-value equal to or smaller than 0.05 were considered differentially expressed. Gene set enrichment analysis (GSEA) was used to determine significantly different gene sets between HRD high and HRD low samples. 'h.all.v7.5.1.symbols' and 'c2.cp.kegg.v7.5.1.symbols' gene sets were downloaded from the Molecular Signatures Database (MSigDB database; Liberzon et al, 2015) for GSEA analysis. The ClusterProfiler R package (Yu et al, 2012) was used for KEGG pathway and MSigDB Hallmark pathway enrichment analysis. Adjusted Pvalue < 0.01 was considered statistically significant. Patient-derived sarcoma cell models Patient-derived sarcoma cell models were authenticated and established as previously described (Bangerter et al, 2022;Chen et al, 2022) from both male and female patients to avoid gender bias. Informed consent was obtained from all subjects. The present study was conducted following regional/cantonal and institutional guidelines and in compliance with the WMA Declaration of Helsinki and after approval by our cantonal ethical review board Zurich (BASEC-2021-00417). Cell culture Patient-derived sarcoma cells were genomically characterized by the FoundationOneâHEME assay and Infinium Human Methylation EPIC 850 k array (Illumina) targeted gene sequencing panel and maintained in DMEM (Gibco) with 10% fetal bovine serum (Gibco). Ovarian carcinoma cells UWB1.289 (ATCC, CRL-2945, RRID: CVCL_B079) were genomically characterized by the FoundationOne â CDX assay targeted gene sequencing panel, which confirmed a pathogenic frameshift mutation in BRCA1 (2475delC). Cells were maintained in 1:1 RPMI-1640 medium (Gibco) and Mammary Epithelial Cell Growth Medium (MEGM Bullet kit, Lonza, CC-3150). All cells were passaged when sub-confluent conditions were reached in cell culture-treated flasks, incubated at 37°C in a humidified atmosphere with 5% CO 2 and routinely tested for Mycoplasma. Functional testing Cells were detached from the cell culture flasks by adding TrypLE Express (ThermoFisher, 12604) followed by a brief incubation at 37°C in a humidified atmosphere with 5% CO 2 . Medium-containing serum was added, cells were collected and centrifuged for 3 min at 500 g at RT. Cell pellets were reconstituted in medium and cells were counted using the Countess automated cell counter (Thermo-Fisher Scientific). Depending on the assay duration, a total of 500-1,000 cells/well were seeded onto collagen-coated 96-well plates (Corning,354407). The following chemotherapeutic and targeted agents were added to the cells 24 h later spanning 6 dose levels: doxorubicin (Selleckchem, S1208), oxaliplatin (Selleckchem, S1224), trabectedin (Selleckchem, S7758), olaparib (Selleckchem, S1060), niraparib (Selleckchem, S2741), adavosertib (Selleckchem, S1525) and cells were subsequently incubated at 37°C in a humidified atmosphere with 5% CO 2 . For short-term treatment, cell viability was measured 4 days later with CellTiter-Glo 2.0 (Promega, G9241) following the manufacturer's instructions. For longer treatments, cells were retreated every 4 days and cell viability was measured eight and 12 days after the first treatment. All experiments were performed in triplicates and repeated. Luminescence readout was performed on white 96-well LumiNunc plates (VWR, 732-2698) for 1,000 ms using the Multimode Plate Reader Infinite 200 Pro (Tecan). All data were normalized to untreated control, a non-linear regression was used to calculate IC 50 and area under the curve in GraphPad Prism (GraphPad Software, Inc). SynergyFinder web application was used to investigate synergistic drug effects (Zheng et al, 2022). Immunofluorescence and immunohistochemistry Cells were seeded into l-slide eight-well chamber slides (Ibidi,80806) or black 96-well plates with clear glass bottom (Thermo-Fisher, 165035). On the next day, cells were treated with 10 nM trabectedin and 100 nM olaparib as single agents or in combination for 6 h at 37°C in a humidified atmosphere with 5% CO 2 . Following three washes in 1x PBS, cells were fixed in 4% paraformaldehyde or cold methanol for 20 min. at RT and washed again three times in 1x PBS. Fixed cells were permeabilized and blocked in 0.2% Triton X-100 (Sigma-Aldrich), 3% bovine serum albumin (BSA; Sigma-Aldrich, A3912) for 1 h at RT under gentle agitation. Immunofluorescence was performed with the following antibodies: rabbit anti-RAD51 (Abcam, ab133534 [EPR4030], RRID:AB_2722613) diluted 1/250 or rabbit anti-cH2A.X (phospho S139; Abcam, ab81299, RRID:AB_1640564) diluted 1/250 in blocking/permeabilization solution o/n at 4°C under gentle agitation. Following three washes in 1x PBS, cells were incubated with donkey anti-rabbit secondary antibodies conjugated to Alexa488 (Jackson ImmunoResearch, for 45 min at RT under gentle agitation. Finally, cells were washed three times in 1x PBS and stored at 4°C. Imaging was performed in the ImageXpress Pico imaging system (Molecular Devices) using the same settings for each experiment. Brightness and contrast were adjusted equally to all images in each experiment using Fiji. A macro was implemented to automatically identify cell nuclei and measure nuclear RAD51 and cH2A.X intensity in each cell. Nuclear staining intensity per cell was normalized to untreated control and fold increase of nuclear RAD51 and cH2A.X intensity was plotted in GraphPad Prism (GraphPad Software, Inc). To evaluate RAD51 expression in patient material, immunofluorescence and immunohistochemistry were performed on deparaffinized, rehydrated sections from archived FFPE patient blocks. Following antibody-specific epitope retrieval, immunohistochemistry was performed on 2 lm thick tissue sections using the automated Ventana Benchmark (Roche) system. Primary antibodies against RAD51 (Abcam, ab133534 [EPR4030], RRID:AB_2722613) diluted 1/500 and HRP-coupled secondary antibodies were used. For RAD51 immunofluorescence, 5 lm thick tissue sections were subjected to heat-mediated antigen retrieval (EDTA buffer,pH 8.4) at 95°C for 20 min and blocked/permeabilized for 1 h at RT. Immunofluorescence was performed with rabbit anti-RAD51 (Abcam, ab133534 [EPR4030], RRID:AB_2722613) antibodies diluted 1/250 incubated o/n at 4°C followed by three washes in 1× PBS, 45 min incubation with donkey anti-rabbit Alexa488 antibodies and three washes in 1× PBS. Coverslips were mounted on top of tissue sections with a few drops of FluoroMount aqueous mounting medium (Sigma-Aldrich, F4680) and protected from light. Imaging was performed in the Leica SP8 confocal microscope using the same settings for all samples in a blinded fashion. Brightness and contrast were adjusted equally to all images in Fiji. Statistical analysis and reproducibility All statistical tests for the in silico studies were done using R. Statistical analysis of CIN to compare HRD high and HRD low samples per sarcoma was performed using the Mann-Whitney U-test. Correlations were calculated following the Spearman's correlation method. Functional drug testing, immunohistochemistry and immunofluorescence assays were repeated multiple times for each cell model and performed in triplicates. Dose-response curves and fluorescent images are representative of multiple experiments. For immunofluorescence image acquisition and analysis, the investigators were blinded by a third party. Unblinding was performed immediately before final data analysis. No experimental method was used to predetermine sample size. No samples were excluded from the analysis. Data are expected to have normal distribution and are shown as The paper explained Problem Genomic instability is a hallmark of many cancers. Aberrant proliferation in cancer cells leads to the accumulation of alterations in genes that belong to the homologous recombination (HR) DNA doublestrand break (DSB) repair pathway. Deficiency in HR-mediated repair (HRR) exacerbates genomic instability and correlates with poor prognosis and development of metastases. Determining HRR deficiency (HRD) in a given tumor is of major clinical relevance as it is associated with therapeutic vulnerabilities. HRD has been widely investigated in certain tumor types such as ovarian and breast cancer but remains greatly unexplored in sarcoma, a rare and heterogeneous group of mesenchymal cancers. Results Specific sarcoma entities are characterized by high levels of genomic instability signatures and a wide range of molecular alterations in HRR genes, while exhibiting a complex pattern of chromosomal instability features. Furthermore, sarcomas carrying HRDness traits exhibit a distinct SARC-HRD transcriptional signature that predicts PARP inhibitor sensitivity in patient-derived ex vivo sarcoma models. Concomitantly, HRD high sarcoma cell models lack RAD51 nuclear foci formation upon DNA damage, further evidencing defects in HR-mediated DNA repair. The WEE1 kinase was discovered as a therapeutic vulnerability for sarcomas with HRDness traits. Finally, we demonstrated clinical benefit of combining DNA damaging agents and inhibitors of DNA repair pathways in patient-derived ex vivo cell models and in a leiomyosarcoma patient. Impact This work provides the most comprehensive analysis of HRDness in sarcoma to date and offers strategies to successfully treat sarcoma patients. The use of an HRR-CIN score or the newly identified SARC-HRD gene expression signature might enhance the identification of patients that benefit from DNA damage and DNA repair-based therapies to personalize treatment for sarcoma patients. mean AE standard deviation (s.d.) unless otherwise reported in the figure legend. GraphPad Prism was used for statistical analysis. Statistical significance was determined as P < 0.05. One-way ANOVA with Sidak's multiple comparisons test was used for determining statistical significance when comparing staining intensities upon drug treatment. One-tailed unpaired t-test was used for determining statistical significance when comparing area under the curve in the functional assays. Expanded View for this article is available online. Figure EV1. HRD score cut-off based on genomic alterations in HRR genes (HRR-CIN). A-D Histograms depicting the frequency of LOH, LST, TAI, and HRD score in sarcoma. E Bimodal distribution of HRR-CIN in sarcoma. F Receiver operating characteristic (ROC) curve of HRD scores from TCGA-SARC cohort using HRR-CIN for binary classification. G Implementation of the Youden index on the ROC curve (F) to select the optimal cut-off value for the HRD score 32 in soft tissue sarcoma. Datasets from TCGA-SARC (n = 247) were used. Source data are available online for this figure. A-C Heatmaps of gains (A), losses (B) and total CIN (C) per chromosome in sarcoma cell models. D Volcano plot showing enrichment of HRR genes in HRD high compared with HRD low sarcoma cell models. E Enrichment score of HRR genes in HRD high compared with HRD low sarcoma cell models. Normalized enrichment P-value < 0.01. F, G GSEA showing hallmark (F) and KEGG (G) pathways enriched in HRD high compared with HRD low sarcoma cell models. Data information: n = 5 HRD high and 5 HRD low sarcoma cell models. Data in (A-C) are mean-centre and scaled. Source data are available online for this figure. AUC for olaparib, niraparib and adavosertib upon 12 days treatment; corresponding dose-response curves depicted in Fig 5I-K. H, I HRD low sarcoma cell models treated for 3 days with five doses olaparib alone and in combination with 1 nM trabectedin. J, K HRD low sarcoma cell models treated for 3 days with five doses adavosertib alone and in combination with 100 nM doxorubicin.	2023-02-14T06:18:07.120Z	2023-02-13T00:00:00.000	{ "year": 2023, "sha1": "8f8d8b687084906efaf05f148f5eace67ba0f8e4", "oa_license": "CCBY", "oa_url": "https://www.zora.uzh.ch/id/eprint/232649/1/ZORA_232649.pdf", "oa_status": "GREEN", "pdf_src": "PubMedCentral", "pdf_hash": "8e3accf6f944bffbe65102c22e4facb21501b385", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Medicine" ] }
5869615	pes2o/s2orc	v3-fos-license	Amphibious Study on a Basilisk Lizard Inspired Robot This study describes the amphibious study of a novel robot, which attempts to emulate the basilisk lizard's ability to run on the surface of water and walk on land. Functionally, the robot uses four bar mechanism as its driving leg with a self-adaptive foot added to its end. Through some hydromechanics calculations and analyses, its water running ability is theoretically verified. And via terrestrial gait planning, the feasibility of land walking is also realized and the performance has been tested by both simulation and experiment. This study opens the door for legged robots to become ambulatory over both land and water in China and its research method has given a good reference for further study of the amphibious robot. INTRODUCTION With the continuous development of robotics and bio-science, study of the amphibious robots has become more and more popular.Amphibian animals, as a transition from aquatic to terrestrial vertebrates, inherit the dual characteristics of both aquatic and terrestrial vertebrates.They are not only endowed with the abilities of crawling on land like terrestrial vertebrates which are capable of obstacle crossing, but also possess the excellent performances of water swimming and running like aquatic vertebrates (Rui et al., 2009).Till now, achievements of the amphibious robots have mostly been focused on the study of their driving mechanism.The development of computer science has offered a chance to test the feasibility of the designs through simulation, which is both time-saving and economical. As is known to all, unlike small and lightweight animals, large creatures cannot reside at rest atop the water.They must strike the free surface with sufficient vigor to generate hydrodynamic forces on their driving legs capable of bearing their weight.Like most large animals, the basilisk lizard rely on a combination of form drag, added mass and gravitational forces generated by vigorous slapping of the free surface for both weight support and propulsion (John and David, 2006).By examining the surface locomotion of the basilisk lizard and elucidating their subtle water-walking technique Glasheen and McMahon characterized the driving stroke in terms of three distinct phases: slap, stroke and recovery (Tonia and Gorge, 2004). Our study is mainly focused on the amphibious design and performance analysis of a basilisk lizard inspired robot.On the basis of the preliminary researches and in the study of hydromechanics theory, an amphibious robot which can move both on land and water was brought forward.The knowledge gained by this research will help expand the limits of legged robot locomotion and also assist to increase the understanding of the basilisk lizard and its ability to walk in amphibious environment. MODELING AND ANALYSIS OF THE ROBOT BASED ON WATER RUNNING Selection of the driving mechanism: Since the real motion trajectory of a basilisk is elliptical as shown in Fig. 1a, Steven Floyd from Carnegie Mellon University found that a four bar mechanism in a Grashof crankrocker configuration could mimic the motion of a basilisk's leg (Steven and Metin, 2008).Figure 1b presents a four bar mechanism and its resultant loop.A simplified foot is added to its end to make its simulative motion result seen more clearly.Given the simplicity and flexibility of the four bar mechanism, the configuration was used as a reference in our design. Design of robot's feet: One can see from Fig. 1b that the simulative trajectory of a four bars mechanism is divided into four phases: O→A, A→B, B→C and C→O.When the driving motor rotates a circle, hydroforces acting on one foot corresponding to each phrase are analyzed in Fig. 2 where F d and F l are defined as the drag force and lift force, respectively.Obviously, averaged over time, the total lift of the system composed by F d and F l must equal the weight of robot.From Fig. 2 one can conclude that the total lift is originated from the Phases O→A and A→B.In other word, the total lift in Phase O→B benefits the stability of the robot for water-running while in Phase B→O it has the opposite effect.Inspired by the configuration of some water-paddling animals such as duck, a novel self-adaptive foot is brought forward (Shu-Wen et al., 2012).This kind of foot contains a combination of three identical "toe" structures and a semi-circle "wrist" structure as seen in Fig. 3.Each "toe" consists of three stems with a hinge structure mounted between every two contiguous stems to constitute a revolute pair.A spring was added on each hinge and its contiguous later stem to ensure the upstream face is "palm" the moment foot contacting water.A piece of elastic material connected the fold joints are used to make up a compliant foot which can increase the effective spring stiffness of the flap bending.During Phase O→B, the direction of the total lift force acting on the "palm" of foot is upward and the foot opens naturally leading to the increase of the total area of its upstream face.As a result, the total lift increases afterwards, bringing in the stability of the system advanced.During Phase B→O, the upstream face is "instep" and all the effects brought by the total lift acting on the foot are contrary to the precedent, the foot closes naturally. Overall modeling of the robot: As the quadrupedal robot shows higher stability than a bipedal one, the quadrupedal structure was applied.Furthermore, the total weight of the body should be limited within a certain range to meet the requirements of water running.Increased mass that the legs can lift is, of course, a positive aspect, whereas the increased power used is negative.Therefore, most parts of the body were frame structured and micro-motors were used to reduce the total weight.After the preliminary modeling, several feasible analyses on water running were done to modify and optimize the original structure.As both F d and F l are proportional to v 2 , then for a robot with fixed weight, to increase the actuating speed v of the driving motors is a good means to rise the total lift.To realize water motion, the configuration of the robot must not only meet the requirements of lift but also satisfy the demand for water dynamic stability.That is to say, one must concern with the moment balance in both forceand-aft and left-to-right directions when the robot is running on water. Considering the moment balance in force-and-aft direction, the resultant of F d and F l is divided into two components, one is a lift force F Liƒt in the fluctuation direction to maintain the body of the robot and the other is a thrust force F Thrust in the anteroposterior direction to push the body forward.Since the self-adaptive foot is applied, the values of F Liƒt and F Thrust are different even when the foot is in the locations the same distance from the surface of water in Phase O→B and Phase B→O.Result shows that as the clockwise resultant moment of Phase O→B is bigger than the anticlockwise resultant moment of Phase B→O, each leg of the robot has a tendency to drive the rear of robot into the river.To solve the above problem, a tail is placed at the back of the body which can generate an anticlockwise moment to counteract part of the surplus clockwise moment, making the body stay upright while running on water.Since the moment generated by its two left-sided legs and the corresponding right-sided ones are equal in value and opposite in direction, the moment balance in the left-to-right direction is self-established.Thus far, the robot is capable of the ability to realize its water dynamic stability after structural modifications and its final configuration is shown in Fig. 4. Actuating speed for water running: As the structure of this robot is confirmed, the chief concern of a free running system is its total lifting ability.As is said above, averaged over time, the total lift of the system must equal the weight of the robot.Unlike quadrupedal walking on land, where the weight is distributed over two or three legs (depending on the gait), when water running, only one leg is producing the majority of the total lift force F Liƒt at a given time (Steven and Metin, 2008).Each leg, therefore, provide a peak lift force greater than the body weight.As is seen from Fig. 2, F Liƒt is mostly generated in Phase O→B, thus during Phrase O→B the peak value of F Liƒt must be greater than the body weight G. As the total lift for water running is decided by the velocity of a single leg and the movement of the leg is generated by the actuating facility, thus the total lift on each foot is determined by the corresponding actuating speed of motor connecting with driving joint all in all.Figure 5 is a kinematics modeling of the driving mechanism where Crank AB acts as the driving part.Assuming the lengths of the Links 2~4 constituted the mechanism are l 2 ~l4 respectively and the homologous inclined angles are θ 2 ~θ4 relative to the coordinate axis x.Point 13 P is the instant center of ground (Link 1) and Link 3. Point E is the mass center of one foot and the distance between Point B and E is l 5 .Through kinematics modeling, the numerical relationship between the actuating speed and its corresponding total lift is finally obtained. From the closed vector equation + = + , one can get the relationship between ω 2 and ω 3 : Assuming the included angle between and is α, the distance between instant center P 13 and Point E is as follows: ) Since the thickness of the foot in this study is far less than its length, the relative motion between foot and water can be regarded as "flow around a flat plat at high attack angles".So the peak value of the total lift should satisfy the following formula: Combining the Eq. ( 1) to (3) and the existing relational expression γ = 90˚ -α + θ 3 together, one can determine the critical actuating speed ω dmin as follows: 4) ANALYSIS AND REALIZATION OF THE ROBOT BASED ON LAND WALKING Study of the terrestrial kinematics: In this study, each leg of the robot has only one driving joint.And every time when it rotates, it affects the displacements of the body in both forward and fluctuation directions.The forward displacement can force the body to move ahead while the fluctuation of the body may result in the instability of the motion.Hence, if the robot moves fast, it is rather difficult to satisfy the requirements for stability in both directions that only terrestrial movement with a low speed is addressed here.Like other quadrupedal robots, our basilisk lizard robot also walks with a time-varying topological structure.When one leg sways, the whole system shows an open chain structure and when all the legs contacting the ground, it turns to be a paralleled closed chain structure of multiple degrees.The result of its motion changes with the difference in landing points. Assuming the robot is walking at a low speed and when the body of the robot walks a distance of ∆x, the rotational angle ∆θ of the driving joint in its homologous supporting leg can be determined via inverse kinematics in Fig. 5 with the following Eq.( 5) to ( 6): Theoretical analysis of land walking stability: Generally, when a robot walks in the static stable state its duty factor β always equals to or above 0.75, so at any moment, there are at least three legs to constitute a supporting polygon as shown in Fig. 6 where Point C is the center of ground reaction forces R a , R b , R c acting at contacting points P a , P b , P c respectively in a plane.Define the axis normal to the plane and let , be the resultant force and moment respectively acting on the body.For a robot whose total mass is m, use as the gravity vector, the coordinate of Point C (Wang, 2007) is decided: From the Eq. ( 7), one can see that the location of Point C is influenced by many factors and is changeable with time.Hence, there exists possibility that sometime Point C lands rightly at one edge of the supporting polygon, then the body is actually supported by two legs making up the edge line and the others can be regarded as "virtual" ones for the reason that they are ineffective for supporting.Obviously, the robot is unstable on this condition, even a rather small external force or moment can result in its deviation from the current state.So in order to make the robot walk stably, the center of ground reaction forces must be within the polygon at any time.Therefore, the robot must walk via its three or four legs supporting the body alternatively with its stability margin S>0 and the duty factor β>0.75 (Gao and Bingcong, 1992). Gait planning of land walking: An ideal gait planning can realize the final posture of the robot completely the same as its original state after a step.Generally, as to a quadrupedal robot, the gaits of its four legs are defined similar to each other with the only difference in phases.In this study, every step of the robot is generated by the rotational joints in its legs.Since the rotational angle of each leg is not always the same, the posture of each leg is different from one another.Under this circumstance, the relative forward displacement of each leg is diverse from each other even when the driving motors rotating a same angle.As the difference of synchronous rotational angles among the supporting legs is quite tiny, the error is neglected here.That the relative span E of each swinging legs equals in value is made as prerequisite for this gait planning. As to land walking, there is no need for the foot to open or close, the included angle between every two stem is made fixed.A simplified model of the robot is established with each leg numbered 1~4 from left-front to right-back.The stability of a robot performs best while its legs swaying in the order 1-4-2-3 (Gao and Bingcong, 1992).Assuming that the rotational angle of each supporting leg is a uniform value θ during threeleg-supporting period and 2θ during four-legsupporting period and then the rotational angle during swinging period is 360°-7θ.If the negative sign is used to express the rotational direction of a driving motor that makes the body backwards, then the original positions of each leg marked 1-4 are determined as follows respectively: 3θ, -θ, -4θ, 0°, each relative to its own lowest point in the motion trajectory. Simulatiive motion and parametric analysis: To verify the accuracy of planned gait for land walking and optimize the performance of the designed robot, a virtual prototype was developed.Before simulative motion, static and dynamic friction coefficients were set to 0.3 and 0.5 and θ, the rotational angle of supporting legs during three-leg-supporting period was set to 10°.Process of simulative motion is shown in Fig. 7. After simulation, a successive of analyses was done to reinforce the feasibility of planned gait.Figure 8 shows that the curves of each leg's displacement in the forward direction are resemble in shape during a single pass which is well accordant with our original intention for gait planning. As in a unchangeable simulative condition, we wished to know if there was an optimum value of a motor's rotational angle θ or stride frequency f.Set the value of stride frequency fixed with the value of rotational angle θ ranging from 8° to 12°, one can get that a larger rotational angle leads to larger body fluctuation as seen in Fig. 9, but larger rotational angle also leads to larger forward displacement seen in Fig. 10.As the mass center of the robot is 130 mm up from the ground in the design and generally the body fluctuation ratio of a legged robot during the stable walking state should not exceed 1%, hence θ = 10° is decided as the optimum value of rotational angle of supporting legs during three-leg-supporting period in the given condition.Figure 11 shows that as θ is fixed to 10°, when the stride frequency is lower or equals to 3 Hz, difference in body fluctuation is tiny, but when the value of stride frequency continuous to grow there exists a radical change in body fluctuation.Since a larger frequency leads to higher performance, here f = 3 Hz is chosen as the optimum value of stride frequency. Experiment on land walking: Experiment of the robot walking on land was performed as a complement to test the simulation results.A quadrupedal simplified robot was established 400 mm in length, 100 mm in width and 10 mm in height with four identical crank-rocker configurations making up its driving legs as seen in Fig. 12.Four DC motor were put inside the body to actuate the driving joints.Since this experiment here was only done on the ground, from the view of simplicity for operation, its self-adaptive feet were replaced by some rubber chucks with a piece of black paper pasted to the surface of each piece, making the results seen more clearly.During the experiment, one could see that the robot walked stably at a low speed.In the process, Parameter E, the relative stride span of a leg was measured 12 mm and Parameter λ, a step length of the robot was 17 mm.The result E<λ satisfied the requirement of a quadrupedal robot walking in the static stable state, which successfully added the veracity and rationality of the planned gait. CONCLUSION In this study, an amphibious basilisk lizard inspired robot is designed.A novel self-adaptive foot based upon the water running ability is modeled.Using four bar mechanism, the robot can move in amphibious environment theoretically.The work not only opens a door but also gives a good reference for the study of bipedal and quadruped robots becoming ambulatory over both land and water.Up to now, ability of the robot to walk on land has already been tested by both simulation and experiment while its ability of water running hasn't been examined experimentally yet.Doing water experiment is rather difficult that it shows far higher demands on both quality of experimental facilities and researchers. Future study will towards the test and verification of the ability for water running.Some preliminary preparations have already been done for water experiment.During the process, some real values of water motion parameters aim to obtain on basis of the theoretical analyses. Fig. 1: Trajectories of the basilisk and simulation(Tonia and George, 2003)	2015-07-14T19:54:51.000Z	2013-04-10T00:00:00.000	{ "year": 2013, "sha1": "e4e7b77ed863244cbb901fb6bf0d027f72752840", "oa_license": "CCBY", "oa_url": "https://www.maxwellsci.com/announce/RJASET/5-3372-3379.pdf", "oa_status": "HYBRID", "pdf_src": "ScienceParseMerged", "pdf_hash": "871f94435c74befb1d3f4401cc8153af524def95", "s2fieldsofstudy": [ "Biology", "Engineering", "Environmental Science" ], "extfieldsofstudy": [ "Engineering" ] }
86630196	pes2o/s2orc	v3-fos-license	TO COMPARE THE FUNCTIONAL BALANCE BETWEEN NORMAL ELDERLY, ELDERLY WITH DIABETES, ELDERLY WITH DIABETIC POLYNEUROPATHY Results: The mean of age, gender and duration since diabetes was calculated in all groups. Berg balance scale scores were calculated and in that high fall risk there were 20(55.56%) participants in elderly with diabetic polyneuropathy. In medium fall risk there was 1(2.78%) participant in elderly with diabetes and 16(44.44%) participants in elderly with diabetic polyneuropathy. While in low fall risk there were 36 (100%) participants in normal elderly, 35(97.22%) participants in elderly with diabetes and 0(0%) in elderly with diabetic polyneuropathy. One way Anova was done and all the three groups were having significant difference in their BBS scores. increases. In 2000, the prevalence of DM was estimated to be 0.19% in people d"20 years old and 8.6% in people e"20 years old [2]. The most frequently-occurring micro vascular complication in diabetes is diabetic neuropathy (DN). Diabetic polyneuropathy is characterized by severe pain, loss of ambulation, balance problems, risk of fall and increased risk of foot ulceration and amputation [2]. Although DPN may be present in up to 10% of patients with type 2 DM at diagnosis, the prevalence of neuropathic symptom increases with duration of disease, with the highest rate of neuropathy found among patients who have had DM for at least 25 years [3,5]. Falls present a substantial health problem among the elderly population. Approximately one-third of community-dwelling people over 65 years of age will experience one or more falls each year (Stelmach and Worringham, 1985;Tinetti et al., 1988;Powell and Myers, 1995;Spirduso, 1995;Hausdorff et al., 1997;Shumway-Cook et al., 1997a). Subsequently, the frequency increases to nearly 40% for those individuals over 80 years of age and affects women more than men (Nickens, 1985;Powell and Myers, 1995) [5]. Shumway-cook and colleagues (1997) reported that the BBS was the best single predictor of fall status in community dwelling older adults without neurologic pathology. This test uses 14 different items, which are rated 0 to 4. Declining BBS scores were associated with increased fall risk [6]. Some studies have trace of evidence that functional balance is impaired in patients with diabetic polyneuropathy [7]. As these patients have distal symmetrical polyneuropathy there are neurological deficits and characterized by loss of position sense, vibration, light touch and sensory ataxia with loss of ankle reflexes. Balance and postural control clearly rely upon the integrity of peripheral sensory information, and patients with diabetic distal neuropathy, because of their altered lower limb proprioception, may be at a higher risk of falling than non-neuropathic subjects [8]. In patients with diabetes, the chance of falling may be increased due to lower extremity neuropathy. Balance is affected by hyperglycaemia which may cause dizziness; on the other hand, there is increase in risk of fall with hypogly-caemic episodes that are linked to tight glycemic control. The consequences of falls are expected to be more severe among elderly with diabetes. (8) In India there are dearth of studies where comparison is being done among elderly without Diabetes, Elderly with Diabetes and elderly with diabetic polyneuropathy on balance issues, so here a need arises to compare balance scores amongst these three groups. Objective of the study: To compare functional balance using Berg Balance Scale among three groups namely elderly without diabetes, elderly with diabetes and elderly with diabetic polyneuropathy. METHODOLOGY This study is a cross sectional study comprising of 108 patients and was divided into three equal groups. Ethical Clearance was obtained from the Ethical committee of S.D.M. college of Medical Science And Hospital. All the patients whose diabetes type-2 and diabetic polyneuropathy confirmed by Medicine department and Neurophysicians of S.D.M. College of Medical science and hospital, Dharwad, were included as per inclusion and exclusion criteria. Age matched elderly patients without diabetes visiting medicine opd were also included in the study. Patient willing to participate were briefed about the study and their written consent was taken. Procedure: Patients were divided into 3 groups namely: Group A-Elderly without diabetes, Group B-Elderly with diabetes and Group C-Elderly with diabetic polyneuropathy. Berg balance scale was administered in the above mentioned three groups. The study was briefly explained to the participants. If one item cannot be assessed in one time then therapist can take three trials of that item and amongst three trials the best one is taken. Berg balance scale consists of 14 functional tests of which 6 are static balance items and 8 are dynamic balance items. This scale requires minimal equipments: 2 standard chairs (1 with arms and 1 without), stepper/steps, ruler, stopwatch and space. BBS completion needs 10-20 min and its score represents the participant's ability to control postural balance. BBS is a 5 point likert type of scale. Each test was scored by the therapist from 0-4 (0 -inability to complete the task; 4 -independent task fulfillment). The overall score is the sum of the obtained scores for each test. Thus, the maximal overall score is 56, and the minimal is zero. The higher score indicates a better functional balance. Scores of 0 to 20 represent balance impairment (high risk of fall), 21 to 40 represent acceptable balance (medium risk of fall), and 41 to 56 represent good balance (low risk of fall). In the majority items, the subject is asked to sustain a given position for a definite time. Comparison of three groups with respect to Berg balance scale scores was done by one way ANOVA Pair wise comparison of three groups amongst each other was done by Tukeys multiple post hoc procedures P value < 0.05 was considered as statistically significant. in Elderly with diabetic polyneuropathy. In 80yrs and above age group there were 4 (11.11%) participants in normal elderly, 5 (13.89%) in elderly with diabetes, 14 (38.89%) in elderly with diabetic polyneuropathy. The prevalence of diabetes mellitus increases with age, mainly it was reported to be prevalent in age group more than 50 years. (10) The factors contributing to diabetes in elderly are 25% of older persons have impaired glucose tolerance (IGT), rises in glucose levels, especially post prandial blood glucose levels that directly correlate with age. As shown in that Unipedal balance is known to be sensitive to aging effects and exhibits significant decrements already by the fifth decade [9]. This study demonstrated that elderly with diabetic polyneuropathy are at high risk of fall compared to other two groups. Also elderly with diabetes are at risk of falls compared to normal elderly without diabetes and the factors contributing to it could be use of hypoglycemic medication, use of insulin, cognitive defects and poor muscle function. RESULTS participants are suffering from diabetes more than 10 years of age. [7]. In the BBS score range of 54 to 56, a 1-point change in the berg balance score was associated with a 6 to 8% increase in fall risk. Below the score of 36, fall risk was close to 100 % [6]. The type 2 diabetic group with neuropathy was at greater risk of falls, and confirming the view that major risk factors for falling are, previous falls history, increasing age, presence of diabetes and increased postural sway [11]. As shown in table 5, when all three groups were compared with respect to BBS by using one way Anova, the p-value was found to be significant. The mean score of BBS was more in normal elderly than elderly with diabetes and elderly with diabetic polyneuropathy. Also the mean score of BBS was more in elderly with diabetes than in elderly with diabetic polyneuropathy. This indicated that the berg balance score is much lowered in elderly with diabetic polyneuropathy than other two groups. The contributing factors for high risk of fall in elderly with diabetic polyneuropathy could be because of poor postural control, affection in somatosensory system, lack of appropriate proprioceptive information from lower extremity, use of hip strategy instead of ankle strategy [5]. One of the study done by Mathew S suggested that DM is an independent fall risk factor among elderly nursing home residents. The reasons for falls were hypoglycemia from inappropriate glycemic control; visual impairments contribute to increased risk of falls. There are numerous studies which explain why there is high risk of falls in elderly with diabetic polyneuropathy. In one of the previous study done by James K. Richardson on "diabetic peripheral neuropathy impairs weight transfer and unipedal balance in the elderly"; they found CONCLUSION In this study it can be concluded that elderly with diabetic polyneuropathy were at high risk of fall than the other two groups; however elderly with diabetics were also at low risk of fall compared to elderly without diabetes though the result was not significant.	2019-03-28T13:33:30.567Z	2019-02-11T00:00:00.000	{ "year": 2019, "sha1": "2c97fcfe6d5c3e52a650c726bda4fd3e9956832c", "oa_license": "CCBYNCSA", "oa_url": "https://www.ijmhr.org/ijpr.7.1/IJPR.2018.195.pdf", "oa_status": "HYBRID", "pdf_src": "Anansi", "pdf_hash": "7869955b0ffd108f8b1796833cdb8f77a059d81c", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [ "Medicine" ] }
641701	pes2o/s2orc	v3-fos-license	Transovarial Transmission of Orientia tsutsugamushi in Leptotrombidium palpale (Acari: Trombiculidae) Transovarial transmission of Orientia tsutsugamushi in colonies of Leptotrombidium palpale was studied in the parent and F1 and F2 generations. Both transovarial transmission and filial infection rates were 100% in the parent and F1 generations of Leptotrombidium palpale. The filial infection rate in the F1 generation was 100%, but it declined to 94.3% in the F2 progeny. The sex ratio of the F1 generation from infected L. palpale was 1∶0.8 (male:female) and the proportion of males was relatively high. This study is the first to report on the transovarial transmission of O. tsutsugamushi in L. palpale. High transovarial transmission rates in L. palpale suggest that this species might be one of the major vectors of tsutsugamushi disease in Korea. Introduction Scrub typhus, better known as tsutsugamushi disease, is an acute and febrile disease caused by Orientia tsutsugamushi infection. This disease, which is transmitted by the bite of infected chiggers was first reported in Korea in 1951 [7]. The incidence of scrub typhus has increased remarkably in Korea. A total of 8,604 cases of scrub typhus were reported in 2012 (http://stat.cdc.go.kr). Seven species-Euschoengastia koreaensis, Leptotrombidium orientale, L. scutellare, L. pallidum, L. palpale, L. zetum and Neotrombicula japonica-are considered to be the major vector species in Korea [6,12,13,14]. Because the larval stage is the only parasitic stage of O. tsutsugamushi, to maintain disease transmission, it is necessary for O. tsutsugamush to be transmitted transstadially through the nymph and adult stages and transovarially transmitted through the eggs to the progenies [2]. The efficiency of transmission of Orientia by infected chiggers is important in determining how the disease is maintained in nature. Previous studies on transovarial transmission occurred in L. pallidum [16] and L. scutellare [1] in Japan. Additionally, it was proven that O. tsutsugamushi was transmitted transovarially through eggs in the infected colonies of L. fletcheri, L. arenicola, L. deliense, L. imphalum, and L. chiangraiensis [3,8,11,15]. It is known that males are aberrant hosts of O. tsutsugamushi [18], and infected males have been recorded previously in only two species, L. pallidum [16] and L. imphalum [8,9]. We studied the transovarial transmission of O. tsutsugamushi in two generations (Parent and F 1 ) of naturally-infected L. palpale colonies. Two parameters, transovarial transmission rate and filial infection rate, were studied. Oviposition and hatching rates in naturally infected and uninfected L. palpale were also compared. Collection of chiggers The animal protocol used in this study was reviewed and approved based on ethical procedures and scientific care by the KCDC-Institutional Animal Care and Use Committee (KCDC-IACUC; KCDC-12-032-1A). Engorged larval chiggers were collected from wild rodents, which were captured in March 2010 from Jangan-myeon, Hwaseong-si Gyeonggi Province, Korea. There was no need for specific permission for using these collecting sites, because these sites were not located at national parks or protected areas and installation of traps was supported by Public Health Center in Hwaseong-si. A total of 50 Sherman live folding traps (36369 inch), baited with a peanut butter spread paper, set up at five points in the collection site with 5 m intervals and collected at next day morning. A total of 20 wild rodents were captured. The captured wild rodents were transferred individually into small cages made of stainless steel and each cage was placed on a petri dish containing water. The fully engorged larvae were collected from the water surface every day. These parent and F 1 generations of trombiculid mites were used in this study. Rearing of chiggers under laboratory conditions The collected engorged larvae were reared in plastic containers (50 mm diameter, 40 mm height) containing plaster of calcium sulfate hemihydrate with charcoal powder (9:1) to maintain the humidity level in the incubator. Deutonymphs and adults were fed with the eggs of Collembola (Sinella curviseta). When chiggers developed into adults, their sexuality was determined by observing the presence of genital setae located in the genital pore by using a stereomicroscope [20]. Males and females were maintained in rearing containers. When the spermatophore in males was observed, the females were placed into the rearing containers for mating. Egg-laying female mites were observed daily. The males were then removed from the rearing container prevent cannibalization of the eggs. After the eggs hatched, the larvae were attached on the ears of mice for feeding. Detection of O. tsutsugamushi in chiggers DNA was extracted from chigger mites using the G-spin total DNA extraction kit (iNtRON Biotechnology, Korea). The 56-kDa genes of O. tsutsugamushi were detected using a nested PCR assay performed as described in [4]. Primers 34 (59-TCA AGC TTA TTG CTA GTG CAA TGT CTGC-39) and 55 (59-AGG GAT CCC TGC TGC TGT GCT TGC TGC G-39) were used for the first PCR, and second PCR primers 10 (59-GAT CAA GCT TCC TCA GCC TAC TAT AAT GCC-39) and 11 (59-CTA GGG ATC CCG ACA GAT GCA CTA TTA GGC-39) were used to amplify a 483-bp fragment. In the first PCR, 5 mL of template DNA of chigger mites was added to the PCR premix (Bioneer, Korea). The cycling conditions used were as follows: 94uC for 5 min followed by 30 cycles of 94uC for 30 sec, 60uC for 2 min, and 72uC for 2 min, and a final extension of 72uC for 10 min. For the second PCR, 2 mL of the first PCR product was amplified by the same procedure as described above, except the use of the second PCR primer pairs as follows. The second PCR products were analyzed by electrophoresis on a 1.5% agarose gel. The second PCR products size was 483 bp. The nucleotide sequence of the nested PCR products was analyzed using the BLAST program of NCBI (http://blast ncbi.nlm. nih.gov) to confirm whether the gene is from O. tsutsugamushi. Both infected and uninfected L. palpale females produced eggs for 16 weeks. Infected females laid 32.666.7 eggs per female and uninfected ones laid 31.567.7 eggs per female. The hatching rate of the eggs from infected females was 64.8614.4% and that in uninfected females was 74.568.3%. The number of eggs from infected and uninfected females were not significantly different (P.0.05), while the hatching rate in the infected cohort was lower than that in the uninfected cohort (P,0.05) ( Table 3). Discussion Successful transovarial transmission in chiggers is important in the epidemiology of scrub typhus [8]. Previous studies on transovarial transmission was conducted in L. pallidum [16] and L. scutellare [1] vector species in Korea. This is the first report on transovarial transmission of L. palpale, which is a vector species of scrub typhus in Korea. Transovarial and filial infection rates in L. palpale are similar to those in L. pallidum [17]. Transovarial and fillial infection rates in L. pallidum were 100% [19] in the F 1 generation, but these rates declined to 97% in the F 2 and 90% in the F 3 . The fillial infection rate rapidly decreased in the succeeding generations. Electron microscopic observations revealed that O. tsutsugamushi did not always invade the oocytes in the ovaries of infected females [5]. Phasomkusolsil et al. [8] recorded that the filial infection rate in F 1 of L. imphalum was 100%, which declined to 62.3% in the F 2 . In this study, L. palpale females laid eggs for 16 weeks, but it was for 28 weeks and longer in L. imphalum [10]. This period might vary with the species and rearing conditions. To date, seven species-Euschoengastia koreaensis, L. orientale, L. scutellare, L. pallidum, L. palpale, L. zetum, and Neotrombicula japonicahave been considered as vectors in Korea [6,12,13,14]. In order to determine the vector species of tsutsugamushi disease, chiggers collected from wild rodents were tested for O. tsutsugamushi infection using an indirect immunofluorescent antibody (IFA) test and polymerase chain reaction (PCR) methods. In this case, chiggers were a possibly temporarily infected through feeding on the fluids of infected wild rodents. In order to determine the vector species more clearly, it is important to investigate whether the unfed larvae collected from the soil were infected by the pathogens or not, or their transovarial transmission should be confirmed through successive rearing. This is the first study describing transovarial transmission of O. tsutsugamushi in L. palpale in Korea. Further studies are needed to confirm transovarial transmission of other species for vector determination and to investigate the distribution of tsutsugamushi disease associated with the vector species.	2018-04-03T00:16:09.807Z	2014-04-10T00:00:00.000	{ "year": 2014, "sha1": "7b25bf58f804fb745a131ff258a358727a373c65", "oa_license": "CCBY", "oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0088453&type=printable", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "7b25bf58f804fb745a131ff258a358727a373c65", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Medicine", "Biology" ] }
216025697	pes2o/s2orc	v3-fos-license	A Multilevel Iteration Method for Solving a Coupled Integral Equation Model in Image Restoration : The problem of out-of-focus image restoration can be modeled as an ill-posed integral equation, which can be regularized as a second kind of equation using the Tikhonov method. The multiscale collocation method with the compression strategy has already been developed to discretize this well-posed equation. However, the integral computation and solution of the large multiscale collocation integral equation are two time-consuming processes. To overcome these difﬁculties, we propose a fully discrete multiscale collocation method using an integral approximation strategy to compute the integral, which efﬁciently converts the integral operation to the matrix operation and reduces costs. In addition, we also propose a multilevel iteration method (MIM) to solve the fully discrete integral equation obtained from the integral approximation strategy. Herein, the stopping criterion and the computation complexity that correspond to the MIM are shown. Furthermore, a posteriori parameter choice strategy is developed for this method, and the ﬁnal convergence order is evaluated. We present three numerical experiments to display the performance and computation efﬁciency of our proposed methods. Introduction Continuous integral equations are often used to model certain practical problems in image processing. However, the corresponding discrete models are often used instead of continuous models because discrete models are much easier and more convenient to implement than continuous integral models. Discrete models are piecewise constant approximations of integral equation models, and they introduce a bottleneck model error that cannot be addressed by any image processing method. To overcome the accuracy deficiency of conventional discrete reconstruction models, we use continuous models directly to restore images, which are more in line with physical laws. This idea first appeared in [1], and was widely used later, such as in [2][3][4]. In addition to making more sense in physics, continuous models can be discretized with higher-order accuracy. This means that the model error will be significantly decreased when compared with piecewise constant discretization, especially in the field of image enlargement. Many researchers have made great contributions to the solution of integral equations, however, many difficulties remain. First, integral operators are compact in Banach space since integral kernels are normally smooth. This will produce a situation in which the solutions of the relevant integral equations do not depend on the known data continuously. To overcome this problem, the Tikhonov [5] and the Lavrentiev [6] regularization methods were proposed to regularize the ill-posed integral The Integral Equation Model for Image Restoration In this section, we first depict the overall process of reconstructing out-of-focus images and then describe some common notations that are used throughout the paper. Finally, in the second subsection, we introduce the approach to formulating our image restoration problem into an integral equation. System Overview We present an overview flowchart for reconstructing an out-of-focus image in Figure 1. This process includes four main parts, that is, modeling, discretization, the parameter choice rule, and solving the equation. For the input out-of-focus image, we formulate it into a Tikhonov-regularized integral equation, which is described in the next subsection. Solving this integral equation necessitates discretization. We propose a fully discrete multiscale collocation method based on an integral approximation strategy. This method works by converting the calculation of the integral into a matrix operation. The next two parts describe the parameter choice rule and solution of the problem. We note that these two parts are actually a whole when executed in practice. However, we describe it in two parts because it is too complicated to describe as a whole. For a clearer presentation, we first display the multilevel iteration method in Section 4 under the condition that we have already selected a good regularization parameter. Then, we describe how this regularization parameter is chosen in Section 5. Some notations are needed. Suppose that R d represents d-dimensional Euclidean space. In addition, Ω ⊂ R 2 and E ⊂ R denote the subsets of R d . L ∞ is a special kind of space of L p when p = ∞, and it is proved to be a Banach space. We use x, including x with upper and lower indices, to denote the solution of the equation. Similarly, we use y, including y with upper and lower indices, to denote the known variable of the equation. Furthermore, the operator K represents the blurring kernel, and K * represents the adjoint operator of K. The notation A ⊕ ⊥ B denotes the direct sum of spaces A and B when A⊥B. The notation a ∼ b means that a and b are in the same order. R(·) represents the range of the operator. Finally, many extended and new notations that are not declared here are defined in the context in which they are first used. Model Definition In this subsection, we describe an integral equation model obtained from a reformulation of the Tikhonov regularization equation for image restoration. In addition, an equivalent coupled system is developed for solving this integral equation quickly and efficiently. Assume that the whole image is a support set. In general, images are usually rectangular. Let Ω ⊂ R 2 denotes the image domain, also the support set. The image restoration can be modeled by a continuous integral equation of the first kind where y is the blurred image and we aim to reconstruct the blurred image y into a clearer image x : Ω → R. Thus, image x can also be called the reconstructed image. K is the linear compact operator defined in terms of a kernel g by Let The images may not be in [0, 1] × [0, 1], but can be shaped to by scaling and shifting. According to [20], the out-of-focus image is usually modeled with the kernel where u:= (u, v),u := (u , v ) ∈ Ω, and σ is the parameter characterizing the level of ambiguity in the kernel. Blurring kernel (3) is a two-dimensional symmetric operator. It can be written as a combination of two univariate integral operators. That is to say, solving Equation (1) is equivalent to solving the following system: In this system, x 2 is the final solution of the reconstruction, and x 1 is the intermediate result. These two equations mean that we can first deal with all the rows of the image (first equation) and then all the columns (second equation). Hence, the problem has changed significantly. We have changed the procedure of dealing with a two-dimensional image to processing the rows of the image first, and then the columns. Furthermore, as can be seen from the two equations in (4), the rows and columns of the image are processed similarly, and both processes can be formulated to a one-dimensional integral equation: For x ∈ X := L ∞ (E), K is the linear compact operator defined by and y ∈ X is the observed function. Hence, we focus on Equation (5) in the following illustration. Ill-posedness makes Equation (5) difficult to solve, but the Tikhonov regularization method can handle this problem. By using the Tikhonov regularization method, we get the following equation: where α > 0 is the regularization parameter, and K * is the adjoint operator of K. It is easy to prove that the operator αI + K * K is invertible. In addition, y δ is usually noisy data, with for a noise level δ. x δ α denotes the solution of Equation (6) when y is replaced by y δ . K is self-adjoint; that is, K * = K. Note that Equation (6) includes K * K, which is defined by a 2-fold integral. This 2-fold integral is tremendously computationally expensive. By letting v δ α := y δ −Kx δ α √ α , the authors of [7] split Equation (6) into a coupled system: These two were proven to be equal in [7], and the advantage is that system (8) only involves one single integral. Thus, we do not solve Equation (6) but instead solve system (8) to reduce the computational difficulties. In the next section, we apply a fully discrete method to solve system (8). Fully Discrete Multiscale Collocation Method In this section, a multiscale method is reviewed first, and then we develop a fully discrete formulation of the multiscale collocation method using an integral approximation strategy. By using this strategy, we turn the computation of the integral into a matrix operation, which will greatly reduce the calculation time. Similarly, the multiscale collocation functionals and the collocation points are important. Suppose that ij is the collocation functional. Meanwhile, we have a sequence of collocation points {v ij : (i, j) ∈ U n } in E. We define an interpolation projection P n : L ∞ (E) → X n , n ∈ N and an orthogonal projection Q n : L 2 (E) → X n , n ∈ N. For n ∈ N, let K n := P n K\| X n . The multiscale collocation method for system (8) is to find the solutions v δ α,n := ∑ (i,j)∈U n v α,n ij e ij and x δ α,n := ∑ (i,j)∈U n x α,n ij e ij of the system √ αx δ α,n − K * n v δ α,n = 0 K n x δ α,n + √ αv δ α,n = P n y δ . For (i, j), (i j ) ∈ U n , after the introduction of three definitions, Note that K n is a dense matrix. The compression of K n is an important factor of our method. Following the compression strategy of [7], we get the compression matrix K n : Thus, substituting K n with K n , we finally need to solve the equation Integral Approximation Strategy The computation of system (11) mainly lies in the integral operator K n . Next, we focus on this problem and develop an integral approximation strategy using the Gaussian quadrature formula to solve this coupled system. Note that the integral operator K is and the orthogonal basis functions {e ij : (i, j) ∈ U n } are all piecewise polynomial functions. Thus, the piecewise Gauss-Legendre quadrature is used here. Integral approximation strategy: With the supports of these basis functions, we divide E equally into µ n parts. Let E q := [ q µ n , q+1 µ n ], for q ∈ Z µ n ; then, E = q∈Z µ n E q . Each basis function is a continuous function in E q , q ∈ Z µ n . Then, we choose m > 1 Gaussian points in each part E q and let γ = 2m − 1. All Gaussian points form a set of piecewise Gaussian points G in order. Let g(n) := \|G\|; then, g(n) = m × µ n . Thus, the integral under the accuracy of γ in E q can be written as where {g t , t = qm + 1, · · · , (q + 1)m} represents a Gaussian point in E q , and ρ t represents the weight corresponding to g t . Further, the integral with an accuracy of γ in E is which can be written as a formulation of vector multiplication Furthermore, as shown in Figure 2, we can use this strategy to approximate K n as a matrix form of where W g(n)×s(n) is a basis function matrix with weights. And it stands for all the basis functions {e ij , (i, j) ∈ U n } act on all piecewise Gaussian points of G with the weight of ρ t . L n denotes the matrix representation of point evaluation functional s , and the details of L n refer to [26]. In order to combine it with the matrix compression strategy, we write Equation (13) in blocks. This is the matrix representation K n , i, i ∈ Z n+1 , which is the approximate value of K n using the integral approximation strategy. Then, Equation (10) becomes a formulation of the fully discrete multiscale collocation equation: where K n is as given in Equation (14). Let A := K * K, A n := K * n K n . Assume that K n is the operator corresponding to the matrix representation K n using the compression strategy, and define A n := K * n K n . Additionally, let K n represent the operator with respect to the matrix representation K n using the integral approximation strategy and the compression strategy, and then define A n := K * n K n . Therefore, corresponding to Equation (11), system (9) becomes By adopting the integral approximation strategy, the computation of the integral becomes the computation of the matrix multiplication (14), which greatly reduces the calculation. Next, we estimate the convergence order of the fast multiscale collocation method using the integral approximation strategy. We should note that parameter c is different in different scenarios below, unless explicitly stated. We assume that there exists a constant M such that K L 2 (E)→L ∞ (E) ≤ M. According to [5,12], for any α > 0, αI + K * K is invertible. We also have the inequality Assume thatx is the exact solution of Equation (1), and y ∈ R(K). Following from [5,27], if hypothesis (H1) holds, then we have the convergence rate Suppose that c 0 > 0. In order to estimate the convergence rate of the integral approximation strategy, we propose the following hypothesis: (H2) For all n ∈ N and some positive constants r, is a Fredholm integral operator with a kernel having the rth derivative, this hypothesis holds true. Following [28], we can write the remark below. The remainder of the Gauss-Legendre quadrature is given as We can conclude that: Note that when m Gaussian points are used, the accuracy of the Gauss-Legendre quadrature is Corresponding to Remark 1, we give the following proposition. Proposition 1. Assume that the integral accuracy m satisfies Then, we can obtain the conclusion that Proof. Assume that r > 2m, the operator K ∈ H 2m [0, 1]. Because the polynomial functions are infinitely differentiable, we can get the result that Ke ij ∈ H 2m [0, 1], for (i, j) ∈ U n . From Remark (1), we gain the following inequality: Furthermore, we can infer from inequality (21) that when all µ n areas are added, then Thus, the proof has been completed. Note that the Gaussian function has an infinite derivative; thus, assumption (19) is easy to satisfy. Lemma 1. Assume that hypothesis (H2) holds, and c 0 is the same parameter as in Theorem 1. If parameters n and γ are chosen from then we can conclude that αI + A n is invertible. In addition, Proof. According to a known result in [29], we conclude that αI + A n is invertible and Estimate (25) follows from the above bound, condition (24), estimates (17), and Theorem 1. Next, we give two lemmas. The proofs are similar to those in [22], so they are omitted here. Lemma 3. Suppose that hypothesis (H2) holds, and inequality (24) is satisfied. Then, for n ∈ N and c > 0, For the remainder of this section, we estimate the error bound for x − x δ α,n ∞ . Theorem 2. Suppose that hypotheses (H1) and (H2) and assumption (19) hold,x ∈ R(K * ), and inequality (24) is satisfied. Then, for c 1 > 0, Proof. From the triangle inequality, we have It is apparent that the estimate in this theorem follows directly from the above bound, inequality (18), and Lemmas 2 and 3. Multilevel Iteration Method In general, we solve Equation (16) while choosing the regularization parameter. When parameter selection is finished, the equation is solved. Note that when executed in practice, these two processes are a whole and occurring simultaneously. However, in order to describe it more clearly, we split it into two processes. In this section, we present the multilevel iteration method (MIM) for a fixed α and assume that this parameter is already well selected. In the next section, we show the regularization parameter choice rule. In this section, we first describe the multilevel iteration method and then present the computation complexity of this algorithm. Finally, the error estimation is then proved. Multilevel Iteration Method After obtaining matrices E n , K n , and y δ , we begin to solve Equation (15). If we just invert this equation directly, it will require considerable time. Thus, we follow a MIM instead of inversion to obtain a fast algorithm. First, we introduce the MIM to the coupled system (16). We now assume that the fixed parameter α is already selected according to the rule in Section 5. With the decomposition of solution domain, for n = k + m and k, m ∈ N + , we can write the solutions x δ α,n ∈ X and v δ α,n ∈ X of system (16) as two block vectors where ( x δ α,n ) 0 ∈ X k , ( v δ α,n ) 0 ∈ X k , and ( x δ α,n ) j ∈ W k+j , ( v δ α,n ) j ∈ W k+j , for j = 1, 2, . . . , m. The operator K k+m also has the following matrix form The MIM for solving the coupled system (16) can be represented as Algorithm 1. Let K L k+m := P k K k+m Q k+m , and K H k+m := (P k+m − P k ) K k+m Q k+m . We split the operator K k+m into two parts, K k+m = K L k+m + K H k+m , which are lower and higher frequencies, respectively. Similarly, we can obtain K * L k+m and K * H k+m by splitting K * k+m using an analogous approach. Accordingly, the coupled system (16) can be written as Algorithm 1: Multilevel Iteration Method (MIM). Step 4 : Stopping criterion. Stop the iteration when Computation Complexity We now turn to studying the computation complexity of this algorithm. Specifically, we estimate the number of multiplications used in the method. As a result, we write the operator Equation (30) in the matrix representation form. First, we introduce the block matrix ]. Moreover, for a fixed k ∈ N, we define the blocks K k 0,0 := K k . Additionally, for s, s ∈ N, we define K k 0,s : We also partition matrix E n in the same way, which we omit here. Then, the matrix representations of the operators K L k+m and K H k+m are We also write down the matrix representations of the operators x δ, α,k+m and v δ, α,k+m at the th iteration as x k,m := [ x i : i ∈ Z m+1 ] and v k,m := [ v i : i ∈ Z m+1 ]. Furthermore, we define y δ k+m := [y δ i : i ∈ Z m+1 ]. Using this block form of the matrix, the solutions of Equation (30) become For a matrix A, we denote by N (A) the number of nonzero entries of A. Let For > 0, we need 2N ( K k,m ) + 2N (E k+m ) multiplications with an inverse operation R −1 k to obtain x i and v i , i = m, m − 1, . . . , 1, 0 from Equation (32). However, in all iterations, the inverse operation R −1 k only needs to be done once; thus, we assume that the inverse operation R −1 k needs M(k) multiplications. Hence, the number of multiplications for computing x i and v i , i = m, m − 1, . . . , 1, 0 from x −1 In addition, we only need to compute the same inverse operation R −1 k to obtain x 0 k,m and v 0 k,m in the first step in Algorithm 1. Therefore, we are now ready to summarize the above discussion in a proposition. Proposition 2. The total number of multiplications required to obtain x k,m and v k,m is given by Note that when we solve the coupled system (16) directly, we need to compute the inverse operation R −1 k+m . However, when we use the MIM, we only need to compute the inverse operation R −1 k . This is the key factor that leads to a fast algorithm. In the next subsection, we estimate the error x − x δ, α,k+m ∞ . Error Estimation From the triangle inequality we only need to estimate x δ α,k+m − x δ, α,k+m ∞ . Let D k,m := K * n K H k+m + K * H k+m K L k+m , F k,m (α) := αI + K * L k+m K L k+m . Lemma 4. Suppose that hypotheses (H1) (H2) and condition (24) hold, then D k,m ∞ → 0, as k → ∞, for m ∈ N + . Then for N ∈ N + , k > N and m ∈ N + , (37) Proof. If these two hypotheses are true, then there exists a constant c 2 > 0 such that From the definition of F k+m (α), (17), and Theorem 1, for any u ∈ X k+m , we have This, together with Equation (38), proves that the first part of (37) is true. Then, obviously, the second part is true. A corollary (cf, Corollary 9.9, page 337 of [10]) confirms that the condition numbers cond(αI + K * L n K L n ) and cond(αI + K n ) have the same order. In other words, the MIM will not ruin the well-condition property of the original multiscale method. However selecting an appropriate k is important, which will influence the result of the iteration process. It follows from Equation (30) Generally, in order to make the iteration process convergence, we would choose k such that and Theorem 3. If hypothesis (H2) and condition (24) hold, let where t k (α) = µ −rk and then for N 0 ∈ N + , k > N 0 , and m ∈ N + , we obtain the result Proof. We prove this result by induction on . First, we prove that Equation (44) holds when = 0. It is apparent that x δ,0 α,k+m = x δ α,k . Therefore, from the result of Lemmas 2 and 3, we can conclude that there exists a constant c, and then we obtain Equation (44) when = 0. Meanwhile, following from Equation (29), we can obtain Subtracting (40) from (46), we have where , and . On the basis of Lemma 4, for N 0 ∈ N + and k > N 0 , and Then, we get We assume that Equation (44) holds for = q − 1, q ∈ N + , and prove that it also holds for = q. Following from Equations (41) and (42), we can obtain From Equation (51) and inequality (52), we obtain which completes the proof of this theorem. Theorem 4. If hypotheses (H1) and (H2) and assumption (19) hold, the parameters n and γ are chosen to satisfy condition (24), and the integer k is chosen to satisfy Equations (41) and (42), then for N 0 ∈ N + , k > N 0 , and m ∈ N + , Moreover, if parameters k and m are chosen on the basis of n := k + m, and the number of iterations satisfies Proof. From the triangle inequality, we have α,k+m ∞ . The combination of Theorems 2 and 4 implies the inequality (54). If the three parameters n, γ, and k are chosen to satisfy conditions (41), (42), and (55), together with m := n − k, as well as inequality (54), we can conclude that which completes the proof. Note that and γ α,k,m, is exponentially decreasing. Therefore, the stopping criterion can be reached at a finite step. With the next remark, we show that the stopping criterion of Algorithm 1 can guarantee the convergence rate of Equation (56). Remark 2. Suppose that hypotheses (H1) and (H2) and assumption (19) hold. The parameters n and γ are selected to satisfy condition (24), and n satisfies nµ −rn α 3/2 ≤ δ α . If the iteration stops when then the estimation error is Proof. From the triangle inequality, we get On the one hand, following from system (30), we can write x δ, α,k+m as On the other hand, Combining Equations (60) and (61), we have From the right inequality of (17) and the error bound (58), we can obtain According to Theorem 2, the above inequality, and also nµ −rn α 3/2 ≤ δ α , we can get the final result: Regularization Parameter Choice Strategies Choosing an appropriate regularization parameter α is an important process in solving the ill-posed integral equation. We present a posteriori parameter choice strategy [14] for our proposed method. For any given α > 0, we choose the parameters k(α), m(α), n(α), γ according to Theorem 4. Following from [14], we assume that there exist two increasing continuous functions, with ϕ(0) = 0, such that we can write Equation (54) as Then, α = α opt := (ϕψ) −1 (δ) would be the best choice. For constants q 0 > 1 and ρ > 0, we let the positive integer N be determined by Obviously, α * := max{α i : α i ∈ M(∆ N )} can be the approximation of the regularization parameter, but the function ϕ(α) involves the unknown smoothness order ν of the integral operator. Therefore, it is infeasible to use M(∆ N ) directly, and a little modification is necessary. We next present a rule for choosing the regularization parameter α. Rule 5. As suggested in [14], we choose the parameter α := α + = max{α j : α j ∈ M + (∆ N )} as an approximation of α opt , where This is a posteriori parameter choice strategy, which does not involve the smoothness order ν. We next present a crucial lemma, which can be found in Theorem 2.1 of [14]. Proof. This lemma can be obtained from [7] with a slight modification. Thus, we omit the details of the proof. Numerical Experiments In this section, three numerical examples are presented for the restoration of out-of-focus images to verify the performance of the fully discrete multiscale collection method and the multilevel iteration method. Matlab was used to conduct our simulations, and all examples below were run on a computer with a 3.00 GHz CPU and 8 GB memory. Using the integral equation necessitates transforming the discrete matrix (the observed image) into a continuous function. We used the method in [1] directly. Assume that the size of the image is ro × co, and the pixels of image are on the grid {(i/ro, j/co) : i = 0, 1, . . . , ro; j = 0, 1, . . . , co}. The function to formulate the image is where h ij is the old pixel value. Assume that s is a positive integer. Then, for l = 0, 1, . . . , s, Then Equation (5) becomes The noise level is defined as δ = y ∞ · e/100. Note that we employ the piecewise linear functions in [26] for simplicity in the following examples. Example 1. In our first experiment, we verified the effectiveness of the integral approximation strategy for the coupled integral equation. We set n = 0 as the initial level and measured the computing time in seconds to generate the coefficient matrix K n of the coupled operator Equation (9) for the range n = 5 to 10 in Table 1. For comparison, we repeated the experiment of the numerical quadrature scheme in [1]. In this example, we set both integral methods to have the same accuracy, γ = 3. For different values of n, T N denotes the computing time of the numerical quadrature scheme in [1], and T I denotes the computing time of the proposed integral approximation strategy. As we can see in Table 1, the proposed integral approximation strategy uses less time to generate the coefficient matrix K n , which proves that the integral approximation strategy is an efficient fast algorithm. Example 2. The second simulation was executed to verify the efficiency of the proposed multilevel iteration method. Figure 3a is the original clear 'building' image with the size 256 × 384, and the blurred image is recovered in Figure 3b with σ = 0.01 in the blurring kernel and noise level δ = 0.01 using the MIM. For comparison, we also conducted experiments using the Gaussian elimination method (GEM) in [28] and the multilevel augmentation method (MAM) in [10]. T GEM , T MAM and T MI M represent the computing time of the Gaussian elimination method, the multilevel augmentation method and the multilevel iteration method, respectively. The value n listed in Table 2 ranges from 5 to 10, and in this case, the continuous intensity function (70) is needed. As shown in Section 2, two one-dimensional integral equations are solved to recover the blurred image. Therefore, the computing time here denotes the summation of the time needed to solve the coupled Equation (8) twice. In our MIM experiments, all processes stopped at the second iteration, which is very fast because of the good selection of the initial values x δ α,k and v δ α,k . These two initial values can be found in Step 1 of Algorithm 1. Figure 3c-e show the reconstructed images of (b) using the GEM, the MAM and the MIM. Meanwhile, Table 2 and Figure 4 exhibit the computing time of these three methods. On the whole, the computing time of the MIM is the least among the results of these methods. All results show the proposed multilevel iteration method requires less computational resources. The difference is obvious, especially when the indicator of the extended dimension n is large. Example 3. In this example, we demonstrate that the performance of the MIM is as good as the alternative method. As shown in Figure 5b-d, we consider the restoration of the 'Lena' image, which has the size 257 × 257. We use the image with σ = 0.01 in the blurring kernel and different noise level δ. Note that when δ = 0, the image is noise free. We introduce the peak signal-to-noise ratio (PSNR) to evaluate the restored images and blurred images. For comparison, we solved the corresponding integral equation by using the proposed multilevel iteration method and the Gaussian elimination method with the piecewise linear polynomial basis functions at n = 8. Tables 3 and 4 list α + obtained from the parameter choice using Rule 1, the PSNR value of the blurred image (PB), the PSNR value of the reconstructed image (PR), and the corresponding time to solve the equation using the GEM and the MIM separately, where the noise level δ ranges from 0 to 0.15. Figure 5e-j show the reconstructed image corresponding to different methods and different noise levels of 0, 0.03, and 0.1. In general, by comparing the numerical experiments in Tables 3 and 4, we can conclude that there is almost no difference in the PSNR value of the reconstructed image using the GEM and the MIM, but more specifically, the MIM performs better in most cases. But more seriously, the MIM performs better in most cases. Example 2, combined with Example 3, proves that the MIM uses less computation time than the alternative methods, while the performance is equally well. Therefore, we can conclude that the multilevel iteration method is an effective fast algorithm to solve the integral equation in image restoration. Conclusions In this paper, we formulate the problem of image restoration as an integral equation. In order to solve this integral equation, we propose two fast algorithms. The first one is the fully discrete multiscale collocation method, which converts the calculation of the integral to a matrix operation. The second one is the multilevel iteration method, which guarantees that the solution has an optimal order. All examples verify that the proposed methods are accurate and efficient when compared with alternative strategies. In the future, we will still focus on finding faster and more efficient methods.	2020-03-05T10:28:26.132Z	2020-03-04T00:00:00.000	{ "year": 2020, "sha1": "b3e995737b8bc00c52b2fcd41fbbefa3f1aef769", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2227-7390/8/3/346/pdf", "oa_status": "GOLD", "pdf_src": "Adhoc", "pdf_hash": "9b84fb9a27a113b262d9086ed3ade90355122a49", "s2fieldsofstudy": [ "Computer Science", "Engineering", "Mathematics" ], "extfieldsofstudy": [ "Computer Science" ] }
264892178	pes2o/s2orc	v3-fos-license	A Population Description of Young Women with Breast Cancer in Newfoundland and Labrador It has been well established in the literature that young women tend to carry more aggressive subtypes of breast cancer than their older-aged counterparts. The objective of this study was to describe the characteristics and outcomes of young women with breast cancer. In this retrospective analysis, data were collected for women under the age of 40 years who were diagnosed with breast cancer between 2008 and 2018 in the province of Newfoundland and Labrador. Specifically, data were collected on demographics, staging, pathological characteristics, treatment, and survival outcomes for young women with this disease. Results demonstrate that most of these women were diagnosed between the age of 35 and 39 years (91.2%). Most women presented with early-stage disease (stage I and II—66.4%), while 24% were stage III and 9.6% presented with stage IV metastatic disease. The prevalence of hormone-receptor-positive disease represented 41.9% of the cohort, with triple-negative and HER2+ measuring 27.7% and 30.4%, respectively. Five-year disease-free survival was 80.5% and overall survival was 82.7%. These findings provoke discussion regarding the intersecting roles of genetics, environment, and lifestyle in a region with some of the highest rates of malignancy in the country. Introduction Breast cancer is the most commonly diagnosed malignancy amongst women in Canada, with incidence rates that increase with age [1].Young women with breast cancer, or those diagnosed before age 40, represent a small but significant subset of this population that present with unique disease characteristics and management challenges.While women under 40 represent only 4% of breast cancer diagnoses [2], female breast cancer is the most common malignancy amongst adolescents and young adults aged 15-39 [3].Breast cancer in this population tends to present at a later stage than it does for older females with a more aggressive tumor biology carrying significant morbidity and mortality rates [4].Young women with breast cancer have higher rates of both local and distant recurrences as well as lower survival rates when compared to older adults [5].Even amongst those who present with early-stage disease, young women have been found to be almost 40% more likely to die from breast cancer compared to those over 40 years [6].This has led to the establishment of specific guidelines for the care of adolescents and young adults with breast cancer in recent years and highlights the importance of high-quality data and evidence to inform best practices [7]. The province of Newfoundland and Labrador was projected to have the highest agestandardized incidence rates (ASIRs) in the country for females in 2021 (542.9 per 100,000).Rates of breast cancer in females specifically are expected to be higher in Newfoundland and Labrador than any other province in Canada (136.6 per 100,000 in Newfoundland and Labrador versus 126.8 per 100,000 within Canada) [1]. Breast cancer is a heterogeneous disease consisting of specific biological and molecular subtypes.The expressions of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) are molecular markers that classify breast cancer subtype, determine the prognosis, and carry significant implications for treatment across all age groups.Tumors that do not express hormone receptors but express HER2, or do not express any receptors at all (also referred to as 'triple negative'), demonstrate more aggressive phenotypes and are known to lead to inferior outcomes and lower overall survival [8].It has been well established in the literature that a greater proportion of young women carry these higher-risk subtypes [9].In addition, tumors that present in younger women are more likely to be a higher grade with more proliferative capacity and increased vascular invasion [10]. It should be noted that even when young patients with breast cancer are matched according to biomarker subtypes and compared to older women, they continue to demonstrate worse survival outcomes [11].Reasons for this discrepancy are thought to be attributable to further biological and non-biological factors.Young women who are diagnosed with breast cancer are more likely to carry familial genetic mutations, which predispose them to developing the disease at an earlier age [12].One study estimated that half of women under the age of 30 diagnosed with breast cancer have germline mutations in BRCA1, BRCA2, or TP53 [13].Women who are genetic carriers of these mutations are also at an increased lifetime risk of developing other malignancies, and their mutational status carries implications that impact both surgical and medical treatment decisions [14]. Beyond biological and lifestyle risk factors, young women present with complex challenges in the face of breast cancer diagnoses that are unique to this cohort.Screening too plays a role.While the Canadian Task Force on Preventative Health Care currently recommends mammography for women starting at the age of 50, there is no screening recommendation for average-risk young women as benefits have not been found to outweigh risks before age 49 [15], and breast masses in young patients are often identified with self-examination [16].While screening protocols exist for patients who are considered high risk (i.e., known carrier of a BRCA1/2, strong family history, or previous thoracic radiation), average-risk young females are not routinely screened for breast cancer [15], potentially contributing to their later-stage diagnoses.Adolescents and young adults with breast cancer report notably higher impacts on their quality of life compared to older women [17] as issues including fertility, body image, and psychosocial factors make treatment and survivorship particularly cumbersome to navigate.It is for this reason that current consensus guidelines emphasize the importance of multimodality care with collaboration between their physicians and allied healthcare team. The objective of this study is to describe the demographic, pathological, and survival outcomes of young women (<40 years) with breast cancer in Newfoundland and Labrador.The aim is to contribute to the literature on this unique and often underrepresented population in the province projected to carry the highest rates of breast cancer in Canada.The analysis of these data sparks discussion pertaining to the intersection of genetic and environmental risk factors calling for careful consideration of the trends demonstrated in this young patient cohort. Materials and Methods A retrospective chart review was performed to obtain population-based data pertaining to young women with breast cancer in the province of Newfoundland and Labrador.Women aged <40 years old at the time of diagnosis between 2008 and 2018 were included.They were required to have had biopsy-proven, invasive breast cancer (i.e., patients with ductal carcinoma in situ (DCIS) were excluded), which was managed within the province.Following ethics approval, patients were identified within the Newfoundland and Labrador Cancer Care Registry (NLCCR) and data were abstracted from electronic medical records.The initial data extraction included 158 patients with 148 patients meeting inclusion criteria after chart review was performed.The ten excluded patients were those who were treated primarily out of province or who did not have an invasive breast cancer diagnosis.Study variables related to patient information that were analyzed during this chart audit included demographic (age at diagnosis, BMI as categorized by the World Health Organization), pathological (tumor stage, grade, and subtype), and treatment-related variables (i.e., neoadjuvant therapy, ovarian function suppression, and up-front surgery).Survival data were collected, including 5-year disease-free and overall survival.Disease-free survival was defined as the absence of local or distant recurrence of breast cancer, or the presence of a new primary 5 years out from their date of diagnosis, while overall survival captured mortality at this timepoint. The electronic database was password-protected and deidentified.Data collection was completed with retrospective chart review by M.M. and S.S. Statistical analyses were performed using the statistical analysis software (Statistical Package for the Social Sciences, IBM SPSS Advanced Statistics 28.0.1.0(5725-A54)) to identify notable trends and descriptive statistics for this patient population.Specifically, frequency analyses and crosstabs were utilized to yield proportions and percentages for the data variables. Results Data were collected on 148 patients who met eligibility criteria and frequency statistics pertaining to each of the variables of interest can be found in Table 1.The majority of patients were within the 35-39 age group (135 patients, 91.2%).Most women in this cohort demonstrated early-stage disease at the diagnosis (stage I or II disease), while 9.6% (14 patients) presented with stage IV, de novo metastatic disease.Biomarkers were noted for each patient, with 62 patients (41.9%) being hormone-receptor-positive (positive for estrogen, progesterone, or both and HER2-negative) and 45 (30.4%) patients had HER2-receptorpositive disease (which was inclusive of those testing positive for hormone receptors in addition to HER2).Triple-negative disease was reported in 41 patients, 27.7% of cases.The body mass index (BMI) was recorded at the time of the diagnosis.Most patients in this cohort (60.5%) fell into categories of overweight (25.2% of patents with BMI between 25.0 and 29.9) or obese (35.3% of patients with BMI greater than 30). With regards to initial surgical management, 56% of women underwent mastectomy and 29.8% had breast-conserving surgery.Thirty-two patients (21.6%) were treated with systemic therapy prior to their initial surgery (neoadjuvant setting) and ovarian function suppression was delivered to 58 patients (41.7%). Five-year disease-free survival and overall survival data were collected for this cohort with the exception of patients diagnosed in 2018 who did not meet this 5-year mark at the time of the analysis.At 5 years, 80.5% of women were disease-free, with no locoregional or systemic recurrences.Five-year overall survival was 82.7%.As demonstrated in Figure 1, the 5-year survival rates were 88.7% for women with hormone-receptor-positive disease, 87.5% for women with HER2-positive disease, and 70.0% for those with triple-negative breast cancer.Figure 2 depicts overall survival by stage at the diagnosis.Those with early-stage disease demonstrated better overall survival at 5 years, while overall survival was only 23.1% for those who had presented with stage IV, metastatic breast cancer.Kaplan Meier survival curves for stage and biomarker status are depicted in Figures 3 and 4, respectively. Discussion In this study of young women with breast cancer in the province of Newfoundland and Labrador, patient demographics, tumor biology, and survival outcomes were evaluated.Several key themes emerged from these data prompting comparison to similar patient cohorts.Points of discussion include prevalence of biomarker subtypes, BMI, and Discussion In this study of young women with breast cancer in the province of Newfoundland and Labrador, patient demographics, tumor biology, and survival outcomes were evaluated.Several key themes emerged from these data prompting comparison to similar patient cohorts.Points of discussion include prevalence of biomarker subtypes, BMI, and survival data. As demonstrated, the prevalence of HER2+ disease and triple-negative disease, the more aggressive biological subtypes of breast cancer, was 30.4% and 27.7%, respectively, in the Newfoundland and Labrador population.A large, North American cohort (n = 46,265) that was evaluated by Murphy et al. of young women under the age of 40 with breast cancer diagnosed between 2010 and 2015 reported rates of HER2+ disease of 26.0% and triple-negative disease of 21.2% [18].A population of young women with breast cancer in France reported a rate of triple-negative disease of 19.1% amongst those aged 35-40, with a HER2+ rate of 16.4% [10].The rates of these aggressive tumor subtypes are notably higher in the Newfoundland and Labrador population of young women with breast cancer.Reasons for this discrepancy may be considered within the context of both genetic and environmental factors.It has been well established that Newfoundland and Labrador genetics stem from a limited founder population, as those of English and Irish decent in the mid-1700s comprise the genetic backbone for the majority of the inhabitants.Scientific medical data from the province have therefore been an attractive resource in the evaluation of complex diseases harboring genetic mutations.A founder effect in Newfoundland and Labrador has been identified in the prevalence of several malignancy-associated Mendelian disorders such as MEN1 (multiple endocrine neoplasia type 1) and HNPCC (hereditary non-polyposis colorectal cancer, also known as Lynch Syndrome) [19].It has therefore been suggested that the Newfoundland and Labrador young female population may carry a higher prevalence of genetic mutations, carrying an increased risk for breast cancer such as BRCA1/2, predisposing them to these more aggressive tumor biologies, such as triple-negative disease. With regards to modifiable risk factors, our cohort was noted to carry a significant burden of obesity at the time of the diagnosis, with 60.5% of patients falling into categories of overweight (25.2%) or obese (35.3%).Newfoundland and Labrador carries the highest provincial obesity rate in the country, with 42% of adults having increased adiposity as of 2021 [20].This may be hypothesized to contribute to the higher rates of aggressive tumor biology seen within this study population.It should be noted however that there is debate surrounding the association between body weight/body mass index (BMI) and risk of a breast cancer diagnosis in young females.Obesity is known to increase breast cancer risk for older women; however, some studies have associated low to moderate BMIs with an increased risk of breast cancer in younger populations.More recent studies evaluating waist circumference however have found central obesity to increase risk in both pre-and postmenopausal populations [21].Increased physical activity, low red meat consumption, and plant-based diets have been associated with a lower risk of breast cancer in the AYA population; however, this is based on limited data from small studies and a further analysis with large, multicenter prospective trials is required [22].There is variability pertaining to the contributory role of other modifiable risk factors for breast cancers as well.For example, while the association between alcohol intake and an increased risk of developing breast cancer in older populations is robust, the role of alcohol as a modifiable risk factor for young patients is unclear [23], reiterating that the role of modifiable risk factors warrants further investigation in young females with breast cancer. The analysis of survival outcomes demonstrated that 80.5% of patients were diseasefree without systemic or locoregional recurrences at 5 years, while the overall survival for this patient population was 82.7%.These represent lower overall survival compared to similar patient cohorts.The American National Cancer Institute (NCI) reports a 5-year overall survival rate of 86.4% amongst female breast cancer patients aged 15-39 between 2013 and 2019 [24].The aforementioned French population study on young women diagnosed with breast cancer between 1980 and 2014 found the overall survival for women between the ages of 35 and 40 to be 92.3% at 5 years [10].That study noted a 5-year disease-free survival rate of 87.6%, compared to the 80.5% found within this study.When broken down by biomarker status, the 5-year overall survival for the Newfoundland and Labrador cohort for hormone-receptor-positive, HER2+, and triple-negative breast cancers was 88.7%, 87.5%, and 70.0%, respectively, reflecting worse outcomes for more aggressive disease pathologies as anticipated.The slightly higher rates of those subtypes (HER2+ and triple-negative breast cancer) in the province may account for the lower 5-year overall survival rate when compared to other North American populations. A notable limitation in this study is the lack of data collection on specific chemotherapy regimens received by each patient.This was in part inaccessible as systemic therapy regimens were available from various EMR systems for some patients and not others.It is not believed however that a lack of access to care was a factor in outcomes and it is presumed that patients were treated according to the standard of care at the time.Another limitation of this study includes the lack of access to universal genetic mutation status amongst this cohort.While some patients had documented mutations, it was not clear whether the entire cohort had undergone genetic assessment.As per the current provincial guidelines (see supplementary materials), any young female diagnosed with breast cancer prior to age 40 warrants a referral to genetics and it is hoped that these data will be available in this regard in the near future.This is of notable importance as mutational status may warrant changes in systemic therapy due to recent advancements in the realm of treatment of BRCA mutated breast cancer with PARP (poly-ADP ribose polymerase protein) inhibitors.Other limitations warrant considerations in guideline protocols and standards of care, which evolved within the global landscape of breast cancer treatment during the study time period.For example, the indications for neoadjuvant systemic therapy or ovarian function suppression, which were incorporated into guidelines in the last several years, were not the standard practice nationally or provincially during the earlier years of this study, which may account for variations in the data.It is imperative that future research in this area explores comprehensive aspects of these patients' care including geographic location (urban versus rural), fertility, mental health and wellness, as well as psychosocial supports. This population description of young women with breast cancer in the province of Newfoundland and Labrador has produced insights into the demographics and outcomes of an often underrepresented and vulnerable population in the province projected to have the highest incidence rates of female breast cancer in Canada.The high proportions of aggressive subtypes of breast cancer and the reduced overall survival associated with those diagnoses that have been demonstrated in this cohort highlight the importance of multidisciplinary care and attention to guidelines, particularly with respect to genetic testing.Detection of genetic mutations influences treatment decisions pertaining to systemic therapy, impacting surgical decision making and family planning.As a result, early genetic testing and referral is essential for young, high-risk women.Fertility considerations in this young cohort are another area that could avail from further assessment given the difficulties that these patients often face when trying to conceive.Most notably, however, this study calls for an extensive investigation into the epigenetic implications within the province of Newfoundland and Labrador that conceivably contribute to higher rates of more aggressive malignancies.This unique intersection of environmental and genetic risk factors may shed light upon pertinent pathways of oncogenes that provoke phenotypic expression of malignancies and that may provide investigators with the questions that must be asked in order to identify and best treat oncology patients. Figure 1 . Figure 1.Five-year overall survival by stage at diagnosis in young women (<40 years) with breast cancer in Newfoundland and Labrador. Figure 1 . Figure 1.Five-year overall survival by stage at diagnosis in young women (<40 years) with breast cancer in Newfoundland and Labrador. Figure 2 Figure 2 Five-year overall survival by biomarker subtype in young women (<40 years) with breast cancer in Newfoundland and Labrador. Figure 2 . 7 Figure 3 Figure 2. Five-year overall survival by biomarker subtype in young women (<40 years) with breast cancer in Newfoundland and Labrador. Figure 3 .Figure 4 Figure 3. Kaplan Meier curve of 5-year overall survival by cancer stage at diagnosis. Figure 4 . Figure 4. Kaplan Meier curve of 5-year overall survival by biomarkers at diagnosis. Table 1 . Patient demographics, disease characteristics, management, and survival outcomes in women <40 years diagnosed with breast cancer between 2008 and 2018, by 5-year overall survival.	2023-11-02T15:28:46.735Z	2023-10-31T00:00:00.000	{ "year": 2023, "sha1": "d4691cca2301f0ddc6bce9a361d2adac751c1504", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/1718-7729/30/11/695/pdf?version=1698740908", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "5d3d348bfac43d79705fb42dc0d9d1d05e09f042", "s2fieldsofstudy": [ "Medicine" ], "extfieldsofstudy": [] }
248796395	pes2o/s2orc	v3-fos-license	Proﬁling the Volatile and Non-Volatile Compounds along with the Antioxidant Properties of Malted Barley : In this work, the stability of antioxidant compounds in malting barley seeds before and after the production of the ﬁnal products is reported. In this reﬂection, the ﬁndings revealed that the process of fermentation had a signiﬁcant impact on antioxidant activity. In vitro, antioxidant capacities were evaluated using DPPH free radical scavenging assay. The results obtained from the spectrophotometric analysis showed that the lowest inhibition value was observed in the samples that were obtained by the classical fermentation process (ABC) and the samples of non-alcoholic beer obtained by the thermal process (NABT), with free radical inhibition capacity values of 8.50% and 5.50%, respectively. The samples of hopped wort (HW) and malted barley seeds extract (BSE) showed very high antioxidant activity with free radical inhibition capacity of 14% and 12.60%, respectively. The obtained extracts were analyzed by gas chromatography and high-performance liquid chromatography, both combined with mass spectrometry detection (GC–MS, HPLC–MS). GC–MS analysis of the SPME extraction showed the presence of 29 compounds with isopentyl alcohol in major concentration (18.19%) in the alcoholic beer; on the other hand, the HPLC–DAD–ESI/MS analysis of the ethyl acetate extract showed the presence of 13 phenolic compounds. Interestingly, the degradation of 3-Hydroxyphloretin 2 (cid:48) - O -glucoside in the ﬁnal products of the non-alcoholic beers was found. Finally, the FTIR analysis was also employed in order to detect the type of efﬁcient groups present in the extracts. Introduction The food and drink processing sectors are undergoing an intense change in production conditions and systems. These changes are due, on the one hand, to modifications in the habits of our modern societies and, on the other hand, to the health requirements of each individual. Currently, one of the main trends in nutrition is the objective of physical health. This wellbeing is achieved by choosing foods that have a positive impact on health. Barley is a herbaceous plant that is among the oldest cultivated cereals [1]. It is eaten raw or roasted and, in ancient times, was also used to make nutritious but very heavy bread. Today, only 33% of barley production in the world is intended for human consumption [2]. It is mainly germinated to make malt for beer and whisky. However, in the last few years, barley grass or grass juice made from young barley shoots has appeared for detox or nutritional purposes. The barley grain is rich in carbohydrates, which is a source of energy. Barley also contains fibers (especially hulled barley) that are good for digestion and bring satiety. Among these fibers, we find beta-glucans (soluble fibers), which play a crucial role in lowering blood cholesterol and bad cholesterol [3,4]. Several investigations have been focused on the study of the phenolic compounds because of their use in medication for a variety of diseases and their application in the conservation of comestible foodstuffs in the food industry [5]. Hence, their biological activities and health benefits also differ e.g., anti-inflammatory [6][7][8], anti-allergic [9], as well anti-carcinogenic properties [10,11]. Additionally, the phenolic compounds have gained the attention of nutritional and therapeutic scientists for their effective exercises and their aptitude to scavenge free radicals and break radical chain reactions. Likewise, the use of high phenols has been correlated with a plethora of various pharmacological activities [12][13][14][15]. Barley has been used as a source of extraction of beer, one of the first popular fermented beverages in the history of mankind; the brewing process of beer has not changed much since its discovery. Generally, the process is based on the natural enzymatic activity that occurs throughout the malting of the seeds, the brewing of the seeds, and the fermentation of the wort [16]. Beer is a complicated combination of ingredients that are fermented from raw material, yeast, malt, and hops, and includes a wide variety of several chemical elements that can act together at all phases of the brewing process [17]. Different groups of composites have been indicated to play a significant role in the flavor properties [18], for example, ketones, higher alcohols, aldehydes, esters, and organic acids, which contribute to the final sensory nature of beer, [19][20][21][22][23][24]. The production of non-alcoholic beer using the thermal process is mostly applied in industry; in this case the beer is degassed and subsequently preheated in a plate heat exchanger. Afterwards, the beer is fed into the stripping section of a rectification column and the product flows through the column at a temperature between 43 • C and 48 • C. In the countercurrent, the product meets rising vapors that cause the selective separation of the alcohol from the product. Afterwards, the alcohol-free beer is introduced into an evaporator where the rectification process takes place [25]. In this study, the stability of antioxidant compounds in malting barley seeds before and after the production of the final products was studied. These stability investigations were carried out in order to determine the most adequate control conditions while avoiding their degradation; also, the study aims to provide information on the impact of the fermentation processes on the products that are metabolized during this stage, which requires a strict control. Samples In the present work, barley seeds were ground and then degreased for 24 h with n-hexane with successive agitation at ambient temperature and were kept at 4 • C until use. Subsequently, the malted barley seeds were subjected to two fermentation methods, namely classical fermentation, and fermentation with a thermal process. The extracts obtained were analyzed by different techniques (Figure 1). namely classical fermentation, and fermentation with a thermal process. The extracts obtained were analyzed by different techniques (Figure 1). Figure 1. Simplified flowchart of the procedure for the preparation of samples for analysis, during different production processes. Extraction Method An ultrasound-assisted extraction (EAU) process was used in order to extract the phenolic compounds (Ultrasonic homogenizer, UIP 1000 hdT, Hielscher Ultrasonics GmbH, Germany). For this aim, 15 g of defatted barley seeds were placed into the sonicator, adding 200 mL of acetone, ethanol, and methanol into each container. The temperature of the ultrasonic water bath was set at 50 • C. The process of extraction took 30 min and was repeated 3 times. The power of the sonicator used was 400 W [26]. Finally, the beer samples from the different fermentation methods were analyzed during the production of alcoholic and dealcoholized beer. The yeast was propagated from a freezing broth maintained at 150 • C [27]. Spectroscopic Analysis FTIR-ATR analysis was carried out as follows: the liquid samples were analyzed by a Fourier transform infrared (FTIR) spectrometer with attenuated total reflectance (ATR), transmission, specular and diffuse reflectance, and Universal ATR, which enables fast analysis of liquids from 450 to 4000 cm −1 (PerkinElmer, Ground Floor, Room G31 Chemical Sciences Building (F10), UNSW Sydney Kensington, NSW 2052, Australia). Spectroscopic Analysis of Polyphenols The spectrophotometric analysis of polyphenols was achieved using a UV-1601 spectrophotometer from Shimadzu (Duisburg, Germany) and was replicated three times for each extract or calibration point (n = 3). Determination of Total Phenolic Content (TPC) The total phenolic content was determined based on a spectrophotometric method using the Folin-Ciocalteu reagent according to the method of Signleton et al. (1999) [29]. This reagent oxidizes the phenolic compounds, which turns the solution blue. The TPC was quantified from a calibration curve prepared with gallic acid standard (y = 1.552x + 0.208, R 2 = 0.962) and expressed as mg of gallic acid equivalents (GAE) per 100 mL of sample (mg GAE 100 mL −1 ). Determination of Flavonoid Content (TFC) Total flavonoid content was performed using the aluminum chloride method (AlCl 3 ) based on the protocol described by Kim et al. (2003) [30]. The TFC in samples was quantified from a calibration curve prepared with catechin standard (y = 2.857x + 0.080, R 2 = 0.999) and expressed as mg of catechin equivalents (CE) per 100 mL of sample (mg CE 100 mL −1 ). Determination of Free Radical Scavenging Potential by DPPH (2,2-Diphenyl-1-Picrylhydrazyl) For the evaluation of free radical inhibition potential in beer samples by DPPH, a method reported in ref. [31] was employed, after minor modifications. Preparation of the diluted sample was carried out as follows: 13.3 mL of degassed beer was diluted with water to 100 mL at a temperature of 20 • C in a volumetric flask and the solution was vortexed thoroughly; then 2.5 mL of diluted beer was added to 20 mL of ethanol and the combination was left to stand for 20 min at 20 • C; afterwards, it was filled up to a volume of 25 mL with attemperated ethanol, the solution was homogenized rigorously, finally the mixture was transferred to a fluted filter, to be filtered, and was retained at 20 • C until use. Preparation of the diluted blank was carried out as follows: 2.5 mL of water was diluted with ethanol in a 25 mL volumetric flask and attemperated for 20 min at 20 • C. The mixture was further filtered through a fluted filter. Preparation of the DPPH stock solution was carried out as follows: 0.06 mM of DPPH methanolic solution (0.0024 g DPPH/100 mL methanol) was attemperated for 20 min at 20 • C. The stock solution should be freshly used. Then, 1.5 mL of diluted sample was added to 1.5 mL of DPPH solution, the mixture was homogenized and kept for 30 min in the dark at room temperature and the absorbance was assessed at 517 nm against diluted blank. A total of 1.5 mL of diluted blank was added to 1.5 mL of DPPH solution and vortexed thoroughly. In addition, it was incubated in the dark for 30 min and the absorbance was measured at 517 nm versus diluted blank. DPPH scavenging potential was expressed as a percentage of free radical scavenging activity (FRSA) with DPPH or as percentage inhibition of the free radical with DPPH, according to the following formula: %I: Percentage of inhibition of free radical DPPH A control: Absorbance of control A sample: Absorbance of sample GC-MS Analyses The achieved findings of the GC-MS study of the SPME extraction revealed the existence of esters, alcohols, etc., (Table 1, Figure 2). A total of 29 compounds were identified. Isopentyl alcohol was the compound that was detected in major concentration (18.19%) in the alcoholic beer and such a compound is the one that mostly affects the aroma in alcohol. It influences the drinkability because the aroma of the beer becomes more pronounced if the concentration of isoamyl alcohol increases. Notably, isobutyl alcohol (0.95%) in the alcoholic beer has an adverse impact on the quality of beer if its concentration exceeds 20% of the total amount of n-propanol, isobutyl alcohol, and isoamyl alcohol. In humans, isoamyl alcohol causes sedative, hypnotic, and anticonvulsant effects similar to the effects of ethanol by ingestion or inhalation and was earlier utilized in medicine for these reasons [32]. Phenolic Profile by HPLC-DAD-ESI/MS In the interest to provide a phenolic characterization of alcoholic and non-alcoholic beer, an HPLC-DAD-ESI/MS method was used. Figure 3 and Table 2 describe the phenolic content of the ethyl acetate extract, which turned out to be the most complex one. A total of 13 compounds were identified and tentatively known based on their retention times, MS data, and comparison together with the information that was earlier described in the literature [33][34][35]. HPLC-MS analysis shows the degradation of antioxidants, such as 3-Hydroxyphloretin 2′-O-glucoside, in the finished product of non-alcoholic beers, depending on the fermentation process on which they are produced. Phenolic Profile by HPLC-DAD-ESI/MS In the interest to provide a phenolic characterization of alcoholic and non-alcoholic beer, an HPLC-DAD-ESI/MS method was used. Figure 3 and Table 2 describe the phenolic content of the ethyl acetate extract, which turned out to be the most complex one. A total of 13 compounds were identified and tentatively known based on their retention times, MS data, and comparison together with the information that was earlier described in the literature [33][34][35]. HPLC-MS analysis shows the degradation of antioxidants, such as 3-Hydroxyphloretin 2 -O-glucoside, in the finished product of non-alcoholic beers, depending on the fermentation process on which they are produced. The final beer product under different conditions was analyzed by GC in order to identify its aromatic compounds (Table 3). The final beer product under different conditions was analyzed by GC in order to identify its aromatic compounds (Table 3). Fermentation, coupled with the thermal process, represents the easiest method to create alcohol-free beer, the application of heat between 35 • C and 60 • C, until boiling, results in the elimination of alcohol and other volatile substances, such as esters and higher alcohols, consequently increasing the turbidity from 0.35 to 2.5 IBC (Table 3) [36]. The degradation and diminution of the solid content of the wort from 11.60 to 3.16 (wt%) shows that the quality of the non-alcoholic product is impacted by the production process. Interpretation of Spectroscopic Analysis The results are reported as the means of triplicates analysis. The data obtained were subjected to one-way analysis of variance (ANOVA) used to compare the difference between the means obtained by each type of sample (Table 4). This analysis showed that there was a significant difference between the means of all of the assays studied (p < 0.05). Table 4. Statistical analysis of the means performed by one-way analysis of variance (ANOVA). The statistical analysis was performed by the statistical analysis software package, which stands for Statistical Package for Social Sciences (IBM SPSS Statistics 23.0, Chicago, IL, USA). Type of Analysis The results of the analyzed samples shown in Figure 4 show that the hopped wort (HW) sample contained flavonoids and total phenolics in higher concentrations (28.65 mg CE 100 mL −1 , 65.12 mg GAE 100 mL −1 , respectively) than the barley seed extract (BSE), which is reflected in their very high antioxidant activity with a free radical inhibition capacity of 14%, Figure 4a. After the different fermentation processes, the highest concentration of flavonoids and total phenolic contents (18.05 mg CE 100 mL −1 , 40.15 mg GAE 100 mL −1 , respectively) was observed in the alcoholic beer by using classical fermentation (ABC), with a higher free radical scavenging capacity than the non-alcoholic beer by using the thermal process (NABT). The lowest concentration of flavonoids and total phenolic contents (10.01 mg CE 100 mL −1 , 35.1 mg GAE 100 mL −1 , respectively) was detected in the non-alcoholic beer by using thermal process (NABT) Figure 4b. The FTIR spectra analysis of the barley seeds and beer are demonstrated in Figure 5, which displays the -OH functions associating phenol and alcohol from 3200 to 3400 cm After the different fermentation processes, the highest concentration of flavonoids and total phenolic contents (18.05 mg CE 100 mL −1 , 40.15 mg GAE 100 mL −1 , respectively) was observed in the alcoholic beer by using classical fermentation (ABC), with a higher free radical scavenging capacity than the non-alcoholic beer by using the thermal process (NABT). The lowest concentration of flavonoids and total phenolic contents (10.01 mg CE 100 mL −1 , 35.1 mg GAE 100 mL −1 , respectively) was detected in the non-alcoholic beer by using thermal process (NABT) Figure 4b. The FTIR spectra analysis of the barley seeds and beer are demonstrated in Figure 5, which displays the -OH functions associating phenol and alcohol from 3200 to 3400 cm −1 , C-H cycloalkane bonds from 2850 to 2925 cm −1 , C=O bonds of amides and aromatic ketones, N-H bonds of primary amines from 1550 to 1650 cm −1 , C=O carboxylic acid from 1400 to 1450 cm −1 , C-O bonds of esters between 1300 and 1450 cm −1 , C-O bonds of primary and secondary alcohols between 1040 and 1090 cm −1 , C-O ethers from 1000 to 1090 cm −1 , Ar-C bonds of aromatics between 850 and 890 cm −1 , and finally, monosubstituted aromatics from 700 to 800 cm −1 . Conclusions Spectrophotometric and chromatographic analysis at different stages of the production of alcoholic and non-alcoholic beer is essential in order to understand how to preserve the equilibrium between the different types of flavors, as well as the stability and evaluation of the antioxidant quality of the obtained product, which is related not only to the nature of the raw materials but also to the technological process, which has a direct impact on the antioxidant potential of the final products. In view of the increasing consumer interest in health issues and the problems associated with excessive alcohol consumption, breweries are being encouraged to expand their range of low-alcohol products. The objective of producing low-alcohol beers can be achieved through the thermal process, the gentle removal of alcohol from ordinary beer, and the limitation of ethanol formation during beer fermentation. Within this basic strategy, there are several methods that change in performance, efficiency, and ease of use. This study provides a comparison of these techniques and provides an evaluation of the sensory properties of the low alcohol and alcohol-free beers that are produced. Conclusions Spectrophotometric and chromatographic analysis at different stages of the production of alcoholic and non-alcoholic beer is essential in order to understand how to preserve the equilibrium between the different types of flavors, as well as the stability and evaluation of the antioxidant quality of the obtained product, which is related not only to the nature of the raw materials but also to the technological process, which has a direct impact on the antioxidant potential of the final products. In view of the increasing consumer interest in health issues and the problems associated with excessive alcohol consumption, breweries are being encouraged to expand their range of low-alcohol products. The objective of producing low-alcohol beers can be achieved through the thermal process, the gentle removal of alcohol from ordinary beer, and the limitation of ethanol formation during beer fermentation. Within this basic strategy, there are several methods that change in performance, efficiency, and ease of use. This study provides a comparison of these techniques and provides an evaluation of the sensory properties of the low alcohol and alcohol-free beers that are produced. Conflicts of Interest: The authors declare no conflict of interest.	2022-05-15T15:05:30.187Z	2022-05-12T00:00:00.000	{ "year": 2022, "sha1": "9f1c9152507ace3d9560f5e94b7b9bfecf8deabc", "oa_license": "CCBY", "oa_url": "https://www.mdpi.com/2297-8739/9/5/119/pdf?version=1652346090", "oa_status": "GOLD", "pdf_src": "MergedPDFExtraction", "pdf_hash": "9e61677f20823431168830728c22e934c6c7d5be", "s2fieldsofstudy": [ "Chemistry", "Agricultural and Food Sciences" ], "extfieldsofstudy": [] }
82601946	pes2o/s2orc	v3-fos-license	Can Hydro-Priming Improve Germination Speed, Vigour and Emergence of Maize Landraces under Water Stress? We evaluated whether hydro-priming could improve vigour characteristics and seedling emergence of local maize (Zea mays L.) landraces compared to two commercial hybrids under water stress at the University of KwaZulu-Natal, Pietermaritzburg. Seeds from local landraces were produced and characterized according to kernel colour, white (Land A) and purple (Land B), and compared to two hybrids, SC701 and SR52, which are popular amongst local farmers. Seeds from each variety were soaked in water for 0 hours (unprimed or control), 12 hours and 24 hours, and germinated in a germination chamber at 25 °C for 8 days. Parameters measured included final germination, mean germination time (MGT) and germination velocity index (GVI). Seedling emergence was performed in seedling trays, using pine bark wetted to 25% or 75% of field capacity (FC), for 21 days in a temperature-controlled glasshouse (25 °C day; 15 °C night; 60% RH). Parameters measured included final emergence, mean emergence time (MET), root and shoot lengths, and leaf area. Priming landraces for 12 and 24 hours reduced MGT by 9% and 7%, respectively, compared to 5% in hybrids for both 12 and 24 hours priming. GVI of landraces was improved by 40% following 12 hours of priming. GVI of hybrids was 11% and 7% slower than landraces after priming seeds for 12 and 24 hours, respectively. Priming seeds for 24 hours improved emergence at 25% FC. Priming seeds for 24 hours reduced MET for all varieties. Priming seeds for 12 and 24 hours increased leaf area by 33.8% and 29%, respectively. Hydro-priming seeds for 12 and 24 hours, respectively, improved GVI, reduced MGT and improved emergence and MET of maize landraces under water stress. Performance of hybrid seeds remains superior to that of landraces even after seed treatment to improve germination and vigour. The positive response of landraces to seed treatment, and improved performance under water stress conditions, suggest that there is a need to identify genes for vigour in landrace maize. Introduction Good crop establishment is essential for efficient water use [1] and is a major constraint to crop production in the semi-arid tropics [2][3][4]. This is particularly true for maize (Zea mays L.) which does not tiller [5]. Good germination and emergence are important for achieving good crop establishment and maximum possible plant populations in the field, more so under adverse growing conditions. As such, speed of germination and emergence is important for successful establishment [6]. Technology that enhances germination and emergence is thus important in mitigating deleterious effects of poor crop establishment due to drought. Such technology would allow farmers to achieve good crop stands and ultimately good yields. Seed priming is one such technology which has been developed to enhance germination characteristics of seeds [7]. Its purpose is to partially hydrate the seeds to a point were germination processes are initiated but not completed [8,9]. Primed seeds exhibit rapid germination and emergence under field conditions [10]. A variety of methods have been used to study the effect of seed priming on germination and growth rate of maize. These include osmopriming (soaking seeds in osmotic solutions such as polyethylene glycol), halopriming (soaking seeds in salt solutions), hydro-priming (soaking seeds in water), hormonal-priming and matri-priming [11][12][13][14]. Priming maize seed using polyethylene glycol (PEG) or potassium salts (K 2 HPO 4 or KNO 3 ) accelerated germination in a chilling germinator (10°C) [15]. Soaking seed in 2.5% potassium chloride (KCl) for 16 hours reduced coleoptile and radicule length, while soaking seed in 20 ppm GA 3 for 30 min improved some germination traits [16]. Hydro-priming (henceforth referred to as priming) is a simple low-cost method of seed priming that requires no sophisticated equipment and gives results which are easy to see [7]. Nagar et al. [17] observed a significant improvement in field emergency and seedling characteristics after hydro-priming maize for 16 hours. In a series of experiments, Harris et al. [3] showed that hydro-priming greatly improved establishment and vigour of upland rice, maize and chickpea, and resulted in faster development, earlier flowering and maturity and higher yields. This simple, low-cost, low-risk intervention also had positive impacts on the wider farming system and livelihoods and the technology proved highly popular with farmers [3,18]. Maize landraces are still being grown by subsistence farmers in KwaZulu-Natal, South Africa under a rainfed system, which according to Rowland [19] is a risky environment. The risk is related to rainfall amount and distribution [7] during the time of planting. Farmers normally sow their maize either in late spring, before the onset of the rains, or with the first rains. The former crop usually suffers from a dry seedbed, resulting in poor emergence. The latter crop may suffer from rains that usually peter out early. In either case, the result is poor crop establishment leading to poor yields due to reduced plant populations. Earlier work [20] showed that landraces may have the same viability but not vigour as hybrids since landraces were slower to germinate and emerge than hybrids. The aim of this study was to evaluate whether hydro-priming can be used to improve germination speed and emergence of local maize landraces under water stress conditions. The performance of landraces was compared to two popular hybrids, SC701 and SR52. Planting Material Seed for the maize landraces was initially donated by local farmers in KwaZulu-Natal, South Africa, and multiplied at the University of KwaZulu-Natal in the previous year (2007). Maize landraces were characterized according to kernel colour, two of which were selected for this study; white (Land A) and purple (Land B). Two hybrids, SC701 and SR52, were used in the study for purposes of comparing the landraces' performance. Seed Priming Procedure Seeds were soaked in distilled water for 12 (P12) and 24 hours (P24), respectively. After soaking, the seeds were surface dried. Laboratory Germination Three replicates of 25 seeds from each variety and priming treatment were germinated between double layered, moistened paper towels [21]. The paper towels were rolled, put into zip-lock bags and incubated in a germination chamber at 25 ℃ [22] for 8 days. Radicule protrusion was the criterion of germination. Observations for final germination percentage, based on normal seedlings, were made according to AOSA [21] guidelines. Root and shoot length, root: shoot ratio, fresh and dry mass were measured. Mean time to germination (MGT) was calculated according to Eq. (1) by Ellis and Roberts [23]: Where: MGT= mean germination time, n = the number of seed which were germinated on day D, and D = number of days counted from the beginning of germination. N1, N2…Nn = number of sowing days at the first, second… last count. Total Soluble Sugars 0.1 g freeze-dried seed tissue was added to a test tube. To each test tube, 10 mL of 80% ethanol was added and homogenized for 30 sec using Ultraturax and incubated for 1 hour at 80 ℃ in a water bath. Thereafter, the test tube were stored at 4 ℃ for 24 hours whereafter they were centrifuged (10,000 rpm for 15 min at 4 ℃). Extract was filtered through glass wool and dried down overnight in a Savant Vacuum drier. Dried samples were diluted up to 2 mL by adding ultra pure water and centrifuged for 15 min and filtered through 0.4 micron nylon syringe filter into sample vials. Soluble sugars were determined using High-Performance Liquid Chromatography (HPLC) (Shimadza) as reported by Aung et al. [25,26]. Seedling Emergence Three replicates of 10 seeds from each variety and priming treatment combination were planted in seedling trays using pine bark as growing media at 25% and 75% field capacity (FC), respectively, over a period of 22 days in a temperature controlled (25 ℃, 60% humidity) glasshouse. Trays were weighed and watered at two-day intervals to maintain field capacities. Data collected included daily emergence for 14 days, leaf area, root and shoot lengths and root and shoot mass (fresh and dry). Mean time to emergence was calculated using Eq. (3) by Bewley and Black [27]: Where MET = mean emergence time, f = number of newly germinating seeds at a given time (day), and x = number of days from date of sowing. Statistical Analysis Data collected was analyzed using GenStat® Version 11 statistical package. Means were separated using LSD (P = 0.05). Final Germination Priming had a highly significant effect (P < 0.001) on final germination. Results for final germination showed there was a significant interaction (P < 0.05) between priming and variety (Table 1). With the exception of Land B, priming did not increase final germination in the other three varieties. Maximum germination (100% for Land A and 98.67% for both hybrid varieties) was achieved in the unprimed treatment. For both priming treatments (P12 and P24), final germination fell by an average 8% in the hybrids compared to 4% in the landraces. Land B attained maximum germination (98.67%) when seeds were primed for 24 hours (P24). Mean Germination Time (MGT) Priming had an effect (P < 0.001) on mean germination time (MGT). For all varieties, priming reduced MGT. There was a highly significant interaction (P < 0.001) between variety and priming in MGT (Table 1). Hybrids germinated faster than landraces when seeds were not primed. The effect of priming on MGT was more pronounced in the landraces than the hybrids. Priming landraces for 12 and 24 hours reduced MGT by 9% and 7%, respectively, compared to a reduction of 5% for hybrids in both cases. 3.1.3 Germination Velocity Index (GVI) In addition, priming had a highly significant effect on germination speed, increasing GVI in all varieties. There was a highly significant interaction (P < 0.001) between variety and priming with respect to GVI (Table 1). Hybrids germinated 5% faster than the landraces when seeds were not primed. However, when seeds were primed for 12 and 24 hours, landraces germinated 11% and 7% faster than the hybrids, respectively. Over-all, priming seeds for 12 hours had the greatest effect on landraces, improving their GVI by 40% when compared to unprimed seeds. 3.1.4 Germination Vigour Traits Furthermore, there was a highly significant interaction (P < 0.001) between variety and priming for germination vigour traits such as root and shoot lengths and fresh mass (Table 1). Root length for landraces fell by 20% (P12) and 9% (P24) as compared to the maximum root length reached when seeds were not primed. Land B, in particular was negatively affected by priming. Hybrids increased root length in response to priming. Their roots were 28% and 7% longer than the landraces when seeds were primed for 12 and 24 hours, respectively. Priming increased shoot length for all varieties. For landraces, seeds primed for 12 and 24 hours, respectively, had about 22% and 10% longer shoots than the unprimed seeds. While for hybrids, primed seeds were 25% (P12) and 19% (P24) longer than unprimed seeds. Overall, landraces responded better than hybrids to priming with regard to shoot length by 7% (P12) and 1.7% (P24). Lastly, priming had a significant effect (P < 0.05) on dry mass. Landraces had a marginal increase (< 1%) when seeds were primed for 12 hours. Hybrids, however, increased dry mass by 25% and 8% when seeds were primed for Can Hydro-Priming Improve Germination Speed, Vigour and Emergence of Maize Landraces under Water Stress? 24 12 and 24 hours, respectively. Total Soluble Sugars There was no significant interaction (P > 0.05) between variety and priming, with respect to total soluble sugars (TSS) (Fig. 1). Hydro-priming did not have a significant (P > 0.05) effect on TSS. Priming resulted in a decline in TSS in both hybrids and Landrace B. However, there were significant differences (P < 0.05) between varieties with respect to TSS. Landraces had higher TSS content compared to hybrids. Overall, based on mean values, landraces had 19% more TSS than hybrids. Seedling Emergence There were no differences (P > 0.05) in seedling emergence (Fig. 2) with respect to variety, priming and field capacity. There was no significant interaction (P > 0.05) between the three treatment factors. SR52 was adversely affected when seeds were primed for 24 hours. Emergence improved under water stress when seeds were primed for 24 hours. There was no significant (P > 0.05) three way interaction with respect to mean emergence time (MET) (Fig. 3). However, there was a highly significant interaction (P < 0.001) between priming and field capacity (Fig. 3). MET was reduced when seeds were primed for 24 hours in all varieties (Fig. 3). Priming seeds for 12 hours improved emergence under optimum conditions (75% FC) and not under water stress (25% FC). Root and Shoot Length There was no significant three way interaction (P > 0.05) between variety, priming and field capacity for all seedling characteristics (Table 2). With respect to root length, priming and field capacity both had significant effects (P < 0.05) while their interaction was also significant (P < 0.05) ( Table 2). Under water stress conditions (25% FC), root length increased by 4% (P24) and 16% (P12) in response to priming. In particular, landraces increased root length under water stress by 4% (P24) and 21% (P12). Although the interaction between priming and field capacity had no effect (P > Fig. 2 Seedling emergence for landraces (Land A and Land B) and hybrids (SC701 and SR52 ) grown at 25% FC and 75% FC after seeds were either not primed or primed for 12 (P12) and 24 (P24) hours. Fig. 3 Mean emergence time (MET) for landraces (Land A and Land B) and hybrids (SC701 and SR52) grown at 25% FC and 75% FC after seeds were either not primed or primed for 12 (P12) and 24 (P24) hours. 0.05) on shoot length (Table 2), priming, on its own, had a highly significant effect (P < 0.001) on shoot length. There were no differences in root and shoot dry mass ( Table 2). Leaf Area Leaf area development showed no significant three way interaction (P > 0.05) between variety, priming and field capacity ( Table 2). Field capacity had a highly significant effect (P < 0.001) on leaf area (Table 2), reducing it by about 23% under water stress. Nonetheless, priming had a significant effect (P < 0.001) on leaf area ( Table 2); leaf area increased by 33.8% and 29% in response to priming seeds for 12 and 24 hours. Landraces increased their leaf area under water stress (25% FC) by 34% (P12) and 6.5% (P24) while the hybrids increased by 48.5% (P12) and 47% (P24), respectively. Discussion Priming of seed has been effectively used to enhance the vigour and emergence of seedlings under both optimal [28,29] and sub-optimal conditions [30,31]. The objective of this study was to determine whether or not hydropriming can be used to improve vigour, with respect to germination attributes and seedling emergence under water stress, in landraces and thus improve crop establishment. Priming had a negative effect on final germination of all varieties. Rapid uptake of water during priming may have caused imbibition injury, resulting in failure of seeds to germinate. There are similar instances in the literature reporting imbibitional injury in seeds, Can Hydro-Priming Improve Germination Speed, Vigour and Emergence of Maize Landraces under Water Stress? 26 including maize [32][33][34][35][36]. Although most of these reports show imbibitional damage at low temperatures, imbibitional damage at higher temperatures, although less severe, can also reduce germination [5]. Priming improved germination speed and reduced MGT. Primed seeds germinated faster and more uniformly than unprimed seeds. Although priming reduced root lengths in landraces, it increased shoot lengths, fresh mass and dry mass; suggesting that a greater part of seed reserves were channeled to the shoots which is crucial for early establishment and photosynthesis. Overall, priming improved vigour of the seeds, with landraces performing well when seeds were primed for 12 hours. These results are similar to others reported in literature [3]. Wahid et al. [37] observed an increase in soluble sugars concentration in response to priming sunflower achenes. They concluded that priming-induced improvements in germination and seedling growth were associated with greater substrate availability for germination. However, contrary to this expectation, results showed an unclear pattern with respect to TSS. In most instances, priming resulted in reduced substrate availability, more so in hybrids. The higher TSS in landraces may explain their positive response to priming. Successful crop establishment determines plant density, uniformity and management options [38] and depends not only on the rapid and uniform germination of the seed, but also on the capacity of the seed to emerge under water stress [39]. Alleviating the deleterious effect of water stress at this stage can increase chances for attaining a good crop [40]. Priming increased seedling emergence under both optimum and water stress conditions. Priming for 12 hours improved emergence of the landraces at 75% FC while priming for 24 hours resulted in better emergence for all varieties at 25% FC. Priming for 24 hours resulted in reduced MET under water stress. Priming also resulted in increased roots and shoots lengths as well as increased leaf area in the landraces. Thus, priming resulted in improved crop establishment and healthier seedlings. Ghassemi-Golezani et al. [14] reported that hydropriming improved seedling emergence rate and percent in lentil; Harris et al. [3] reported enhanced seedling establishment and early vigour of upland rice, maize and chickpea after hydropriming; Kibite and Harker [41] reported that seed hydration improved uniformity of seedling emergence of wheat, barley and oat seeds. Conclusion Good crop establishment is a prerequisite for successful crop production especially under water stress conditions. Seeds responded better to priming for 12 hours when conditions were optimum while priming for 24 hours improved emergence, reduced MET and improved seedling characteristics under water stress. Farmers may use hydro-priming, combined with a slightly higher seeding rate, when planting early and late in the season. Hydro-priming maize landraces for various periods of time may be used a low-cost technology to enhance emergence and vigour characteristics of landraces under both optimum and water stress conditions. However, there is need for further research to evaluate whether these initial benefits may contribute to improved yields under water stress.	2019-03-19T13:05:23.520Z	2011-05-28T00:00:00.000	{ "year": 2011, "sha1": "7d9aef930a70d91873c0bdc3c1bf788f0692586f", "oa_license": "CCBYNC", "oa_url": "http://www.davidpublisher.org/Public/uploads/Contribute/55d4397221bd3.pdf", "oa_status": "HYBRID", "pdf_src": "Anansi", "pdf_hash": "5b007b3027495c429b1b94a93db5dc9fc4327d59", "s2fieldsofstudy": [ "Agricultural And Food Sciences" ], "extfieldsofstudy": [ "Biology" ] }
181998979	pes2o/s2orc	v3-fos-license	ANALYSIS AND IMPLEMENTATION OF UBUNTU AND MIKROTIK BASED SERVER  Abstract — Internet is a primary requirement in this modern era because it is one of the information’s media and communication. Due to its rapid advancement, internet is now a main infrastructure which must be possessed by an organization, institution (education, government, service provider and others). Besides internet, other infrastructure which is also a primary requirement in an institution is a centralized storage or server. Regarding internet’s availability and server, PT Indonesia Terbit Media faces some setbacks such as the unavailability of sufficient bandwidth management and unavailability of centralized information storage. To solve those setbacks, a MikroTik router is offered to be implemented in the company’s network. Before doing research, observation is held in the company to gather information’s regarding current network. Along with observation, focus group discussion is held with the staffs. After information’s are gathered, current network is analyzed then a new topology is proposed. After the proposal is approved, testing phase is initiated to know the contributions of newly proposed network in solving the mentioned setbacks. Results from the implementation are such as bandwidth management is applied in new network which is resulting in stable internet’s speed even though a user is downloading a quite large file and Ubuntu-based centralized server. I. INTRODUCTION Batam is one of the industrial cities, which needs fast and accurate information.PT Indonesia Terbit Media is one of the companies, which is engaged in journalism and provides information about Batam and it is served in the format of a website [1].Fast internet connection is required to support those necessities.Besides the internet, other infrastructure, which is the main requirement in a company, is a centralized storage or usually called as server.[2] A large scaled company or business entity usually use a physical server as its centralized storage [3].Whereas for the small to intermediate-scaled company mostly uses PC as the centralized storage because it is fairly cheaper than the physical server (which is usually more expensive).[4] Server is usually used by the staff or workers to keep important information related to company's privacy such as data, transaction and others [5].Each staff or Januardi Nasir are with the Department of.Technique, Universiti Putera Batam, Batam, Indonesia (email : januardinasir@gmail.com) worker is usually given permission according to their own department so no one from certain department can get an access through other department's data.[6] The operating system, which is commonly used by server, is Windows because it is easy to operate.There are few servers, which uses Linux as their operating system because it is more secure.[7] II.RESEARCH METHOD 3) .MikroTik RB-750-R2 router is directly connected to modem (internet connection from ISP) [9].Wi-Fi router and server are connected to MikroTik router where bandwidth management will be implemented for both local network and wireless network [10].Local network consists of four laptop computers and a shared printer which are connected to a TP-LINK switch. IV. RESULTS AND ANALYSIS 1) SSH (Secure Shell) 2) Mail Server Wget command is used to send request to webpage of www.indonesiaterbit.co.id and after a few seconds, the request is accepted or responded by the web page.[13] 3) Web Server Wget command is used to send request to webpage of www.indonesiaterbit.co.id and after a few seconds, the request is accepted or responded by the web page.[14] 4) DNS Server Dig command is used to check the query time of a certain domain.For the first dig, the query time is 4991 msec.After trying the second dig, the query time is increasing to 119 msec.9 above shows the download speed using Internet Download Manager is reaching up to 3 Mbps which causes this situation to slow down the browsing, streaming, upload and download process in other devices [15].But after bandwidth management is implemented, download speed is limited up to 260 kbps as shown in figure 14, resulting in other devices are not experiencing excessive delay during browsing, streaming, upload and download. V. CONCLUSIONS Implementation of Ubuntu as a centralized storage or server succeed and works like it supposed to be and MikroTik-based bandwidth management with queue tree method effectively increases the quality of service in network's traffic by limiting bandwidth for each device within the network. Figure 2 Figure 2 Proposed Network Topology Figure 3 Figure 3 Remote login to server using PuTTY Figure 4 Figure 4 Remote login to server established Figure 5 Figure 5 Testing web server using wget command Figure 6 Figure 6 Testing web server using wget command Figure 7 Figure 7 Testing DNS server using dig command Figure 8 Figure 9 Figure 8 Download speed before bandwidth management Figure Figure9above shows the download speed using Internet Download Manager is reaching up to 3 Mbps which causes this situation to slow down the browsing, streaming, upload and download process in other devices[15].But after bandwidth management is implemented, download speed is limited up to 260 kbps as shown in figure14, resulting in other devices are not experiencing excessive delay during browsing, streaming, upload and download.	2019-06-07T20:44:05.063Z	2019-02-28T00:00:00.000	{ "year": 2019, "sha1": "5608233645b3d73362577ba317c290f9584f4671", "oa_license": "CCBYNCSA", "oa_url": "http://jurnal.unmer.ac.id/index.php/jeemecs/article/download/2928/pdf", "oa_status": "GOLD", "pdf_src": "Anansi", "pdf_hash": "0c471fd6e68f8cfc7a4e496b7f357f4ba63b85a0", "s2fieldsofstudy": [ "Computer Science" ], "extfieldsofstudy": [ "Computer Science" ] }
119530337	pes2o/s2orc	v3-fos-license	Dynamic phase transition properties of kinetic Ising model in the presence of additive white noise Using Monte Carlo simulations based on the Metropolis algorithm, we investigate the dynamic phase transition properties of kinetic Ising model driven by a sinusoidally oscillating magnetic field in the presence of additive white noise. We calculate equilibrium and dynamic properties such as the temperature dependence of average magnetization and magnetic specific heat, as well as the period dependence of dynamic order parameter and scaled variance. After determining the critical period at which order-disorder transition takes place, we perform finite size scaling analysis to extract the exponent ratios, and discuss the variation of these properties in the presence of noisy magnetic field. As a general result, we show that for a noisy system, DPT does not fall into a universality class of the conventional dynamic (and also equilibrium) universality class of the Ising model. I. INTRODUCTION Dynamic phase transition (DPT) properties of kinetic Ising model have been investigated in detail during the past three decades [1][2][3]. Up to now, comprehensive theoretical efforts clarified several aspects of DPT. For instance, fluctuations of dynamic order parameter and energy in kinetic Ising model have been found to exhibit a singularity behavior at the transition temperature in the presence of oscillating magnetic fields [4][5][6]. For large field amplitude and small lattice size, the system may exhibit a stochastic resonance behavior [7,8] accompanied with a discontinuous transition observed in the thermal variation of the order parameter [9]. Besides, the studies regarding the dynamic hysteresis process of kinetic Ising model [3,10] revealed that a dynamic symmetry breaking originates as a consequence of a competition between two time scales namely, the period of the dynamic order parameter and the relaxation time of the system resulting in a DPT between dynamically ordered and dynamically disordered phases. For a given field period P , the relaxation time depends on the several system parameters such as the temperature T , the magnetic field amplitude h 0 , and the exchange interactions acting in the system. In several works, modified versions of the problem such as the kinetic Ising model with next-nearest neighbor interactions [11], kinetic Blume-Capel (BC) model [12], as well as site and bond diluted systems [13,14] have been handled. On the other hand, for a Co(4Å)/Pt(7Å) multilayer system with strong perpendicular anisotropy, an example of DPT has been observed by Robb et al. [15]. Besides, very recently, DPT has also been experimentally observed for uniaxial ferromagnetic films by Berger et al. [16] and Riego et al. [17]. The experimental results reported in Ref. [17] have also been verified by Ref. [18] using numerical and theoretical tools. Moreover, regarding the universality class of the DPT observed in kinetic Ising model, two (2D) [19][20][21][22][23][24][25] and three (3D) [26,27] dimensional models have been widely investigated, and it has been concluded that the critical exponents of the kinetic Ising system belongs to the same universality class of the equilibrium model. Although the kinetic Ising model has been widely investigated in the literature, very few works have paid attention on the effect of randomness on the DPT properties of the model. Among them, Acharyya [28] studied the kinetic Ising model in the presence of a randomly varying (in time but uniform in space) magnetic field. For this system, ordered phase disappears with increasing randomness whereas for small random fields, the system remains in a dynamic asymmetric (ordered) phase. In the presence of a rectangular random field distribution, the same author reported the existence of a tricritical point at T = 0 [29]. Haussman and Ruján [30] investigated a kinetic Ising ferromagnet in the presence of a fast switching, random external field which was realized according to a bimodal type of random field distribution. They found a novel type of first order phase transition in their system which was related to dynamic freezing. The fourth order cumulant (i.e. the Binder cumulant) and dynamic magnetization-reversal transition analysis of kinetic Ising system in the presence of pulsed magnetic fields have been studied by Chatterjee and Chakrabarti [31] where they found that the transition to fall in a mean-field-like universality class. Meanwhile, Crokidakis [32,33] studied the random field kinetic Ising model where the local magnetic fields change sign randomly with time where the type of the random field is selected to be a double Gaussian type. According to his results, 2D system exhibits continuous (discontinuous) phase transition for a bimodal (double Gaussian) random field whereas the transition for the 3D system becomes discontinuous for a bimodal field distribution with large field values. Apart from these, very recently Akinci [34] modeled a kinetic Ising system in the presence of both periodic and randomly fluctuating magnetic fields. As a source of randomness, the author considered the Gaussian white noise. Effect of the white noise on the DPT properties of the system has been investigated, and it has been shown that there exists a white noise induced DPT in the system. Indeed, we encounter the noise (such as paper crumpling) in our every day life, mostly as an unwanted effect. It can also be observed in several ubiquitous systems including rotary motion of biological bacteria populations [35][36][37], voltage fluctuations across the resistance of electrical circuits [38], seismic activity during earthquakes [39], and magnetization flips [40], as well as random motion of magnetic domain walls [41] of magnetic systems. Depending on the nature of the noise (additive or multiplicative), effect of the noise on a particular property of a system may not always be of destructive kind but it may also play a constructive role. Such behavior manifests itself as noise induced ordering and disordering transitions and reentrant phenomena in stochastic systems [42][43][44][45][46][47][48][49][50]. To the best of our knowledge, there does not exist any work regarding the DPT properties of kinetic Ising model in the presence of noise. Therefore, it would be interesting to pursuit the answer for the question if there is any dynamic phase transition in the kinetic Ising system in the presence of both periodic and noisy magnetic fields. For this aim, in addition to the conventional time dependent periodic part of the external magnetic field, we also consider the additive white noise acting on the lattice sites of the system. For this aim the paper is organized as follows: In Sec. II, we present out model. The results and related discussions are given in Sec. III. Finally, Sec. IV is devoted to our concluding remarks. II. MODEL AND SIMULATION DETAILS In this work, we consider a kinetic Ising model defined on a square lattice with lattice coordination number q = 4. The following Hamiltonian defines the dynamic behavior of the system where the first term denotes the ferromagnetic (J > 0) exchange between nearest neighbor spins and the last summation which stands for the Zeeman energy contribution is carried over all the lattice sites. Within the framework of the Monte Carlo simulation method, the system defined by Eq. (1) can be handled by several local spin update schemes such as where ∆E T = ∆E J + ∆E H is the local energy variation after flipping the spin S i at the site i. In the present work, Eq. (1) has been treated by Metropolis Monte Carlo scheme [51] with periodic boundary conditions on a L × L square lattice. The time dependent magnetic field acting on the site i is given by where h 0 and ω respectively correspond to the amplitude and the angular frequency of the periodic magnetic field, and h r is a random magnetic field which can be called as the additive Gaussian white noise. At each time step of the simulation, a random value h r is drawn from the following normal probability distribution where σ is the width of the random field distribution. The simulation procedure can be outlined as follows: At first, we start by a random configuration of spins, and randomly visit the lattice sites (random sweeping) in one Monte Carlo step per spin (MCSS). The period P = 2π/ω of the periodic part of the external field is defined in terms of MCSS. For the thermal variation of magnetic properties, the simulations run over 2000 periods of the external field cycle where 50% of them were discarded for thermalization. On the other hand, for the calculations performed at constant temperature, our simulations have been carried out over 2 × 10 5 field periods where 10% of them have been discarded to allow the system to reach equilibrium. Once the value of h r is assigned at a given simulation time, each spin on the lattice interacts with this noisy magnetic field during one complete sweep of the lattice sites in one MCSS. During the simulation for a particular process, we have monitored a variety of quantities such as • the time series of the magnetization • the averaged magnetization M(T ) and specific heat C(T ), • dynamic order parameter which can be defined as time averaged magnetization over the k th cycle of the periodic magnetic field, • period average of the dynamic order parameter where N k is the number of the complete cycles of the periodic field. • We have also calculated the scaled variance, and the fourth order cumulant (i.e. Binder cumulant) corresponding to the dynamic order parameter Q which are respectively defined as We have calculated the relations given between Eqs. In order to investigate the DPT properties, we have also fixed the field amplitude as h 0 = 0.3J for convention [7]. We also set k B = 1 throughout the work. T c , and the magnetic specific heat exhibits a sharp cusp. It is well known that this model undergoes a continuous phase transition [52]. However, as the randomness takes place with increasing σ, the magnetization decreases from its saturation value to zero at gradually lower temperature region. Indeed, for moderate σ values, it is clear from Fig.1 that the system does not exhibit critical behavior since the C(T ) peak becomes rounded for large σ. Hence, we can conclude from this figure that the equilibrium Ising model in the presence of additive white noise does not exhibit conventional order-disorder transitions with increasing randomness. Next, we discuss the DPT properties in the presence of additive white noise. For this aim, defining the half-period parameter t 1/2 = 0.5P is a convenient selection in the literature. To out knowledge, DPT properties of kinetic Ising system in the presence of sinusoidally oscillating magnetic field have not been investigated using Metropolis scheme. Hence, in we present the dependence of the order parameter \|Q\| L , and the scaled variance χ Q L as a function of the half period t 1/2 in Fig. 2b and 2c, respectively. By keeping in mind the pronounced finite size effects, it is apparent that \|Q\| L evolves from saturation value to zero with increasing t 1/2 . In addition to this, χ Q L plotted for large system sizes such as L = 256 exhibits a prominent peak around the critical value t c 1/2 . In order to precisely measure the critical value t c 1/2 , we have calculated the Binder cumulant U L within a narrow region of the half period such as 80 ≤ t 1/2 ≤ 100. The results are displayed in Fig. 3a from which we observe a perfect intersection of U L curves corresponding to different values of linear lattice size L. Our numerical data yield U * L = 0.61 and t c 1/2 = 92. Note that the value t 1/2 = 95 at which χ Q L diverges does not coincide with the value obtained from Binder cumulant analysis. We also underline that our estimated value U * L = 0.61 for the kinetic Ising model can be compared to that obtained for the equilibrium model U c L = 0.61069 [53]. The critical period value P c = 2t 1/2 = 184 is a new value in the literature. Using the scaling relations for the dynamic order parameter and scaled variance [20], we have also extracted the critical exponent ratios β/ν and γ/ν at P c = 2t 1/2 = 184. Fig. 3b. Our estimate for the exponent ratios β/ν = 0.134, and γ/ν = 1.749 are clearly very close to those obtained for the equilibrium counterpart of the 2D Ising model [52]. Combining our results in the following scaling [25] relation we get 2(β/ν) + γ/ν = 2.018 ≈ d. The obtained results have been shown in Based on the above discussions, we can clearly say that DPT observed in the present system in the absence of white noise falls into the same universality class with its equilibrium counterpart. The critical values obtained in the present analysis have been presented in the In order to investigate the influence of the additive white noise on the DPT properties of the kinetic Ising model, we have plotted in Fig. 4 the variation of dynamic order parameter \|Q\| L , and the scaled variance χ Q L as functions of the noise parameter σ for a square lattice with L = 256, and half period value t 1/2 = 50. The simulated random field distributions within 0.0 ≤ σ ≤ 0.3 are also depicted in this figure. One can clearly deduce from Fig. 4b that the order parameter \|Q\| L evolves from unity to zero with increasing randomness. Besides, the variance χ Q L passes through a sharp maximum at a critical randomness σ c . Evolution of Q as a function of field period P also supports the possible presence of disorder induced phase transition in the system. However, there is a distinction which shows itself for large randomness such as σ = 0.3 where Q(p) randomly oscillates between states ±1. The snapshots of the system corresponding to σ values given in Fig. 4c have been presented in Fig. 5. Fig. 5a shows that in the presence of weak randomness such as σ = 0.1, the system exhibits a long range order where the great majority of the spins pointing in the spin-↑ direction. Form Fig. 5b, we see that large domains of the same oriented spins are formed for moderate values of σ. According Fig. 5c, although the dynamic order parameter tends to zero for large randomness, instead of nucleated droplets, we observe large domains of spins, hence a short range order originates in the system. Due to these large fluctuations and strong stochastic behavior for large randomness, we will restrict our subsequent investigations to weak noise case. Fig. 6, in order to compare the dynamics of the system, we plot the time series of the magnetization m(t) as a function of time (in terms of MCSS) for σ = 0.0 (without-noise) and σ = 0.1 (without-noise) cases, respectively. It is clear from Fig. 6 that the noisy input (h(t)) causes a noisy response (m(t)) in the system. In Fig. 7, we further analyze the influence of σ on the time series of the order parameter Q (Eq. 7), as well as on the half-period t 1/2 dependence of dynamic quantities \|Q\| L and χ Q L . From Fig. 7a, it seems that a DPT in the presence of noisy input field (σ = 0.1) may take place between dynamically ordered and dynamically disordered phases. According to Figs. 7b and 7c, \|Q\| L evolves from saturation value to zero with increasing t 1/2 whereas χ Q L exhibits a divergent behavior around the critical half period value. In order to clarify whether the results presented in Fig. 7 indicate a true dynamic phase transition or not, we have performed finite size scaling analysis given by Eqs. (11) and (12) when the noise parameter is selected as σ = 0.1. The results are given in Fig. 8. From this figure, we can observe that χ Q L curves exhibit size dependent maximum, i.e. a divergent behavior around t c 1/2 . Regarding the Binder cumulant analysis, as a consequence of possible correction-to-scaling effects [19], and the large fluctuations due to the presence of noisy magnetic field, we can not identify a distinct intersection point U * L for different L. Hence, we have estimated the location of the intersection by examining and crossing the successive couples of U L curves with different L values such as U 256 with U 180 , and U 180 with U 128 , and so on. Averaging over the obtained values, we roughly estimate the location of critical half period as t c 1/2 = 73. Using this value, as well as the value obtained from the peak value of χ Q L , we estimate the critical exponents as β/ν = 0.047, γ/ν(t c 1/2 ) = 2.201, and γ/ν(peak) = 2.196 for the kinetic Ising model in the presence of white noise with σ = 0.1. These results are displayed in Fig. 8. Since the obtained critical exponent values are definitely different from those obtained for clean system (i.e. in the absence of noise), we can conclude that even in the presence of weak randomness, DPT observed in the system does not fall into a universality class of the conventional kinetic (and also equilibrium) Ising model. and (c), we present \|Q\| and χ Q L as a function of half period t 1/2 for σ = 0.1 for various system sizes. Each figure has been plotted for h 0 = 0.3J and T = 0.8T c . that the DPT of kinetic Ising model driven by a sinusoidally oscillating magnetic field in the absence of white noise is in the same universality class with its equilibrium counterpart On the other hand, in the presence of a weak noise such as σ = 0.1, the obtained critical exponents β/ν = 0.047, γ/ν(t c 1/2 ) = 2.201, and γ/ν(peak) = 2.196 indicate that the DPT observed in the system does not fall into a universality class of the conventional dynamic (and also equilibrium) universality class of the Ising model. We hope that the results presented in this work stimulates further interest in research of the DPT in stochastic systems.	2018-07-09T12:42:45.000Z	2018-07-09T00:00:00.000	{ "year": 2018, "sha1": "db4ed0f327af1a090d86ff8eaab1672413a53fcc", "oa_license": null, "oa_url": null, "oa_status": null, "pdf_src": "Arxiv", "pdf_hash": "db4ed0f327af1a090d86ff8eaab1672413a53fcc", "s2fieldsofstudy": [ "Physics" ], "extfieldsofstudy": [ "Physics" ] }
17179778	pes2o/s2orc	v3-fos-license	Redox control of senescence and age-related disease The signaling networks that drive the aging process, associated functional deterioration, and pathologies has captured the scientific community's attention for decades. While many theories exist to explain the aging process, the production of reactive oxygen species (ROS) provides a signaling link between engagement of cellular senescence and several age-associated pathologies. Cellular senescence has evolved to restrict tumor progression but the accompanying senescence-associated secretory phenotype (SASP) promotes pathogenic pathways. Here, we review known biological theories of aging and how ROS mechanistically control senescence and the aging process. We also describe the redox-regulated signaling networks controlling the SASP and its important role in driving age-related diseases. Finally, we discuss progress in designing therapeutic strategies that manipulate the cellular redox environment to restrict age-associated pathology. Biological theories of aging Why do we age? The answer to this perennial question has encouraged the scientific community to pursue hypotheses that can delineate the mechanisms behind the human body's functional decline with age. However, despite medical advances there remain challenges with respect to delineating controls that regulate the span of human life and health. Elucidation of these control mechanisms may lead to better-targeted interventions for the gamut of diseases propagated by age. Ultimately, amelioration of age-associated pathologies and comorbidities would limit age-related frailty and improve the well-being of our aging population. Biological aging is the progressive manifestation of accumulated cellular damage with age, and is determined by environmental or genetic factors [1][2][3][4]. The process of aging is complex and can be explained by a number of complimentary theories. The wear and tear theory, proposed by August Weismann in 1882, compares organisms to machines, suggesting that cells 'wear out' with time and accumulate damage through excessive consumption of fat, sugar, or exposure to UV radiation, leading to declines in functional efficiency of organs. However, this theory disregards the ability of cells to repair damage. The stochastic theory which is a by-product of the wear and tear theory, postulates that aging is due to the accumulation of damage in cells resulting from failure of wound repairing mechanisms, infections, UV radiation, or environmental stress [5]. Evolutionary theories of aging support the concept that specific genes implicated in longevity are involved in the stability and maintenance of somatic cells, and are susceptible to mutations with age. Medawar's mutation accumulation theory links aging to natural selection, hypothesizing that there is no selection pressure on the aging population, as there is no known evolutionary mechanism to eliminate a population of mutations that cause deleterious effects only in aged species [6]. This suggests that detrimental late-acting alleles may accumulate with age and exert adverse effects on survival. The antagonistic pleiotropy model suggests that a pleiotropic gene that selectively increases fitness and survival during the early stages of life can still induce adverse late-acting effects in the population [7]. In the context of cellular aging, this could apply to replicative senescence that suppresses tumor formation early in life, but may promote cancer in later stages. The disposable soma theory, put forth as an extension of the antagonistic pleiotropy model, highlights the balance between somatic maintenance and repair versus reproduction and suggests that with age extensive maintenance would be necessary as cells can accumulate multiple lesions in parallel [8,9]. The rate of living theory puts across the idea that organisms that metabolize oxygen more rapidly have a higher energy expenditure and typically have shorter lifespans that can be extended by caloric restriction [10][11][12][13]. According to the cross-linking (or glycosylation) theory of aging, cross-linked proteins or sugar moieties can bind to DNA causing replicative damage or an age-related decline in protein turnover that can be linked to the loss of functional proteins, further promoting age-associated pathologies [14]. While all of the aforementioned theories have been examined and laboriously tested, the mitochondrial free radical theory of aging, later termed the oxidative stress theory (OST), is currently one of the most popular correlative theories of the aging process. OST explains aging at the molecular level and results from failure to maintain oxidative defenses, mitochondrial integrity, proteostasis, barrier structures, DNA repair, telomeres, immune function, metabolic regulation, and regenerative capacity (Fig. 1). More than half a century ago, Gerschman introduced that oxygen free radicals, commonly considered to be too reactive to exist in biological systems, are generated in situ in response to radiation and oxygen poisoning and are responsible for the associated toxicities [15,16]. Later, Denham Harman proposed that hydroxyl and hydroperoxyl radicals generated endogenously from oxygen-consuming metabolic processes play crucial roles in the aging process [17]. This hypothesis coalesced from a number of observations: (i) indications that free radicals are produced in vivo; (ii) free radicals cause pervasive damage to macromolecules in vitro; and (iii) the deleterious effects of exposure to irradiation and hyperoxia are alleviated by antioxidant administration. Free radicals are highly reactive atoms or molecules with one or more unpaired electron(s) in their outermost shell and can be formed when oxygen interacts with certain molecules. They can be formed endogenously by natural biological processes, or generated upon exposure to external stimuli. Reactive oxygen species (ROS) collectively describes a number of reactive molecules, such as superoxide (O 2 •- ), hydroxyl radical (OH • ), hydroperoxyl radical (HO 2 • ), nitric oxide (NO • ), nitrogen dioxide (NO 2 • ), and peroxyl (ROO • ), produced as by-products during the mitochondrial electron transport of aerobic respiration or by oxidoreductase enzymes. Also, hydrogen peroxide (H 2 O 2 ), ozone (O 3 ), singlet oxygen ( 1 O 2 ), hypochlorous acid (HOCl), nitrous acid (HNO 2 ), peroxynitrite (ONOO -), dinitrogen trioxide (N 2 O 3 ), and lipid peroxide (LOOH), are oxidants that can easily lead to free radical reactions in cellular systems [18]. Expanding knowledge of the mechanisms of ROS generation, intracellular sources, antioxidant defenses, and the chemistry of ROS-induced oxidative damage, was critical to improve understanding of the homeostasis between pro-oxidants and anti-oxidants, thereby evolving the free radical theory into the OST of aging [19,20]. The "structural damage-based" hypothesis driving this theory is that age-associated functional losses are due to the accrual of oxidative damage to macromolecules such as lipids, DNA, and proteins by free radicals, and that the progressive oxidant/anti-oxidant imbalance leads to the consequent disruption of redox-regulated signaling mechanisms. Beckman and Ames divided this hypothesis into "strong" and "weak" versions: The strong version proposed that oxidative damage determines life span, while the weak version correlated oxidative damage to age-associated pathologies [21]. Although widely accepted, the OST has been severely refuted in recent years; most criticisms try to discredit the theory pointing at failure to increase longevity by increasing antioxidants and the production of fewer free radicals in certain long-living species [22][23][24][25][26]. Recently, a critical idea has emerged that ROS at non-toxic levels function as signaling molecules that induce protective defenses against age-dependent damage, and that peroxisomes may be implicated in the regulation of aging [27]. Currently, the two major traits correlating organismal aging and longevity to the OST of aging are considered to focus primarily on the species-specific low mitochondrial ROS generation rates at complex I of the electron transport chain (ETC) in longlived animals, and low levels of fatty acid unsaturation on cellular and mitochondrial membranes [28][29][30]. Cellular senescence Cellular senescence, also termed replicative senescence, was first proposed four decades ago [31,32] and has since been proven to be a significant tumor constraining mechanism that halts the proliferation of primary mammalian cells after a finite number of population doublings [33][34][35][36][37] allowing the organism to antagonize the potentially detrimental effects of uncontrolled growth. Proliferation is checked by persistently arresting cell growth at the G1- [38][39][40][41][42], G2- [43][44][45], G2/ M- [46] or S-phase of the cell cycle leading to failure of DNA replication. So far, senescence-associated growth arrest has been shown to depend functionally on the tumor-suppressor pathways controlled by p16 INK4a and pRB (retinoblastoma protein), as well as by p53. Progressive telomere attrition due to persistent DNA damage responses [47] has been attributed to be one of the clocking mechanisms that keeps track of the number of times a cell divides, providing an appropriate trigger for the onset of telomere-induced senescence through the p53 pathway [34,[48][49][50][51][52]. ROS can induce a senescent growth arrest in vitro, by triggering the DNA damage response (DDR) pathway, with ATM or ATR kinases blocking progression of the cell cycle through stabilization of p53 and transcriptional activation of the cyclin-dependent kinase (CDK) inhibitor p21 [53]. However, in response to persistent DNA damage, p16 INK4a is activated via p38-MAPK-mediated mitochondrial dysfunction and ROS production. Nuclear lamin B1 downregulation triggers modifications in chromatin methylation and induces senescence progression with the formation of highly condensed regions of chromatin called senescence-associated heterochromatin foci [54]. These foci are enriched in chromatin modifications that associate with genes required for maintenance of senescence-associated growth arrest. Senescence can also be induced by cell-type specific stress independent of telomere shortening, resulting in increased expression of the tumor suppressor p16, in which case it is termed stress-induced senescence [42,[55][56][57]. This includes chronic signaling by anti-proliferative cytokines that increase cellular oxidative stress and potentiate senescence [58][59][60]. Oncogene-induced senescence (OIS), characterized by aberrant DNA replication and the associated DNA damage response, is affected by activated oncogenes or the loss of tumor-suppressor genes in healthy cells [45,[61][62][63][64] and upregulation of the CDK inhibitors p15 INK4b , p16 INK4a , and p21 CIP1 [65]. Therapy-induced senescence (TIS) has been described in tumor cell lines in response to induction with selective preoperative neoadjuvant chemotherapeutics or radiation agents, and is considered crucial to mitigate toxicity-related side effects, thereby presenting a novel therapeutic target for cancer [66,67]. Senescence-Associated Secretory Phenotype (SASP) Cellular senescence is a significant tumor constraining mechanism that halts cellular proliferation in response to damage that occurs during replication. However, senescent cells acquire a deleterious, irreversible senescence-associated secretory phenotype (SASP) involving secretion of soluble factors (interleukins, chemokines, and growth factors), degradative enzymes like matrix metalloproteases (MMPs), and insoluble proteins/extracellular matrix (ECM) components [33,68] that can alter tissue microenvironments and affect neighboring epithelial cells in a paracrine fashion to promote tumor progression [65,69,70]. Also termed the senescence-messaging secretome (SMS) [71], these factors promote shedding of membrane-associated proteins, degradation of signaling molecules, and/or ECM processing, thereby disrupting normal tissue framework and function [72]. Morphologically, senescence is characterized by cellular enlargement and flattening with an accompanying increase in cell volume, increased size of organelles including Golgi, lysosomes and nucleus, appearance of vacuoles in the cytoplasm and ER and an increase in the number of cytoplasmic microfilaments. The senescent phenotype has been long associated with driving organismal aging, and has recently been linked to development and tissue repair. The SASP is primarily a persistent DDR coupled as a positive feedback loop with the senescent stimuli ROS, prompting telomere-dependent or -independent signaling mechanisms to facilitate growth arrest. The non-cell-autonomous activities mediated by SASP serve to deplete stem and progenitor cells and demonstrate that the heterogeneous functional networks of senescence are formed by multiple effector pathways involving highly dynamic signal amplification [73]. The SASP can also be uncoupled from the senescenceassociated cell-cycle arrest, as it exemplifies a perpetual state of damage in comparison to growth arrest. SASP has been characterized by analysis and conservation of factors between human and mouse cells cultured under physiological oxygen conditions [74] and its occurrence has been seen in vivo in mice and humans [75,76] and amongst diverse proliferative cell types (fibroblasts, epithelial cells, endothelial cells, astrocytes, etc.) [76][77][78]. The SASP components that modulate neighboring cells can lead to the activation of signaling cascades involved in several disease processes. It is sufficient for a given tissue to be comprised of less than 20% of senescent cells to exert SASP-mediated systemic effects [79]. The gamut of downstream effects initiated by SASP include tumorigenesis (paracrine), immunomodulation (paracrine), senescence amplification (paracrine and autocrine), transformation of the tissue microenvironment (paracrine), and perturbation of stem cell niche [80]. Continued exposure to senescent cells has been shown to potentially promote senescence in healthy bystander fibroblasts via gap junctionmediated cell-cell contacts and processes implicating ROS [81]. While a deluge of evidence points out to the role of ROS in inducing senescence, Lawless et al. employed an empirical stochastic step model of replicative senescence to suggest that increased mitochondrial ROS generation in senescent cells is a repercussion of SASP rather than the reverse [82]. The proteins of the SASP component are broadly conserved, although differences exist among cell types [76]. The contribution of non-protein factors such as nucleotides, bradykines, prostenoids, or ceramides to SASP's effects is also relatively unexplored. The composition of the SMS may vary dynamically with time after the initiation of senescence and partly depends on the senescence-inducing mechanism. This 'late-induced' senescence phenomenon propagates by forming cytoplasmic chromatin fragments with DNA lesions that eventually undergo lysosome-mediated proteolysis causing overall histone loss, thereby illustrating perpetual genomic and epigenomic remodeling in senescence [76]. Beneficial effects of SASP The SASP is fundamentally a response to genomic damage that allows damaged cells to communicate their compromised state to neighboring cellsand stimulates tissue repair and/or regeneration after insult by attracting immune cells and inducing local inflammation. However, it has been indicated that senescence is not consistently followed by immune cell infiltration and inflammation [83] as observed in highly persistent senescent cells in human melanocytic nevi. The SASP also includes several chemokines and cytokines that activate immune cells to specifically target and eventually clear senescent cells from tissues [84,85]. SASP factors such as IL-6, IL-8, protease inhibitor plasminogen activator inhibitor-1 (PAI-1), and pleiotropic protein insulin-like growth factor binding protein-7 (IGFBP-7) are also im-plicated in tumor suppressive growth arrest of cells [75,86,87]. The SASP-produced MMPs also limit fibrosis during wound healing [88] or following liver injury [89,90]. Additionally, developmentally programmed and transiently-regulated senescence is established to be vital in promoting tissue remodeling, embryogenesis and patterning [91][92][93]. Finally, the deterioration of the immune system with age coupled with the kinetics and efficiency of senescent-cell clearance determines the switch of senescence from a temporal programmed process to a persistent stochastic response. The persistent stochastic response involves a multitude of concurrent signals that are paramount to the progression of chronic age-related pathologies. SASP in disease Cellular senescence and consequently SASP factors are involved in several acute and chronic pathological processes (Fig. 2). In a murine model, this association was supported by the delayed onset of agerelated diseases when senescent cells were genetically ablated [94]. Considering that the SASP components differ in character from cytokines to growth factors and proteases the effects they can convey individually or more importantly collaboratively in different tissues may vary as well. Byun et al. have proposed that senescent cells have a definitive role in age-associated pathologies including their ability to modulate young cells through 1) release of inflammatory SASP factors i.e. IL-1; 2) secretion of autocrine or paracrine factors that modulate the activity of other senescent cells or disease-prone young cells (EMT activation by IL-6 and IL-8); 3) expression of SASP receptors (IL-17); and 4) release of anti-inflammatory molecules such as IL-10 [95]. The SASP can promote distinct disease pathologies by perpetuating a proinflammatory state and promoting cell transdifferentiation. For example, various cardiovascular risk factors including obesity, hypertension, diabetes and atherosclerosis that compromise metabolism are associated with an inflammatory state and increased cellular senescence and SASP [96]. Vascular calcification, another risk factor for cardiovascular disease, is linked to a SASP-driven osteoblastic transdifferentiation of senescent smooth muscles cells [97], while atherogenesis Schematic representation of diseases propagated by age. Cellular senescence and the associated secretory phenotype have been implicated in initiation and progress of several acute and chronic pathological maladies driven by age-associated inflammation (inflammaging) and oxidative stress-mediated damage. AMD -Age-related macular degeneration; COPD -Chronic obstructive pulmonary disease). Although cellular senescence can serve as a tumorigenic restrictive mechanism, it can also promote tumor growth and tumor invasiveness, through SASP-mediated regulatory effects [104,105]. A known pathway for SASP-promoted cancer metastasis is the induction of epithelial to mesenchymal transition (EMT); a process that can have both beneficial wound healing effects, and pathological fibrotic and invasiveness-promoting ones [106]. EMT is also a crucial process that can elicit fibrosis in Crohn's disease and graft loss after kidney transplantation [107,108]. Moreover, decreased renal function due to both acute and chronic kidney injury has also been associated with cellular senescence and SASP [109,110]. Though the SASP signature of specific pathological processes still remains to be elucidated, great attention is increasingly being paid to the effects of both biological and stress-induced senescence in different tissues. Both the beneficial and deleterious effects of SASP suggest a possible use of the implicated factors as therapeutic targets for disease control. In addition, the oxidative stress-mediated regulation of biological aging and age-associated pathologies has become a promising target for therapeutic strategies against age-associated maladies. Here, we review the recent progress in understanding the role of the oxidative stress and the cellular redox environment in senescence, with a focus on redox-control of senescence-regulatory factors. The effects of oxidative stress on cellular senescence at a cellular and nuclear level are depicted in Figs Mammalian target of rapamycin pathway The mammalian target of rapamycin (mTOR) protein is a highly conserved large protein kinase of the phosphatidylinosital 3 kinaserelated kinase (PI3KK) family, and an intracellular target of rapamycin, a pharmacological immunosuppressant approved by FDA (Food and Drug Administration, USA) in therapies against various types of cancers [111,112]. The conserved TOR complexes, mTORC1 and mTORC2, can be differentiated by their distinct associated proteins, Raptor and Rictor, respectively. mTOR complexes play crucial roles in mediating nutrient signaling, autophagy and growth regulation (mTORC1) and also regulate cell survival and spatial organization of the cytoskeleton (mTOR2) [113,114]. Inhibition of mTORC1 by rapamycin may both counter damageinducing senescence mechanisms and enhance repair pathways, and as a consequence, result in extended lifespan in model organisms [115]. Over the past decade, the impact of mTOR signaling on cellular aging has gained traction with several studies performed in multiple cellular systems that argue for a pivotal role of mTOR in the senescent program [112,116]; however, little is known with respect to how cellular redox state contributes to mTOR regulatory function in senescence. Cellular stresses, such as hypoxia, or low energy status, which can be sensed by the AMP-sensitive kinase (AMPK) inhibit mTOR signaling [111]. Conversely, elevated mTOR activity in response to excess nutrients leads to increased oxidative stress by enhancing mitochondrial oxygen consumption. Cysteine oxidation enhances phosphorylation of S6K, which is a direct readout of mTORC1 activation [117]. Conversely, treatment with reducing agents effectively suppresses mTORC1 activation via inhibition of S6K phosphorylation. Furthermore, oxidants destabilize the mTOR-Raptor interaction but not the mTOR-Rictor interaction suggesting that a redox-sensitive mechanism may underlie amino acid-dependent mTORC1, but not mTORC2 regulation [118]. These studies also indicate that the phosphorylation of tuberous sclerosis complex (TSC2) tumor suppressor protein regulates Rheb GTPase activity which plays an essential role in mTORC1 regulation in response to cellular redox potential. GRp58 (also known as ERp57), a widely expressed protein disulfide isomerase regulating protein-protein interactions through a redox-based mechanism acts via its two thioredoxin-like domains and has shown to be an mTOR-interacting protein [119]. These findings suggest that a redoxsensitive mechanism regulates the activity of mTORC1, and the interaction between Raptor and proteins with redox-sensing abilities are potentially involved in the assembly and regulation of mTOR complexes. In contrast, Liu et al. demonstrated that mTOR regulation under hypoxic conditions is independent of cellular redox change [120] as H 2 O 2 -scavenging by catalase failed to attenuate the hypoxic hypophosphorylation of p70S6K and 4EBP1. More recently, regulation of the mTORC2 complex formation and stability has been demonstrated to be redox sensitive [121], as knockdown of p22phox inhibits NADPHdependent superoxide generation and decreases downstream Rictorassociated mTORC2 complex activity without affecting Raptor-associated mTORC1 complex. This likely occurs through redox-control of Rictor expression and its subsequent interaction with mTORC2. mTORC1 also regulates mRNA translation and protein synthesis under conditions that favor growth, and there is evidence to suggest that regulation of mRNA translation can modulate longevity in several model organisms [122]. The cellular redox status may likely play a role in global reduction in mRNA translation through TOR signaling and would be advantageous to the aging process allowing for better maintenance of protein homeostasis. Taken together, these observations indicate that approaches that exploit the redox-sensitive nature of both mTOR complexes might prove useful in the design of therapeutics that target age-associated disorders. IL-1alpha Pro-inflammatory members of the interleukin-1 (IL-1) family of cytokines (IL-1α and β) are important mediators of the host defense response to infection, but can also aggravate inflammation that is central to the progression of many disease processes [123]. IL-1α, one of the upstream regulators of SASP, is a dual-function cytokine which is rapidly expressed not only upon stimulation but is present constitutively in healthy cells. It is synthesized as a precursor protein that is subsequently cleaved into two functional fragments that have distinct subcellular localizations by proteases such as calpain [124], CTL/NKgranzyme-B, mast cell chymase, or neutrophil elastase [125]. However, IL-1α cleavage by calpain is rare and only serves to increase the potency of the mature form of the protein in activating pro-inflammatory mediators of SASP. Basal and tumor necrosis factor (TNF)-induced expression of IL-1α has been shown to be regulated by superoxide via modulation of mitochondrial superoxide dismutase (SOD2) that in turn influences the cellular redox homeostasis [126]. Their findings indicate a critical role for a superoxide-reactive intermediate of mitochondrial origin in regulating IL-1α mRNA stability both constitutively and in response to TNF. The IL-1α proximal promoter contains binding sites for the transcription factor AP-1 [127], that has previously been established to be activated by an H 2 O 2 -dependent mechanism [128], again suggesting the redox-dependency of IL-1α induction. In exploration of the mechanistic drivers of IL-1α, redox-engineered fibrosarcoma cells were used to demonstrate that the cellular redox state mediates IL-1α expression and processing through the Ca 2+ dependent protease calpain and alterations in calcium (Ca 2+ ) homeostasis [129]. Steady state levels of intracellular hydrogen peroxide (H 2 O 2 ) significantly promoted IL-1α expression and nuclear translocation, where it serves as a pro-inflammatory mediator by increasing NF-κB activity, thereby further delineating the role of IL-1α in ROS-mediated inflammation. Similarly, in serially cultured senescent human fetal lung fibroblasts, the redox-dependent processing of IL-1α by calpain has been shown to be critical to the regulation of SASP [70]. Senescence-associated upregulation of the mature form of IL-1α promoted metastatic progression of neighboring healthy epithelial cells, triggering an epithelial-mesenchymal transition and was reversed by antioxidant treatment, implying a redox-driven control mechanism. More recently, Laberge et al. have shown that mTOR drives the SASP and that treatment with the mTOR inhibitor rapamycin partly suppresses the secretory phenotype and tumor-promoting ability via translational inhibition of IL-1α [130]. Despite its role in the regulation and amplification of SASP, there exist few studies that describe aberrant activation mechanisms for IL-1α, thus understanding these mechanistic controls is necessary to allow successful therapeutic intervention to reduce the deleterious effects of the normal aging process. Matrix metalloproteinase regulation Matrix metalloproteinases (MMPs) are a group of endopeptidases with important roles in wound healing, tissue remodeling and cellular growth [131,132]. Abnormally high MMP expression is associated with age-related and chronic diseases such as cancer, Alzheimer's, atherosclerosis, osteoarthritis, and lung emphysema [132]. The mitochondrial redox control of MMPs has been previously reported, where ROS are shown as key regulators of MMPs including MMP-1, −2, −7, and −9, [133][134][135] while also being upregulated by high levels of MMP-2 [136]. In particular, the association between MMP-1 and ROS and the significance of these interactions in degenerative disease etiology is well established. MMP-1, cleaves the structural connective tissue components collagen type I, II and II, and is detected only at low levels under physiological conditions, but can be abnormally high in several ageassociated pathologies [132,137]. The increased expression of MMP-1 can be both age-and ROS-dependent [128,138,132]. ROS and age associated MMP-1 expression involves the recruitment of the primary transcription factors Ets-1/AP-1 (c-Jun/c-Fos) to the distal promoter region of MMP-1, along with the differential recruitment of chromatinmodifying proteins P/CAF and HDAC2. These coordinated events mediated by ROS are responsible for induction of an active MMP-1 promoter complex. Multiple studies also show that while oxidants such as H 2 O 2, and antioxidant inhibitors such as aminotriazole can enhance MMP expression [139], treatment with antioxidant agents such as catalase, Vitamin A, Vitamin E, trans retinoic acid, resveratrol, and Nacetyl cysteine can block or decrease MMP expression in different cell lines [140][141][142][143][144][145][146]. More recently, ROS-activated MMPs in the wall of cerebral vessels has also been shown to be exacerbated with age in a mouse model that recapitulates cerebromicrovascular alterations present in elderly humans [147], adding to the growing body of literature supporting age-associated redox-driven MMP induction. This redox sensitive regulation of MMPs provides a definitive rationale for the use of antioxidant-based therapies in the treatment of degenerative disorders associated with aberrant matrix destruction propagated by age. IGF Signaling and FOXO regulation The insulin/insulin-like growth factor-1 (IGF-1) signal transduction pathway controls various proliferative, survival and metabolic signaling networks. IGF-1 in association with its downstream effectors was the first evolutionarily conserved longevity regulatory network identified from yeast to humans [148,149]. Impaired insulin/IGF-1 signaling lowers the oxidative burden and the associated oxidative damage in cells which contributes to increased longevity [150][151][152]. Forkhead box O (FOXO) transcription factor family members (FOXO1, FOXO3a, FOXO4, and FOXO6 in mammals; DAF-16 in C. elegans; DFOXO in D. melanogaster) are evolutionarily conserved AKT substrates that regulate genes involved in cell cycle, apoptosis, DDR, metabolism, oxidative stress defense mechanisms, and aging [153]. The activity of FOXO proteins is primarily regulated by signaling in response to IGF receptor engagement in a redox-dependent fashion [154]. FOXO family members also confer protection from oxidative stress through transcription of antioxidant genes, SOD2, catalase, peroxiredoxin 3, and members of the sestrin family [155][156][157]. The ROS-induced formation of cysteine-thiol disulfide-dependent com-plexes of FOXO with the p300/CBP (CREB-binding protein) acetyltransferase has also been implicated in the modulation of biological activity of FOXO4 [158]. Overall, FOXOs serve as stress sensors and appear to influence both pro-(inducing senescence and apoptosis) and anti-aging (ROS scavenging) mechanisms. The insulin/IGF-1 pathway turns from a growth-promoting regulator in early stages and into a senescence-inducing pathway during the later stages of life [169,170]. A detailed characterization of the regulatory role of ROS in insulin/IGF-1-mediated signaling at all stages in life is crucial to design therapeutics targeting age-related disorders. Calcium (Ca 2+ ) Calcium is a ubiquitous secondary signaling molecule regulating a disparate range of physiological process, including muscle contraction, gene transcription, and cell proliferation [171]. Comprehensive reviews by Ureshino et al., and Farfariello et al. summarize the Ca 2+ signaling networks that may mediate senescence via multiple regulatory pathways including proliferation and cell cycle arrest, apoptosis, autophagy, alterations in membrane potential, altered telomerase activity, and SASP [172,173]. The role of Ca 2+ as a key signaling intermediate involved in SASP regulation has only recently emerged [70]. Ca 2+ signaling is central to driving morphological changes that are the hallmarks of senescence. Senescence-associated increase in cell volume is normally countered by regulatory volume decrease (RVD) which preserves the structural integrity of the cell [174]. Senescenceassociated cell volume changes lead to membrane stretching or osmotic swelling-based activation of TRP vanilloid 4 (TRPV4) or TRP melastatin 7 (TRPM7) channels that mediate Ca 2+ influx from the extracellular space, subsequently activating RVD counter-regulatory signals [175,176]. Mitochondrial Ca 2+ importers maintain homeostatic regulation of the electron transport and increased Ca 2+ influx decreases overall ATP production, elevating cytosolic NADH, decreasing sirtuin activity leading to senescence [177]. Similarily, recent work by Wiel et al., identified two calcium channels, inositol 1,4,5-trisphosphate receptor, type 2 (ITPR2) localized in the ER and the mitochondrial calcium uniporter (MCU), as novel regulators of senescence program by increasing Ca 2+ uptake [178]. In aged human fetal lung fibroblasts, senescence-associated increases in basal intracellular Ca 2+ is implicated in downstream activation of the Ca 2+ -dependent protease calpain, and the subsequent regulation of IL-1α [70]. Treatment of senescent fibroblasts with BAPTA-AM, a Ca 2+ chelator or calpain inhibition reduces the senescence-associated expression of IL-8 and IL-6, respectively. These observations suggest there exists both a redox and Ca 2+ -dependent process in the regulation of the prominent SASP factors IL-6 and IL-8. During OIS and replicative senescence, ITPR2 triggers Ca 2+ release from the endoplasmic reticulum (ER), with subsequent mitochondrial Ca 2+ accumulation via MCU channel, leading to reduced mitochondrial membrane potential, ROS accumulation, and senescence. Conversely, intracellular Ca 2+ reservoirs in the mitochondria are released in response to stress, and facilitate activation of cAMP-responsive element binding protein 1 (CREB) that upregulates p21 CIP1/WAF1 expression, thereby inhibiting cell proliferation [179]. Sharov et al. have shown that age-dependent loss in approximately 2.8 mol of cysteine /mol of sarcoplasmic reticulum (SR) Ca 2+ ATPase (SERCA) as a result of oxidation, may in part be responsible for the age-dependent decline in SERCA activity by about 40% [180]. In a similar model employing young (5 month old) and aged (21 month old) transgenic mice, myocyte-specific catalase overexpression limited oxidative modification of SERCA and preserved SR Ca 2+ , SERCA activity and myocyte relaxation [181]. The ability of catalase overexpression to preserve SERCA activity strongly suggests that the post-translational modifications of SERCA in the senescent heart are H 2 O 2 -dependent. Though the role of Ca 2+ dysregulation in physiological and organ-specific aging processes has been explored, further work is needed to decipher the connection between Ca 2+ dynamics and oxidative damage-induced senescence. Sirtuins Silent information regulator (Sir2) genes and their functional orthologs are conserved from bacteria to humans and constitute a family of NAD + -dependent protein histone deacetylases (HDACs) called sirtuins (SIRT). Initially described as gene silencers, sirtuins are implicated in a wide variety of physiological processes including apoptosis, lipid metabolism, cellular response to stress, mitochondrial biogenesis, fatty acid oxidation, insulin production, and inflammation. SIRT regulates the lifespan-extending effects of caloric restriction improved glucose tolerance, inhibit of degenerative disorders, improve endothelial function, promote regression of atherosclerotic plaques and prevent cancer [182]. The sirtuins class III family is comprised of seven human sirtuins (SIRT1-7) that exhibit deacetylation, demyristoylase, ADP-ribosylation, lipoamidase, demalonylation, desuccinylation, or deglutarylation activity depending on the substrate. SIRT 1-7 are localized in various sub-cellular compartments in mammalian cells: SIRT1, 6, and 7 are localized mainly in the nucleus [183][184][185], SIRT2 in the cytoplasm [186], SIRT3, 4, and 5 are active in the mitochondria [187]. As sirtuin activity requires NAD + as a cofactor, cellular redox status-based NAD + /NADH ratio can further affect sirtuins [188] at the transcriptional or post-transcriptional level of activity [189,190]. Recent work has established a role for some sirtuins in cellular senescence. SIRT1 is the most widely studied and well-characterized sirtuin family member and is involved in the regulation of DNA repair and apoptosis, mitochondrial biogenesis, glucose and insulin homeostasis, and cell stress responses. However, our knowledge pertaining to the molecular redox control of SIRT1 is limited. A pro-oxidative shift in the cellular environment has been shown to induce SIRT1 expression in neural progenitors both in vitro and in vivo [191]. Additionally, the SIRT1 promoter is directly trans-activated by hypoxia-inducible factor (HIF) transcription factors in response to a hypoxic environment [192]. SIRT1-mediated protection against oxidative stress-induced cellular damage involves induction of SOD2 expression via deacetylation and modulation of FOXOs, p53, p21, and proteins involved in DNA damage and repair in distinct cell types [193][194][195]. This fits well with the observation that oxidative stress promotes acetylation of p53 by removing the inhibitory effect of SIRT1 on p53, activating the downstream target p21, and inducing premature senescence followed by pro-tumorigenic IL-6 overexpression [196]. A similar trend is observed in the interaction of SIRT1 with RelA/p65 protein in the NF-κB complex which deacetylates lysine 310 and potentiates the transactivation capacity of the NF-κB complex [197]. Exposure to cigarette smoke oxidatively decreases SIRT1 levels and concomitantly increases NF-κB-dependent release of pro-inflammatory mediators in MonoMac6 cells and in mouse lungs [198,199]. The role of SIRT1-FOXO3 axis in cellular adaptation to hypoxia-associated oxidative stress has also been elucidated by Kume et al. who demonstrate that hypoxia inhibits SIRT1 activity and prevents nuclear translocation of FOXO3, subsequent autophagy, and cell cycle arrest leading to an increase in mitochondrial oxidative damage in kidneys of aged mice [200]. Interestingly, there are ambiguities in the effect of SIRT1 on FOXO activity with variations according to gene or function; SIRT increases transcription of genes involved in cell cycle arrest and resistance to oxidative stress and represses genes promoting FOXOdependent cell death [201]. Similar to the studies described above, ionizing radiation-induced ROS also promotes cellular senescence in articular chondrocytes by negative post-translational regulation of SIRT1 through the ROS-dependent activation of p38 [202]. In contrast, SIRT1 has been implicated in promoting oxidative stress through IRS-2/Ras/ERK signaling downstream of insulin/IGF-1 receptors [203]. There also is a strong link between the insulin/IGF-1 longevity pathway and SIRT1 activity in senescence. The IGF-1/SIRT1/p53 interplay involves prolonged IGF-1 treatment which inhibits SIRT1 deacetylase activity, resulting in increased p53 acetylation, stabilization, and activation leading to premature cellular senescence [204]. Ectopic expression of SIRT1 effectively abrogates IGF-1-induced cellular senescence, thereby linking IGF-1-SIRT1-p53 to cellular senescence. Recently, SIRT1 has also been shown to dissociate chromatin structure and epigenetically repress the expression of SASP factors IL-6 and IL-8 [205]. However, oxidative stress or DNA damage-mediated can dissociate SIRT1 from the promoter regions of IL-6 and IL-8 increasing their levels systemically and further expanding senescent cell populations. The RNA-binding protein HuR has been shown to stabilize SIRT1 mRNA by binding its 3′ untranslated region in a manner that is inhibited by redox-dependent post-translational modifications. Overall, alterations in SIRT1 activity in response to oxidative stress appear to promote genomic instability, enhance inflammation and senescence and offers an additional target for therapeutics that limit the process of senescence. SIRT3 acts as a tumor suppressor gene, owing to its deacetylation role which enhances the activity of two significant targets, SOD2 and isocitrate dehydrogenase 2 (IDH2), the latter of which generates NADPH for the glutathione pathway of ROS detoxification [206][207][208]. SIRT3 is also essential for the caloric restriction-mediated mitigation of oxidative damage by regulating the glutathione antioxidant system, thereby regulating senescence-associated pathologies propagated by oxidative stress [209]. Lack of SIRT3 leads to activation of Akt signaling suggesting a potential role in increasing cellular ROS levels and as a driver of senescence, by affecting mitochondrial function and ATP generation, ROS detoxification, and the mitochondrial unfolded protein response (UPR). Both SIRT3 transcript and protein levels are induced by the generation of mitochondrial ROS [210]. In mice fed with high fat diets for a prolonged 12-week period, evidence of hepatic redox stress, and an associated reduction in SIRT3 transcript and protein levels has been established [211]. SIRT3 is involved in cancer progression, neurodegenerative and cardiovascular disease as well as metabolic syndromes [212], and is likely a critical participant in many age-related disease processes. SIRT6 is another key member of the sirtuin family with varied functions in DNA damage repair, maintenance of genomic stability, inflammation, tumor suppression and participates in longevity regulation [213]. The promise of employing targeting strategies against SIRT6 for limiting age-associated disease processes by impeding cellular senescence and delaying premature aging [214] or through apoptosis engagement has been established [215]. Recently, Pan et al. demonstrated that SIRT6 deficiency in human adult stem cells promotes premature and accelerated cellular senescence and is accompanied by elevated ROS, disrupted redox homeostasis, and increased sensitivity to oxidative stress. SIRT6 complexes with and transcriptionally coactivates nuclear factor erythroid 2-related factor 2 (NRF2), a critical regulator of antioxidant responses, thereby modulating stem cell redox homeostasis, providing a unique target for constraining this senescence-associated oxidative-stress based stem cell attrition [216]. The best-known actions of SIRT6 as a tumor-suppressing protein include its inhibitory effects on c-Jun by blocking IGF-Akt signaling, and Myc, while suppression of chronic inflammation is achieved by repressing TNF-α and NF-κB activity [217], thus enabling varying levels of senescence enforcement. Telomere maintenance The ends of linear eukaryotic chromosomes are protected from damage by the inclusion of tandemly repeated DNA sequences and specialized proteins, called telomeres. In mammalian species, the telomeric sequence TTAGGG extends in a 5' to 3' direction with a Tloop structure at the end comprised of a G-rich 3' overhang and associated proteins that offers telomeric protection. The repeats and the bound proteins form a complex structure called sheltrin, that camouflage chromosomal ends from being recognized as damaged subsequently preventing DNA end-joining, recombination, and repair processes. Over the course of cell division, chromosomal attrition occurs, as the 5' to 3' nature of the DNA replication machinery results in incomplete replication of the lagging parent strand. In eukaryotes, this is resolved by the DNA polymerase enzyme telomerase, which adds telomeric repeat sequences to the ends of chromosomes de novo and compensates for their erosion. Telomere-associated proteins may also localize to other sub-nuclear or sub-cellular sites, and interact with other associated proteins to regulate telomere length [218]. Senescence is usually triggered when the terminal restriction fragment of telomeres shorten from an average length of 15-20 kb to 4-7 kb, by contributing to a persistent DDR that propagates and maintains senescence-associated proliferation arrest, with dysfunctional telomeres, and damaged nuclear foci termed DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS) [219]. While telomere attrition has mostly been attributed to senescence induction, certain studies suggest that DNA damage, and the ensuing damage to telomere structure may be sufficient to trigger senescence independent of telomere shortening [220,221]. Also, the complex molecular T-loop structure at the tips of telomeres that are modulated differently in different cell populations, may also actively determine the cellular proliferative capacity, independent of telomere length. Comprehensive evidence establishes the theory that progressive telomere loss contributes to replicative cellular senescence and is an indicator of age-related disease. Telomere maintenance is regulated by genetic factors and is additively determined by telomerase activity, cell turnover rate, and stress-inducing non-genetic environmental elements. The G-rich composition of telomeres renders them highly susceptible to oxidative damage with the formation of oxidative damage lesions 8-oxo-2'-deoxyguanosine (8-oxo dG) [47,222] which can induce telomere uncapping in a cell cycle-dependent manner by titrating out essential factors such as protection of telomeres protein 1 (POT1). POT1 protects against cellular senescence by promoting telomere elongation, 3′ overhang protection and inhibits chromosomal aberrations [223]. Telomeric DNA is defective in the repair of oxidantinduced single-strand breaks, therefore telomere shortening resulting from unrepaired or residual oxidative damage determines the occurrence of mutations, and unrepaired nucleotides that interfere with the replication fork [53]. This persistent stress-induced telomeric damage is characteristically independent of telomerase activity and telomere length. The repair deficiency in telomeres can be attributed to the binding of telomeric repeat binding factor 2 (TRF2) that makes the strand breaks inaccessible to DNA repair enzymes [224], inhibits ATM kinase phosphorylation [225], and interacts with polymerase β [226], thereby negatively impacting DNA repair. This also suggests that telomere-driven senescence occurs as a potential tumor-suppression mechanism in response to accumulating genomic damage, and not after a set number of cellular divisions as clocked by telomeres. Mechanistically, telomeric foci contain multiple DDR factors signaling through ATM to p53, up-regulating p21 and causing cell cycle arrest at the G1 phase. Telomere-and DNA damage-independent up-regulation of p16 was also observed, suggesting that distinctive senescence effector pathways are not mutually exclusive and can develop in parallel, resulting in a mixed cell population, or an individual cell responding to collective signals [42]. Recently, analysis of 3D chromosomal architecture using combined biochemical, cytological, and computational techniques has revealed significant decreases in volume, and increases in chromosome compaction during cellular senescence [227]. This unique senescent chromosomal signature is attributed to an increase in short-range chromatin contacts and genome-wide contraction of chromosome arms. It is hypothesized that the structural 3D organization of chromosomes may regulate senescence-associated cell cycle arrest. Mathematical models have been employed to quantitatively predict the role of telomere shortening in senescence. One such model assessed rates of telomere loss occurring from oxidative damage and activity of C-strand specific exonuclease, and the possible interactions between these two mechanisms [228]. The model predicts that under lower oxidative burden the end-replication problem and C-strand processing are major contributors to telomere loss; however under normoxic or hyperoxic conditions, it is predicted that single strand breaks induce shortening. Another model incorporates a shortening factor and autonomous telomere loss and assumes that senescence incidence is proportional to telomere length [229]. A simple negative feedback regulation model of telomere reduction is proposed, suggesting that telomere erosion is dependent on telomere length. Ectopic expression of telomerase alone [230], or in combination with p16 INK4A /pRb inactivation [231] can also prevent telomeric lossdependent senescence in primary fibroblasts and extend replicative lifespan. Likewise, studies suggests that loss of telomeres correlates with depletion of antioxidant enzyme levels and decreases in oxidative burden by antioxidant treatment might limit the telomere shortening rates and delay cellular senescence. However, a critical review and meta-analysis on human data performed by Simons reveals no causal involvement of telomere length in the aging process, suggesting that thorough investigation of the mechanistic in vivo role of telomeres is crucial to assess their function in senescence [232]. Tumor suppressors p53, p21, p16 The G1 phase of the cell cycle is the main checkpoint at which DDR pathways activate cellular senescence as a protective mechanism to induce cell cycle arrest. [38,233]. Both the p16 INK4a /pRB and p53/p21 tumor suppressor pathways induce cell growth arrest through senescence activation [234,235]. Both p16 and p53 display compensatory interactions [236,237] and both have a role in senescence initiation and maintenance [79,238,239]. p21 and p16 are cyclin-dependent kinase inhibitors involved in preventing cell cycle progression by preventing pRB phosphorylation [37,240]. In general terms, DNA damage activates DDR, a p53/21dependent response that is originally transient but which can become persistent if the damage is severe enough, and which can also be reversed through the p16/pRB pathways [219,241]. After DDR activation, the p53/p21 pathway deactivates the cyclin E-CDK2 complex responsible for pRB phosphorylation [242], while the p16/pRB pathway inhibits pRB phosphorylation by deactivation of kinase CDK4/6 [239]. Preventing pRB phosphorylation allows pRB to retain its association with the transcription factor E2F1, preventing the expression of E2F1 target genes that are necessary for the cell transition from G1 to the S phase [239]. p53 has been shown to both promote and inhibit cellular senescence [243,244] partly through its up and down regulation of ROS, and its effects on mTOR. Also, a positive feedback loop between p53 and ROS has been shown to promote senescence in human fibroblastsa loop that can be counteracted by the cell cycle mediator cullin-4B (CUL4B) via p53 ubiquitination in stressed cells [245]. Moreover, ROS promotes p53 acetylation and induction of premature senescence by inhibition of the deacetylase SIRT1 in fibroblasts [246]. In epithelial cancer cells, the transcription factor FOXp3 accelerates p53-mediated senescence while also increasing ROS levels and p21 expression, demonstrating the intricate link between ROS and these pathways [247]. It has also been suggested that ROS can induce senescence through activation of the p16 INK4a /pRB pathway by activating p38 MAPK and extracellular regulated protein kinase (Erk) [248], although in extrinsically induced senescence models, the same pathway was engaged [249]. While there is evidence of the regulatory role of ROS in the p16 INK4a /pRB and p53/p21 pathways that lead to senescence and certain pathologies, much remains to be elucidated to fully understand the implication of oxidative stress as both an effector and affected agent in cellular senescence. Summary The complexity of the aging process and the drive to comprehend its molecular underpinnings, steers focus towards approaches to improve organismal healthspan and lifespan. Despite the transition from cell-culture curiosity to a potential regulator of cancer and aging, cellular senescence remains enigmatic and continues to raise a variety of complex of questions. Oxidative stress plays a major role in several pathways that lead to cellular senescence and the associated secretory phenotype. Although initially intended to be a protective response to oncogenic insult, cellular senescence induced by DNA-damaging agents and ROS, can potentially elicit harmful effects and can drive many agerelated diseases. While the ROS-driven SASP has proven to be beneficial in certain conditions, it is also associated with perpetuating a pathological inflammatory state. The ability to genetically ablate senescent cells has clearly established a causal role for senescence in many degenerative disease processes, and while there is much unknown with respect to the role of oxidative stress in such processes, the evidence compiled here suggests that precise ROS-mitigation may limit senescence and senescence-associated pathologies. However, translation of these findings into relevant human interventions is at present restricted due to our incomplete understanding of both the basic molecular biology of senescent cells in vivo and the etiology of senescence-associated pathologies. While the long-standing notions of the free radical theory of aging postulating a causal role of ROS in the aging process (Harman, 1956) seem to fit with several studies, there are a number of observations that suggest that the cellular effects of ROS, with regard to inducing senescence, do not unequivocally translate into organismal aging. Therefore, efforts should be made to escalate research directed at resolving the basic biology of cellular aging and the redox-control of these mechanisms to generate integrated therapeutic strategies to improve human health and lifespan.	2018-04-03T00:44:23.038Z	2016-11-16T00:00:00.000	{ "year": 2016, "sha1": "88a1532c53ac90ce1066eee4450d42839d38809d", "oa_license": "CCBYNCND", "oa_url": "https://doi.org/10.1016/j.redox.2016.11.005", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "fcd434025e2f192dc8046bee420cba620208a119", "s2fieldsofstudy": [ "Biology" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }
16060604	pes2o/s2orc	v3-fos-license	Dominant Negative Effect of Mutated Thyroid Stimulating Hormone Receptor (P556L) Causes Hypothyroidism in C.RF-Tshrhyt/wild Mice C.RF-Tshrhyt/hyt mice have a mutated thyroid stimulating hormone receptor (P556L-TSHR) and these mice develop severe hypothyroidism. We found that C.RF-Tshrhyt/wild heterozygous mice are also in a hypothyroid state. Thyroid glands from C.RF-Tshrhyt/wild mice are smaller than those from wild-type mice, and 125I uptake activities of the former are significantly lower than those in the latter. When TSHR (TSHR(W)) and P556L-TSHR (TSHR(M)) cDNAs were cloned and co-transfected into HEK 293 cells, the cells retained 125I-TSH binding activity, but cAMP response to TSH was decreased to about 20% of HEK 293 cells transfected with TSHR(W) cDNA. When TSHR(W) and TSHR(M) were tagged with eCFP or eYFP, we observed fluorescence resonance energy transfer (FRET) in HEK 293 cells expressing TSHR(W)-eCFP and TSHR(W)-eYFP in the absence of TSH, but not in the presence of TSH. In contrast, we obtained FRET in HEK 293 cells expressing TSHR(W)-eCFP and TSHR (M)-eYFP, regardless of the presence or absence of TSH. These results suggest that P556L TSHR has a dominant negative effect on TSHR(W) by impairing polymer to monomer dissociation, which decreases TSH responsiveness and induces hypothyroidism in C.RF-Tshrhyt/wild mice. Introduction The thyroid stimulating hormone receptor (TSHR) is a member of the G protein-coupled family of receptors, whose main function is to regulate hormone synthesis, secretion and cell growth in thyroid glands [1]. TSHR knockout (TSHR-KO) mice have small thyroid glands, and show severe hypothyroidism with no detectable thyroid hormone and elevated TSH [2]. KO mice die within 1 week of weaning, unless fed a diet supplemented with thyroid hormone. In contrast, heterozygotes of TSHR-KO mice have normal circulating thyroid hormone and TSH levels, and are apparently unaffected. C.RF-Tshr hyt/hyt mice (Tshr hyt/hyt mice) represent another model of hypothyroidism resulting from a TSHR mutation in the fourth transmembrane domain. A proline to leucine mutation at codon 556 results in plasma membrane targeting, but defective TSH binding and receptor function [3,4]. Furthermore, it has been reported that the mutation did not alter TSHR expression levels [3]. At present, TSHR is detectable in a variety of cell types, including the adipose tissues [5], bone [6,7], thymus heart, lymphocytes and retro-orbital fibroblasts [1]. Using Tshr hyt/hyt mice, we previously reported that TSHR in brown adipose tissue is involved in the regulation of thermogenesis [8]. In the course of these studies, we found that Tshr hyt/wild mice were also in a hypothyroid state. As heterozygotic TSHR KO mice are unaffected and maintain a euthyroid state, the evidence prompted us to study the reasons why Tshr hyt/wild mice exhibit hypothyroidism. Animals and Cells All studies were approved by the Animal Research Committee of the University of Yamanashi. C.RF-Tshr hty/wild mice were obtained from The Jackson Laboratory (Bar Harbor, ME), and were bred to generate experimental animals. Mice were kept in an SPF mouse room void of thyroid hormone supplementation. All mice were aged 70-84 days at the start of the experiments. Determination of TSHR genotype was carried out as described previously [8]. Rectal temperature was measured using a digital thermometer (TD-300; Shibaura Electronics Co., Ltd., Tokyo, Japan). Fluorescence resonance energy transfer (FRET) HEK 293 cells (10 6 cells) were transfected with 0.5 mg of pcDNA-TSHR(W or M)-eCFP and the same amount of pcDNA-TSHR(W or M)-eYFP by electroporation. After 72 h of culture, using eCFP as a donor (D) and eYFP as an acceptor (A), emission FRET studies in regions of interests (RoI) in single living cell were carried out with or without 1 mU/ml TSH using an Olympus IX80 microscope equipped with emission and excitation FRET filter wheels controlled by Metamorph software (Molecular Devices Japan, Tokyo, Japan). FRET was performed in RoIs Covalent cross-linking of TSHR and Western blot analysis TSHR-expressing cells were cultured in 10 mM carbonate buffer containing 0.15 M NaCl with or without 1 mU/ml TSH for 1 h. Dimethyl suberimidate (DMS) (Pierce, Rockford, IL) was added to the medium at a final concentration of 2 mg/ml, followed by further incubation for 30 min. After addition of Laemmli buffer, samples were electrophoresed on 12.5% SDSpolyacrylamide gels, transferred to nitrocellulose membrane, and stained with TSHR polyclonal antibody (14450-1-AP; Protein-Tech Group, Inc., Chicago, IL) at a dilution of 1:500. Assay for thyroid hormones, 125 I-TSH binding activity and TSH binding activity Serum-free T3 and free T4 levels were assayed using the ECLusis system (Roche Diagnostic Co., Tokyo, Japan). Mouse TSH was assayed using the Rat TSH ELISA kit (AKRTS-010; Shibayagi, Gunma, Japan). 125 I uptake by thyroid glands was measured by administering 10 4 Bq of 125 I-Na (GE Healthcare, Tokyo, Japan) into the peritoneal space. After 24 h, mice were anesthetized with pentobarbital, the thyro-tracheal unit was resected, and radioactivity was measured with a gamma counter (Autowell Gamma System, ARC-380; Aloka, Tokyo, Japan). 125 I-TSH binding activities in the cells were determined using the methods of Mizutori et al. [9] with 125 I-bovine TSH (Cosmic Co., Tokyo, Japan) and bovine TSH (Sigma-Aldrich, Inc., St. Louis, MO). Statistical analysis Statistical analysis was carried out by one-way ANOVA and Student's t-test. Function of and morphological changes in thyroid glands from Tshr hyt/wild mice We previously reported that rectal temperature is a sensitive marker of thyroid status in mice [8]. We noticed that rectal temperature of Tshr hyt/wild mice at room temperature (36.960.20uC) was significantly lower than that of wild-type mice (38.160.19uC) (p,0.001). When Tshr hyt/wild mice were exposed to cold (4uC), rectal temperature rapidly dropped to 30.760.77uC at 120 min, whereas that of wild-type mice was 37.660.71uC (p,0.001) ( Table 1), suggesting that Tshr hyt/wild mice are in an overt hypothyroid state. Thyroid glands in Tshr hyt/wild mice were smaller than those in wild-type mice (Fig. 1a, b). When the mean maximum diameter of the left and right lobes was measured, the value in Tshr hyt/wild mice (2.5760.08 mm, n = 6) was significantly smaller than that in wild-type mice (3.6560.07 mm, n = 7) ( Table 1). Free T4 and free T3 levels in Tshr hyt/wild mice were significantly lower than those in wild-type mice, and serum TSH levels in the former were higher than those in the latter. 125 I uptake activity of Tshr hyt/wild mice was significantly lower than that in wild-type mice. Anti-thyroglobulin autoantibodies were not detected in either Tshr hyt/wild mice or wild-type mice (Table 1). Microscopically, thyroid glands in Tshr hyt/wild mice consisted of larger follicles with thin thyroid epithelial cells (Fig. 1c, d). Collins and Capen reported that thyroid follicular cells became more columnar and follicular lumens became smaller after TSH treatment [10]. These results, as well as thyroid functions, suggest that despite the high TSH concentrations in sera, TSH signals in thyroid glands of the Tshr hyt/wild mice are impaired, which may lead to overt hypothyroidism. Receptor function of P556L-TSHR (TSHR(M)) In order to clarify the mechanisms of hypothyroidism in Tshr hyt/wild mice, we studied the effects of TSHR(M) on the function(s) of wild-type TSHR (TSHR(W)). TSH dose-dependently increased cAMP production in HEK 293 cells expressing TSHR(W), but had no effect in HEK 293 cells expressing TSHR(M) (Fig. 2a), as reported previously by Gu et al. [4]. When equal amounts of pcDNA-TSHR(W) (0.5 mg) and pcDNA-TSHR(M) (0.5 mg) were transfected into HEK 293 cells, cAMP production in response to TSH decreased to about 20% of that in HEK 293 cells transfected with TSHR(W) (0.5 mg) and pcDNA (0.5 mg) (Fig. 2b). Figure 2c shows the 125 I-TSH binding activities of these cells. We observed high-affinity TSH binding activity in HEK 293 cells expressing TSHR(W) (Ka = 35 mU/ml), and no binding was observed in HEK 293 cells expressing TSHR(M). Maximal TSH binding and affinity for TSH in HEK 293 cells coexpressing TSHR(W) and TSHR(M) (Ka = 42 mU/ml) were nearly the same as in those expressing TSHR(W) (Fig. 2c). These results suggest that TSHR(M) has a dominant negative effect on TSHR(W) functions. The protein-protein interactions of TSHR were also observed in a cross-linking experiment. HEK 293 cells expressing TSHR(W)-eCFP and TSHR(W)-eYFP were cultured with or without TSH, followed by cross-linking with dimethyl suberimidate (DMS). Anti- TSHR antibody recognized two bands at 250 and 120 kDa. In the presence of TSH, the 125-kDa band was dominant, but in the absence of TSH, the 250-kDa band was stained more strongly than the 120-kDa band. When HEK 293 cells expressing TSHR(W)-eCFP and TSHR(M)-eYFP were cultured and crosslinking agent was added to the medium, the 250-kDa band was dominant, irrespective of the presence or absence of TSH (Fig. 3f). Discussion The hyt/hyt recessive trait mouse, which provides a reproducible model for studying inherited hypothyroidism, was initially described by Beamer et al. [11]. Hyt/hyt homozygous animals can easily be differentiated by their obviously smaller size, and few differences in size and in serum thyroid hormone levels are observed between the hyt/wild and the wild/wild mice. Thus, many studies, even after identification of a point mutation in the TSHR gene [3], have used hyt/hyt mice as hypothyroid animals, and hyt/wild and the wild/wild groups as euthyroid mice [12,13]. In the present study, we determined the genotypes of Tshr hyt/hyt , Tshr hyt/wild and Tshr wild/wild mice by direct sequencing of the gene, and found that serum-free T4 levels in Tshr hyt/wild mice are significantly lower than in Tshr wild/wild mice. In addition, rectal temperature, size of the thyroid gland, and 121 I uptake activity of Tshr hyt/wild mice indicated overt hypothyroidism. However, these results are inconsistent with the report of TSHR KO mice by Marians et al. [2]. TSHR KO/KO mice show severe hypothyroidism with no detectable thyroid hormone, and die within 1 week. No significant differences in thyroid hormone and TSH, or in growth and development, are observed in TSHR KO/wild mice and TSHR wild/wild mice. As P556L TSHR has been reported to properly integrate into the plasma membrane [4], we hypothesized that this mutant receptor has a dominant negative effect on the wildtype receptor. Indeed, a co-expression study of mutant and wild-type receptors in 293 cells demonstrated that mutant receptors impaired TSH-induced cAMP production by wild-type receptors. Using TSHR differentially tagged with RFP and YFP, Latif et al. previously demonstrated by FRET that TSH-induced activation of the TSHR would result in disruption of the dimer [14]. However, Urizar et al. were unable to confirm these results in a bioluminescence resonance energy transfer (BRET) experiment [15]. We obtained FRET from TSHR(W)-eCFP to TSHR(W)-eYFP only in the absence of TSH, and from TSHR(W)-eCFP to TSHR(M)-eYFP in the presence of TSH. Therefore, our results support the hypothesis of Latif et al. and suggest that the hyt mutation, the P556L mutation in the fourth transmembrane domain, interferes with TSH-induced dissociation of dimer to monomer. As a consequence thereof, our results support the hypothesis proposed by Latif et al. [14] that only monomers should contribute to G protein activation and the release of second messengers. Recently, Latif et al. also reported that a tyrosine residue, Y116, in the extracellular domain of TSHR plays an important role in stabilizing multimer formation of the receptor [16], but the present data suggest that the fourth transmembrane domain also confers monomer-multimer dissociation of TSHR. In addition to entrapment of wild-type TSHR in endoplasmic reticulum by the mutant receptor, which induces TSH resistance in humans [17], this is the first report linking dominant negative mutations of a G protein-coupled receptor integrating in plasma membrane to an abnormal endocrine phenotype in mammals, and provides an explanation for hypothyroidism in C.RF-Tshr hyt/wild mice.	2017-04-13T02:42:51.817Z	2012-08-16T00:00:00.000	{ "year": 2012, "sha1": "f21a49cb752e5f1e336bb243e89e104c3b604faf", "oa_license": "CCBY", "oa_url": "https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0042358&type=printable", "oa_status": "GOLD", "pdf_src": "PubMedCentral", "pdf_hash": "f21a49cb752e5f1e336bb243e89e104c3b604faf", "s2fieldsofstudy": [ "Medicine", "Biology" ], "extfieldsofstudy": [ "Biology", "Medicine" ] }

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