The ovine Major Histocompatibility Complex (MHC) harbors clusters of genes involved in overall resistance/susceptibility of an animal to infectious pathogens. However, only a limited number of ovine MHC genes have been identified and no adequate sequence information is available, as compared to those of swine and bovine. We previously constructed a BAC clone-based physical map that covers entire class I, class II and class III region of ovine MHC. Here we describe the assembling of a complete DNA sequence map for the ovine MHC by shotgun sequencing of 26 overlapping BAC clones.DNA shotgun sequencing generated approximately 8-fold genome equivalent data that were successfully assembled into a finished sequence map of the ovine MHC. The sequence map spans approximately 2,434,000 nucleotides in length, covering almost all of the MHC loci currently known in the sheep and cattle. Gene annotation resulted in the identification of 177 protein-coding genes/ORFs, among which 145 were not previously reported in the sheep, and 10 were ovine species specific, absent in cattle or other mammals. A comparative sequence analyses among human, sheep and cattle revealed a high conservation in the MHC structure and loci order except for the class II, which were divided into IIa and IIb subregions in the sheep and cattle, separated by a large piece of non-MHC autosome of approximately 18.5 Mb. In addition, a total of 18 non-protein-coding microRNAs were predicted in the ovine MHC region for the first time.An ovine MHC DNA sequence map was successfully assembled by shotgun sequencing of 26 overlapping BAC clone. This makes the sheep the second ruminant species for which the complete MHC sequence information is available for evolution and functional studies, following that of the bovine. The results of the comparative analysis support a hypothesis that an inversion of the ancestral chromosome containing the MHC has shaped the MHC structures of ruminants, as we currently observed in the sheep and cattle. Identification of relative large numbers of microRNAs in the ovine MHC region helps to provide evidence that microRNAs are actively involved in the regulation of MHC gene expression and function. The sheep is one of the major domestic animal species for human meat protein, milk, and its wool is a source of industrial fiber. The Major Histocompatibility Complex (MHC) of the sheep, also designated as ovine Lymphocyte Antigen (OLA), harbors clusters of immunological genes involved in overall resistance/susceptibility of the animal to infectious diseases -3. A numStudies of the ovine MHC also help to provide valuable information on comparative genome evolution in mammals. The extreme high level of polymorphism observed for MHC loci may be a result of the evolutionary consequences of intensive interactions between infectious pathogens and the host defensive system . HaplotyNotch4 and Btnl2 was successfully closed by addition of two more overlapping BAC clones [We previously constructed a BAC-clone-based physical map of the ovine MHC for Chinese merino fine-wood sheep , a valueC clones .Here we describe our work on sequencing of the entire ovine MHC by shotgun sequencing of the 26 BAC clones, assembling of the sequence data into a finished DNA sequence map as guided by the physical map, and the sequence analysis that resulted in identification and annotation of 177 genes and 18 microRNAs in ovine MHC region.FJ986852 - FJ985877 . Preliminary experiments confirmed the mRNA transcripts for 4 of the predicted ovine-specific genes (data not shown). The distribution of these novel genes seems to be random throughout the ovine MHC region. It is interesting to notice that a multiple DQ loci (DQ cluster) were identified, each with different orientation of transcription, when compared with those of other sheep breeds [Sequence analysis resulted in the identification and annotation of 177 protein-coding genes/ORFs in the ovine MHC  in the available bovine sequence contributed negatively to the homology score. For virtually any locus currently identified in bovine MHC, a homologous match could be identified in the ovine MHC, including those in the class IIb region Figure . It is nThe hypothesis that cattle and sheep shared an ancestral chromosome was previously proposed in the studies of cattle -44. Detabutyrophilin-like (Btnl ) cluster at the boundary between the ovine class IIa and III  in platypus, and is duplicated extensively in mammals that have more litter sizes. This might be an indication that milk production was closely associated with the function of MHC in mammals, due to the apparent need for mammals to protect their offspring from microbial infections via milk ingestion. Taken together, we propose a hypothesis that, formation of the Btnl loci is associated not only with the gene duplication of immunological loci, but also with the emergence of mammals in evolutionary history.Our sequence analysis also identified a I Figure . Banal ioduction . Comparis Figure . Btnl isA complete ovine MHC sequence map was assembled by successful shotgun sequencing of 26 overlapping BAC clones. This makes the sheep the second ruminant species for which the MHC sequence is available for evolutionary and functional studies. Gene annotation resulted in the identification of 177 genes, among which 145 were identified for the first time, and 10 were ovine-species specific. In addition, a total of 18 microRNAs coding sequences were predicted in the ovine MHC for the first time. Comparative analysis revealed a remarkable conservation of MHC sequence between sheep and cattle, supporting the hypothesis that the two species shared an ancestral chromosome that shaped the ruminant MHC as currently observed. Identification of a relatively large number of micro RNAs in the ovine MHC region helps to provide evidence that micro RNAs are actively involved in the regulation of MHC gene expression and function.E. coli stock containing the target BAC clones were used to prepare the BAC clone DNA, which were solicited to form randomized small DNA fragments between 0.5 - 2.0 kb. After cloning of the small fragments into the plasmids, random DNA sequencing was performed with an ABI 3730 automated DNA sequencers  to generate the randomized short DNA sequence reads.Shotgun sequencing libraries were constructed individually for each of the 26 BAC clones following the modified protocols described by Celera Genomics Group . BrieflyThe short random DNA sequences generated by the sequencing were assembled into full-length sequence utilizing the Prep program  for each of the BAC clones. Resequencing was performed when necessary for gaps detected during the sequence assembly, including sequencing by primer walking of the PCR-amplified fragments for regions showing low level of accuracy. Blast alignments  of the rhttp://genome.lbl.gov/cgi-bin/WGVistaInput.The assembled ovine MHC sequence was analyzed using an automatic Ensemble pipeline  with modKL carried out experiments, data analysis and gene annotation; JFG carried out conformational experiments; HBL and GL carried out construction of shotgun libraries; HTB assisted in manuscript writing, CFC and PPT performed data checking and technical assistance. RZM supervised the studies and wrote the manuscript. All authors read and approved the final manuscript.


Heavy Menstrual Bleeding (HMB) is common and existing treatments often ineffective. The MRC funded DexFEM trial aims to investigate whether oral dexamethasone  reduces HMB.Our integrated approach to the development of a Bayesian response-adaptive dose-finding study uses SAS for data handling, generating scripts and executing analysis in WinBUGS. The clinical aims are twofold: (i) to determine if Dex is efficacious in reducing HMB compared with placebo and, if so, (ii) to identify the optimal dose for further phase III study. Patients are randomised to placebo or one of several active Dex doses. Randomisation probabilities change as outcome data accumulate from patients already recruited. This design ensures that maximum information is gathered on the dose-response curve, and also that fewer women are randomised to inferior doses. Development of an adaptive design cannot invoke standard frequentist concepts of power. We estimate the dose-response curve in a Bayesian Normal Dynamic Linear Model.Several competing design options are considered including: the number of doses; the criterion for adapting the randomisation; the length of the run-in before adaptation commences; the frequency with which randomisation probabilities are adapted. Through extensive simulation work we assess the performance of candidate designs under a variety of plausible scenarios for dose-response curve shape and outcome measure variance, and decide on a final design. Adaptive designs offer flexibility and efficiency and are likely to be used more in the future. Our integrated approach is a practical, powerful tool for the development of such designs.


In western medicine emerging research evidence suggests a distinct psychological/biological phenomenon that manifests itself in 'out of control' sexual behaviour. Historically, this behaviour has been described as hypersexuality and compulsive sexual behaviour. Recently it is popularly called 'sexual addiction'.The aim of this study is to obtain a clear understanding of the diagnostic and treatment issues experienced by healthcare providers who deal with the concept of ´sexual addiction' in clinical practice.A qualitative approach was used. Data collection includes: pilot-study, focus-groups, questionnaires and interviews involving 87 adult participants consisting of 43 treatment providers who work with this phenomenon in clinical practice and 44 self-identified 'sexual addicts'. Ethical approval was obtained from the schools' ethics committee and consent was given by the participants. Interpretative Phenomenological Analysis (IPA) and Thematic Analysis (TA) are used for data analysis.Treatment providers report the presentation of a clinical condition popularly referred to as 'sexual addiction'. The main characteristics of this condition are intense sexual urges, recurring fantasies and out of control sexual behaviour. Treatment providers who clinically deal with this are working in the areas of medicine, sexual health, psychiatry, psychology and psychotherapy. The major clinical challenge is the lack of agreement on criteria and the absence of a scientific classification for this condition. Other concerns include diagnosis, treatment, disclosure and understanding the aetiology of 'sexual addiction'. Additionally, there is a high incidence of dual addiction and co-morbid psychological conditions among those who present with 'sexual addiction'. There is a lack of clarity whether the 'sexually addictive' behaviour is the primary condition or whether it is a symptom of an underlying condition. In addition to a catalogue of negative consequences the 'sexual addict' is vulnerable to contracting sexual disease. Treatment is a multifaceted combination of medication, psychotherapy, 12-step fellowship programmes and education. There is a wide range of opinion among treatment providers on the concept of 'sexual addiction'.Empirical research is required to investigate and critique the concept of 'sexual addiction'. Diagnostic criteria and classification need to be determined. Clinical training is necessary to diagnose, assess and treat this behaviour.


Ophiostoma novo-ulmi continues to be a serious threat to the American elm (Ulmus americana) in North America. Extensive studies have been conducted in North America to understand the mechanisms of virulence of this introduced pathogen and its evolving population structure, with a view to identifying potential strategies for the control of Dutch elm disease. As part of a larger study to examine the genomes of economically important Ophiostoma spp. and the genetic basis of virulence, we have constructed an expressed sequence tag (EST) library using total RNA extracted from the yeast-like growth phase of O. novo-ulmi (isolate H327).The highly aggressive pathogenic fungus A total of 4,386 readable EST sequences were annotated by determining their closest matches to known or theoretical sequences in public databases by BLASTX analysis. Searches matched 2,093 sequences to entries found in Genbank, including 1,761 matches with known proteins and 332 matches with unknown  proteins. Known proteins included a collection of 880 unique transcripts which were categorized to obtain a functional profile of the transcriptome and to evaluate physiological function. These assignments yielded 20 primary functional categories (FunCat), the largest including Metabolism , Sub-cellular localization , Protein synthesis , Transcription , Biogenesis of cellular components , Cellular transport, facilitation and routes , Classification unresolved , Cell rescue, defence and virulence  and the unclassified category, or known sequences of unknown metabolic function . A list of specific transcripts of interest was compiled to initiate an evaluation of their impact upon strain virulence in subsequent studies.O. novo-ulmi transcriptome. The expression profile obtained from the yeast-like growth phase of this species will facilitate a multigenic approach to gene expression studies to assess their role in the determination of pathogenicity for this species. The identification and evaluation of gene targets in such studies will be a prerequisite to the development of biological control strategies for this pathogen.This is the first large-scale study of the  Ulmus americana) has been a favoured urban tree for planners and landscape architects in many North American cities, providing shade along innumerable streets and boulevards. The elm is a particularly popular choice in northern climates because of its resistance to extremes of weather and harsh urban growing conditions, while its abundant crown foliage is large enough to span a city street .A transcription profile was created for the WH was the supervisor for the project, conducted data analysis and database creation and was responsible for the preparation and submission of the manuscript. CB and LB were co-Principal Investigators on this project and assisted with data analysis as well as providing input to the manuscript. The experimental work was conducted by SB, MP and PB. Additional data analysis and revision of the manuscript was done by VJ and RH. All authors have read and approved the final manuscript.Identified genes and FunCat assignments.xls (excel spreadsheet).Click here for file


Coronal fractures of permanent dentition are the most frequent type of dental injury. Fractured anterior teeth are usually treated with conventional post and core and crown techniques, after being treated endodontically. If the original tooth fragment is retained following fracture, the natural tooth structures can be reattached using adhesive protocols. Fiber-reinforced post makes the reattachment of the crown esthetically possible with minimal preparation and reduces the possibility of tooth fracture during function. This paper presents the therapeutic approach of reattachment of crown fragment to the tooth at the cervical and middle third levels. Fracture of anterior teeth by trauma is the most frequent type of injury in the permanent dentition, especially among children and adolescent affecting up to 25% of this patient population . If the Despite the recent advances in adhesive materials and restorative technique there is no restorative material that can reproduce the esthetic and functional needs as much as the natural dental structures . VariousResin-based restorative materials are frequently used in restoration of the fractured teeth. Because of the poor mechanical resistance of these materials, different approaches developed to strengthen the composite resin, such as fiber posts . Tooth-c A crown-root fracture involving the biologic width needs to be approached carefully, as placing the margins within the biologic width frequently leads to gingival inflammation, clinical attachment loss, and bone loss . HoweverA 23-year-old male patient was referred to the Department of Conservative Dentistry and Endodontics, with the complaint of fractured maxillary central incisors because of road traffic accident. The patient's medical history was noncontributory. Initial examination revealed the horizontal fracture on teeth no. 11 and 21, and associated pulp exposure . A diagnAfter local anesthesia, the coronal fragments were separated with minimum force and were stored in saline to prevent dehydration. Single visit root canal treatments were carried out on both the teeth, and only the apical thirds were obturated by gutta percha with sectional obturation technique . The posA 21-year-old female patient attended the Department of Conservative dentistry and Endodontics, 2 days after an automobile accident. The patient had multiple lacerations on the face and fractured crown. After the general medical, dental, and traumatic incident histories were reviewed, clinical and radiographic examinations were conducted. A complicated crown fracture was observed in relation to left maxillary central incisor, tooth no. 21 . The croLocal anesthesia was administered and fractured segment was gently removed and stored in saline. One visit root canal treatment was performed. The fractured segment was reattached as described in case report 1. Since adjacent teeth had mobility, they were splinted with 19-gauge stainless steel wire and light cure composite resin . At the A 14-year-old patient reported to the Department of Conservative dentistry and Endodontics, 2 days after sustaining crown fracture to his maxillary right central incisor during sports activities. After the general medical, dental and traumatic incident histories were reviewed and clinical and radiographic examinations were conducted. Clinical examination revealed a fracture of the middle third of the crown, exposing the pulp with tooth no. 11 . The remLoss of tooth tissue in the anterior region in a young patient may create severe aesthetic and emotional problems . FunctioReattachment of the crown fragment to a fractured tooth influences esthetic by retaining natural translucency and surface texture and is the first choice for crown fractures of anterior teeth. Once the original fragment is reattached, the natural appearance will be restored instantly. Also, this procedure is relatively simple, atraumatic, and inexpensive. This technique requires only a thin layer of composite resin and restores the original form and color of the tooth that often provides the best aesthetic result. Several case reports show that even subgingival tooth fractures can be restored successfully . StudiesIn our cases we have used reattachment technique of the autogenous tooth fragment to the crown, with or without fiber reinforced post. Although the use of pre-fabricated post does not mechanically strength the endodontically treated teeth, but it helps in retention of the coronal restoration .Fiber reinforced post has demonstrated negligible root fracture. In addition, the fiber reinforced post can be used with minimal preparation because it uses the undercuts and surface irregularities to increase the surface area for bonding. Thus, it reduces the possibility of tooth fracture during function or traumatic injury . The useThe rule of nature is to conserve as much of tooth structure as possible. Preservation of the remaining natural and healthy tooth structure will have a positive psychological impact on the patient. With the advancements in the chemistry of dentine adhesives and composite resins having remarkable bond strength, the success rate has been enhanced. Reattachment of fractured fragment and reinforcing it with fiber post is a viable technique that restores function and esthetics with a conservative approach. Although unsupported by the laboratory studies and by many clinical trials, results of multicenter clinical studies have indicated a more favorable prognosis when the dentist is well equipped with the knowledge about the clinical situation, and advancements in the material sciences.


Hypoglycemia is one of the most important complications of diabetes treatment. The risk of severe hypoglycemia is higher in elderly patients, those having comorbidities such as vascular disease or renal failure, pregnant women and in children with type 1diabetes. Moreover, in type 2 diabetes, progressive insulin deficiency, longer duration of diabetes, and tight glycemic control increase the risk of hypoglycemia as much as type 1 diabetes.Episodes hypoglycemia may lead to impairment of counter-regulatory system, with the potential of development of hypoglycemia unawareness. So, hypoglycemia may increase the vascular events even death in addition to other possible detrimental effects. Glycemic control should be individualized based on patient characteristics with some degree of safety. Recognition of hypoglycemia risk factors, blood glucose monitoring, selection of appropriate regimens and educational programs for healthcare professionals and patients with diabetes are the major issues to maintain good glycemic control, minimize the risk of hypoglycemia, and prevent long- term complications. Diabetes is a chronic disease that requires continuous medical care and patient self-management education to prevent acute complications and reduce the risk of long-term complications . The preIn Middle East, internationally published data on national estimation of type 2 diabetes prevalence are spare. In Iran a prevalence of 7.7% in people younger than 65 years is reported . DiabeteThe overall objective of type 2 diabetes’s management is to achieve and maintain blood glucose control and reduce the risk of long-term complications. Many studies have shown that modern management with intensive glycemic control can limit, delayed or even prevent the chronic complications of diabetes. However this intensive diabetes treatment could be associated with an increased risk of hypoglycemia , particuHypoglycemia is a true medical emergency which requires prompt recognition and treatment to prevent organ and brain damage. The spectrum of symptoms depends on duration and severity of hypoglycemia and varied from autonomic activation to behavioral changes to altered cognitive function to seizures or coma. The short and long term complications include neurologic damage, trauma, cardiovascular events and death . Severe In this review we will give an overview of the current literature on impact of hypoglycemia on diabetes management and try to provide guidance to health care providers to safely and effectively assist the diabetic patients.For diabetic patients hypoglycemia is a fact of life. Approximately 90% of all patients who receive insulin have experienced hypoglycemic episodes. The reported incidence of hypoglycemia varies considerably among studies, however in general patients with type1 diabetes have an average of two episodes of symptomatic hypoglycemia per week and one episode of severe hypoglycemia once a year. An estimated 2–4% of deaths of this population have been attributed to hypoglycemia ,12.It should be noted that the incidence of reported hypoglycemia is usually under- estimates because of difficulty to determine. The detection of hypoglycemia would require continuous blood glucose measurements. In this regard asymptomatic episodes of hypoglycemia could be missed unless detected by monitoring of blood glucose. On the other hand, the incidence of symptomatic episodes is difficult to determine because they are rarely reported. Additionally in some clinical trials hypoglycemia is not a primary outcome which makes it difficult to estimate the incidence of hypoglycemia. While the episodes of severe hypoglycemia are a small fraction of the total hypoglycemic, estimation of this event is the most reliable since severe hypoglycemia events are better documented . The freThe incidence of hypoglycemia also could be affected by how tight glycemic control is performed. The Action to Control Cardiovascular Risk in Diabetes (ACCORD) reported a threefold increase in severe hypoglycemia and coma in intensively treated patients versus conventionally treated patients .Glucose is an obligate metabolic fuel for the brain under physiological conditions. Because the brain cannot synthesize glucose, maintenance of brain function requires a virtually continuous supply of glucose from the circulation. In normal situation redundant glucose counter-regulatory mechanisms effectively prevent or rapidly correct hypoglycemia .When hypoglycemia happens, a decrease in insulin secretion is the first response. Other hormones including glucagon and epinephrine are also secreted promptly after falling plasma glucose levels and both induce a rapid increase in glucose production .Hypoglycemia typically arises when abnormalities in the mechanisms involved in glucose homeostasis is existed. In patients with type 1 diabetes secretion of both counter-regulatory hormones of insulin and glucagon is severely interrupted. The third defense mechanism, the epinephrine response to hypoglycemia, is also become progressively impaired in type1 diabetic patients . The freHypoglycemia-associated autonomic failure (HAAF) in type1 diabetes apparently results from recent antecedent hypoglycemia that caused by both defective counter-regulatory response and hypoglycemia unawareness. Thus hypoglycemia caused HAAF may play a role in the vicious cycle of recurrent hypoglycemia ,21.The mechanisms of defect in glucose counter-regulation in type 2 diabetes are the same as type 1diabetes which includes both defect in hypoglycemia counter-regulation mechanism and deficiency of sense of hypoglycemia . Similar’s physiological need exists. The following reasons could account for hypoglycemia in diabetics: Iatrogenic, Diet changes and infections.In general, hypoglycemia in diabetic patients occurs when an imbalance between insulin/hypoglycemic agent’s intake and bodyDiabetes medications including insulin and sulphonylureas are among the most common causes of hypoglycemia in diabetic subjects . The lonHypoglycemia may also result from little foods intake or increase in activity in relation to medication and food intake. Other causes such as alcohol consumption, some drugs, stress and infections should also be considered in diabetic subjects. Alcohol may contribute to the severity of hypoglycemia by inhibiting gluconeogenesis.Hypoglycemia could also be a symptom of severe organ illnesses. Liver diseases such as hepatitis and cirrhosis as well as kidney diseases often cause hypoglycemia, mostly because of the major role that these organs play in glucose production and maintenance blood sugar levels.Hypoglycemia unawareness and a history of previous severe hypoglycemia episodes are risk factors for severe hypoglycemia. Impaired symptomatic awareness is associated with a 6-fold and 9-fold increased risk of severe hypoglycemia in patients with type1 and type 2 diabetes respectively ,20. PatiIn type 2 diabetes progressive insulin deficiency and duration of insulin therapy increase the risk of hypoglycemia as in type 1 diabetes, and the risk of hypoglycemia is highest in those with type 2 diabetes who have received insulin for more than 10 years . MoreovePregnancy is associated with a high risk of severe hypoglycemia in diabetic subjects. Hypoglycemia in pregnant women may lead to severe morbidity and even death . In womeAlthough intensive metabolic control can prevent diabetic complications in pregnant woman and her offspring, it may increase the risk of hypoglycemia . HistoryTight metabolic control with focus on reduction of glucose fluctuations during pregnancy improves the pregnancy outcome. Appropriate self-management training including carbohydrate counting, hypoglycemia awareness, and clear insulin dose adjustment instruction, are important in diabetic-pregnant women in order to maintain near-normoglycemic state without episodes of severe hypoglycemia particularly in early pregnancy stage.Hypoglycemia is a common problem in old people with diabetes. Aging modifies the cognitive, symptomatic, and counter-regulatory hormonal responses to hypoglycemia . The effIn the elderly subjects, episodes of hypoglycemia are more likely to be followed by changes in the blood brain circulation which may further increase the risk of neurological damage in this population ,36.Severe hypoglycemia has a considerable impact on wellbeing, productivity and quality of life in old people with diabetes . ManagemHypoglycemia is one of the most common acute complications of insulin therapy in children and adolescents with diabetes. The incidence of hypoglycemia is reported to be between 3 and 27 episodes per 100 patient- year in children with type 1diabetes . The recImpaired hypoglycemia awareness is a significant problem for children with type 1diabetes and a major risk factor for development of severe hypoglycemia . ChildreTherefore regular glucose monitoring and detection of the risk factors of hypoglycemia may help to reduce the severity and frequency of hypoglycemia in children and adolescent with diabetes.Hypoglycemia can cause severe morbidity and even death, usually depending on its severity or duration.A possible link between hypoglycemia and acute vascular events like angina, myocardial infarction, and acute cerebrovascular disease has been proposed by several studies ,43. In aet al.[However some controversies exist in this issue. The Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial could not establish the direct role of hypoglycemia in increased cardiovascular events , while aet al. suggesteet al.,48. Chanet al.. While cet al., recurreet al..In addition hypoglycemia could potentially increase the sudden death through inducing either ischemic or other electrocardiographic changes (increased QTc), which in turn may predispose the diabetic subjects to ventricular arrhythmias ,52..[A new research suggests a possible association between severe hypoglycemia and cognitive dysfunction. Åsvold et al. suggeste. found that risk factors for cognitive impairment in children with diabetes include: hypoglycemia, duration of diabetes and poor glycemic control [Many studies showed that the development of diabetes in the first stages of life is associated with an increased risk of neurocognitive dysfunction regardless of whether or not severe hypoglycemia was present . It has control .In type 2 diabetes, one longitudinal cohort study in elderly patients revealed that severe hypoglycemia episodes are associated with an increased risk of dementia in this population, although the impact of mild episodes on dementia risk remains unknown .A recent study found that severe hypoglycemia causes brain damage in cortex and the hippocampus regions and the extent of damage was closely correlated to the presence of seizure-like activity. The results were indicative of elevation of sensitivity of the cortex to the damaging effects following an episode of severe hypoglycemia .These researches remind us to be particularly alert for the child or adult who experience severe hypoglycemia early in life. This population is more likely to manifest developmental delays and cognitive dysfunction . Their ret al. found the positive association between severity/ frequency of hypoglycemic episode and greater fear of hypoglycemic events [Studies reported that severe hypoglycemia can have a significant impact on patients' health-related quality of life, treatment satisfaction, and cost of diabetic management. The wellbeing of patients may be affected both directly from the effects of hypoglycemia and indirectly from fear of recurrence ,61. Marr events . As a reWhile achieving and maintaining the optimal glycemic control is one of the principal aims of prevention and management of diabetes complications, hypoglycemia remains a major challenge . ObviousDiabetes self-management, supported by education and empowerment, is a basic part of diabetes care to achieve successful health-related outcomes ,67. SeveIn addition, blood glucose monitoring (BGM), using any of the widely available self- monitoring blood glucose (SMBG) or interstitial glucose sampling using continuous glucose monitors (CGM); is an important part of management of diabetes; especially for people who experience hypoglycemic episodes . BGM proIt is important that in patients with a history of recurrent hypoglycemia, the time of episodes be identified and the treatment regimen be adjusted accordingly . With a Continuous subcutaneous insulin infusion (CSII) with a rapid-acting insulin analog improves the glycemic control and reduces the rate of hypoglycemia compared with multiple daily insulin injection .Patients on oral anti-diabetic drugs are also at risk for developing hypoglycemia. Agents such as metformine, dipeptidyl peptidase-4 inhibitors, and thiazolidines are preferable to sulfonylureas in minimizing hypoglycemic risks .Glycemic goals should be individualized with some degree of safety particularly for patients with long duration of diabetes; patients who have a high risk of severe hypoglycemia development, and/or subjects with multiple co-morbidities ,75,76. IHypoglycemia is a major limiting factor in tight glycemic management of diabetes and may increase vascular events in addition to other possible detrimental effects. Glycemic control should be individualized based on patient characteristics with some degree of safety. Recognition of hypoglycemia risk factors, blood glucose monitoring, selection of appropriate regimens, education programs for healthcare professionals and patients with diabetes are the major issues to maintain good glycemic control, minimize the risk of hypoglycemia, and prevent long- term complications.The authors declare they have no competing interests.Dr. Shafiee participated in the drafting of the manuscript, Dr. Mohajeri-Tehrani participated in the study design, Dr. Pajouhi critically revised the manuscript, and Dr. Larijani was main supporter of the study and contributed in the writing of specific sections of the manuscript. All authors read and approved the final manuscript.


The Pliensbachian-Toarcian (Early Jurassic) fossil record is an archive of natural data of benthic community response to global warming and marine long-term hypoxia and anoxia. In the early Toarcian mean temperatures increased by the same order of magnitude as that predicted for the near future; laminated, organic-rich, black shales were deposited in many shallow water epicontinental basins; and a biotic crisis occurred in the marine realm, with the extinction of approximately 5% of families and 26% of genera. High-resolution quantitative abundance data of benthic invertebrates were collected from the Cleveland Basin , and analysed with multivariate statistical methods to detect how the fauna responded to environmental changes during the early Toarcian. Twelve biofacies were identified. Their changes through time closely resemble the pattern of faunal degradation and recovery observed in modern habitats affected by anoxia. All four successional stages of community structure recorded in modern studies are recognised in the fossil data . Two main faunal turnover events occurred: (i) at the onset of anoxia, with the extinction of most benthic species and the survival of a few adapted to thrive in low-oxygen conditions (Stages I to 0) and (ii) in the recovery, when newly evolved species colonized the re-oxygenated soft sediments and the path of recovery did not retrace of pattern of ecological degradation (Stages I to II). The ordination of samples coupled with sedimentological and palaeotemperature proxy data indicate that the onset of anoxia and the extinction horizon coincide with both a rise in temperature and sea level. Our study of how faunal associations co-vary with long and short term sea level and temperature changes has implications for predicting the long-term effects of “dead zones” in modern oceans. The expansion of oxygen minimum “dead zones” in modern oceans is at the top of the list of emerging environmental challenges Hypoxia can occur at a variety of temporal and spatial scales. Only the smallest temporal and spatial scales may be readily observed or recreated experimentally st century Rocks spanning the Pliensbachian-Toarcian interval of the Early Jurassic (∼185-181 Ma) are an archive of natural data from one of these past episodes of global warming and anoxia. Temperatures are estimated to have increased by 2–3.5°C in subtropical areas Marine ecosystems were adversely affected by these climate-driven environmental changes, and early Toarcian strata record a major extinction of marine organisms, particularly amongst the infaunal benthos Despite the importance of this event in the early evolution of modern marine ecosystems, and as a potential analogue for current environmental concerns, there is no quantitative palaeoecological study of community-level changes in marine macro-invertebrates through the Pliensbachian-Toarcian interval. Previous studies have mostly involved the collection of presence/absence data of fossil macro-invertebrates, in order to identify extinction horizons by analysing the stratigraphic distribution of species The main aim of this study is to analyse how benthic communities changed through an interval of past global warming characterized by the development of widespread hypoxic and anoxic conditions in marine settings. In particular we aim to assess (i) how alpha and beta diversity  changed through the studied interval; (ii) how benthic communities responded to sea level, oxygen concentrations and temperature variations; and (iii) how this fossil event compares to the recorded patterns of faunal succession in modern communities affected by hypoxic and anoxic conditions.This study is focused on the Pliensbachian-Toarcian sections of the northern Cleveland Basin (UK) because of its well-preserved fossil record of macro-invertebrates, and the abundance of published geochemical, sedimentological and stratigraphical data, which are useful in interpreting environmental change. In the Early Jurassic, the Cleveland Basin was situated at a palaeolatitude of 30–40°N exaratum subzone) consists of dark grey, organic-rich shales, laminated on a millimetre-scale, which contain regular horizons of centimetre to metre scale calcareous concretions. Total organic carbon content is very high, ranging between 5% and 15% falciferum subzone) laminations are less pronounced and the content of organic matter is lower bifrons and variabilis zones) consists of dark grey, non-bituminous shales, with abundant calcareous nodules The overlying lower Toarcian Whitby Mudstone Formation is subdivided into three members. The lower Grey Shale Member consists of light to dark grey silty mudstones with horizons of calcareous and sideritic concretions No permits were required for the described study, which complied with local guidelines in relation to responsible collecting from foreshore exposures.Coastal sections were sampled at Staithes, Port Mulgrave, Runswick Bay, Kettleness and Saltwick Bay . Rock exFor each sample alpha diversity was measured using the Simpson index of diversity (1-d), taxonomic diversity (Δ) and taxonomic distinctness (Δ*), which are appropriate measures of diversity for species abundance data when sample size is not homogeneous For multivariate elaboration, species occurring in only one sample were removed, and samples containing less than 20 individuals were excluded. The resulting culled data set includes 47 species, 115 samples and 11936 individuals . Percentage data were used because the volume of each sample was not consistent and the absolute numbers of individuals are thus not comparable between samples Nonmetric multidimensional scaling (nMDS) with the Bray Curtis similarity matrix, and detrended correspondence analysis (DCA) are among the best ordination methods for detecting ecological gradients in modern and ancient marine environments e.g., . Althouga priori groups of samples a priori groups of samples) was retained . This indicates that samples do not group according to the two different sampling methods and therefore the two datasets are comparable. The homogenous nature of the combined dataset can also be confirmed by nMDS ordination  was carried out to test the degree of differences between dination .a priori divided into groups, do not differ from each other in multivariate structure. A detailed description of the methods can be found in sensuHierarchical agglomerative clustering , with the unweighted pair-group average cluster model, was applied to recognise those species that tend to co-occur in samples and to group together samples of similar taxonomic composition. The similarity profile test (SIMPROF) was applied to determine significant difference between the clusters Although lithology it is not an absolute palaeoenvironmental indicator, it may be used as a general guide to palaeoenvironment as parameters such as grain size broadly correlate with depositional energy and, frequently, with water depth 18O curve based on belemnite, bivalve and brachiopod calcite was derived from literature data published on the Cleveland Basin 18O were interpreted in terms of relative, rather than absolute, changes in palaeotemperature, because other factors such as species-specific effects , the index falls to zero over a very short stratigraphic distance, which indicates a change to communities dominated by a single taxon. In the falciferum and bifrons zones, the value of the index fluctuates between 0 and 0.4, but, although average values are higher in the bifrons zone, never returns to the same evenness distribution as in the lower part of the section. Taxonomic diversity (Δ) records a similar trend to the Simpson index  ranges from 0.4 to 0.8 in the ribution . In the on index , with th subzone . ThereafThe results of nMDS ordination demonstrate that samples group according to their stratigraphic units (i.e. members), with little or no overlap between most of them . The strtenuicostatum subzone. The greatest differences are between samples from the semicelatum and the exaratum subzones, and from the falciferum and commune subzones  without reference to lithology. There is a good degree of overlap in the ordination space between groups of samples (subzones) belonging to the same stratigraphic unit (Member) . This isBivalves dominate the dataset, representing 96.4% of the total number of individuals. They are followed by brachiopods (1.2%) and gastropods (1.2%), and all the other categories together  are less than 1% in abundance. Cluster analysis shows that at low values of similarity (<20%) samples are subdivided into two main clusters . One cluIn the lower part of the section biofacies are not restricted to single Members , 3. The The main axis of the ordination (nMDS1) mostly reflects temporal changes through the studied section, with samples from the Penny Nab, Kettleness and Grey Shale Members on the left side of the diagram, and samples from the Mulgrave and Alum Shales on the right . Ordinatsemicelatum subzone, reaching minimum values at the base of the exaratum subzone. This main drop in diversity corresponds to the extinction horizon recognised by Little The diversity analyses show that during deposition of the Cleveland Ironstone Formation and of the majority of the Grey Shale Member a diverse benthic population was present . Diversiclevelandicum subzone, which indicates that the benthic community underwent progressively increasing stress before the semicelatum subzone. In fact, a decrease in the hierarchical level of diversity of species is expected with increasing levels of environmental perturbation clevelandicum subzone Both taxonomic richness and distinctness, however, record a progressive decline from the earlier exaratum subzone, and coincide with the interval of maximum water mass restriction and stratification in the basin, deposition of organic-rich sediments and high runoff rates exaratum subzones, where water mass restriction was less intense falciferum zone, taxonomic distinctness is relatively high compared with taxonomic diversity, indicating that the assemblages comprise relatively few, taxonomically unrelated species. Bivalves dominate the samples in term of abundance, with either rare brachiopods , echinoderms  or gastropods  also occurring periodically. A relaxation of stressed conditions from the bifrons zone is testified by a general increase in alpha diversity.Minimum values of alpha diversity persisted for most of the semicelatum and the exaratum subzones and coincides with the large drop in alpha diversity of the extinction event. The second occurs between the falciferum and the commune subzones, corresponding to a large increase in taxonomic distinctness  and Pseudopecten equivalvis biofacies  represent undisturbed communities, characterise the lower part of the studied section, and indicate a high energy, clear water and well oxygenated environment. Biofacies A has a large compositional variability, as shown by the low SIMPER average similarity value , as do small infaunal suspension feeding bivalves , with respect to biofacies of Stage III. There is a reduction in the diversity of deep infaunal suspension feeders, which suggests a decrease in sediment oxygen levels. There is also an increase in the abundance of the opportunistic detritus feeding gastropod Levipleura blainvillei, which was probably feeding on organic material, microbes and bacteria tenuicostatum zone In the Bositra radiata  in the semicelatum subzone mark a dramatic change in faunal composition and reflect disturbed communities living just before the extinction. Most authors agree that Bositra radiata was an epifaunal suspension feeding bivalve adapted to live in low-oxygen conditions  signals the return to oxygenated, bioturbated sediments in the Alum Shale Member, and is mostly characterized by newly evolved species, such as the shallow infaunal deposit feeding bivalve Pseudomytiloides dubius, Meleagrinella substriata), but mostly new species from surviving lineages . Post-extinction species largely belong to the same genera as the species becoming extinct, and their appearance is therefore due to the origination of new species and/or immigration of closely related taxa. Aberhan As observed by Harries & Little Levipleura blainvillei and Ptychomphalus expansus, which disappear from the Cleveland Basin after the extinction, occur in the falciferum zone of the adjacent East Midlands Shelf Pholadomya ambigua occurs in the falciferum zone of central Spain Pseudopecten equivalvis and Camptonectes subulatus, are found in middle-upper Toarcian sediments of Spain and Greenland In the studied section, local extinction among deposit- and suspension-feeding infaunal bivalves, semi-infaunal bivalves, gastropods and brachiopods was 100% at the species level, whereas epifaunal bivalves and crinoids, were less affected by the extinction  The pattern of faunal degradation and recovery discussed above demonstrates that oxygen concentration played an important role in marine community dynamics through the studied interval. However, sea level and palaeotemperature also varied through the section, and likely had a role in controlling faunal change. All three factors co-vary to some extent, as water temperature is a key factor controlling sea-level changes Ordination can extract high resolution palaeoenvironmental signals from quantitative data, especially when palaeoecological analyses are integrated within a sequence stratigraphic framework . In contexaratum subzone). Superimposed on this longer term deepening are a number of transgressive-regressive cycles at the base of, and within, the Grey Shale Member up to the semicelatum subzone. In the Mulgrave Shale Member the new curve shows minor fluctuations without substantial changes in the relative water depth, followed by shallowing within the Alum Shale Member.The resulting curve  shows stIn the lower part of the section  there is no evidence for oxygen restriction, and changes in biofacies are strongly linked to changes in relative water depth. The distribution along nMDS2 of biofacies A to D, which correspond to Stage III communities that lived under normally oxygenated conditions, seems to be mainly controlled by water depth: biofacies A only occurs in the shallowest sediments, whereas biofacies B, C and D are associated with deeper water . In the 18O isotopes derived from belemnites, bivalves and brachiopod calcite from the Cleveland Basin, especially in the Cleveland Ironstone Formation and lower Whitby Mudstone Formation , coinciding with a sharp sea level rise, and culminating in the extinction horizon  the recovery of the benthic fauna was still not complete. Thus, even though the events described in this study occurred over almost 5 Ma Similarly, in our case-study, ecological recovery took longer than the pattern of degradation; was hindered by recurrent hypoxic events; and started with the re-colonization by opportunistic taxa. Stressed conditions increased from the This study demonstrate how high quality, quantitative, palaeoecological analyses of benthic invertebrates integrated within a sequence stratigraphic and environmental framework can advance our understanding of the response of past ecosystems to large-scale environmental changes.semicelatum subzone). A second faunal turnover event occurred between the Mulgrave and Alum Shale members, when newly evolved taxa replaced the impoverished fauna during recovery.Analyses of alpha and beta diversity demonstrate that a net decrease in diversity coupled with high faunal turnover characterizes the extinction horizon at the top of the Grey Shale Member . Under slightly less restricted conditions, the community was a little more diverse . Under renewed oxygenated conditions new species colonized the sediments .Twelve biofacies were identified, representing a community replacement sequence that is similar to the successional stages recognised in modern ecosystems affected by anoxia. Benthic communities moved from an undisturbed, climax community , through gradually increasing stressed conditions, characterized by episodic hypoxia, increased nutrient supply, and increased oxygen stress in the sediments . At the onset of long-term hypoxia only disturbed, pioneer communities were present , with the occurrence of the possibly chemosynthetic bivalve Ordination of samples coupled with sedimentological and palaeotemperature proxy data demonstrated that variations in benthic communities correlate with long and short term sea level and temperature changes. The onset of anoxia and the extinction horizon coincide with both a rise in palaeotemperature and sea level. Unlike the situation in modern settings affected by anoxia, this study demonstrates that when the duration of warming-related hypoxia and intense anoxia is long enough (∼900 ka in this case), and extinction occurs, the benthic system will move to a new stable state characterised by newly evolved species.Figure S1Location map of the study area. (A) Schematic structural map of the Cleveland Basin, modified from (PDF)Click here for additional data file.Figure S2Detailed stratigraphic section. Stratigraphic log of the studied composite section with details of ammonite zones and subzones, lithology, bed numbers (following (PDF)Click here for additional data file.Figure S3Comparison of nMDS and DCA ordination of samples. (A) nMDS ordination with labels as samples numbers. (B) nMDS ordination with labels as bed numbers. (C) DCA ordination with labels as sample numbers. Note that nMDS and DCA produce similar clusters of samples, however the DCA ordination has a triangular shape derived by the compression of values on axis 2.(PDF)Click here for additional data file.Figure S4nMDS plots showing the distribution of samples collected with two different sampling methods. (A) nMDS ordination of axis 1 versus axis 2. (B) nMDS ordination of axis 2 versus axis 3. In green samples collected by SD, RJT and MEC; in blue samples collected by CTSL. Note, even though collected with two different sampling methods, the samples show a very high degree of overlap. This, together with the results of the ANOSIM test  discussed in the text, indicates that the dataset is homogenous.(PDF)Click here for additional data file.Table S1Species-level abundance distribution of the collected samples. Taxonomic composition and abundance of each collected sample. Additional information include the associated lithostratigraphic unit, the subzone, the bed number, the stratigraphic height and the sampling method.(XLSX)Click here for additional data file.Table S2Taxonomic information for the species listed in  Information on the taxonomic hierarchy  for each species used to calculate taxonomic diversity (Δ) and taxonomic distinctness (Δ*).(XLSX)Click here for additional data file.


Two main models have been proposed to explain how the relative size of neural structures varies through evolution. In the mosaic evolution model, individual brain structures vary in size independently of each other, whereas in the concerted evolution model developmental constraints result in different parts of the brain varying in size in a coordinated manner. Several studies have shown variation of the relative size of individual nuclei in the vertebrate brain, but it is currently not known if nuclei belonging to the same functional pathway vary independently of each other or in a concerted manner. The visual system of birds offers an ideal opportunity to specifically test which of the two models apply to an entire sensory pathway. Here, we examine the relative size of 9 different visual nuclei across 98 species of birds. This includes data on interspecific variation in the cytoarchitecture and relative size of the isthmal nuclei, which has not been previously reported. We also use a combination of statistical analyses, phylogenetically corrected principal component analysis and evolutionary rates of change on the absolute and relative size of the nine nuclei, to test if visual nuclei evolved in a concerted or mosaic manner. Our results strongly indicate a combination of mosaic and concerted evolution (in the relative size of nine nuclei) within the avian visual system. Specifically, the relative size of the isthmal nuclei and parts of the tectofugal pathway covary across species in a concerted fashion, whereas the relative volume of the other visual nuclei measured vary independently of one another, such as that predicted by the mosaic model. Our results suggest the covariation of different neural structures depends not only on the functional connectivity of each nucleus, but also on the diversity of afferents and efferents of each nucleus. In recent years, there has been an increased interest in understanding the principles and processes that govern brain evolution A possible drawback of the use of major subdivisions of the brain is that they do not represent functional units; each region contains multiple independent motor and sensory pathways. This means that the size of these different regions of the brain is the result of a complex combination of multiple selection pressures and constraints affecting several motor and sensory pathways. Selective hypertrophy of neural structures related to sensory e.g. , and motThe visual system of birds is a good candidate to study the covariation of the relative size of nuclei that belong to the same pathway or sensory modalities. In birds, like in all vertebrates, projections from the retina go to several retinorecipient nuclei, which give rise to several parallel visual pathways. The main retinorecipient structure is the optic tectum (TeO), a multilayered structure that in pigeons receives more than 90% of retinal projections and forms part of the tectofugal pathway . The tecGallus domesticus), Imc is composed of two different cells types; one cell type projects to Ipc and SLu, and the other cells project to TeO (Taeniopygia guttata), these two cells types are segregated in two subdivisions, which are identified as the external (Imc-ex) and internal (Imc-in) Imc  phylogenetically corrected principal component analysis and, ii) evolutionary rates of change, on the absolute and relative size of the nine visual nuclei. Previous studies In all cases, the specimens were provided to us dead. Some of these species were collected dead from window strikes and culling operations in Australia by ANI under collection permits issued by the Victorian Department of Natural Resources and Environment. Other species were provided by other researchers, all of which had the correspondent capture/handling permits and/or ethics approval from their respective institutions. This includes Dr. Catherine Carr which had approval from the University of Maryland institutional animal care and use committee (IACUC), Dr. Lainy Day which had approval from the University of Mississippi IACUC, Dr. Ken Welch Jr. which had approval from the University of California, Riverside IACUC, and Dr. Tim R. Birkhead, who obtained specimens from local hunters in West Woodyates, Dorset, United Kingdom. Other specimens were provided by the Healesville Sanctuary , the Springvale Veterinary Clinic , the Melbourne Zoo  and the Alberta Institute for Wildlife Conservation  staff. In all of these cases, the specimens died from causes unrelated to this project.ad libitum, and supplemented with mealworms (Tenebrio molitor) daily. Tissue collection began by euthanizing birds with CO2, followed by immediate removal of the eyes for a different study and the head  for this study.Some of the songbird specimens in this study were captured in Tippecanoe County, Indiana, USA using mist-nets and live traps by BAM and EF-J. Authorization to capture these birds was obtained from the Indiana Department of Natural Resource and the U.S. Fish and Wildlife Service. Capture and study of all animals did not involve endangered or protected species. The Purdue Animal Care and Use Committee (protocol #1201000567) approved all capturing, handling, and experimental procedures with the birds see . Birds wFAST Cooled mono 12-bit camera  and OPENLAB Imaging system  attached to a compound light microscope . For some brains, images of full sections were obtained with a digital slide scanner  with a 20× objective.We measured the relative volume of Ipc, Imc, SLu, ION, LM, GLv, nBOR, nRt and TeO in 100 specimens representing 98 species . Some ofhttp://rsb.info.nih.gov/ij/) and volumes were calculated by multiplying the area in each section by the thickness of the section (40 µm) and the sampling interval. For those species represented by more than one specimen  species as independent data points, and (2) phylogenetic generalized least squares (PGLS) to account for phylogenetic relatedness Non-phylogenetically corrected statistics and post-hoc tests were performed using the software JMP . Additionally, we calculated phylogeny-corrected 95% prediction intervals To compare patterns of evolution among the different nuclei, we used maximum likelihood values for the lambda (λ) and alpha (α) parameters phyl.pca functions of the PHYTOOLS package phyl.pca functions of the PHYTOOLS package which performs a PCA that takes into account the phylogenetic relationships among species. As with the previous analyses, we used two different phylogenies To test how the relative size of the nuclei vary with respect to each other, we used a correlation based principal components approach taking into account the phylogenetic relationships among species, using the All multivariate analyses included 94 of the 98 species because four species did not have a recognizable ION see  and the Surnia ulula), SLu is divided into dorsal and ventral portions that are separated by a bundle of fibers that courses dorsal to Ipc, but ventral to the lateral part of the mesencephalic reticular formation, towards the brachium conjunctivum  scale with negative allometry against brain volume ; Fig. 6AWe also examined the size of the isthmal nuclei relative to the size of the TeO. Imc and Ipc scaled with isometry or positive allometry with the TeO, while SLu scaled with isometry with TeO ; Fig. 6COrder also had a significant effect on the relative size of all of the other visual nuclei. Differences in the relative size of ION among orders were not different from those previously reported . GLv and10-transformed volumes of each visual nucleus departs significantly from a Brownian motion model using maximum likelihood estimates of α and λ. When the absolute size of the visual nuclei was used, none of them differed significantly from a Brownian model of evolutionary change . Although there were some minor differences in the loadings between the two phylogenies . This strongly suggests that PC1 describes variance in the different structures' volumes resulting from differences in brain size. In other words, evolutionary changes in brain size explain about 80% of the variance in the absolute size of the visual nuclei. TeO, nRt Imc, Ipc and SLu had the largest loadings in PC1, which indicates a strong correlation between the volumes of these structures and overall brain size. In contrast, the lower loadings of the other visual nuclei, particularly GLv and ION, suggest a weaker correlation between the volume of these two nuclei and whole brain size. PC2 explained around 7% total variance , suggesting that the size correction removed most, but not all, effects of variation in brain size. PC2 explains about 15% of the variance with a strong loading of GLv and LM. Finally, PC3 explained about 10.5% of the variance with a strong loading of ION.Because the relative size of some of the nuclei departs from a Brownian motion evolutionary model, we performed a PCA using both a Brownian motion model and Pagel's λ model of evolutionary change. We found no major differences in the estimated values between the two models with either of the phylogenies used , S5. WheThis is the first study to assess variation of the relative size of the isthmal nuclei in birds. In recent years, the isthmotectal system has received increased attention, especially in birds, as a model to study visual spatial attention and competitive stimulus selection Our results indicate that evolutionary changes in the size of SLu are distinct from that of the other isthmal nuclei. Although bivariate allometric coefficients and loadings of SLu in PC1 of the relative size PCA suggest that the relative size of SLu varies more closely with Imc, Ipc, TeO and nRt, other lines of evidence suggest that the relative size of SLu is more independent. First, the differences among orders in the relative size of Imc and Ipc closely follow the variation in relative size of TeO and nRt, but relative SLu size did not significantly vary among orders. This suggests that the variation in relative size of SLu is different from that of Imc and Ipc (and TeO/nRt). Second, while the evolutionary rate of the relative size of Imc, Ipc, TeO and nRt do not differ significantly from a Brownian motion model (see below), that of SLu clearly does . The difInterestingly, while we found no differences in size of SLu relative to the brain among orders , owls haIn both PCAs, the second principal component explained around 15% of the variation and GLv and LM were loaded in the same direction, suggesting they vary in relative size together and therefore may have shared functions. Groups like gallinaceous birds and pigeons, which have relatively large LM and GLv, have likely driven this covariation of LM and GLv sizes. In a previous study, Iwaniuk and Wylie Variation in the relative size of the TeO and nRt among orders were similar to what has been reported before Previous studies that tested for differences between mosaic and concerted models of evolutionary change in the brain did so by examining allometric scaling trends e.g. . AlthougOur study also examined both absolute sizes and phylogenetically corrected relative sizes whereas previous studies have only examined one or the other Our results strongly suggest a combination of mosaic and concerted evolution in the relative size of nine nuclei of the visual system of birds. Across the 98 species of birds we examined, the relative size of the isthmal nuclei (particularly Imc and Ipc) and components of the tectofugal pathway (TeO and nRT) vary together, but the relative volumes of ION, nBOR and ION vary independently of one another in more of a mosaic manner. This pattern is supported by several lines of evidence. First, the bivariate allometric coefficients between Imc, Ipc, SLu, TeO and nRt are all close to 1 , indicatThe low degree of covariation in the relative sizes of GLv, nBOR and LM from TeO suggested by our results is somewhat surprising given that all three nuclei receive projections from the retina. Iwaniuk et al. 2010;  suggestePrevious studies have suggested that functionally and anatomically related neural structures should vary together We think our study further emphasizes the need for future research to consider variation of neural pathways as a whole and not isolated neural structures, particularly when the relative size of a neural structures in being correlated with a particular ecology or behavior. Our study shows that a combination of multivariate statistics and rates of evolution constitute a robust method to study patterns of evolutionary change in neural pathways.Table S13) of the magnocellular and parvocellular portions of nucleus isthmi , the nucleus semilunaris (SLu), the isthmo optic nucleus (ION), the ventral part of the geniculate nucleus (Glv), the nucleus of the basal optic root (nBOR), the nucleus lentiformis mesencephali, the optic tectum (TeO) and the Brain for each species.List of the species surveyed, sample sizes and volumes (mm(DOC)Click here for additional data file.Table S2Results of least-squares linear regression performed on the log-transformed volume the magnocellular and parvocellular portions of nucleus isthmi , the nucleus semilunaris (SLu), the isthmo optic nucleus (ION), the ventral part of the geniculate nucleus (Glv), the nucleus of the basal optic root (nBOR), the nucleus lentiformis mesencephali, the nucleus rotundus (nRt) and the optic tectum (TeO) against the log-transformed brain volume minus the volume of the respective nuclei are provided using both species as independent data points (‘no phylogeny’) and two models of evolutionary change, Brownian motion (PGLS) and Ornstein-Uhlenbeck (OU) with two different phylogenetic trees.(DOCX)Click here for additional data file.Table S3Results of least-squares linear regression performed on the log-transformed volumes of the magnocellular and parvocellular portions of nucleus isthmi , the nucleus semilunaris (SLu), the isthmo optic nucleus (ION), the ventral part of the geniculate nucleus (Glv), the nucleus of the basal optic root (nBOR), the nucleus lentiformis mesencephali, the nucleus rotundus (nRt) and the optic tectum (TeO) against the log-transformed brain volume minus the volume of the respective nuclei with the order of each species as a covariate. Results are provided using both species as independent data points (‘no phylogeny’) and two models of evolutionary change, Brownian motion (PGLS) and Ornstein-Uhlenbeck (OU) with two different phylogenetic trees. Values for regression of the log-transformed volume of Imc, Ipc and Slu against the log-transformed TeO volume are also provided.(DOCX)Click here for additional data file.Table S4P values for the λ and α parameters were determined from likelihood ratio tests against an unconstrained Brownian motion model. Values for the relative size using Livezey and Zusi  of eight visual nuclei using two different phylogenies. si 2007;  phylogen(DOCX)Click here for additional data file.Table S5Loadings, eigenvalues and cumulative amount of variation explained by four of the components (PC's) obtained from a PCA analysis using the log-transformed volume or the relative size  of nine visual nuclei. Values obtained using Livezey and Zusi 2007;  phylogen(DOCX)Click here for additional data file.


Spatiotemporal pattern formation in neuronal networks depends on the interplay between cellular and network synchronization properties. The neuronal phase response curve (PRC) is an experimentally obtainable measure that characterizes the cellular response to small perturbations, and can serve as an indicator of cellular propensity for synchronization. Two broad classes of PRCs have been identified for neurons: Type I, in which small excitatory perturbations induce only advances in firing, and Type II, in which small excitatory perturbations can induce both advances and delays in firing. Interestingly, neuronal PRCs are usually attenuated with increased spiking frequency, and Type II PRCs typically exhibit a greater attenuation of the phase delay region than of the phase advance region. We found that this phenomenon arises from an interplay between the time constants of active ionic currents and the interspike interval. As a result, excitatory networks consisting of neurons with Type I PRCs responded very differently to frequency modulation compared to excitatory networks composed of neurons with Type II PRCs. Specifically, increased frequency induced a sharp decrease in synchrony of networks of Type II neurons, while frequency increases only minimally affected synchrony in networks of Type I neurons. These results are demonstrated in networks in which both types of neurons were modeled generically with the Morris-Lecar model, as well as in networks consisting of Hodgkin-Huxley-based model cortical pyramidal cells in which simulated effects of acetylcholine changed PRC type. These results are robust to different network structures, synaptic strengths and modes of driving neuronal activity, and they indicate that Type I and Type II excitatory networks may display two distinct modes of processing information. Synchronization of the firing of neurons in the brain is related to many cognitive functions, such as recognizing faces, discriminating odors, and coordinating movement. It is therefore important to understand what properties of neuronal networks promote synchrony of neural firing. One measure that is often used to determine the contribution of individual neurons to network synchrony is called the phase response curve (PRC). PRCs describe how the timing of neuronal firing changes depending on when input, such as a synaptic signal, is received by the neuron. A characteristic of PRCs that has previously not been well understood is that they change dramatically as the neuron's firing frequency is modulated. This effect carries potential significance, since cognitive functions are often associated with specific frequencies of network activity in the brain. We showed computationally that the frequency dependence of PRCs can be explained by the relative timing of ionic membrane currents with respect to the time between spike firings. Our simulations also showed that the frequency dependence of neuronal PRCs leads to frequency-dependent changes in network synchronization that can be different for different neuron types. These results further our understanding of how synchronization is generated in the brain to support various cognitive functions. Neuronal synchronization is thought to underlie spatiotemporal pattern formation in the healthy To demonstrate the universality of the frequency-dependent effects on the neuronal PRC, we consider a reduced model neuron described by the Morris-Lecar equations Our results provide important insight into differential changes in the propensity for network synchronization induced by the external modulation of neuronal frequency. As neuronal firing frequency changes, the changes in network spatiotemporal patterns depend upon the response characteristics of the individual cells in the network.We used the Morris-Lecar model The Type I and Type II neuronal models share the parameter values in situ and simulates the effects of cholinergic neuromodulation The cortical pyramidal model neuron we employed was motivated by recent computational and experimental findings, as reported in with Activation of the with with where in silico that eliminating this current is sufficient to switch the model neuron's PRC from Type II to Type I The slow, low-threshold For both neuronal models, In all network simulations, the number of neurons is 200, and the synapses are exclusively excitatory. The network connectivity pattern is constructed using the Watts-Strogatz architecture for Small World Networks s is the same for all synapses within a given simulation, and we set Synaptic current is transmitted from neuron We employ two different methods to modulate network firing frequency in our simulations. The first is to simply modulate the supra-threshold value of In order to model more biologically relevant environmental inputs, we also run simulations of cortical pyramidal neuronal networks in which frequency is modulated by stochastic input. All neurons are given the same constant sub-threshold baseline current, plus square current pulses randomly delivered to each neuron at a specified frequency any value, not just zero. The MPC between a pair of neurons, We monitor phase-synchronization of neuronal firing in our simulations using the mean phase coherence (MPC) measure, where B, which is 0 for random spiking and approaches 1 for perfect locking at phase zero between all neurons, for a large number of total spikes and neurons. Calculation of B requires a time-ordered list of the spike times of all neurons over the duration of the entire simulation i and i+1, which do not necessarily (and probably do not) correspond to spikes of the same neuron, B is then defined asWe quantify phase-zero synchronization of a network by calculating the bursting measure where We first investigate the underlying cellular basis of the differential frequency effects on Type I and Type II PRCs. We show that the relative activation levels and timing of hyperpolarizing, potassium currents in relation to depolarizing currents play a crucial role in shaping the phase response of a neuron. We then show that individual neuronal spiking frequency modulates network synchrony in significantly different ways for networks consisting of Type I or Type II cells. Specifically, synchrony in Type I networks is affected very little by frequency modulation near threshold, whereas in Type II networks, synchrony falls dramatically as frequency increases above firing threshold. This effect is due to the disparity in the frequency-modulated attenuation of the PRCs of the two cell types. We first show this effect in excitatory networks composed of Morris-Lecar model neurons, and then investigate it in depth for excitatory networks consisting of model cortical pyramidal cells under acetylcholine modulation.In both models, increasing frequency by increasing the constant applied current results in an attenutation of phase responses . This atIn both models, phase delays exist in the Type II parameter regimes because there is a voltage interval in which activation of an outward, hyperpolarizing current is greater than activation of the inward, depolarizing current. In the Type II Morris-Lecar model, the steady state activation curve of the fixed dynamics of the delay-inducing shrinking interspike interval. Phase advances are less sensitive to frequency modulation since the instantaneous dynamics of the inward currents in both models can directly track the faster cycle trajectory.As firing frequency increases, the cycle trajectory passes through this We further illustrate this point by modulating the speed of the gating variable controlling the delay-inducing potassium current in each model. quencies . The deprequency , phase dA similar dependence of phase delay amplitude on the rate of the gating variable requency , but theIn the cortical pyramidal neuron model, the amplitude of phase delays also depended on the rate of the tivation , gated bThese results imply that appropriate selection of the rate of variables gating the intracellular currents mentioned above permits the recovery of specified PRC delay depths for different levels of external current. B, versus the re-wiring parameter for different network frequencies. In Type I networks, increased neuronal firing frequency had little effect upon synchronization, while synchronization of Type II networks substantially decreased with increased neuronal firing rates. We analyzed network activity patterns in large-scale (N = 200) excitatory networks composed of Morris-Lecar model neurons with Type I and Type II PRCs under different network connectivity regimes. As described in the We first investigated synchronization properties of networks driven with constant applied currents, as in the Morris-Lecar network simulations. Every cell was driven with a constant current, We observed sharp differences between responses of the Type I and Type II networks to frequency modulation. As shown in B varied with the re-wiring parameter and synaptic weight in both types of networks. Note that synaptic weights were much higher in Type I than in Type II networks because Type I networks required greater coupling in order to reach appreciable levels of synchronization. The left panels in B between the high- and low-frequency networks for each network type, revealing the fundamental difference in synchronization response of the two types of networks. The right panel in s greater than approximately To show that these results were robust to network structure and coupling strength, lone see , while dlone see .time to synchronization. In the right panel of B in Type II networks decreased above approximately B diminished. This effect is displayed in the tightly-packed values of B shown in In the high-coupling regime, where differences in steady-state synchronization between high- and low-frequency Type II networks were diminished, we investigated whether frequency might still affect the Finally, B between high- and low-frequency Type I networks were again very small for virtually all values of the network re-wiring parameter and coupling strength  tend to synchronize better than neurons with Type I membrane dynamics, when coupled with excitation. Previous theoretical work indicates that when excitatory networks are driven with constant current, those composed of Type I neurons will not synchronize as well as those composed of Type II neurons In this study, we focused on the implications for network synchronization of the observed frequency-dependence of PRCs. Our results suggest that the severe attenuation of the phase-delay region of Type II PRCs at increased firing frequencies contributes to the observed decline in network synchronization at such frequencies. Frequency-dependent modification of PRCs has been investigated before in complex, multi-compartment neuronal models The frequency-dependent synchronization which we have described in this paper could potentially be involved in any cognitive process, functional or pathological, which involves spatiotemporal pattern formation of neuronal populations. For example, cholinergically-induced switching between sensitivity and insensitivity to frequency modulation could be important in proper memory consolidation during slow wave and REM sleep, two states that are characterized by differing levels of acetylcholine in cortical and hippocampal regions. Frequency-mediated synchrony could also play a part in the binding of signals from multiple sensory modalities. Gamma oscillations (20–80 Hz) in cortical networks are believed to be generated by synchronous activity of fast-spiking interneurons At the same time, the importance of our results is not confined to these examples alone. Our findings point to the possibility that Type I and Type II excitatory networks function in two separate coding regimes, with Type I networks functioning in the rate coding regime and Type II networks functioning in the temporal coding regime, effectively acting as low-pass filters. Further experimental investigation into the interplay between cellular properties, frequency, and network synchronization is clearly required.


Malnutrition is a global health problem affecting more than 300 million pre-school children worldwide. It is one of the major health concerns in India since around 50% of children below the age of two suffer from various forms of malnutrition. The gut microbiome plays an important role in nutrient pre-processing, assimilation and energy harvest from food. Consequently, dysbiosis of the gut microbiota has been implicated in malnutrition.Metagenomics approach was adopted to investigate the gut microbiome sampled from 20 rural Indian children with varying nutritional status. The changes in the abundances of various taxonomic and functional groups were investigated across these gut microbiomes. A core set of 23 genera were observed across samples, with some showing differential abundances with varying nutritional status. One of the findings of the current study is the positive/negative associations of specific taxonomic and functional groups with the nutritional status of the children. Notable alterations in the architecture of the inter-microbial co-occurrence networks were also observed with changes in nutritional status. A key example is the clustering of potentially pathogenic groups into a distinct hub in severely malnourished gut. Our data does not demonstrate causality with the microbiome patterns that we observed, rather a description of some interesting patterns, whose underlying mechanism remains to be uncovered.The present study envisioned interrelationships between the pattern of gut microbiome and the nutritional status of children. The cause of this pattern needs to be explored. However, insights obtained from the present study form the basis for further metagenomic investigations on larger population of children. Results of such studies will be useful in identifying the key microbial groups that can be utilized for targeted therapeutic interventions for managing severe acute malnutrition. The human gut microbiome, collective genomes of all the microbes residing in the gastrointestinal tracts, provides several metabolic functions that are not encoded in our own genome. Examples of these functions include fermentation of dietary polysaccharides, anaerobic metabolism of proteins and peptides, biosynthesis of vitamins, absorption of ions and regulation of a number of host metabolic pathways In its journey down the human alimentary canal, food is intercepted by trillions of microbes  with the following accession numbers SRR1067674, SRR1087919, SRR1068216, SRR1067721, SRR1068217, SRR1068218, SRR1068219, SRR1068596, SRR1068597, SRR1067720, SRR1067719, SRR1087910, SRR1087911, SRR1067716, SRR1087913, SRR1087914, SRR1087915, SRR1087916, SRR1067717, SRR1087918. Data was anonymized immediately after collection of the samples. The study was approved by the institutional review board of the Society for Health and Demographic Surveillance. Written informed consent was obtained from parents of the children in presence of another third person. The process of obtaining consent was not documented. The institutional review board had approved the procedurefor obtaining consent.The details of the 20 subjects considered for this study, along with the methodology for sample collection, DNA extraction, pyrosequencing and quality filtering of sequenced metagenomic reads are described in Supporting Information S1 in The nutritional status of each of the 20 children considered in this study was quantified using the WHO recommended (2009) three nutritional Z-scores namely, height for age (referred to in this study as Z-score1); weight for age (referred as Z-score2) and weight for height (referred as Z-score3). The formulae used for the calculation of these Z-scores, as recommended by WHO Based on the cumulative nutritional index, the 20 gut metagenomes were divided into three groups. These groups were (a) apparently healthy (AH)  with cumulative nutritional indices greater than −6, (b) borderline malnourished (BL)  and (c) severely malnourished (SM) .et al.A database containing taxonomic information on known bacterial groups was created from the available data from the fully sequenced genomes at the NCBI database as well as from the bacterial/archaeal genomes sequenced as part of the Meta-HIT and the Human Microbiome Project consortiums. The database contained 2352 fully/partially sequenced bacterial genomes. Reads in each metagenome were subjected to BLASTN search The abundances of different taxonomic groups  in each gut microbiome were obtained by normalizing the number of reads assigned to the group in the corresponding gut metagenome by the number of sequenced reads in the entire metagenome In order to identify taxonomic groups having positive or negative influence on nutritional status, Spearman rank correlations were computed between the ranked abundances of different groups in each metagenome and the cumulative nutritional index of the corresponding child. Taxonomic groups showing statistically significant correlations with at least one Z-score measure were suggested to influence the nutritional status of the children. The statistical significances of these associations were judged using the ReBoot Method 2), which can be defined as the proportion of variance associated with or accounted for each of the main effects, interactions, and error in an ANOVA study, was also measured.In order to identify taxonomic groups that were specifically over-abundant in one of the three groups of microbiomes , the abundances of the microbiota across the three groups were compared, using both parametric (ANOVA) and non-parametric  tests. Furthermore, to reduce the chances of Type I error, multiple test corrections were performed using the Benjamini-Hochberg FDR method. Given the lower sample size in the current study, this method was chosen over the Bonferroni method since the power of Bonferroni corrections (for datasets with limited sample size) is usually low and is likely to miss out on bona fide relationships. Further, Tukey-Kramer's test was used for post-hoc analysis of difference of means  in ANOVA. This method considers all possible pairs of means while controlling the family-wise error rate . The Effect size (measured by ηSequences in each metagenome were first subjected to a BLAST search (with thresholds of identity >65% and query coverage >90%) against the eggNOG database The relative abundances of the different COG groups in each gut microbiome were obtained by dividingthe number of reads assigned to the group in the corresponding gut metagenome by the metagenome size. These abundance values were then rank normalized across the 20 samples.In order to identify functional groups showing positive or negative associations with nutritional status, Spearman Rank correlations were calculated between the abundances of functional groups in each metagenome and the nutritional indices of the corresponding child. The COG groups showing significant positive or negative correlations (with P<0.05) were further analyzed.The abundances of the various carbohydrate active enzyme families For the metagenomes under study, pairwise correlations betweenthe rank normalized abundances of different genera were first obtained. Subsequently, pairs of genera having a correlation coefficients of P-value less than 0.05 (obtained using the ReBoot method) were linked by edges. Using this strategy, networks of co-occurring genera were generated and visualized using Cytoscape The anthropometric data along with the sample metadata corresponding to the 20 children are provided in Table S2 in Across the 20 microbiomes, 72% of the sequences (ranging from 61.8% to 86.6%) could be assigned at the phylum level  were observed to be present across all 20 gut microbiomes , Streptococcus , Shigella , Enterobacter  and Veillonella , wereobserved to have significant negative correlation with the nutritional index of the children , Faecalibacterium , Butyrivibrio  had significant positive correlations with nutritional index , indicated five taxonomic groups that were significantly different across the three categories  of microbiome . Several64 and 112 COG groups were observed to have significant positive  and negative correlation  with the cumulative nutritional indices, respectively  positive correlations with at least one of the nutritional indices . While mThe number of virulence factor homologs detected in the microbiomes was observed to show a progressive increase with decreasing cumulative nutritional index  , were observed to be characterized by distinct network architecture.Streptococcus (belonging to the group G4) was observed to be associated with beneficial genera like Faecalibacterium (belonging to group G1). Similarly the genera Enterobacter (belonging to G4) was observed to be associated with genera Eubacterium, Roseburia and Dorea (from group G1).The co-occurrence network of the AH group of metagenomes were characterized by four distinct connected groups of genera. Interestingly, although genera belonging to the groups G1 and G4 were observed to have contrasting trends in their abundance patterns, some of themshowed strong positive associations amongst each other . For exaEsherichia, Shigella, Streptococcus, Veillonella) were observed to be placed together in a single connected hub. Furthermore, the average node density of the co-occurrence networks obtained for the borderline metagenomes was also observed to be higher as compared to that obtained for the apparently healthy metagenomes. This probably indicates that with a progressive decrease in nutritional status, there may be an increasing degree of functional interdependence among genera .Distinct changes were observed in the genera co-occurrence network with a progressive decrease in the nutritional status. For example, in the borderline (BL) group of metagenomes, genera belonging to the group G4  belonging to the group G4, the second hub was constituted by the commensal and the beneficial genera , belonging to G1, in addition to Prevotella and Paraprevotella. These results suggest that the gut microbial community in SM group is likely to be characterized by the presence of two  groups of genera. The average node degree within these two hubs were also observed to be high , indicating a strong inter-dependency within the members in each group.The increasing interdependence among the genera is observed to be even more pronounced in the co-occurrence networks obtained for the severely malnourished group of metagenomes. The networks of co-occurring generain the severely malnourished (SM) microbiomes were observed to contain two dominant hubs of genera. While one hub was primarily constituted by the likely pathogenic genera  identified the average number of neighbors to have significant negative correlation with the nutritional status  indicatiet al.The onset and progression of malnutrition has been attributed to a variety of causal factors. These include inadequate nutrient intake, prolonged enteric infections due to sub-standard hygiene, metabolic disorders and the variations in the taxonomic and functional composition of the gut microbiota. Previous studies have provided the early snapshots of the taxonomic and functional dysbiosis in the gut microbiome of malnourished children Roseburia, Faecalibacterium, Butyrivibrio, Eubacterium and Phascolarctobacterium, have been suggested to have positive influence on the nutritional status of the children. These genera have earlier been reported to be a part of the normal commensal microflora of the human gut Roseburia and Butyrivibrio are known producers of short chain fatty acids (SCFA) in the intestine Faecalibacterium, Roseburia and Eubacterium have been shown to possess anti-inflammatory properties The present study indicates that the impaired nutritional status is not only due to the abundances of likely pathogenic microbial groups, but also a result of depletion of several commensal genera. These genera, namely, The present study revealed that certain functional categories (COG groups) are positively/negatively correlated with nutritional status. The identified positively correlated functional groups are likely to aid in better utilization of the nutrients in the gut, thereby helping in maintenance of the overall well-being of the child. On the other hand, the negatively correlated functional groups may initiate the infection process which might indirectly lead to malabsorption of nutrients. The higher number of virulence genes identified in the children with lower nutritional index further corroborates this finding.A key finding of the current study is the identification of three groups of carbohydrate active enzymes  families (in the gut microbiomes) showing positive associations with the nutritional status of the children. These groups were observed to be dominated by CAZyme families that preferentially degrade peptidoglycans and complex plant carbohydrates. Dietary fibers, chiefly constitutedby complex carbohydrates, cannot be completely degraded by enzymes coded by the human genome. These complex carbohydrates can only be degraded by the enzymes encoded by the gut microbiota. The human host can harvest energy from such recalcitrant carbohydrates only after bacterial enzymes have pre-processed them. Such ‘energy harvest’ in the gut becomes particularly crucial for subjects with lower nutritional status.Identification of distinct changes in genera co-occurrence networks with progressive decrease in the nutritional status of the children is a key finding in the current study. The results indicate that with decrease in the nutritional status, there is a likely increase in functional interdependence among the various microbes residing in the gut. It was also interesting to observe the associations of probable pathogenic groups with potentially beneficial ones in healthy gut microbiomes. Clustering of potentially pathogenic groups into a distinct hub in severely malnourished gut probably suggests how these groups may help each other in a cooperative manner to a create an imbalance in microbial populations. This in turn may lead to the creation of milieu that is progressively detrimental in maintaining the nutritional homeostasis of the gut. Functional associations within microbial groups may modulate specific functional behaviors of its constituent members in terms of their expressed genes and proteins. Thus, the observed changes in the architecture and composition of networks of co-occurring microbial groups under different nutritional settings add further insights into the gut microbial environments associated with the nutritional status. The functional interdependence of the network could be used in future for therapeutic interventions to address the challenge of clinical management of severe acute malnutrition. The network analysis suggests that one can modulate the gut microbiome by disrupting certain key players (genera) in order to achieve conditions that could result in the regression of the disease phenotype.Considering the pilot nature of the study and the limited sample size, the results obtained in the study should be interpreted with due caution. However, the present findings open up an interesting premise which furthers our understanding of the role of gut microbiota in causation of malnutrition under nutrient limited conditions. Further, given that the geographical location, age and diet of most of the children (considered in the current study) were similar, it was assumed that the observed trends  could provide a rough indication of the changes in the microbiome with varying nutritional status. However, in order to obtain a definitive picture of the underlying changes associated with either increase or decrease of nutritional status, a longitudinal study is absolutely necessary. We further acknowledge that ‘unacknowledged heterogeneity’ remains as there could be numerous genetic and environmental factors affecting the composition of the gut microbiome The present study describes patterns wherein the gut microbiome varies in response to nutritional status. Although the biological bases of such patterns may be inferred to a certain extent, the universality and the biological mechanisms governing the occurrence of these patterns remains to be experimentally verified. Although limited by sample size and range of nutritional status of selected subjects, the present study opens up avenues for in-depth analyses of gut microbiomes on much larger populations belonging to diverse geographies as well as nutritional status. Such studies will help in better understanding the role of microbial groups in host nutrient assimilation and how they interact, which are evolving as important determinants of systemic conditions like malnutrition. Much comprehensive and well designed functional studiesare required for formulating a microbial basis of therapy for severe acute malnutrition.File S1Combined supporting information. Additional data including details of study population, sample collection, DNA extraction, pyrosequencing, quality filtering, abundances of carbohydrate active enzyme families and virulence factors in the gut microbiomes are provided in supporting information in File S1. Supplementary Figures and supplementary Tables are also available in File S1.(PDF)Click here for additional data file.