The fifth Millennium Development Goal (MDG-5) aimed to improve maternal health, targeting a maternal mortality ratio (MMR) reduction of 75% between 1990 and 2015. The objective of this study was to identify all maternal deaths in Suriname, determine the extent of underreporting, estimate the reduction, audit the maternal deaths and assess underlying causes and substandard care factors.A reproductive age mortality survey was conducted in Suriname (South-American upper-middle income country) between 2010 and 2014 to identify all maternal deaths in the country. MMR was compared to vital statistics and a previous confidential enquiry from 1991 to 1993 with a MMR 226. A maternal mortality committee audited the maternal deaths and identified underlying causes and substandard care factors.In the study period 65 maternal deaths were identified in 50,051 live births, indicating a MMR of 130 per 100.000 live births and implicating a 42% reduction of maternal deaths in the past 25 years. Vital registration indicated a MMR of 96, which marks underreporting of 26%. Maternal deaths mostly occurred in the urban hospitals (84%) and the causes were classified as direct (63%), indirect (32%) or unspecified (5%). Major underlying causes were obstetric and non-obstetric sepsis (27%) and haemorrhage (20%). Substandard care factors (95%) were mostly health professional related (80%) due to delay in diagnosis (59%), delay or wrong treatment (78%) or inadequate monitoring (59%). Substandard care factors most likely led to death in 47% of the cases.70 per 100.000 live births) and eliminate preventable deaths include improving data surveillance, installing a maternal death review committee, and implementing national guidelines for prevention and management of major complications of pregnancy, childbirth and puerperium.Despite the reduction in maternal mortality, Suriname did not reach MDG-5 in 2015. Steps to reach the Sustainable Development Goal in 2030 (MMR ≤  Reducing maternal mortality is one of the major challenges to health systems worldwide. United Nations’ Millennium Development Goal 5 (MDG-5) called for a 75% reduction of the maternal mortality ratio (MMR) between 1990 and 2015. The global MMR fell from 385 deaths per 100.000 live births in 1990 to 216 in 2015, corresponding to a decline of 44%. A vision of ending all preventable maternal deaths has emerged in 2015, being one of the Sustainable Development Goals (SDGs); it aims to reduce the global MMR to less than 70 deaths per 100.000 live births by 2030. Achievement of this target will require robust information systems with high-quality data, specifically on causes of death, as it is of great importance in informing decision-makers and ultimately reducing maternal mortality [per 100.000 live births in 1991-1993, suggesting a 31% decrease instead of the 25% increase as suggested by the UN [UN’s Maternal Mortality Estimation Inter-Agency Group reports that Suriname is one of the few countries with an increase in MMR from 127 in 1990 to 155 in 2015 . Howevery the UN , 5. HoweMaternal health outcomes are strongly associated with higher capital levels, suggesting that an increase in Gross National Income (GNI) per capita should correspond with a reduction in maternal mortality . SurinamAccording to WHO-estimates, Suriname MMR 155) belongs to the four worst performing countries in Latin America and the Caribbean  55 belong.Therefore, the aim of the study is first to identify all maternal deaths in Suriname from 2010 to 2014, second to determine whether maternal deaths were accurately registered and classified, third to assess the reduction of maternal deaths in 25 years, fourth to perform an in-depth audit of the deaths and finally to determine the level of substandard care.A reproductive age mortality survey (RAMoS) was conducted, using different methods to identify maternal deaths nationwide in Suriname between January 1st 2010 and December 31st 2014.Suriname is a multi-ethnical South American country with a population of 541,638 served by four referral hospitals in the capital, Paramaribo, and one hospital near the western coast, Nickerie. In addition to general practitioners, Regional Health Services (RGD) and Medical Mission (MZ) are responsible for primary healthcare. RGD comprises of 43 facilities serving the whole coastal area and the Medical Mission has 56 health posts throughout the interior. Figure According to the ICD-MM a pregnancy-related death is the death of a woman while pregnant or within 42 days of termination of pregnancy, irrespective of the cause . A materMaternal deaths in Suriname are identified mainly by the collection of death certificates and sporadic informing in the hospitals. No independent surveillance systems are adapted to investigate deaths in women of reproductive age. Notification of death is compulsory by law. However burial can take place without the official death certificate, when there is ‘an act of death’ . The death certificate is filled in afterwards and often received with a delay (>3 months) and in 15% not received at all. In addition the death certificate lacks a pregnancy checkbox . IdentifThe RAMoS consisted of different steps. First, case records of maternal deaths from 2010 to 2014 identified by vital registration were collected. Second, all medical records of deceased women aged 10 to 50 years in our study period were collected from the archives of all hospitals and the primary health care institutions . Third, The Central Bureau of Civil Affairs provided a list of all deceased women in the country between 2010 and 2014 with an offspring in the preceding year. Fourth, an inventory was performed in the largest mortuary . Fifth, obstetric health care professionals in all facilities were asked their knowledge on local maternal deaths in the past 5 years.Medical records were collected and examined extensively and in case of an incomplete file involved health care professionals were interviewed. Verbal autopsy with family member(s) was performed when maternal deaths occurred outside of the hospital. This was conducted according to the WHO-instrument on verbal autopsy . All avaMDR: Clinical summary form tool [An elaborate clinical case summary of every pregnancy-related death was made according to the FIGO-LOGIC orm tool . InformaWHO guidelines on applications of ICD-MM [. Substandard care factors were analysed according to an adapted version of the FIGO-LOGIC MDR Grid analysis of clinical case management form [An expert committee, consisting of different obstetricians, an internal medicine specialist or anaesthesiologist and midwives, audited all pregnancy-related deaths with two authors (LK and KV) presenting and moderating the sessions. When no consensus was achieved, external expert opinion (JR and HK) was sought. The committee reviewed the cases and agreed to a mode of death, underlying cause, contributing factors and classified each death using f ICD-MM . Substanent form . Due to Data were manually entered into IBM SPSS version 21.0  for analysis. All maternal deaths were individually analysed and cross-linked with registered maternal deaths by civil registration. Causes, contributing factors and substandard care factors were recoded into categorical variables.Of the 1335 deceased women of reproductive age between 2010 and 2014, 71 were pregnancy-related and 65 were maternal deaths  and by misclassification in 65% (n = 31) (Table Underreporting occurred by misidentification in 26% n = 7 and by n = 17) occurred in the hospitals in 88% (n = 15) or at home in 12% (n = 2). The causes of these hospital-deaths were infectious diseases in 87% (n = 13), admitted and deceased on non-obstetric wards. These death certificates did not indicate or suggest that the woman was or had been pregnant. Maternal deaths, which were identified by vital registration but were classified incorrectly, consisted of deaths without the cause mentioned on the death certificate (n = 9), non-obstetric diseases (n = 13), deaths complicated with more than one diagnosis (n = 8) and cases in which the mode of death was reported on the death certificate rather than the underlying cause (n = 17).The maternal deaths not identified by vital registration .The women in Suriname who died during pregnancy, childbirth or puerperium lived in a rural coastal area or in the rural interior in respectively 15 and 17% Fig. . Maternan = 45) of the deceased women. Socially insured women were maroons or creoles in 75% (n = 34) of the cases. Anaemia (Hb ≤ 6.0 mmol/L) complicated 45% of the cases. Post-mortem investigation was performed in 3% (n = 2) of maternal deaths.Social insurance, indicating the (near) poor, covered 69%  and obstetric haemorrhage .Of the 65 maternal deaths, 41 63%) were due to direct causes, 21 (32%) due to indirect causes and three (5%) maternal deaths were classified as unspecified because the cause of death was unknown  caused by uterine atony (29%), retained placenta (23%), ruptured uterus (15%), vaginal / cervical tear (8%), and unspecified causes (10%). Underlying cause of all ante partum haemorrhages was placental abruption .Obstetric haemorrhage was mainly due to postpartum haemorrhage  accounted for 14% of maternal deaths. However, hypertensive disorders, such as pregnancy induced hypertension and pre-eclampsia, were diagnosed in 30% of all maternal deaths. Though not the underlying cause of death, they were commonly classified as a contributing factor.n = 8, 12%) were four probable amniotic fluid embolisms, one obstructed labour, one suicide by intoxication at 24 weeks, one case of acute fatty liver of pregnancy with consequently hepatic encephalopathy and multi-organ failure. The underlying cause of one case remained unknown as the woman died without any reported symptoms within a few hours after caesarean section for foetal indication.The remaining other causes of direct maternal deaths (Sepsis occurred either due to direct obstetric complications (9%) of which one third had puerperal sepsis while being HIV positive or due to medical conditions aggravated by the pregnancy  and therefore were classified as indirect maternal deaths (18%).n = 9) concerned two cases of endocarditis resulting in heart failure, one pulmonary bleeding caused by idiopathic thrombocytopenia, one case of end-stage renal failure due to diabetes and one woman, diagnosed with pre-existent hypertension, died due to a cerebrovascular accident.The other non-sepsis indirect maternal deaths (n = 56/59) of the cases , inadequate treatment (78%) or poor monitoring (59%). Blood transfusion was unavailable in 10 of 31 cases (32%) when this was required. An ICU bed was not available when requested in 11 (24%) of 45 cases. The committee agreed that in 47% of the maternal deaths substandard care factors certainly (21%) or most likely (26%) led to death.The MMR in Suriname is 130 per 100,000 live births between 2010 and 2014. Mungra et al. reported a MMR of 226 between 1991 and 1993, which indicates a 42% reduction in maternal deaths and an improvement in underreporting from 64% to 26% . A compaThough our study suggests that, over the years, there is a growing reliability on identification of maternal deaths, the underreporting rate in Suriname (26%) is still higher than reported in Jamaica (20%), Argentina (9.5%) and Mexico (13%) –19.The underreporting due to misidentification of maternal deaths in Suriname can be explained by numerous facts: first, physicians are not obliged to report maternal deaths. Second, part of the death certificate (including the cause of death) is not always available as it is not obliged to be completed before the burial takes place. Third, the death certificate does not include a pregnancy checkbox and finally no active enquiry or RAMoS is performed. The effectiveness of a pregnancy check box on death certificates has proven to be effective in identifying pregnancy-associated mortality , 21. MisSocial insurance as a marker indicating poverty was found in the majority of maternal deaths (69%), while less than half of the general population had social insurance. A difference in ethnicity is seen between the general female population  and the maternal deaths . Similar to two decades ago, obstetric haemorrhage is the most common direct cause of death, which is lower than reported in low-income countries (27%) and higher than in high-income countries (16%) , 19. AltIndirect maternal deaths 32%), in particular non-obstetric sepsis (18%), accounted for a greater part of the maternal deaths in our study compared to the 27% of other Latin American and Caribbean countries [2%, in paThe most striking finding of our survey is that the majority of maternal deaths occurred in hospital (85%) with the most important substandard care factor being delay in diagnosis (59%) and delay in treatment by health care providers, and less frequently due to patient delay (15%). This finding necessitates actions such as training and retaining skilled staff and implementation of evidence-based guidelines. Another important finding is that most of the deaths occurred postpartum, indicating that improvements can be made in the care provided in the period after birth. Patients should be provided with more information. We advise more frequent and qualitative better postnatal checks and if necessary home-visits should be performed. Lastly, a great number of maternal deaths occurred on the wards (30%) and the monitoring of patients is found to be inadequate in 59% of these cases. Implementation of an early warning system for timely interventions in order to reduce serious adverse events has been proven effective and is recommended .Limitations of this study are indwelled in its retrospective nature. Though we performed a robust enquiry, maternal deaths could have been missed, especially if they occurred outside of health care facilities or during early pregnancy. In addition, not all case files were available, records were often incomplete and post-mortem investigations were generally not performed. This affected the quality of the classification of causes and evaluation of substandard care during the maternal death reviews. Finally, but most importantly, due to lack of national data on characteristics of the pregnant population, pregnancy and delivery, we were not able to perform multivariate analysis and assess risk factors.Suriname has a high MMR compared to other Latin American countries and the Caribbean with similar or lower income economies. We highly recommend (1) to improve national data surveillance, (2) install a maternal mortality committee to review all maternal deaths, (3) implement an early warning score and national guidelines on postpartum haemorrhage and eclampsia and (4) improve postnatal care strategies. Lastly, as maternal mortality is merely the tip of the iceberg, severe morbidity research should be conducted to assess and prevent severe obstetric complications and make progress to reach the SDG of a MMR <70 in 2030.


Weight response data including feed conversion ratios (FCR), carcasses without giblets (WOG), wing, skin, white meat were collected during processing. Extracted DNA from cecal contents was utilized for microbiome analysis via an Illumina Miseq. In conclusion, white meat yield of Biolex® MB40 supplemented group exhibited significant improvement compared to both negative control (NC) and BMD50 supplemented groups. In addition, antibiotic significantly decreased level of Lactobacillus in 2 wk compared to other groups. A significantly higher percentage of Campylobacter was observed from the 4 wk old birds treated with antibiotic BMD50 compared to the NC and prebiotic group. Retention of broiler performance and improvement of white meat yield suggest that the prebiotic MB40 appears to be a potential alternative to replace the antibiotic growth promoter.Prebiotics are defined as fermentable food ingredients that selectively stimulate beneficial bacteria in the lower gastrointestinal tract of the host. The purpose of this study was to assess growth performance of broilers and the cecal microbial populations of an antibiotic, BMD50, supplemented birds compared to broiler chickens fed the prebiotic, Biolex Bifidobacteria and Lactobacillus [Bifidobacteria and Lactobacillus thus, leading to increased SCFA production which in turn inhibits colonization and growth of pathogens [In 1995, Gibson and Roberfroid first defined prebiotics as non-digestible food ingredients that selectively promote beneficial bacteria such as bacillus –8. Prebibacillus . For exaathogens –16.® MB40 , consists of 1,3–1,6- β-D-glucan and mannanoligosaccharides (MOS) which are derived from the cell walls of Saccharomyces cerevisiae, and several studies have shown their positive effects on growth performance of broilers [The commercial prebiotic evaluated in this study, Biolexbroilers , 18. Accbroilers , supplembroilers  reportedSalmonella utilizes this type-1 fimbriae thus, reduction of Salmonella concentration is expected upon introduction of MOS. Spring et al. [et al. [Salmonella reduction in broilers by adding mannose to their diets. In a previous report Lee et al. [Salmonella from the same cecal samples used for the current study. In addition to improvement of growth performance and suppression of Salmonella colonization, supplementation of MOS also exhibited elevation of immunoglobulin A (IgA) and immunoglobulin G (IgG) level [One of the distinct features to the MOS is the ability to bind to mannose-specific type-1 fimbriae of pathogen therefore, prevent colonization of pathogens . Receptog et al.  and Oyof [et al. , 23 obsee et al.  detectedG) level , 26.et al. [The current study evaluated microbial populations of individual birds in each group using Next Generation Sequencing (NGS) approaches on samples collected from the study by Lee et al.  where poet al. . Therefoet al. , the obj® MB40. Since the birds were raised in an off-campus commercial farm, the current study was exempted from review by the University of Arkansas Institutional animal care and use committee (IACUC). No researchers at the University of Arkansas were involved directly in any aspects of the chicken growth part of the study. Therefore, authors did not need permission to access the farm since samples were collected by farm employees and subsequently transported to the laboratory at the University of Arkansas. In addition, the National chicken council (NCC) guidelines were  followed by the commercial cooperators to ensure internal animal welfare. Ten birds from each treatment were chosen for sampling of cecal contents as described previously [Three houses were assigned for each treatment and each house contained 15,300 Hubbard x Cobb straight run broilers  . The bireviously .® Genomics for analysis of variance (ANOVA). P-values less than 0.05 were interpreted as significant differences among treatments.The twenty birds that resided in the respective pens were weighed at 14, 28, 40 and 53 days of age and recorded. In addition, carcasses without giblets (WOG), wing, skin breast and tender weights were measured from randomly selected 100 birds  that were located in the house but outside the pens at 53 days of age. Birds that lost tags or were condemned by the USDA inspector were exempted from statistical analysis. Finally feed conversion ratios (FCR) were determined by feed intake and body weight at 28, 40 and 53 days of age. White meat yields were calculated by dividing sum of breast and tenders by live weight of randomly selected 100 birds within each house. Recorded data were imported by Microsoft Excel and JMP-1 for the preparation of a sequencing library targeting the V4 region of 16S rRNA [TM Normalization kit . The library was constructed by combining 5 μL of each normalized aliquot samples for further assessment. Library concentration and product size were confirmed using a KAPA Library Quantification Kit  via a quantitative PCR  and an Agilent 2100 Bioanalyzer system . The 20 nM of pooled library aliquot and the 20 nM of PhiX control v3 were combined with 0.2 N fresh NaOH and HT1 buffer and mixed a second time with 5% of the PhiX control v3. The 600 μL of the mixture containing pooled library aliquote, PhiX control v3, NaOH and HT1 buffer was subsequently loaded onto a MiSeq v2 Reagent cartridge to conduct sequencing.Cecal contents (200 mg) from each birds were collected for DNA isolation utilizing QIAamp DNA Stool Mini Kit . The concentration of extracted DNA was diluted to 10 ng μL16S rRNA . Based o® Genomics for ANOVA and a P-value of 0.05 to determine significant differences.Sequencing read files were processed using Quantitative Insights into Microbial Ecology (QIIME) pipeline (version 1.9.0) . Each ofet al. [The current study is the continued analysis of previously published research of Biolex® MB40 by Lee et al.  where fiet al. [et al. [et al. [et al. [et al. [et al. [et al. [et al. [Growth performance including weight of the birds, FCR, weight of the parts and white meat yields of broilers are considered important since they directly relate to the market value of the bird , 31. Aveet al.  and Midi [et al.  where no [et al.  however, [et al.  which ag [et al.  and Pelí [et al.  when bro [et al.  reported [et al.  observed [et al.  speculat [et al.  and Rosh [et al.  have hypet al. [® MB40, observed similar results of pasture flock broiler performance with no significant differences occurring in FCR and average bird body weights among treatment groups. However, the prebiotic MB40 results did match the responses seen with antibiotic fed birds therefore, prebiotic MB40 potentially could replace the benefit seen with the antibiotic supplement. Significant weight gains of the birds were observed in the study by Roshanfekr and Mamooee [Another study by Park et al. , on the  Mamooee , when suWhen comparing poultry processing responses including chicken part weights and white meat yield, only the white meat yield was significantly increased by supplementation of prebiotic MB40 compared to negative control group and antibiotic treated group . AccordiLactobacilli and Bifidobacteria [Lactobacilli and Bifidobacteria but also improves glucose homeostasis, leptin sensitivity and intestinal homeostasis [Lactobacilli and Bifidobacteria such as Faecalibacterium prausnitzii and Akkermansia muciniphila which in turn benefits the host [When prebiotics were first introduced, the only microorganisms that were noted as beneficial bacteria were bacteria . Howevereostasis , 44. In the host –48.et al. [Barnesiellaceae which is a microorganism that commonly occurred in all groups at wk 4 according to previous DGGE-band sequencing analysis [Bacteroides dorei and Bacteriodes rodentium only in the negative control group for wk 2 and 4 and were less frequent by 6 wk. Mollicutes levels were low in 2 and 4 wk but significantly higher levels were detected in 1 and 6 wk. However, the level of Bacteroidaceae increased significantly in 2 wk and decreased afterwards.According to Lee et al. , antibioanalysis . Howeveranalysis . Out of analysis –50. Bactanalysis  which derss genes and concluded that V3 yielded the best DGGE profiles. For microbiome sequencing, generally V3-V4 or V4 are preferred due the coverage of both bacteria and archaea found in gut and fecal samples [While both methods, DGGE-band sequencing and NGS results were somewhat similar there were differences as well. Such differences in outcomes between DGGE profiles and NGS microbiome sequencing of broiler cecal samples has been observed by our group in previous work . Therefo samples , 53–55.  samples  observedCampylobacter however, 90% of Campylobacter were found in birds with 4 and 6 wk of age which indicates Campylobacter colonizes within birds in their later phases of growth. Recovery of Campylobacter among all microorganisms were averaged 0.3%. Several studies have shown that colonization of Campylobacter can be suppressed by introduction of Lactobacillus and Bifidobacteria [et al. [Campylobacter and Lactobacillus however, no statistical relationship was found in the current study where a significantly higher level of Campylobacter was observed at 4 wk in the antibiotic treated group compared to the negative control and prebiotic treated group  and MB40 (T2) were compared. Consistency of body weight, FCR and parts yields among prebiotic, antiobiotic and control groups was also observed in previous studies –65. The Lactobacillus level occurred in birds receiving antibiotic BMD50 supplementation at 2 wk. No significant reduction of chicken performance was observed thus, MB40 could be a viable alternative of in-feed antibiotic supplementation when addressing concerns related to the presence of antibiotic resistant bacteria.Overall, commercial prebiotic, MB40 significantly improved white meat yield in conventionally raised broilers but was not accompanied by significant enhancement in other aspects including body weight, FCR and parts weight. According to the NGS approach to microbiome analysis a significant decrease in


Emblica officinalis (EOT) – a fruit that has been traditionally used for the treatment of numerous disorders for centuries. Significant alterations in MDA, GSH, TBARS, GPx, mitochondrial ATP, and mitochondrial membrane potential levels were observed in rats fed HSCD, which indicated presence of oxidative stress. Moreover, classic signs of cognitive impairment and deficits in spatial learning and memory were observed in the neurobehavioral tests. E. officinalis tannins exhibited good affinity to Nrf2 receptors in in silico studies, significantly reversed the changes in the aforementioned biomarkers of oxidative stress which were altered in the model group, as well as improved the performance of rats in Morris water maze task. Our results also reflected that EOT supplementation significantly increased the expression of Nrf2 in the CA1 region of hippocampus and cortex. Additionally, TUNEL assay indicated that EOT supplementation led to reversal of DNA fragmentation and apoptosis caused by HSCD. Immunohistochemical analysis and western blot further revealed a surge in the nuclear location of Nrf2. Through our study, we have demonstrated that cognitive impairment can be caused in rats via HSCD as a result of the oxidative stress induced by the same. Additionally, we have investigated a novel mechanism of action for EOT (which strongly suggests to be via the Nrf2–ARE pathway) and demonstrated that it has the potential to be used in the treatment of cognitive impairment.Modern diets containing high quantities of salt and cholesterol have exhibited to cause a considerable effect on our health. Such diets, when consumed in the long term, have also shown to be a precursor to several disorders such as the metabolic disorder and consequently, various other diseases, including cognitive deficits. In the present study, we used a high salt and cholesterol diet (HSCD) to induce cognitive impairment in rats and also investigated the pharmacological action of tannins enriched fractions of  Dementia is a clinical syndrome characterized by a progressive decline in memory in addition to impairment or difficulties in cognitive functions such as language (aphasia), executive function , or skilled movements (apraxia). It is estimated that about 135 million people would suffer from dementia by the year 2050 and that the global cost of dementia care would increase to one trillion United States dollars by the year 2030 . DementiDiets containing high amounts of salt and cholesterol (a precursor to the aforementioned risk factors) have been implicated in causing cognitive impairment . One hypIncreased oxidative stress is a consistent feature in various forms of dementia. Significant damage due to oxidative stress is also commonly seen in peripheral blood during the neurodegeneration process . OxidatiRecent research studies have underscored the potential role of the Nrf2-ARE pathway in numerous neurodegenerative disorders. As mentioned earlier, decrease in Nrf2 in the brains of Alzheimer’s patients is a common feature and is considered to be the possible reason behind the increased vulnerability of neurons to stresses in these patients . Most ofEmblica officinalis Gaertn. (Amla or Indian gooseberry) is a plant, which grows in the tropical regions of India, Indonesia, China, and the Malay Peninsula. It is a member of the Euphorbiaceae family, and the fruit of this plant has traditionally been used for the treatment of a multitude of ailments in the Ayurveda, Unani, Tibetan, Siddha, Arabic and various other folk systems of medicine. The fruit contains several tannins such as emblicanin A, emblicanin B, pedunculagin, punigluconin, gallic acid, rutin, etc. In a study by E. officinalis (EOT) fruits normalized alterations in frontal cortical and striatal superoxide dismutase, glutathione peroxidase, catalase, and lipid peroxidation induced by chronic stress via chronic unpredictable foot-shock induced perturbations. In another study, EOT reversed amnesia caused due to scopolamine administration as well as reduced oxidative stress induced by it. In addition, it also reversed the increased acetylcholinesterase (AchE) levels in the brain  fruits were provided as gift samples by Indian Herbs Research and Supply Company . The tannins were extracted as per the method given by E. officinalis, following which, column chromatography was performed over Sephadex LH-20 employing methanol and methanol-water as the eluent. The concentration of the various tannins was as follows: emblicanin A (37%), emblicanin B (33%), punigluconin (12%), pedunculagin (14%), rutin (3%), and gallic acid (1%) (Tannins enriched fractions of cid (1%) . PiracetThe experimental protocol was approved by Institutional Animal Ethics Committee of Jamia Hamdard (Hamdard University), New Delhi, India (Registration no. JH/993/CPCSEA) as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals. Female Wistar rats in the range of 150–200 gm of body weight were issued from Central Animal House Facility, Jamia Hamdard, New Delhi and housed in standard polypropylene cages (six rats each cage) and had access to commercial standard pellet diet . The rats were maintained under controlled room temperature (23 ± 2°C) and relative humidity (60 ± 5%) with 12 h light/12 h dark (day/night) cycle in the departmental animal house.Suspension of EOT and PCT were prepared by triturating the weighed amount of EOT (100 and 200 mg/kg) and PCT (200 mg/kg) in 0.5% carboxy methyl cellulose (CMC) suspension (w/v) in normal saline, respectively . High saad libitum for 8 weeks to induce cognitive impairment (Table 1). The rats were observed for behavioral parameters (Moris water maze test) and then immediately sacrificed for histopathological examination and estimation of biochemical parameters .Prior to the commencement of experimental studies, animals were fed with standard rat food pellets for 2 days. Then, animals were fed a HSCD pairment . After tin silico studies were performed to evaluate the predominant binding mode as well as the binding affinity of the chief EOT constituents with the target receptor  was estimated using the colorimetric method as described by 2O2 at 340 nm (-1 mg-1 protein.Glutathione peroxidase (GPx) activity in brain was assayed spectrophotometrically through the glutathione/NADPH/GR system, by the dismutation of Ht 340 nm . GPx actg for 10 min. Thereafter, supernatant was transferred to other test tubes and placed in a boiling water bath for 10 min. The absorbance of pink color produced was measured spectrophotometrically at 535 nm. The TBARS content was calculated by using a molar extinction coefficient of 1.56 × 105 M-1 cm-1 and expressed as nmol of TBARS formed/hr/mg of protein.The thiobarbituric acid reactive substances (TBARS) test was used to determine malondialdehyde (MDA) – a biochemical produced during the lipid peroxidation process . 0.1 mL n = 6) were carefully dissected as per anatomical guidelines and placed in a glass Dounce homogenizer containing five times the volume of isolation buffer . A low-speed spin (1300 g for 5 min) was performed to remove unbroken cells and nuclei after the homogenization process. The supernatant was transferred to fresh tubes, topped off with isolation buffer and spun down again at 13,000 × g for 10 min. The supernatant was discarded, and the resultant mitochondrial pellets were suspended in 500 μl of isolation buffer with 1 mM EGTA  and 0.1% digitonin (in DMSO) was added to the pellets to disrupt the synaptosomes. After 5 min, isolation buffer containing 1 mM EGTA was used to bring the samples to a final volume of 2 mL, after which it was centrifuged at 13,000 × g for 15 min. Next the pellets were resuspended in isolation buffer without EGTA  and was centrifuged at 10,000 × g for 10 min. Isolation buffer without EGTA was used to suspend the final mitochondrial pellet to yield a final protein concentration of approximately 10 mg/mL and was immediately stored on ice. In order to normalize the results, the concentrations of protein were estimated with all the samples on the same micro well plate using a BCA protein assay kit.Preparation of Isolated Mitochondria: The rats were euthanized with isoflurane after completion of behavioral studies and the procedure given by A 200 μM stock solution of JC-1  was made employing dimethyl sulfoxide (DMSO) as the solvent. The assay buffer consisted of mitochondrial isolation buffer along with 5 mM pyruvate and 5 mM malate. 150 μl of assay buffer and 20 μl  of mitochondria were added to the wells of a 96-well black, clear bottom microplate (Corning). 1 μM JC-1 was added and gently stirred. Aluminum foil was used to cover the microplate, and the microplate was left for 20 min at room temperature before observation. Fluorescence  was then measured .Production from isolated mitochondria, Mitochondrial ROS production was measured following incubation of isolated mitochondria with 25 μM 2,7- dichlorodihydrofluorescein diacetate for 20 min and then the DCF fluorescence  was read as previously described . In shorLuminometric method [using ATP bioluminescence assay kit ] was used to determine ATP levels according to the provided protocol. Mitochondrial samples were assayed for ATP content using the ATP dependence of the light emitting luciferase-catalyzed oxidation of luciferin. ATP concentration was calculated according to a standard curve and related to protein content .For immunohistochemical analysis of Nrf2 protein, paraffin sections of the brains were deparaffinized in xylene followed with acetone for 5 min each. A graded series of ethanol was used to rehydrate the samples. The samples were then washed with double distilled water, after which, antigen retrieval was performed by citrate buffer (pH 6). Three changes of section were done with TBS buffer solution. These sections were then blocked with 1.5% normal goat serum for 1 h. Sections were then incubated overnight at 4°C with purified goat polyclonal antibody raised against a peptide mapping at the N-terminus of Nrf2 of human origin . Biotinylated anti-goat rabbit secondary antibodies and avidin-biotin-peroxidase complex was used to detect immunoreactivity. Immunoreactive signal was developed using diaminobenzidine as a substrate for 2 min. A Meiji microscope enabled with lumenera camera was used to take the photomicrographs. The images were analyzed with lumenera analyze 3 software. All immunohistochemical samples were analyzed in a blinded fashion. For the quantification of the protein expression semi automatically, Image J 1.49 software was used to estimate the volume fraction of immune-reactive cells within the tissue sample. The range of pixel intensities of images was in between 0 and 250. Values 0 and 250 indicates the darkest and lightest shade of the image color, respectively .g for 1 h at 4°C. The soluble fraction was collected as the TBS-soluble fraction, and the pellet was resuspended in 15 volumes of 1% Triton X-100/TBS (TBSX). After incubation for 30 min on ice, the tube was centrifuged at 100,000 g for 1 h at 4°C. The supernatant was removed as the TBSX-soluble fraction, and 70% formic acid (FA) was added to the pellet, followed by centrifugation at 100,000 g at 4°C for 1 h. The FA fraction was neutralized with 20 volumes 1 M Tris-base (pH 11), and subsequently aliquoted and stored at -80°C. The total protein level of TBS-, TBSX-, and FA- was determined using the BCA protein assay kit  and the Bradford protein assay kit . For western blotting, equal quantities of protein extracts were subjected to SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes  (Frozen brain was homogenized with 15 volumes (w/v) of TBS buffer, containing phosphatase and protease inhibitor cocktails , followed by centrifugation at 100,000  States) . After bTUNEL assay, which is used to detect the apoptosis, was performed according to the manufacturer’s instructions (Roche Inc.) . BrieflyP-value < 0.05 was considered statistically significant. Error bars represent the standard error of the mean (SEM). All statistical tests were performed using the Prism software package .Results were expressed as the mean ± standard error of mean (SEM). The statistical significance of difference between groups was determined using one-way analysis of variance (ANOVA) followed by Tukey’s test. E. officinalis tannins and piracetam were investigated through docking study using Schrodinger programme Maestro 10.5. The ligands were docked into the active site of Nrf2 binding domain (PDB 2FLU) generated by running the site map program and free binding energy, docking scores, interacting amino acid residues H-bonds and stacking amino acid residues pi–pi bond were determined. Emblicanin A – one of the chief constituents of EOT – was well located inside the binding site and the results had revealed that emblicanin hydroxyl and keto group interact in the Nrf2 binding sites with the amino acid Ser508, Arg415, Arg483, Ser555, Ser363, Arg336, Tyr572, Arg380, Tyr334, Ala556. The standard drug piracetam showed two hydrogen bonds, i.e., Tyr334 and Arg336 with the keto and amine group present in the drug similar to the ligand emblicanin. Furthermore, hydrophobic interaction was observed with the amino acids Ile461, Tyr525, Hie575, Phe577, Gly603, Gly364, and Gly509 with emblicanin A. The more hydrogen as well as hydrophobic binding with the drug emblicanin A as compared to the standard piracetam resulted in higher docking score. Other tannins such as punigluconin and gallic showed even better affinity toward Nrf2, the results of which are present in Table 2 and Figure 2.The theoretical affinity of Figure 3). A decrease in escape latencies was observed in the acquisition trial. Rats in TC group had significantly greater escape latencies than normal group (###p < 0.001). Rats administered EOT200 had significantly lesser escape latencies than rats in the PCT200 group ($$$p < 0.001). Similarly, consistent results were observed when other parameters such as target quadrant path length, number across the platform in probe trial and target quadrant time in probe trial were observed. In summary, no significant difference was observed in rats administered 200 mg of tannins enriched fractions of E. officinalis in the Morris water test when compared to rats in the positive control group.Positive results were obtained on the evaluation of neurobehavioral parameters using Morris water test . The results obtained were consistent with previous findings. EOT administration dose-dependently ameliorated the attenuated the levels of GSH and GPx (∗∗∗p < 0.001). 200 mg dose of tannins enriched fraction of E. officinalis was significantly more efficacious than 200 mg piracetam in increasing GSH and GPx levels ($$$p < 0.001).Levels of reduced glutathione (GSH) and glutathione peroxidase (GPx) were also estimated. GSH is produced in the body via the reduction of glutathione disulphide by nicotinamide adenine dinucleotide phosphate (NADPH) in the presence of glutathione reductase which acts as the catalyst . GSH is Table 3, a significant increase in TBARS level was observed in the TC group, which indicates oxidative stress (###p < 0.01). Moreover, it was observed that TBARS levels significantly decreased as the amount of EOT administered was increased (∗∗∗p < 0.001). Furthermore, EOT200 was found to be significantly more effective in controlling high TBARS level when compared to PCT200 ($$$p < 0.001).Thiobarbituric acid-reactive substances (TBARS) assay is a commonly used analytical procedure to estimate oxidative damage. The TBARS assay helps in estimating malondialdehyde, which is produced by the degradation of polyunsaturated lipids by ROS . As seen###p < 0.001). This decline in SOD levels was significantly reversed in rodents administered EOT (∗∗∗p < 0.001).Superoxide dismutase (SOD) catalyzes the dismutation of superoxide radicals generated as a result of oxidative stress. Hence, estimation of SOD is also an important analytical test to investigate oxidative damage. Administration of HSCD led to a significant decrease in SOD levels in the model group . Decrease in ROS was also observed in the group administered 200 mg of piracetam, but the diminution was relatively lesser than the EOT groups (∗p < 0.05). EOT200 group was significantly more effective in reducing ROS than the PCT200 group ($$p < 0.01).Reactive oxygen species and the resulting oxidative stress have been implicated to be a contributing factor in numerous diseases . As reprFigures 4B,C indicates that there was a significant drop in the MMP and mitochondrial ATP in the TC group with respect to the NC group (###p < 0.001). A significant amelioration of reduced MMP and mitochondrial ATP levels was observed in EOT100 and EOT200 groups (∗∗∗p < 0.001). 200 mg piracetam was also able to significantly increase MMP and mitochondrial ATP levels . However, in both cases, tannoid principles of E. officinalis were significantly more effective in reversing the decreased MMP and mitochondrial ATP levels than piracetam.Mitochondrial membrane potential (MMP) and mitochondrial ATP were estimated as a measure of the oxidative stress generated. Figure 5). It was observed that Nrf2 proteins were mostly present in cellular plasma, and dark brown staining was seen in the positive cells. The NC group showed low levels of Nrf2 expression in both cortices as well as the CA1 region of the hippocampi. However, in the tissue samples obtained from rats in the TC group, a significant enhancement in the Nrf2 immunoactivity was observed in both cortices as well as hippocampi. Moreover, it was observed that supplementation with EOT and administration of PCT200 in the treatment groups resulted in further increase in the expression of Nrf2. Additionally, densitometric analysis further confirmed these observations.In order to further assess oxidative stress, immunohistochemical analysis was employed to evaluate Nrf2 expression in cortex tissues and the CA1 region of the hippocampi .In our findings, the protein expression of Nrf2 in cytoplasmic fractions was detected to gradually decrease when treated with PCT200, EOT100, and EOT200, respectively, when compared to the TC group, indicating the translocation of Nrf2 to nucleus  significantly increased to 185777 (###p < 0.001) in the TC group, in comparison with normal control (iMFI = 6238). EOT100, EOT200, and PCT200 treated groups showed a significant reduction in iMFI levels with 39091, 7350, and 38188, respectively (∗∗∗p < 0.001). However, EOT200 was significantly more capable in controlling DNA fragmentation than PCT200 ($$$p < 0.001).The terminal deoxynucleotidyl transferase [TdT]-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) assay is a commonly used analytical method to evaluate DNA fragmentation and hence, apoptosis. In the present study, flow cytometry was used to conduct the TUNEL assay. HSCD resulted in nuclear DNA fragmentation as evidenced by an increase in dUTP-FLOUS labeling. As shown in E. officinalis (EOT) to ameliorate this oxidative stress-induced cognitive impairment by their action on the Nrf2-ARE pathway.In the present study, we have investigated the role of HSCD in causing cognitive impairment. Moreover, we have tried to investigate the potential of supplementation by tannins enriched fractions of Western dietary habits consisting of high levels of salt and cholesterol have time and again exhibited their capacity to cause cognitive impairment. Numerous epidemiological studies have shown that high levels of saturated fatty acids in the diet lead to cognitive deficits via various mechanisms such as oxidative stress, inflammation, insulin resistance, reduction in brain-derived neurotrophic factor levels, reduction in integrity of dendrites in the hippocampus . Obesityin silico studies, we found that tannins enriched fractions of E. officinalis had good binding affinity. These in silico results translated well to in vivo studies as it was observed that EOT was able to reverse alterations in GPH, GPx, MDA, and SOD levels, which were induced due to HSCD. Increase in oxidative stress in the brain has been observed in case of mild cognitive impairment. Moreover, an elevation in oxidative stress is an established characteristic of Alzheimer’s disease (AD) and also been proposed to be used as a possible predictor for AD and other forms of dementia  is a transcription factor responsible for the regulation of expression of numerous anti-oxidative and cytoprotective genes. Nrf2 is inhibited by Kelch-like ECH-associated protein 1 (Keap1) and undergoes degradation through the ubiquitin-proteasome pathway. Exposure of cells to ROS, toxins, or electrophilic radicals leads to post-translational modification and inactivation of Keap1 which ultimately leads to attenuation of Keap1-mediated degradation of Nrf2. This causes translocation of Nrf2 proteins in the nucleus of the cells and heterodimerization of Nrf2 with small Maf proteins (sMaf), following which binding between the heterodimers and the ARE takes place in the target genes . Throughdementia . The cendementia . Bringindementia . Furthery of EOT .Furthermore, evaluation of neurobehavioral parameters conducted using the Morris water maze (MWM) task. The MWM task aids in the evaluation of spatial learning which is assessed through multiple, repeated trials whereas determination of reference memory is done by checking the time spent by the rodent in the quadrant where the platform was initially placed. Good spatial learning and cognition are associated with reduced escape latencies, increase in time spent in the target quadrant, as well as increase in the number of times the rodent crosses the point where the platform was placed in the acquisition trial. The results obtained from the study reflected that rats fed HSCD had poor performance in the MWM task and administration of EOT led to significantly better performance, however, there was no significant difference between the performance of the EOT groups and the PCT group. Improved performance in behavioral tests and the amelioration of cognitive deficit was consistent with previous studies evaluating the potential of EOT as well as other cognition enhancing agents .Significant alterations in mitochondrial ATP and MMP were observed in rats fed HSCD. Accumulating evidence has shown that mitochondrial dysfunction and oxidative stress play a pivotal role in the pathophysiology of numerous neurodegenerative diseases mostly because of the fact that mitochondria have a crucial part in regulating apoptosis as well as in the aging process . MultiplPast research has emphasized on the potential role of Nrf2 activation in controlling oxidative stress, apoptosis, and autophagy . HencefoE. officinalis led to significant amelioration of cognitive deficits. Our studies also indicated that activation of Nrf2-ARE pathway by EOT was the reason behind this neuroprotective effect. However, it cannot be assumed that EOT would be equally effective in humans as it was in our in vivo studies on rats. Hence, we believe that EOT supplementation needs to be explored further to control progression of dementia and reduce cognitive impairment. Individual efficacy of each of the EOT constituents also needs to be evaluated in order to produce a viable pharmacotherapy.Our results indicated that HSCD was associated with significant cognitive impairment in rats. Moreover, supplementation with tannins enriched fractions of Designed the experiments: AN, IH, and MA. Performed the experiments: IH. Analysis and interpretation of data: AN, IH, and MI. Drafting the manuscript: IH, TM, and MI. Editing of manuscript: AN, MZA, and DV.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TMC4 (transmembrane channel-like 4) exon 1 (p.Gly17Glu) and 500 bases- downstream of MBOAT7 gene (TMC4/MBOAT7), in the genetic risk for developing NAFLD in a case-control study. Our sample included 634 individuals (372 patients with NAFLD diagnosed by liver biopsy and 262 control subjects); genotyping was performed by a Taqman assay. Genotype frequencies in controls  and patients  were in Hardy-Weinberg equilibrium; minor allele frequency 40.8%. Our sample had 84–99% power if an additive genetic model is assumed for estimated odds ratios of 1.3–1.5, respectively. We found no evidence of association between rs641738 and either NAFLD  or the disease severity (p = 0.61). Low levels of MBOAT7 protein expression were found in the liver of patients with NAFLD, which were unrelated to the rs641738 genotypes. In conclusion, the role of rs641738 in the pathogenesis of NAFLD is inconclusive.Current knowledge on the genetic basis of nonalcoholic fatty liver disease (NAFLD) suggests that variants contributing not only to the disease predisposition but histological severity as well are located in genes that regulate lipid metabolism. We explored the role of rs641738 C/T located in  PNPLA3 (patatin-like phospholipase domain-containing 3) has been consistently associated with increased liver fat content and NAFLD severity, including fibrosis, across different populations around the world3. The risk effect of rs738409 on developing NAFLD is the strongest ever reported for a common variant modifying the genetic susceptibility of the disease, representing ~5.3% of the total variance3 and a moderate odds ratio (OR) of NAFLD and NASH of ~ 3.33. Likewise, the missense p.Glu167Lys (rs58542926) variant located in TM6SF2 (transmembrane 6 superfamily member 2) gene, while protecting against cardiovascular disease (CVD)5, has been associated with a modest risk of liver fat accumulation (OR ~2.13)5, NAFLD, and the NAFLD severity11.Current understanding of the genetic basis of nonalcoholic fatty liver disease (NAFLD) suggests that variants contributing not only to the disease susceptibility but also histological severity are located in genes that regulate lipid metabolism. Specifically, the missense p.Ile148Met (rs738409) variant located in TMC4 (transmembrane channel-like 4) gene and intergenic downstream of MBOAT7 gene has been associated with a modest risk of developing NAFLD (OR ~1.37), NASH, and fibrosis12. However, these findings were based on a large report involving patients of European descent12. Nonetheless, the authors observed that the effect of rs641738 was restricted to European-Caucasian individuals, while not being significant in African American and Hispanic population12. Unfortunately, the association of rs641738 and NAFLD could not be replicated in other populations around the world, including Europeans from different cohorts, except for a small study that included cases-only (n = 125)13. For instance, a recent study including a large sample (n = 515) of patients with NAFLD recruited from several centers across Germany showed that rs641738 was associated with a marginal effect on liver fibrosis (p = 0.046) without any effect on NAFLD or liver function test14. Similarly, results yielded by analyzing the data pertaining to a small cohort of patients that underwent bariatric surgery in two European centers failed to confirm any association of rs641738 and NAFLD15. Likewise, studies from Asia failed to find an association of the variant with NAFLD or NASH18.Interestingly, a missense  variant located in exon 1 of MBOAT7 (membrane-bound O-acyltransferase domain-containing 7) locus (https://www.ncbi.nlm.nih.gov/snp/rs641738). Likewise, data from the genome assembly shows this variant located in exon 1 of the TMC4 genomic region  , shows that rs641738 as a transcript variant of TMC4 locus. For that reason, previous reports refer to rs641738 as MBOAT7 variant or TMC4/MBOAT7 (https://www.ncbi.nlm.nih.gov/snp/rs641738).The rs641738 variant is mapped 500 bases downstream of In addition, speculations on the putative biological role of MBOAT7 in the pathogenesis of NAFLD still persist because the protein encoded by this gene is a lysophosphatidylinositol acyltransferase, which has specificity for arachidonoyl-CoA as an acyl donor.Combined, available evidence suggests that associations of rs641738 with NAFLD and NASH remain to be either confirmed or refuted. Hence, we performed a hospital-based case-control study to explore the association between rs641738 and NAFLD, including adult patients in whom the histological disease severity was confirmed by liver biopsy.In addition, we explored the protein expression pattern of MBOAT7 in the liver of patients with NAFLD to provide evidence of whether the protein encoded by this locus might be involved in the biology of the disease.Clinical and biochemical features of patients and controls are disclosed in Tables n = CC: 84, CT: 137, TT: 41, p = 0.22) and patients  were in Hardy-Weinberg equilibrium (HWE). The minor allele frequency (MAF) in our sample was 40.8%, in line with that reported in the 1000 Genomes Project for the T allele in all populations (37%) and Europeans (44%) .Genotype frequencies in controls . The variant was associated with neither NASH nor the disease severity (p = 0.61). No association was found with fibrosis status (fibrosis yes/no) (p = 0.95), lobular inflammation (p = 0.46), or NAFLD- NAS score (p = 0.25). However, in univariate analysis we observed a significant association with circulating triglycerides (TG) (p = 0.004). The rs641738 was not associated with glucose metabolism, HOMA-index, total, HDL, LDL-cholesterol or other MetS components.The association analysis of rs641738 and NAFLD showed no effect of the variant on the susceptibility of NAFLD  in the two studied groups are shown in Fig. MBOAT7 in the biology of NAFLD, we further explored whether the protein encoded by this gene is expressed in the liver.In order to provide evidence supporting a putative role of http://www.proteinatlas.org/ENSG00000125505-MBOAT7/tissue). Furthermore, we found no differences in the liver MBOAT7 expression pattern between rs641738 genotypes   Fig. .Figure 312 or liver fibrosis (OR 1.41)12 in European American population; in fact, our entire sample is composed of individuals of self-reported European ancestry. In addition, for polymorphisms with minor allele frequencies >0.2 (like the one observed for rs641738 MAF 0.40), the ORs are expected be in the range of 1.1–1.519. However, our study is underpowered for ORs 1.2–1.25 and for all histological features analyses.In this study, we explored the role of the missense rs641738 variant in the susceptibility of NAFLD and the disease severity. We did not find statistically significant differences in genotypic or allelic frequencies for the variant in either the predisposition of NAFLD or NASH, or other related histological features. Genotype frequencies in controls and cases were in HWE, and sample size estimation showed at least 84% power for the additive genetic model even if a very modest effect (OR: 1.3) is considered. Power calculation based on a OR of 1.3 is justified by previous evidence of association of the variant and NAFLD (OR 1.37)12, our study suggests that it is highly unlikely that rs641738 plays a role in the genetic susceptibility of NAFLD, at least in our population. A note of caution regarding the lack of association of the variant and liver fibrosis in our sample should be added because it could be explained by insufficient power.In contrast to some reports in the literature indicating a significant association of the variant with NASH, liver damage and fibrosis in individuals of European descent but not other ethnicities14, while a marginal but positive effect of the variant on liver fibrosis  was observed.Likewise, Krawczyk and coworkers failed to detect an association of the rs641738 and NAFLD or liver function test20; specifically, there were large studies that included well characterized patients diagnosed by liver biopsy17. Interestingly, these studies showed a negative association of the variant with NAFLD20 and NASH or liver fibrosis17.Meanwhile this manuscript was under the peer review process, several reports on the role of rs641738 were publishedA detailed summary of the available evidence is shown in Table While the reasons behind these discrepancies are unclear, several potential explanations should be considered.et al. stratified the data by ethnic groups of the population-based Dallas Heart Study (DHS), and observed a positive significant effect (p = 0.019) of rs641738 on HTGC content (continuous variable) that was restricted to African Americans. In contrast, association with hepatic steatosis (NAFLD as a disease trait) remained significant in European Americans  but not in African Americans12. The biological reasons behind such discrepancies, while interesting, are certainly hard to explain.The first explanation relates to putative discrepancies at the population level and the design of the extant studies on the effect of rs641738 on either hepatic steatosis or hepatic triglyceride content (HTGC)—as measured by liver spectroscopy—both of which contribute to inconsistencies among different datasets. For example, in their analyses, Mancina et al. showed that rs641738 was associated with NASH and the disease severity in European population; however, genotype frequencies deviated from HWE (p = 0.017)12. Nevertheless, HWE is statistically a null hypothesis as it assumes there is no evolution in the population. In fact, disease-associated allele can be deviated from HWE in a disease population (cases) but not in controls.A second explanation could be a false positive association between the variant and NAFLD ascribed to deviations from HWE or insufficient genotyping accuracy. Mancina et al.’s study12. Nevertheless, this possibility is hard to conceive, as the variant was not associated with NAFLD in patients from other European countries, including Germany15; although the results of the German study could be a remote example of this situation.A third but yet unexplored explanation relates to a putative gene × environment interaction, the occurrence of which seems to be limited to the European cohort of Mancina 12, except for one study from Germany15; hence, the biological meaning of this observation remains unknown.In our sample we observed that rs641738 was associated with triglyceride levels and so, it might indirectly regulate intermediate steps of fatty acid (polyunsaturated fatty acids-PUFA and PUFA-containing TG) biosynthesis. Still, the association of the variant with TG was not observed among individuals included in previous reportsMoreover, a distant but noteworthy explanation could pertain to disparities in the minor allele frequency (MAF) among different populations around the world, including our sample. However, the frequency of the risk allele in our population is comparable to that reported in the 1000 Genomes Project for Caucasians (44%); hence, it seems unlikely that discrepancies among studies arise from racial differences. It is still possible, however, that this variant may bare a population-specific association with NAFLD. It is also likely that rs641738 may work via an interaction with other injuries or unknown environmental factors, especially when these factors may be distributed differently among different populations.MBOAT7/2 kb upstream variant of TMC4.A final plausible explanation is that rs641738 is not necessarily the causal variant; thus, other SNPs in strong linkage disequilibrium (LD) could explain an impact on the phenotype. Exploration of variants in high LD with rs641738 shows at least five SNPs,  and TMC4 (ENSG00000167608.7) in the liver tissue are shown in Table Analysis of eQTLs (expression quantitative trait loci), which denote correlations between genotype and tissue-specific gene expression levels, shows that rs641738 is associated with eQTLs in the liver Tables  and 6 anue Table . Complet21; this point could explain some of the above mentioned observations.The observation that rs641738 is associated with eQTLs in non-liver tissues, including fat, might reinforce the possibility of unexplored associations between the variant and the disease. For example, a recent study showed that associations between common gene variants and NAFLD are uncovered by adiposity degree22. Then, a putative yet uncovered in cis effect of rs641738  might explain participation of the variant in lipid metabolism by regulating MBOAT7 expression.Information regarding protein expression is much limited. We have specifically assessed the protein expression pattern of MBOAT7 in the liver of patients with NAFLD and we observed no evidence of robust immunoreactivity. Similarly, we failed to observe any association between liver-MBOAT7 expression and rs641738 genotypes   family highly expressed among epithelia 23. Interestingly, there is evidence supporting the presence of chloride channels not only in the plasma membrane of hepatocytes but in multiple intracellular compartments as well24. The involvement of ion channels in the pathogenesis of NAFLD and/or in pathways associated with hepatic fibrogenesis remains elusive; nevertheless, it certainly represents an interesting path for future research. In fact, this observation could explain previously reported associations of rs641738 and fibrosis in patients with chronic hepatitis C and B26, and NAFLD14. Still, the exact effect/mechanisms by which the variant could regulate liver fibrogenesis remains uncertain. We reinforce the importance of precision in identifying the genomic location and the biological function of a given variant, as this would increase not only the understanding of the genetic component of NAFLD but also its relationship with the disease pathogenesis.On the other hand, 20 or Germany14. Nevertheless, the association with liver histology, including fibrosis was only observed in patients with NAFLD of European ancestry14; this finding could not be replicated in studies that included Asian population17. Hence, larger studies are required before any definitive conclusion can be reached.In conclusion, the rs641738 is not associated with NAFLD in our population. The association of the variant and NAFLD as disease trait could not be replicated in population-based or hospital based studies from AsiaThe study included a sample of 634 unrelated individuals, of which 262 were controls subjects and 372 were patients who have histopathologic-proven features of NAFLD. Patients and controls were selected from two different hospital-based settings, including a cross-sectional study of patients diagnosed with NAFLD and Metabolic Syndrome (MetS) in the Liver Unit, Hospital Abel Zubizarreta, Buenos Aires, Argentina, and an independent cohort of morbid-obese patients that underwent bariatric surgery in the Surgery Department, Hospital de Alta Complejidad en Red El Cruce, Buenos Aires, Argentina.All investigations performed were conducted in accordance with the guidelines of the 1975 Declaration of Helsinki. Informed and written consent for study participation from all individuals was obtained in accordance with the procedures approved by the ethical committee of our institution .Exclusion criteria: Secondary causes of steatosis, including alcohol abuse , total parenteral nutrition, hepatitis B and hepatitis C virus infection, and the use of drugs known to precipitate steatosis were excluded. In addition, patients with any of the following diseases were excluded from participation: autoimmune liver disease, metabolic liver disease, Wilson’s disease, and α-1-antitrypsin deficiency.Control subjects that matched patients with NAFLD-MetS were selected from subjects attending our hospital for check-up purposes whose age and sex matched the NAFLD patients. In addition to the standard heath examination, all non-obese control individuals were subjected to a liver ultrasonographic (US) examination. They were included in the study if they did not have evidence of fatty change or biochemical abnormalities. Furthermore, control subjects were confirmed not to have any of the features of the metabolic syndrome as defined by the National Cholesterol Education Program Adult Treatment Panel III and did not abuse alcohol.In the population of morbid obese patients, control subjects were obese patients who also underwent bariatric surgery and had not features of NAFLD demonstrated in the liver biopsy.The case participants and the controls were selected during the same study period from the same population of patients attending the above mentioned institution, and all of them share the same demographic characteristics .Health examinations included anthropometric measurements, a questionnaire on health-related behaviours and biochemical determinations.The disease severity was assessed by liver biopsy that was performed before any intervention with ultrasound guidance and a modified 1.4-mm-diameter Menghini needle  under local anesthesia on an outpatient basis or during bariatric surgery. All liver biopsies were evaluated by the same pathologist.et al., based on the percentage of hepatocytes containing macrovesicular fat droplets27. NASH and NAFLD Activity Score (NAS)28 were defined as reported previously; a NAS threshold of 5 was used for further comparisons with variables of interest, NASH was defined as steatosis plus mixed inflammatory-cell infiltration, hepatocyte ballooning and necrosis, Mallory’s hyaline, and any stage of fibrosis, including absent fibrosis28.A portion of each liver biopsy specimen was routinely fixed in 40 g/l formaldehyde (pH 7.4), embedded in paraffin, and stained with hematoxylin and eosin, Masson trichrome, and silver impregnation for reticular fibers. All the biopsies were at least 3 cm in length and contained a minimum of 8 portal tracts. The degree of steatosis was assessed according to the system developed by Kleiner The genetic analyses were done on genomic DNA extracted from white blood cells. Genotyping of rs641738 was performed using a TaqMan genotyping assay  according to manufacturer’s instructions. To ensure genotyping quality, we included DNA samples as internal controls, hidden samples of known genotype, and negative controls (water). The overall genotype completion rate was 100%.29 as we explained elsewhere2. We found no evidence of stratification in our sample because the cases and the controls showed similar Q values and the Structure program assigned a similar distance to clusters with no further improvement in the fitting model by adding up to four clusters (the ln of likelihood was maximum for K = 1). Moreover, all the participants in this study self-reported a Caucasian ethnicity as a surrogate of ancestry, which is consistent with the observed MAF.To account for possible population stratification, we used a collection of 13 SNPs at different loci  and then analyzed the data with the Structure program Version 230 and assuming a prevalence of NAFLD of 0.30, minor allele frequency (MAF) T = 0.40 and an odds ratio (OR) of 1.3–1.5, our sample had 84–99% power, respectively, for the additive genetic model.Using the CaTS power calculator for genetic association studies2O2 in PBS for 20 min at room temperature to block endogenous peroxidase. Following microwave heat-induced epitope retrieval in 0.1 M citrate buffer at pH 6.0 for 20 min, the slides were incubated with a dilution of 1:100 of rabbit polyclonal antibody for Human Anti-MBOAT7 . Immunostaining was performed using the VECTASTAIN Elite ABC Kit  detection system. Subsequently, slides were immersed in a 0.05% 3,3′-diaminobenzidine solution in 0.1 M Tris buffer, pH 7.2, containing 0.01% H2O2. After a brown color developed, slides were removed and the reaction was stopped by immersion in PBS. Negative controls were carried out with rabbit serum diluted to the same concentration as the primary antibody. MBOAT7 immunostaining was evaluated in a blinded fashion regarding any of the histological and clinical characteristics of the patients. The extent of staining was scored according to its amount and intensity by a 4-point scoring system as follows: 0 = no staining, 1 = positive staining in less than 20% of cells, 2 = 21–50% of positive cells, and 3 = positive staining in more than 50% of cells. The sections were observed in bright field microscopy with a microscope Axiostar plus  at a magnification of X400. As control tissue we used a sample of testis retrieved from the collection of tissues of the Pathology Department.Four-micrometer sections were mounted onto silane coated glass slides to ensure section adhesion through subsequent staining procedures. Briefly, sections were deparaffinized, rehydrated, washed in phosphate buffer solution (PBS), and treated with 3% HU or Kruskal-Wallis tests. The Cochran–Armitage test for trend was used in the categorical data analysis to assess the presence of association between the variant and disease severity and a regression analysis for an ordinal multinomial distribution (Probit as the Link function) with disease severity as the dependent (response) variable coding controls; NAFL and NASH subjects as 0, 1, and 2, respectively; age, HOMA, and BMI as continuous predictor variables; and sex and rs641738 genotypes  as grouping variables. Moreover, logistic regression analysis was included for the evaluation of the association between genotypes and histological disease severity . To assess the association between genotypes with NAFLD or quantitative traits, we used a chi-square test and logistic regression or ANCOVA and multiple regression, adjusting for co-variables, such as age, HOMA, BMI, and rs738409. For ordinal multinomial analysis, logistic analysis, or ANCOVA, we adjusted for co-variables that were not normally distributed through log-transformation. Correlation between two variables was done using the Spearman’s rank correlation test. The CSS/Statistica program package version 6.0  was used in these analyses.Quantitative data were expressed as mean ± SD unless otherwise indicated. As a significant difference in SD was observed between the groups in most of the variables and the distribution was significantly skewed in most cases, we chose to be conservative and assessed the differences between the groups using nonparametric Mann–Whitney All data generated or analyzed during this study are included in this published article.


Little is known about the epidemiology of bronchiolitis as a clinical diagnosis and its impact on emergency department visits and hospitalizations in tropical and semitropical regions. We described the epidemiology of bronchiolitis emergency visits and hospitalizations, its temporal trend and geographic distribution in Puerto Rico between 2010 and 2014.International Classification of Diseases, Ninth Revision code 466, 466.11, and 466.19. Chi-square and one-way ANOVA compared sex, age, diagnosis, and severity across the years. Joinpoint Poisson regression analysis evaluated the temporal trend distribution of bronchiolitis hospitalizations per calendar year. A P value less than 0.05 was statistically significant.We performed a retrospective descriptive analysis of a representative sample of privately insured children with bronchiolitis from January 2010 to December 2014. Data was provided by the largest private health insurer in Puerto Rico and identified children < 24 months of age with bronchiolitis by P = 0.0015).During the study period, the annual proportion of emergency department visits and hospitalizations due to bronchiolitis increased from 3 to 5%, and 26 to 38%, respectively. The annual incidence rate of hospitalizations was 3.2 per 1000 privately insured children < 24 months. Non-RSV bronchiolitis was the most frequent diagnosis (51%). Hospitalizations occurred year-round, but increased significantly from August through December. Most children hospitalized resided in the metropolitan San Juan (35%) and surrounding urban areas. Total hospital charges decreased from $3.78 to $3.74 million, with an average cost per hospitalization of $4320.12 (11.3% increase; This is the first study that evaluates the epidemiological characteristics of bronchiolitis in a primarily Hispanic population, living in a tropical country, and using data from a privately insured population. We found a small but significant increase in proportion of emergency visits and hospitalizations. Temporal trend shows year-round hospitalizations with an earlier seasonal peak and longer duration, consistent with Puerto Rico’s seasonal rainfall throughout the study period. Further studies are needed to elucidate whether this epidemiologic pattern can also be seen in publicly insured children and whether Hispanic ethnicity is a risk factor for increased hospitalizations or is related to health disparities in the US healthcare system. Bronchiolitis constitutes an important public health burden in the pediatric population worldwide –4, as thPast studies on bronchiolitis hospitalizations have focused on those caused by the respiratory syncytial virus (RSV), the most common etiologic agent . In tempLittle is known about the epidemiology of bronchiolitis as a clinical diagnosis, with disregard as to the etiologic agent, and its impact on emergency visits and hospitalizations in tropical and semitropical regions like Puerto Rico. Therefore, the objectives of this study are to (1) describe demographic characteristics of emergency department visits due to bronchiolitis, (2) describe demographic and clinical characteristics of patients hospitalized with bronchiolitis, (3) examine temporal trend distribution of bronchiolitis hospitalizations, and (4) describe geographic distribution of bronchiolitis hospitalizations in Puerto Rico.This retrospective descriptive study analyzed secondary data provided by the largest private health insurer in Puerto Rico. During the study period, two thirds of the population was covered under public health insurance; approximately one third under private health insurance. The insurer under study served an estimated average of 680,139 participants per year from 2010 to 2014, over 50% of privately insured Puerto Ricans on a yearly basis.The insurer provided two aggregated and de-identified data sets: (1) children under 24 months of age who visited an emergency department in Puerto Rico from January 1, 2010, to December 31, 2014; and (2) children under 24 months of age with a primary or secondary diagnosis for acute bronchiolitis upon hospitalization in Puerto Rico from January 1, 2010, to December 31, 2014. Due to insurer data extraction procedures and codification, the databases for the emergency department visits and hospitalizations were mutually exclusive and independently analyzed.International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) codes for acute bronchitis and bronchiolitis (ICD-9-CM: 466), acute bronchiolitis due to RSV (ICD-9-CM: 466.11), and acute bronchiolitis due to other infectious organism (ICD-9-CM: 466.19). An emergency visit due to bronchiolitis was defined as any patient < 24 months evaluated at the emergency department with a discharge diagnosis of bronchiolitis based on the ICD-9-CM code. A hospitalization due to bronchiolitis was defined as any patient < 24 months admitted with a primary or secondary diagnosis of bronchiolitis upon hospitalization based on the ICD-9-CM code. Severe bronchiolitis was defined as a hospitalization that required admission to the Pediatric Intensive Care unit (PICU).The diagnosis of bronchiolitis was defined using the Emergency department data included (1) total visits per year by age group (months) and sex, and (2) total visits due to bronchiolitis per year by age group (months) and sex. Hospitalization data included (1) total number of patients hospitalized per year by age group (months) and sex, (2) number of hospitalizations due to bronchiolitis per year, (3) number of cases by diagnosis (ICD-9-CM code), (4) number of hospitalizations per month of admission, (5) number of hospitalizations per unit , (6) mean length of stay per month, (7) hospital charges per day based on number of patients hospitalized per year, and (8) number of hospitalizations per health insurance region based on municipality of residence.Daily precipitation (inches) was monitored and provided by the National Oceanic and Atmospheric Administration from the primary weather station at the San Juan International Airport . Total hThe primary outcome measures were (1) annual proportion of emergency department visits due to bronchiolitis, (2) annual proportion and annual incidence rate of bronchiolitis hospitalizations, and (3) temporal trend distribution of bronchiolitis hospitalizations per calendar year. Other outcomes of interest included disease severity, hospital charges, and proportion of bronchiolitis hospitalizations by geographic region.P value less than 0.05 was established as statistically significant. Analyses used STATA version 11 .Data was analyzed using descriptive statistics, including frequency distribution and measures of association. Categorical variables were analyzed using frequency and percentages; continuous variables were analyzed using means and standard deviation. To estimate the burden of bronchiolitis in this population, we calculated the annual proportion of bronchiolitis emergency visits among total emergency visits per year by age group, and the annual proportion of bronchiolitis hospitalizations among total hospitalizations per year by age group. The denominator of sex and age group distribution for bronchiolitis hospitalizations differed from ICD-9-CM codes and hospital unit due to post-adjustment measures provided by the insurer for billing purposes that included multiple admissions incurred by a single patient, coding, billing, and claim processing. To estimate the annual incidence rate of bronchiolitis hospitalizations per 1000 insured participants by age group, we used the insurer’s enrollment data from June 2014; taking into consideration an adjustment for the annual fluctuation in health insurance enrollment with a stable descent of 13% since 2012 to estimate the insured population per year. To estimate the burden of economic inflation and facilitate direct comparisons between years for hospital charges in Puerto Rico and the USA, we used the medical care component of the Consumer Price Index . Chi-squThis study was approved by the University of Puerto Rico, Medical Sciences Campus (IORG000223) Institutional Review Board.P < 0.001). Infants < 12 months of age represented 68% of all bronchiolitis emergency visits. Subgroup analyses showed that the 3.1- to 6-month age group was most frequently diagnosed with acute bronchiolitis  and represented 21% (SD 2.5%) of all bronchiolitis emergency visits. During the study period, the annual proportion of bronchiolitis emergency visits among sexes (P = 0.15), and age groups (P = 0.116) were relatively constant.A total of 48,886 emergency department visits of children < 2 years were identified from 2010 to 2014, of which 2281  were due to bronchiolitis (Table P < 0.001). Bronchiolitis hospitalizations were most frequent in males  and children < 1 year of age  (Table P < 0.001). We estimated the annual incidence rate of bronchiolitis hospitalizations as 3.2 per 1000 privately insured children < 24 months of age.A total of 13,833 hospitalizations were reported from 2010 to 2014, of which 4986 were due to bronchiolitis; corresponding to 37% (SD 6%) of all hospitalizations for children < 24 months . The proportion of severe bronchiolitis hospitalizations  and the mean length of stay  remained stable throughout the study period.Of the 4986 children, 91% had a single hospitalization, 8% had two hospitalizations, and 1% had three or more (P = 0.0015). Total hospital charges per year for bronchiolitis hospitalizations decreased from $3.78 to $3.74 million , followed by the urban area of Bayamón .We identified emergency department visits and hospitalizations due to bronchiolitis within a representative sample of privately insured children < 2 years between 2010 and 2014. Our study shows an annual proportion of emergency visits due to bronchiolitis of 5% (SD 1%) and an annual proportion of bronchiolitis hospitalizations of 37% (SD 6%). We estimated an annual incidence rate of 3.2 bronchiolitis hospitalizations per 1000 privately insured children < 2 years of age within the general population. Bronchiolitis hospitalizations occurred year-round, increasing significantly as of August through December, and taking place more frequently in the main Metropolitan area of Puerto Rico.During the study period, we identified more than 2200 emergency visits by children with bronchiolitis. Our data shows a similar annual proportion of bronchiolitis emergency visits to other studies in the USA. A study by Hasegawa et al.  based onPrevious studies have shown that in the USA, an increase in bronchiolitis hospitalizations was observed during the 1990s , 7 and eDuring the study period, the proportion of RSV bronchiolitis hospitalizations varied significantly from 27 to 45%, with a 23% annual proportion increase from the year 2010 to 2011. One Canadian study reported the emergence of a new RSV variant, namely ON1 genotype (subgroup A), in late 2010 , associaJoinpoint regression analysis from 2010 to 2014 showed bronchiolitis hospitalizations year-round, with an increase during September through December. During the years 2011 and 2012, there was an increase in bronchiolitis hospitalizations from July through October, consistent with the reported RSV epidemic . This waThe decrease in total hospital charges during the study period is associated with a decrease in bronchiolitis hospitalizations and total hospital days. The cost of bronchiolitis hospitalizations were on average over 50% higher than the US $2815 geometric mean, reported by Hasegawa et al. [In our study, most hospitalizations occurred in patients from the two main metropolitan areas, which together make up over half of bronchiolitis hospitalizations during the study period. This finding most likely reflects the socioeconomic status of privately insured population, with a higher number of subscribers living in the metropolitan area and surrounding urban and suburban municipalities, greater number of hospitals, urbanization, and industrialization . One stuThere are several limitations of this study that should be considered. We used a database of admission-data level based on ICD-9-CM codification for which we might have overestimated bronchiolitis episodes by including early asthma and bronchitis or underestimated the frequency of bronchiolitis hospitalizations by not including misdiagnosed cases or those that developed bronchiolitis after hospitalization. This is an observational study based on aggregated and stratified data obtained for billing purposes. Changes in enrollment or discontinuation of private healthcare coverage by the insurer, as well as discrepancies in diagnostic-coding by physicians, hospitals, and post-adjustment measures for billing purposes could explain variations in bronchiolitis hospitalizations throughout the study period. The lack of individual patient characteristics and clinical information prevents us from determining and comparing annual incidence of bronchiolitis hospitalizations and its relationship with other risk factors and outcomes of interest, such as sociodemographic status, age-specific diagnosis, repeat hospitalization and PICU hospitalization, temporal distribution of bronchiolitis based on ICD-9-CM code, and presence of co-morbidities. Furthermore, we lacked information about the ethnicity of children included in the study but note that the study is based in a tropical country with a primarily Hispanic population. However, a small portion of children of non-Hispanic ethnicity might have been included in the study. The codification of the etiology within this study did not specify the method by which RSV laboratory testing was confirmed. Because there are differences in sensitivity and specificity for RSV testing, we may have over- or under-reported RSV hospitalizations. However, increases seen in the proportion of RSV hospitalizations were consistent with RSV outbreaks reported by the Puerto Rico Department of Health. We also lack information about influenza outbreaks, which could have affected the proportion of non-RSV bronchiolitis cases that were hospitalized. Because the insurer’s data on emergency visits and hospitalizations were mutually exclusive and analyzed separately, we cannot determine hospitalization rate after an emergency visit for bronchiolitis. Lastly, the study only included data from the private insurer. Procuring and including a representative sample that includes children covered by public health insurance or other insurance companies could strengthen future studies.This is the first study to evaluate the epidemiological features of bronchiolitis in Puerto Rico by using representative data of a privately insured population between 2010 and 2014 from the largest insurer in Puerto Rico. This allowed us to determine population-based emergency department visits and hospitalization distributions due to bronchiolitis with decreased health disparities in comparison to public health insurance holders. November and December 2014 were the only months to overlap with the publication of the updated bronchiolitis management guidelines by the American Academy of Pediatrics, which allowed us to evaluate epidemiologic distributions without influence to providers’ decisions in hospitalizing children with bronchiolitis.Between 2010 and 2014, we found that the proportion of emergency department visits due to bronchiolitis is similar to that reported in the USA, while bronchiolitis hospitalizations among children in Puerto Rico was found to be twice as high. Further studies are needed to elucidate whether Hispanic ethnicity is a risk factor for increased frequency of hospitalization or is related to health disparities in the US healthcare system. Temporal trend shows year-round hospitalizations with an earlier seasonal peak and longer duration, consistent with Puerto Rico’s seasonal rainfall. This locally acquired data establishes the seasonality of bronchiolitis in Puerto Rico according to our epidemiology and helps us better define the pediatric population that has greater susceptibility so that interventions and prevention tools may be developed to prevent and control transmission of this disease in the tropical environment of Puerto Rico. Further studies are needed to elucidate whether this epidemiologic pattern can also be seen in publicly insured children and whether Hispanic ethnicity is a risk factor for increased hospitalizations or is related to health disparities in the US healthcare system.