Prosopis cineraria pods on HMG – CoA reductase and regression potential of atherosclerotic plaque.The HMG-CoA reductase is key enzyme of cholesterol biosynthesis which potentially contributes in management of hypercholesterolemia. The present study was designed to assess the inhibitory effect of phytoconstituents of an ethanolic extract of Prosopis cineraria pods was conducted using LCMS, GCMS and FTIR analysis. Further, in-vitro, in-vivo and in-silico assessments were performed.Healthy, adult male, albino rabbits in which hypercholesterolemia was induced by supplying the high fat diet and a supplement of cholesterol powder with coconut oil (500 mg/5 ml/Day/kg body weight) for 15 days, were used as a disease model. Phytochemical analysis of an ethanolic extract in-silico evaluation was made using appropriate docking software and results also indicated as significant interactions of the identified compounds with the target enzyme. Treatment of rabbits with the ethanolic extract of P. cineraria pod resulted in significant (P ≤ 0.001) reductions in total cholesterol, LDL cholesterol, VLDL cholesterol, and triglyceride. Accordingly, reductions were occurred in atherosclerotic plaque, intima and media of aortal wall along with lumen volume of the aorta significantly increased (P ≤ 0.001).The in-vitro assessment of HMG -CoA reductase activity indicated a 67.1 and 97.3% inhibition by the extract and a standard drug (Pravastatin), respectively. Additionally, an Prosopis cineraria pod contains potent bioactive phytocompounds might be inhibit HMG – CoA reductase and have regression potential of atherosclerotic plaque.It can be illustrating that the ethanolic extract of  Appropriate diets and dietary supplements have the potential for use in the management of various metabolic syndromes and their complications . The fasSeveral pharmacological agents are available, however, to manage hypercholesterolemia and atherosclerosis. One of the most widely used agents used for the therapeutic treatment of atherosclerosis are HMG-CoA inhibitors, known as statins , 5. The Prosopis cineraria, has been reported by several researchers to possess numerous medicinal properties, such as antidiabetic, hypoglycemic, anticancer, anti-inflammatory, anti-asthmatic, as well as a myriad of other pharmaceutical properties [Prosopis cineraria is a key ingredient panchkuta, a local Indian food dish with proposed healthy attributes without side effects. Therefore, a study was conducted to evaluate the inhibitory effect of an ethanolic extract of Prosopis cineraria pods on HMG-CoA reductase, and the antioxidant and anti-atherosclerotic potential of the extract. In-vitro, in-vivo, and in-silico assessments of the extract were conducted.For example, the desert plant, operties , 9. Studoperties . NotablyProsopis cineraria (commonly known as Sangari) were obtained from a local herbal shop and authenticated by an expert in botany. The dried pods were ground with a mortar and pestle and a 70% ethanolic extract was obtained by soxhlation. A sticky extract was obtained and stored under desiccated conditions. Atorvastatin, a commonly prescribed statin, was purchased from a local pharmacy and used for comparative purposes (control). All chemicals were of reagent grade and purchased from Loba Chemie.Pods of LC-MS based metabolomics is a methodology used for characterizing the chemical fingerprint of herbal plant extracts. In our analysis, several mobile phase sequences were analysed to obtain the most comprehensive elucidation of chromatographic peaks .Prosopis cineraria was conducted using a standard protocol. The sample was injected into a gas chromatograph interfaced with a mass spectrometer (GC-MS) [GC-MS analysis of the ethanol pod extract of  (GC-MS) .− 1. The spectrum was characterized using the attenuated Total Reflectance (ATR) technique [An FTIR Spectrophotometer  equipped with a standard detector and a germanium beam splitter, which was interfaced to a computer and analytical software, was utilized for the analysis. The KBr pellet technique was used to obtain a spectrum was in the mid IR region of 400–4000 cmechnique .The inhibitory effect of the plant extract on HMG-CoA activity in vitro was determined using an HMG-CoA reductase assay kit (Sigma-Aldrich), which is based on a spectrometric measurement. The concentration of a standard HMG-CoA reductase stock solution was 0.50–0.70 nM. Different concentrations  of the ethanolic extract were mixed with a reaction mixture containing NADPH, HMG-CoA substrate, and HMGR. Pravastatin (Sigma Aldrich co.) was used as a positive control and distilled water served as a negative control , 14. Inh:12.44 = εmM - the extinction coefficient for NADPH at 340 nm is 6.22 mM–1 cm–1. 12.44 represents the 2 NADPH molecules consumed in the reaction.TV = Total volume of the reaction in ml (1 ml for cuvettes and 0.2 ml for plates)V = Volume of enzyme used in the assay (ml)0.6 = Enzyme concentration in mg-protein (0.50–0.70 mg P/ml)LP = Light path in cm (1 for cuvettes and 0.55 for plates).WhereNew Zealand, white, male rabbits, weighing approximately 1.5 ± 0.2 kg, were used as an animal model. They were acclimatized for 10 days in cages under controlled environmental conditions consisting of a 12-h light/dark regime and a temperature of 23 ± 2 °C. Food was supplemented with green leafy, seasonal vegetables and water. Hypercholesterolemia was induced by oral administration of 500 g of cholesterol powder mixed with 5 ml of coconut oil for 15 days along with a high fat diet [n = 5) and the experimentation period was 60 days.Group 1: Control, treated with only distilled water for 60 days.Group 2: Hypercholesterolemic diet for 60 days.P. cineraria pod extract (400 mg/kg/day) for 45 days after induction of hypercholesterolemia for 15 days.Group 3: Treatment with ethanolic Group 4: Treatment with atorvastatin (0.25mg/kg) for 45 days after induction of hypercholesterolemia for 15 days.Animals were divided into four groups  were removed, fixed in formalin. and processed for histopathological examination. Planimetric studies of the aorta wall, lumen volume and atherosclerotic plaque were conducted using a Camera Lucida .Serum was separated by centrifugation and stored at -20 °C. After thawing, assessments of total cholesterol , triglycSerum LPO (lipid peroxidation) was determined by measuring thiobarbituric acid reactive substances (TBARS) and expressed as malondialdehyde (MDA) content, following the method of Ohkawa . CatalasMolecular docking of HMG-CoA reductase (PDB:1DQ9) with Pravastatin  and the dominant phytocompounds in the ethanolic extract, i.e. Prosopilosine (Pubchem CID: 45269734), Prosogerin A (Pubchem CID: 44257586), Cinerin C (Pubchem CID: 101485482), β- Sitosterol (Pubchem CID: 222284), and Gallic Acid (Pubchem CID: 370) were conducted using Autodock 4.2. Water molecule and other co-factors were removed from the target molecule, while hydrogen atoms were added. Ligand was also prepared and docked into the active/ binding site of the target molecule ..Results of the biochemical assessments, organs weights, and planimetric studies are expressed as a mean ± standard error of the mean (SEM) and statistical differences were determined by ANOVA and post hoc mean separation testsThe different concentrations of plant extract and the standard inhibitor exhibited a 67.1 and 97.5% level of inhibition of HMG-CoA activity, respectively, as indicated by the product rate per minute, were calculated using the described formula  in the hypercholesterolemic group compared to the control group (distilled water). However, a significant reduction in MDA content was observed in the rabbits treated with the ethanolic extract. A significant increase (P ≤ 0.001) in serum SOD and catalase was also observed in the group of hypercholesterolemic rabbits treated with the ethanolic plant extract, relative to the untreated group of hypercholesterolemic rabbits.  exhibited significant affinity and molecular interactions with the target protein as indicated by the binding energy, number of H-bonds and bond lengths Table a-e.TablDietary approaches are frequently used for managing health problems, with specific diets being recommended for the therapeutic treatment of various ailments . HistoriProsopis cineraria, whose pods are used to make an herbal extract which is rich in potent phytochemicals [Prosopis cineraria already have been previously demonstrated to have anti-atherosclerotic activity [Prosopis cineraria pods resulted in a significant reduction in plaque formation, as well as improvements in their lipid profiles. These results were similar to the those obtained by Ram and co-workers who demonstrated the anti-atherosclerotic activity of Prosopis cineraria and Acacia senegal bark extracts [in-silico evaluation conducted in previous studies [In this regard, the desert region of western Rajasthan, India has a native plant, hemicals , 9. Barkactivity , 33. In extracts , 33. It extracts –36. Seveextracts , 37. The studies , 39. The studies , 40.Perilla frutescens extracts exhibit a strong antioxidant capacity and avert the oxidation of LDL in vitro and in vivo [P. cineraria pods resulted in a significant elevation in catalase and SOD levels, which may have been induced by the free radical scavenging activity properties of the phytoconstituents present in the plant extract. Our results also indicated that LPO (lipid peroxidation) was reduced in rabbits treated with the extract; which again confirms its role in scavenging reactive oxygen species (ROS). It is possible that these effects may be directly due to the free radical scavenging potential of the phytoconstituents in the ethanolic extract [Prosopis cineraria pods inhibits HMG – CoA reductase activity and reduces the level of atherosclerotic plaque in hypercholesterolemic rabbits.Additionally, oxidative stress and the production of free radicals have been shown to induce degenerative changes that promote endothelial dysfunction and are thus involved in the pathogenesis of atherosclerosis and are also linked to other metabolic syndromes , 42. In  in vivo . Additio in vivo . It appe in vivo . Hyperch in vivo . In the  extract . CollectProsopis cineraria pods can reduce serum cholesterol when fed to hypercholesterolemic rabbits. The extract also reduced the level of atheromatous plague in the aorta. Notably, the extract can improve antioxidant enzyme activity and inhibits HMG-Co reductase activity. Therefore, the use of an ethanolic extract of Prosopis cineraria pods should be further investigated as an alternative therapeutic agent to statins and other drugs, but one that does not have adverse side effects for the treatment of cardiovascular diseases.This present study demonstrated that an ethanolic extract of


The objective of this study is to describe the changes in prescribing practices of antibiotics to treat acute pyelonephritis (APN) in Korea.The claim data base of the Health Insurance Review and Assessment Service in Korea was used to select patients with ICD-10 codes N10  or N12  as the primary discharge diagnosis during 2010–2014. Consumption of each class of antibiotics was converted to Defined Daily Dose (DDD)/event.P = 0.002), but remained stable for outpatients (P = 0.066). The use of parenteral antibiotics increased for inpatients (P < 0.001), but decreased for outpatients (P = 0.017). As for the the antibiotic classes, 3rd generation cephalosporins (3rd CEPs) was the most commonly prescribed (41.4%) for inpatients, followed by fluoroquinolones (FQs) (28.5%); for outpatient, FQs (54.8%) was the most commonly prescribed, followed by 3rd CEPs (13.1%). The use of 3rd CEPs (P < 0.001), beta-lactam/beta-lactamase inhibitors (P = 0.007), and carbapenems (P < 0.001) increased substantially for the treatment of hospitalized APN patients. In particular, carbapenems use increased 3.1-fold over the 5 years.Throughout the five-year period, the average antibiotic consumption were 11.3 DDD per inpatient event and 6.0 DDD per outpatient event. The annual average antibiotic consumption increased for inpatients (Prescription of broad-spectrum antibiotics increased much for the treatment of APN in Korea during 2010–2014. Enterobacteriaceae are common uropathogen, and Escherichia coli is the most common among them. Uropathogens from APN have become resistant to several important antibiotics such as trimethoprim/sulfamethoxazole (SXT), fluoroquinolones (FQs) and the 3rd generation cephalosporins (3rd CEPs) and antibiotic resistance of uropathogens resulted in unfavorable clinical responses in community-acquired APN [rd CEPs among E. coli for community-acquired APN were 27.8, 21.3 and 9.3%, respectively, during 2010–2012 [Acute pyelonephritis (APN) is one of the most common community-acquired bacterial infections, which usually require antibiotic treatment . The annired APN . In Kore010–2012 . AntibioThe aim of this study is to describe the changes in prescribing practices of antibiotics used to treat APN during 2010–2014, using the National Health Insurance claim data in Korea, which may give a clue to the overall changes in prescribing practices of antibiotics used to treat common bacterial infections.International Classification of Diseases, Tenth Revision (ICD-10). Usually, there is a 1–2-month lag in billing for health insurance claims. We obtained the claim data for January 2010 to July 2015.The National Health Insurance System of Korea covers almost the entire population, including low-income families receiving medical aid: 98% of the population was covered in 2014 . We obtaWe included patients whose primary discharge diagnosis was APN from 2010 to 2014: ICD-10 codes N10  or N12 . Two or more claims during a 14-day period were regarded as a single event. The claims for the Veteran Health Insurance were excluded because beneficiaries of the Veteran Health Insurance could claim multiple times with a single event. The events with zero admission/visit day or zero medical cost and the events without antibiotic prescription were also excluded, as well as patients under 15 years of age  classification system, which does not include antifungal agents nor anti-tuberculosis agents. Systemic agents with per oral or parenteral administration route were included while topical agents were excluded. The consumption of each class of antibiotics was converted to Defined Daily Dose (DDD), following ATC classification system of the World Health Organization (WHO) . As for P < 0.05. All analyses were performed using SAS enterprise guide version 6.1 .Linear regression models with monthly measured variables were used to assess the trend of antibiotic consumption over time or age. Statistical significance was defined as A total of 845,656 APN events were identified during 2010–2014 in Korea . Most cases  met N10 code  in ICD-10. One out of 4.1 patients were hospitalized .P = 0.002). On the other hand, the average amount of antibiotic consumption per outpatient event was 6.0 DDD, which remained stable throughout the study period . Males received more antibiotics than females in both inpatient  and outpatient events . As for the antibiotic use by age, more antibiotics were prescribed for older patients  , but not in outpatient events . As for hospital type, the average amount of antibiotics usage was the highest among patients admitted to tertiary hospitals in both inpatient and outpatient events , followed by secondary hospitals .P < 0.001), but it mostly decreased for outpatients . As for per oral antibiotics, no significant change was observed in both inpatients (P = 0.075) and outpatients (P = 0.453).Figure rd CEPs  was the most commonly prescribed, followed by FQs  and beta-lactam/beta-lactamase inhibitors (BL/BLIs) . For outpatients, FQs  was the most commonly prescribed antibiotics, followed by 3rd CEPs  and 2nd generation cephalosporins (2nd CEPs) .Figure rd CEPs (P < 0.001), BL/BLIs (P = 0.007), and carbapenems (P < 0.001) increased every year over the study period; particularly, carbapenems use increased 3.1-fold, and 3rd CEPs use increased by about 25%. In contrast, the use of FQs (P = 0.010), 1st generation cephalosporins (1st CEPs) (P < 0.001), aminoglycosides (AGs) (P = 0.004), and penicillins (P = 0.014) declined every year. There was no noticeable change in antibiotic usage among the remaining classes including 2nd CEPs (P = 0.067), 4th generation cephalosporins (4th CEPs) (P = 0.162), and SXT (P = 0.575).Table rd CEPs (P = 0.049) and carbapenems (P = 0.001) increased gradually (Table st CEPs (P = 0.002), AGs (P = 0.002), and penicillins (P = 0.001). There was no noticeable change in antibiotics usage among the other antibiotics: 2nd CEPs, P = 0.124; BL/BLIs, P = 0.294; FQs, P = 0.555; SXT, P = 0.538.For outpatients, the use of 3 P = 0.00 increaseEnterobacteriaceae. Because the causative organisms of APN are easy to recognize, physicians can readily respond to antimicrobial resistance when they prescribe antibiotics in treating APN. Given that the local guideline for empirical therapy of urinary tract infections (UTI) did not change during the study period, APN is an appropriate disease to examine changes in antibiotic prescribing patterns under the presence of antibiotic resistance.APN is one of the most common infectious diseases caused by rd CEPs increased among APN patients from 1997 to 2001 [In the literature, two studies examined changing antibiotic prescription patterns in APN or UTI , 9. In t to 2001 . Howeverrd CEPs, BL/BLIs, and carbapenems were more prescribed in treating APN during the 5-year period than the traditional antibiotics. This probably happened for the following reasons. Firstly, the physicians were concerned about increasing multidrug-resistant pathogens in UTI. Secondly, resistance to traditional antibiotics in treating APN increased enough. Thirdly, broad-spectrum antibiotics occur more easily to the physicians’ mind.In Korea, broad-spectrum antibiotics such as 3E. coli isolated from blood in large Korean hospitals was 23.9% in 2006–2007 and then increased to 30.8% in 2011 [E. coli to FQs had increased from 21.3% in 2010–2011 to 33.5% in 2017–2018 among community-acquired APN patients [E. coli isolated from female uncomplicated cystitis, the study also demonstrated an increase in FQs resistance [FQs have been widely accepted as a first-line empirical antibiotic for APN in many countries , 11. How in 2011 . Similarpatients . In discrd CEPs  gradually increased. Carbapenems are generally used in hospital-acquired infections caused by multi-drug resistant organisms, however, our data implicates that those broad-spectrum antibiotics are more and more commonly used in mostly community-onset relatively simple infection.We found that carbapenems use increased by 3.1-fold among inpatients (from 0.28 to 0.87 DDD/event) and by 2.1-fold among outpatients (from 0.02 to 0.04 DDD/event), respectively. Also, the proportion of carbapenems use relative to FQs  and that to 3Considering the high prevalence of APN and ecological impact of the extensive use of carbapenems, this is a worrisome finding. Nationwide education and prescription control of broad-spectrum antibiotics for the treatment of common but not serious infections such as community-acquired APN should be reinforced. Other antibiotics which would replace carbapenems for the treatment of APN caused by extended-spectrum beta-lactamase (ESBL) producing organisms such as piperacillin/tazobactam or gentamicin should be studied more and recommended for the treatment of less severe APN patients , 19.rd CEPs and carbapenems increased in Korea since the last decade [Broad-spectrum antibiotic consumption is a common problem in many countries. Based on the data from the European Surveillance of Antibiotic Consumption (ESAC) projects, broad-spectrum penicillins and BL/BLIs were used more frequently than traditional penicillins throughout the EU countries . Carbapet decade . To breat decade . In Frant decade , 24. Fort decade . In addiThe main strength of the present study is documenting the increasing trend of broad-spectrum antibiotics use in treatment of APN which is a relatively simple community-acquired infection with identified pathogens in most cases. In addition, the present study is based on high-quality data source: the National Health Insurance data, which is the most representative health data in Korea. Using a large number of samples, our results show the overall changes in prescribing practices of antibiotics in a common bacterial infection clearly. Even though the reliability of diagnosis may be somewhat questionable as in other studies using administrative data, still there is no doubt that the data quality is high. APN has homogeneous pathology and simple diagnostic criteria to ease analyzing a large data set based on the entire population . We alsoDespite the above strength of this study in terms of data quality, there are also some limitations in this study. Firstly, administrative data do not contain laboratory-level microbiological data, and with the administrative data alone, we could not correlate the level of antimicrobial resistant pathogens with the increasing use of broad-spectrum antibiotic consumption. Also, due to lack of microbiological data, we could not differentiate complicated versus uncomplicated APN cases. Secondly, only the main discharge diagnosis of APN was included. Some cases might have been coded as sub-discharge diagnosis under other ICD-codes such as Gram-negative sepsis and adult respiratory distress syndrome. Thirdly, detailed information on the total duration and amount of each antibiotic use was not available, which made it impossible to analyze combined antibiotic therapies and calculate precise treatment duration for each event that are essential to judge the appropriateness of antibiotic therapies. Fourthly, using Korean data alone is certainly insufficient to shed light on the global antibiotic resistance trend in APN. Despite this limitation, learning about Korean experiences on antibiotic usage might be informative for other countries.rd CEPs, BL/BLIs and carbapenems were prescribed than traditional antibiotics for the treatment of APN in Korea.During the 5 years from 2010 to 2014, substantially more broad-spectrum antibiotics such as 3


Methanobrevibacter smithii, Methanosarcina mazei, and Methanomicrococcus blatticola, were transplanted into Apoe−/− mice fed with high choline/TMA supplemented diet. Similar to C57BL/6 mice, following initial provision of the MA, there was progressive attrition of MA within fecal microbial communities post-transplantation during the initial 3 weeks of the study. In general, plasma TMAO concentrations decreased significantly in proportion to the level of MA colonization. In a subsequent experiment, use of antibiotics and repeated transplantation of Apoe−/− mice with M. smithii, led to high engraftment levels during the 9 weeks of the study, resulting in a sustained and significantly lower average plasma TMAO concentrations (18.2 ± 19.6 μM) compared to that in mock-transplanted control mice . Compared to control Apoe−/− mice, M. smithii-colonized mice also had a 44% decrease in aortic plaque area , and 52% reduction in the fat content in the atherosclerotic plaques , although these differences did not reach significance. Gut colonization with M. smithii leads to a significant reduction in plasma TMAO levels, with a tendency for attenuation of atherosclerosis burden in Apoe−/− mice. The anti-atherogenic potential of MA should be further tested in adequately powered experiments.A mechanistic link between trimethylamine N-oxide (TMAO) and atherogenesis has been reported. TMAO is generated enzymatically in the liver by the oxidation of trimethylamine (TMA), which is produced from dietary choline, carnitine and betaine by gut bacteria. It is known that certain members of methanogenic archaea (MA) could use methylated amines such as trimethylamine as growth substrates in culture. Therefore, we investigated the efficacy of gut colonization with MA on lowering plasma TMAO concentrations. Initially, we screened for the colonization potential and TMAO lowering efficacy of five MA species in C57BL/6 mice fed with high choline/TMA supplemented diet, and found out that all five species could colonize and lover plasma TMAO levels, although with different efficacies. The top performing MA,  The gut microbiome is now recognized as a mediator of numerous host physiological processes. Changes in the gut microbiome have been causally linked to several metabolic, inflammatory, and cardiovascular diseases, including atherosclerosis3. Systemic concentrations of the gut microbe-derived metabolite, trimethylamine-N-oxide (TMAO), is associated with atherosclerosis and major adverse cardiovascular events6. Choline diet dependent enhancement in atherosclerosis could be prevented by either gut microbiota suppression with broad spectrum antibiotics6, or in more recent studies, administration of an inhibitor of microbial choline trimethylamine (TMA) lyase activity and therefore TMAO generation 7, indicating a causal link between TMAO and atherosclerosis. In this study, we tested a novel microbiota-based approach to reduce systemic exposure of TMAO.Atherosclerotic vascular disease is the leading cause of death in the US6, carnitine4, phosphatidylcholine5, betaine8, and trimethyllysine9. TMA is converted to TMAO by the host hepatic flavin monooxygenase 3 (FMO3)10. Multiple phylogenetically distinct bacteria are involved in TMA production11. There are many microbial families, genus, and species which possess microbial enzymes that can make TMA. There have been three microbial enzyme genes/sources of TMA thus far identified; the cutC/D11, the cntA/B gene12, and the YeaW/Z gene13.Gut microbes generate TMA from ingested precursors, including choline14, also known as the fish odor syndrome, which severely reduces the ability to convert TMA to TMAO. Consequently, the affected individuals excrete large amounts of odorous TMA in their urine, sweat, and breath15. Although the disorder is not known to affect patient health, it can have profound social and psychological consequences. Development of a probiotic that can catalytically consume TMA could also serve as a therapeutic approach for treating this genetic disorder.In humans, FMO3 gene mutations cause the inherited disorder primary trimethylaminuria (TMAU)18. Methanobrevibacter smithii is the dominant methanogen in the human gut, detected in 95.7% of individuals, whereas Methaomassiliicoccus luminyensis is detected only in 4% of individuals19. Certain MA such as Methanosarcina species are known to use methylated amines as growth substrates21. A catabolic microbe that literally consumes TMA preventing/intercepting TMA prior to when it can be absorbed and converted into TMAO, would serve as a therapeutic intervention. However, up until now this potential has largely remained a theoretical concept. However, up until now this potential has largely remained a theoretical concept. In this study, we tested the effect of MA colonization on blood TMAO level and atherosclerosis burden in the atherosclerosis prone Apoe−/− mouse model.Methanogenic archaea (MA) represents a distinct group of anaerobic archaea that produce methane as the end-product of their anaerobic respiration. The abundance and diversity of gut MA in humans is highly variable and limited to a few speciesAll methods were performed in accordance to guidelines and regulations, and under approval of the Institutional Biosafety Committee of The George Washington University.Methanobrevibacter smithii, strain PS (DSM-86), (b) Methaomassiliicoccus luminyensis, strain B10 (DSM-25720), (c) Methanosarcina mazei, strain S-6 (DSM-2053), (d) Methanoimicrococcus blatticola, strain PA (DSM-13328) and (e) Methanohalophilus portucalensis, strain FDF-1 (DSM-7471) were obtained from DSMZ (Germany). M. smithii and M. luminyensis were selected because these are indigenous human gut MA22. For comparison, we selected M. blatticola (isolated from the hindgut of cockroach Periplaneta americana)23, M. mazei (isolated from a laboratory digester)24, and M. portucalensis 25.Selected species of known human gut and non-gut MA including: (a) 10. Prior to transplantations, endogenous gut microbiota were suppressed using an oral cocktail of poorly absorbed antibiotics previously reported to suppress TMA and TMAO levels6  administered in drinking water ad lib for three weeks, refreshed 3 times per week. Each mouse was inoculated with a single gavage of 108 MA 24 hours after the antibiotic treatment was completed. The test groups (n = 3/group) received a 0.1 ml gavage of corresponding MA in liquid growth media. The control groups (n = 3 each) received a 0.1 ml gavage of liquid growth media. Following transplantations, mice were supplemented with choline  and TMA  in drinking water ad lib, refreshed 3 times per week. The negative control group (NC) received untreated water. Both choline and TMA were given to animals since antibiotic treatment depletes most of the TMA-generating bacteria. All animals received standard chow diet for the entire study. Blood plasma and fresh stool samples were collected from the mice on days 2, 10, and 30 post-transplantation and stored at −80 °C, until processed. In a second study, 4-week-old female C57BL6J mice with Apoe−/− background (n = 5/group) (Jackson Laboratory) were treated with antibiotics, transplanted with M. smithii, M. mazei, and M. blatticola and fed with high TMA/choline diet as described above to determine the impact of MA on TAMO concentration. In a third study, we examined the effect of sustained high colonization with M. smithii on TMAO in Apoe−/− mice (n = 5/group). The experiment was performed as described above, except that M. smithii transplantation was repeated every three weeks, until the end of the study. One group of mice (n = 5) was maintained throughout the study on an antibiotic regimen (0.5 g/L vancomycin and 1 g/L ampicillin) to which MA are resistant, in order to chronically suppress the endogenous gut microbiota and enhance MA colonization  were randomly selected for transplantation with one of the MA or sham, and placed on high TMA/choline diet. Because of the small sample size (n = 3/group), considering that the male mice have lower plasma TMAO levels, we used only female mice in this pilot study26. Arch344f (ACGGGGYGCAGCAGGCGCGA)26 and Arch806r (GGACTACCCGGGTATCTAAT)27 primers were used to target the archaea 16 S rRNA gene present in MA. Q-PCR was performed for detection and quantification of microbes using SYBR Green PCR Master Mix  in an Applied Biosystems 7900HT Sequence Detection system (Applied Biosystems)28. PCR products amplified from the bacterial and archaeal 16 s rRNA genes were cloned and grown in transformed E. coli to establish a standard curve, as described previously29. The accuracy of the q-PCR assay was confirmed through melting curve analysis and agarose gel electrophoresis. Calibration curves were obtained using 10-fold serial dilutions of known concentrations of cloned DNA samples that were prepared in the laboratory.Gut colonization by the MA was determined using quantitative real-time PCR (q-PCR). Briefly, microbial DNA was extracted from stool samples using the Qiagen DNeasy PowerSoil kit. The stool specimens were tested for the levels of engraftment by q-PCR as previously described30 essentially as previously described31. Briefly, for each reaction, 2 μL of extracted DNA was combined with 12.5 μL 2X Phusion Hot Start II High-Fidelity PCR Master Mix (Thermo Fisher Scientific), and 100 nM each of forward and reverse primers. The primers were designed containing the Illumina linker adaptor, a unique index sequence, followed immediately by a variable sequence spacer (0–6 bases) and 16 S rRNA gene primers  cutoff of 1.0. Non-overlapping high quality paired end reads were retained for downstream analysis by linking them artificially with 15 Ns. High-quality sequences were taxonomically assigned using the Ribosomal Database Project (RDP) Naïve Bayesian Classifier (version 2.9)34 with a bootstrap cutoff of 80%. PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States)35 was used to infer the abundance of metabolic pathways from 16 S rRNA gene sequencing data. The predicted functions (KOs) were collapsed into hierarchical KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways using the categorize_by_function step provided in the PICRUSt pipeline.The V4 region of the 16 S rRNA gene was PCR amplified using barcoded primers36. Briefly, the UHPLC-MS/MS system consisted of an Acquity UPLC I-class sample mannager , Acquity UPLC I-class binary solvent mannager, and a TSQ Quantum Ultra triple quadrupole mass spectrometer . For protein precipitation, plasma (50 µL) was combined with an internal standard (200 µL of d9-TMAO 0.5 µg/mL) in methanol. After centrifugation, 20 µL of supernatant was diluted with 100 µL of 75:25 acetonitrile:methanol and 5.0 µL of this mixture was injected onto the UPLC-MS/MS system. The standard curve ranged from 0.010–5.00 µg/mL (0.13–66.6 µM). The within-run and between-run precision (percent coefficient of variation) was <15%.Ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to measure plasma TMAO concentrations as described previously with minor modification37. Two animals from the negative control group and one of the transplanted animals died prior to obtaining samples for atherosclerosis evaluation.At the end of each study, mice were euthanized, and the heart was removed just proximal to the aortic arch. The heart was cut transversely at the level of the atria and placed ventricle down into a tissue mold in optimal cutting temperature compound (OCT compound) and stored at −20 °C until sectioning. Multiple cryosections at 10 μ thickness were taken of the aortic sinus and aortic root and stained for fat content with Oil-Red-O (ORO). Six sections from each heart were selected for image analysis, including the first section that contained the three leaflets of the aortic valve and the next 5 sections at 40 μ intervals along the aortic root distal to the valve over a total distance of 200 μ. The area of the plaque and the content of ORO was quantified by image analysis (ImageJ software) and calculated as an average of the area of plaque found in each of the 6 individual aortic valve sections taken for analysisM. smithii, M. mazei, and M. blatticola, M. luminyensis, M. portucalensis, and control), the time main effect , and the species x time interaction Values are presented as mean ± SD, and those with a value of P < 0.05 were considered significant. Data analysis was performed using SAS 9.4.We used analysis of variance and Student’s t test to compare mean plaque size between groups. A random effects mixed model was used to examine the species main effect  on day 30 post-transplant , M.mazei , M. blatticola , M. luminyensis , and M. portucalensis , compared to the positive control mice . TMAO concentrations showed a rebound increase from day 10 post-transplantation, which paralleled the decline in MA colonization levels.The plasma TMAO concentration was markedly elevated in the group of mice that were on the choline/TMA-supplemented water (positive control) compared to the negative control mice fed with regular water . On day 2 post-transplant, plasma TMAO concentrations were significantly lower in mice colonized with ice Fig. . TMAO coM. smithii, M. mazei, and M. blatticola, were selected and tested in Apoe−/− mice. In general, engraftment of the 3 transplanted MA in Apoe−/− mice were similar to that obtained in C57BL/6 mice, ranging from 0.6 0 × 107 to 3.0 × 107 copies/mg stool on day 2 post-transplant , M. mazei (7.4 ± 6.1 μM) and M. blatticola (5.8 ± 0.6 μM) were significantly lower on day 2 compared to positive control mice (98.5 ± 26.5 μM)  Fig. . The plaM. smithii leads to sustained high colonization level and persistent reduction in TMA concentration in Apoe−/− mice. In one group of repeatedly transplanted mice (n = 5/group) we also examined whether chronic suppressive antibiotic therapy further enhances colonization with M. smithii. As shown in Fig. M. smithii led to high engraftment levels throughout the study period. High and stable gut colonization with M. smithii led to lower average plasma TMAO concentrations (18.2 ± 19.6 μM) throughout the study period compared to that in positive control mice   and lower average TMA concentrations .In order to improve gut colonization efficacies, we tested whether repeated transplantation with 01) Fig. . The groM. smithii were 2.4% and 2.2% in weeks 4 and 8, respectively in mice that were not on antibiotics, and 64% and 84% in weeks 4 and 8, respectively, in mice that received antibiotics.16 S gut microbiome analysis of the stool samples from mice in the last experiment demonstrated that the baseline and pre-antibiotic microbiome profile of the mice consist of 53% Bacterioidetes, 43% Firmicutes, and 4.9% Verrucomicrobia Fig. . HoweverBacteroides at the baseline, while Clostridium and Turicibacter trended higher among the remaining bacteria in antibiotic-treated mice  than the positive control mice , but the differences did not reach statistical significance (p = 0.34)  compared to that of the positive control mice , which was also not statistically significant (p = 0.10).When examined for aortic root plaque size by histology, in vivo TMA-metabolizing efficacy of several gut and not-gut associated methyl-trophic methanoarchaea of the genera Methanomicrococcus (Methanomicrococcus blatticola); Methanosarcina (Methanosarcina mazei), Methanohalophilus , and Methanomassiliicoccus (Methanomassiliicoccus luminyensis). The ability of all of these species to grow on TMA in culture has been previously demonstrated39. Also included in this study, was M. smithii, a member of Methanobrevibacter genus which is the most predominant methanoarchaea in the human gut. Unlike the other methanoarchaea used in this study, M. smithii is not known to grow on TMA in culture. However, a recent study demonstrated that many essential genes involved in methanogenesis, including methyltransferases are present and significantly up-regulated in vivo when M. smithii was co-colonized with a prominent human gut symbiont40.In this study, we investigated the M. smithii, a normal inhabitant of the human gut. Unlike M. smithii, the other indigenous human gut methanoarchaea, M. luminyensis, was very poor at gut colonization in mice and lowering blood TMAO levels. This observation is however, in accordance with the reports on low prevalence of M. luminyensis which is detected only in 4% of individuals, compared to that of M. smithii which has a high prevalence of nearly 96%19.We show that gut colonization with MA, consistently and significantly reduces plasma TMAO concentrations in two different mouse models that were maintained on a high choline and TMA diet. The reduction in plasma TMAO concentrations, in general paralleled the abundance of MA in the gut. We initially screened five species of MA isolated from gut and non-gut environments and noted that the colonization efficacy and the TMAO lowering efficacy is highest with M. smithii colonization led to distinct change in microbiome profile and functional changes in gene expression. Compared to Apoe−/− mice fed with high choline/TMA diet, mice colonized with M. smithii had 44.2% lower atherosclerotic burden and 52.2% reduced fat content in the atherosclerotic plaques, but these differences did not reach statistical significance possibly due to the small sample size.16 s rRNA sequencing showed that antibiotics, choline/TMA and 6. TMAO originates from the microbiota-dependent breakdown of dietary phosphatidylcholine to TMA. Functional studies have shown that TMAO decreases expression of bile acid transporters in the liver and reduces synthesis of bile acids from cholesterol4. TMAO also inhibits reverse cholesterol transport and promotes accumulation of cholesterol in macrophages through increasing cell surface expression of proatherogenic scavenger receptors CD36 and Scavenger Receptor A6, thus, creating foam cells that subsequently accumulate in the endothelial wall causing inflammation and plaque formation. Another mechanism related to atherosclerosis is the increase in thrombosis mediated by TMAO41.Increased systemic exposure of TMAO is associated with atherosclerosis and major adverse cardiovascular events7. Targeting gut microbial production of TMA using a structural analog of choline, 3,3-dimethyl-1-butanol, inhibits TMA formation and lowers TMAO concentrations in mice fed a high-choline or L-carnitine diet7. 3,3-dimethyl-1-butanol inhibited choline diet-enhanced endogenous macrophage foam cell formation and atherosclerotic lesion development in Apoe−/− mice7. In this study, we took a different approach and aimed at depleting TMA as it is being formed by MA. In vitro studies have shown that certain MA can utilize TMA as a substrate for growth38. However, this is the first in vivo study that examines the utility of MA in decreasing plasma TMAO concentrations.It is therefore, not surprising that there is considerable interest in treatments designed to lower TMAO concentrationsApoe−/− mice. In the final experiment, we examined the effect of repeated transplantation with M. Smithii, and chronic suppression of endogenous bacteria by antibiotics, on colonization efficacy, TMAO concentrations and atherosclerosis level in Apoe−/− mice.We used a logical step-wise approach including screening, discovery and validation. For our initial screening, we chose normal C57/BL6 mice to evaluate the gut colonization capability and TMAO lowering efficacy of five selected species of MA. Subsequently, we tested the TMAO lowering efficacy of the three top performing MA in M. smithii was evident, but the colonization level of the other human gut MA, M. luminyensis, was poor. In the second experiment, a significant reduction in plasma TMAO concentrations were also observed in Apoe−/− mice transplanted with M. smithii (90.4%), M. mazei (92.5%), and M. blatticola (94%) on the 2nd day, compared to non-transplanted control animals fed with high choline/TMA diet. Our initial experiments in both C57BL/6 and Apoe−/− mice revealed that, even after a 3-week-long antibiotic depletion of the gut bacteria, colonization was only transient with a single dose of MA. In a 3rd experiment, we show that repeated transplantations significantly improved the gut colonization levels, which was further improved by antibiotic suppression of endogenous gut bacteria. There was a corresponding sustained reduction in TMAO concentrations. 16 S sequencing analysis confirmed that maintaining the transplanted mice on antibiotics greatly enhances M. smithii engraftment, but depleted Firmicutes and enriched Proteobacteria.Our initial study in C57BL/6 mice showed that all MA tested were able to colonized the gut and reduce TMAO concentrations irrespective of their original habitat, but with large inter-individual variations. Efficient gut colonization by M. smithii because it is known to be the most predominant MA in the human gut. M. smithii thrives in the distal intestine through its versatility in consuming the fermentation products made by saccharolytic bacteria, and by its ability to produce surface glycans and adhesion-like proteins40. M. luminyensis also colonizes human gut, although at much lower levels compared to M. smithii19. In human gut, TMA-metabolizing MA depend on the gut microbiota for converting choline to TMA which serves as a carbon-source for MA. In return, MA facilitates the continuation of bacterial fermentation process in the gut by metabolizing and removing H2.We chose to test Apoe−/− mice demonstrate a total cholesterol level >500 mg/dL, with fatty streaks first observed in the proximal aorta at 12 weeks of age, and fibrous plaques appearing at 20 weeks of age42. A Western diet43, or a choline-rich diet6, induce a marked increase in plaque size and aggressive plaque morphology. We found that gut colonization with M. smithii resulted in a tendency for reduced atherosclerosis burden in Apoe−/− mice fed with high choline/TMA diet. The results were not statistically significant, possibly due to small number of animals studied. In addition, we cannot exclude the possibility that factors other than TMAO contribute to the pathogenesis of atherosclerosis, which were unmodified by this intervention44.On a chow diet, Apoe−/− mice.Since our data on the lowering of plaque characteristics were not statistically significant, either this study needs to be repeated in a much larger cohort of animals, or it could be concluded that lowering TMAO (as significant as our study demonstrates), does not lower TMAO-induced atherosclerosis in We acknowledge that this is a pilot study in small number of animals and that the study could have benefited from measurement of TMA and choline concentrations; however, the study has a number of strengths. Strengths include: (a) first study to explore the utility of gut colonization by MA to lower plasma TMAO concentrations; (b) the evaluation of several candidate gut and non-gut MA in two mouse models; (c) the examination of single vs. repeated transplantation effect of MA; and, (d) the determination of MA engraftment efficiency after suppression of the endogenous gut microbiome.While we do not know and did not investigate the factors that support increased engraftment level of MA in mice maintained on antibiotics, we speculate that there may be other bacteria and/or bacteria-derived factors that would either compete with or inhibit MA gut colonization. Clearly, the use of antibiotic in this study was to enhance gut colonization in our mouse model and facilitate the study of MA impact on blood TMAO levels, and not a recommended method to enhance the MA gut colonization in human. Future studies are needed to identify the exact mechanism and factors that support or compete with MA engraftment in human gut, and the consequence of increased MA abundance on the human host physiology and health.46. M. smithii has been shown to enhance energy retrieval by another sacharolytic bacterial species when co-transplanted into germ-free mice47. A study of fecal DNA extracts from patients with colorectal cancer, polypectomised, irritable bowel syndrome and control subjects found no significant association between these diseases and the presence of MA48. There is also no conclusive evidence that MA is associated with colon cancer50.To date, the impact of methanogens on human physiology and health is poorly understood. Association between MA and inflammatory bowel disease, colorectal cancers and obesity has been reported, however, there is no convincing evidence supporting the pathogenic properties of the MA, beyond occasional reports of constipationM. smithii, an endogenous human gut MA, was most effective in lowering plasma TMAO levels in Apoe−/− mice fed with high choline/TMA, and showed a tendency to attenuate atherosclerosis. The anti-atherogenic potential of the MA should be confirmed in studies using larger number of animals. The field of microbiome research is still nascent, but is evolving rapidly. Therapeutic use of specific toxin-degrading gut commensal microbes as probiotics to lower the levels of uremic toxins such as TMAO is a novel concept, but if proven effective, it will have a significant impact on cardiovascular disease and progression of chronic kidney disease. Safety of MA should be tested in pilot studies prior to launching efficacy studies in human subjects.To summarize, our study shows that gut colonization with certain MA reduces plasma concentrations of TMAO. Among the MA tested,


P = 0.017, P = 0.002 and P = 0.019, respectively) at 6 months after surgery. One months postoperatively, complications were higher in the HHR-LNF group than in the HHR group , and there was no difference between the two groups at 6 months. By the end of the 2-year follow-up, HHR-LNF group showed a significantly lower reflux syndrome frequency-intensity score and greater percentage of satisfaction compared with HHR group . Laparoscopic HHR should be combined with a fundoplication in GERD patients with HH. HHR-LNF is safe and effective, not only improve reflux-related symptom, but also reduce the incidence of complications.Gastroesophageal reflux disease (GERD) is often associated with hiatal hernia (HH). However, the need for fundoplication during hiatal hernia repair (HHR) remains controversial. The objective of this study was to evaluate the effect of HHR with concomitant laparoscopic Nissen fundoplication (HHR-LNF) in HH patients. A total of 122 patients with symptomatic HH were randomized to receive either HHR (n = 61) or HHR-LNF (n = 61). The measures of evaluating outcomes included DeMeester scores (DMS), complications, Reflux Diagnostic Questionnaire and patients’ satisfaction 24 months following surgery. Despite comparable values in both groups at randomization, the DMS, total numbers of reflux episodes and percentage of time with pH < 4 were significantly higher in HHR group than in HHR-LNF group ( Over time, that procedure has been a posterior gastroprexy as described by Hill2 with little or no fundoplication. Ultimately, a fundoplication is performed either through the chest (Belsey Mark IV) or abdomen involving varying degrees of encirclement of the distal esophagus by the fundoplication. Laparoscopic hiatal hernia repair (HHR) has been shown to provide good short- and long-term results in gastroesophageal reflux disease (GERD)4, and may reduce the laparoscopic antireflux surgery complications5. Fundoplication is also an important component of laparoscopic antireflux surgery performed for medication-refractory GERD6.The history of antireflux surgery over the last 48 years  is still controversial. A blinded randomized controlled study showed laparoscopic repair of paraesophageal hiatal hernias should be combined with a fundoplication to avoid postoperative GERD and concomitant esophagitisDespite the above history of surgical advancement, we feel a re-examination of diaphragmatic crural approximation alone and combined with the best procedure to date, a 360° fundoplication as performed would be meaningful and necessary.In this study, laparoscopic HHR (the effect of hernia reduction and only repair of diaphragmatic crura) was compared to HHR with concomitant fundoplication in terms of relative frequency and severity of symptoms before laparoscopic antireflux surgery, and DeMeester scores (DMS), relative symptom improvement, patients’ satisfaction and complications after antireflux surgery.15. Patients were excluded if they had history of previous esophagogastric surgery, achalasia, Zollinger-Ellison syndrome, or malignant tumor. Patients failing to answer more than 40% of the questionnaire and those having a serious intercurrent event  during the course of the study were also excluded from the final analysis.Between December 2013 and December 2015, 136 consecutive patients with symptomatic HH were considered for inclusion in this study. Patients were eligible for enrollment in this study if they had persistent symptoms despite daily use of proton-pump inhibitors (omeprazole 20 mg twice a day) for at least 3 months before surgery17. The following parameters were also evaluated: (1) the total number of reflux episodes, (2) total time for pH < 4, (3) time percentage of upright position pH < 4, (4) time percentage of supine position pH < 4, and (5) number of times pH < 4 for lasting 5 minutes. The HH were classified according to internationally accepted criteria5.All patients underwent preoperative diagnostic evaluation with endoscopy, barium sulfate swallow radiology, and esophageal manometry. GERD was diagnosed if the patient had typical heartburn and regurgitation, endoscopic evidence of esophagitis or abnormal esophageal pH, reflux events and DMS (composite score) ≥ 14.725. Esophagitis was graded according to the Los Angeles (LA) classification18.HH was classified into four types according to international standards, including type I (sliding), type II , type III (mixed), and type IV (mixed with others organs rather than only gastric hernia sac content)15. The patient was sedated and placed in a reverse-trendelenburg position, with the lower extremities abducted. The operating surgeon stood between the legs of the patient, with the first assistant on the left side of the patient and the second assistant on the right side of the surgeon. A veress needle was inserted close to the rib cage, and a pneumoperitoneum was created. The primary 10-mm trocar was inserted at the upper edge of the navel. Four more trocars were then inserted in the upper abdomen under direct visualization. The liver was retracted, and the esophageal hiatus was exposed. The stomach was repositioned, and the hernia sac was dissected and reduced from the mediastinum using a harmonic scalpel. A 3–4 cm of esophageal reposition was dissected intra-abdominally. The diaphragmatic crurae were exposed, and sutured 2–3 times intermittently with non-absorbable lines to reduce the esophageal hiatus. When the hernial sac was >5 cm in size, a horseshoe-shaped polypropylene mesh patch was fixed around the esophagus from behind with 8–12 staples toward the diaphragm. If 360-degree Nissen fundoplication was added. A full-circle valve was formed from the posterior and anterior aspects of the fundus and sutured together using three separate nonabsorbable 2–0 sutures.The surgical technique of laparoscopic surgical procedure was performed as described in our previous studyDuring postoperative follow-up, objective evaluation included 24-hour esophageal pH monitoring and esophageal manometry at 6 months, as well as gastroscopy at 12 months. The 24-hour esophageal pH monitoring was assessed by DMS and percentage of time with pH < 4. Esophageal manometry was performed for measuring hypotonia of the lower esophageal sphincter and HH. The lower esophageal sphincter resting pressure (LESP), using mid-respiratory gastric pressure as zero reference, was measured mid-respiratory resting pressure in the high-pressure zone. Gastroscopy was used to identify recurrent HH and esophagitis. The criterion for gastroscopic diagnosis of recurrent HH was cephalic displacement of the proximal margin of the gastric mucosal folds by more than 2 cm in relation to the hiatus.19. Frequency of symptoms was graded as 0 (none), 1 (less than once per week), 2 (once or twice per week), 3 (three or four times per week), 4 (five or six times per week), and 5 (more than six times per week). Severity was graded as 0 (none), 1 (slight), 2 (mild), 3 (moderate), 4 (severe), and 5 (extremely severe). The sum of the frequency score and the severity score was designated as the symptom score.Questionnaires were completed at admission to investigate the baseline symptom scores, and mailed questionnaire were used during follow-up. Heartburn and regurgitation were the most typical symptom of GERD, and the occurrence of these symptoms were considered to indicate recurrence. A 6-point scale was applied to assess the severity and frequency of heartburn and regurgitation according to the Reflux Diagnostic Questionnaire (RDQ)20.Patients, satisfaction with the result of surgery was also assessed. Four grades were possible: excellent (complete resolution of symptoms); good (symptoms occurring once per month or less frequently); fair (symptoms occurring weekly or less frequently); and poor t test as appropriate. All statistical tests were two-sided. P < 0.05 was considered statistically significant.Data analysis was performed using SPSS version 13.0 software  and GraphPad InStat, version 3.06 . Continuous variables were expressed as median or means ± standard deviations. Comparisons were made between HHR patients and HHR-LNF patients and between preoperative and postoperative status using the chi-squared test, the Wilcoxon paired-samples test, or the The Ethics Committee of Zhengzhou University approved the study. The committee that approved the research, confirm that all research was performed in accordance with relevant guidelines/regulations. Written informed consent was obtained from all participants and/or their legal guardians.Of the total 136 patients, 14 patients did not meet inclusion criteria. The remaining 122 patients were randomly assigned into a HHR group (n = 61) or a HHR-LNF group (n = 61). The study flow was shown in Figs P < 0.001). Table P = 0.305). One months postoperatively, patients in the HHR-LNF group experienced complications such as dysphagia, abdominal bloating, abdominal pain were higher than in the HHR group . However, the DMS, total numbers of reflux episodes and percentage of time with pH < 4 were significantly higher in HHR group than in HHR-LNF group  HHR patients and 63.33% (38/60) HHR-LNF patients were available for esophageal manometry and 24-hour pH monitoring. In some patients, esophageal manometry and 24-hour pH monitoring could not be performed again because of intolerance of the procedure and inspection fees. Significant improvement in DMS, the total numbers of reflux episodes, the total time for pH < 4, percentage of time with pH < 4, percentage time for pH < 4 in the upright position, percentage time for pH < 4 in the supine position, number of times for pH < 4 lasting 5 minutes and LESP were seen in both groups (2/59 HHR P < 0.001). Compared with the preoperative findings, the frequency of esophagitis was significantly lower after HHR-LNF (P = 0.005). The frequency of esophagitis was high after HHR without LNF, although the change was not statistically significant .Preoperatively, Table P < 0.001). The other 18.2% (10/55) HHR-LNF patients and 52.8% (28/53) HHR patients continued to be symptomatic and needed medication or repeat surgery. Two HHR-LNF patients were rehospitalized for laparoscopic revision . 20 HHR patients were rehospitalized for LNF. HHR patients had a significantly higher reoperation rate than HHR-LNF patients (P < 0.001). No major complications or deaths occurred during the study.81.8% (45/55) of HHR-LNF patients were fully or partially satisfied with the surgical results, but only 47.2% (25/53) in HHR patients , as reflected by the RDQ scores and the decrease in frequency and severity of GERD symptoms such as heartburn and regurgitation.15.At follow-up 12 months after surgery, gastroscopy revealed esophagitis in 44.64% of LNF patients vs. 13.79% of HHR-LNF patients. Both the frequency and the severity of esophagitis were significantily greater in HHR group, which were the samilar as a previous studyet al.27 found significant improvement in preoperative symptoms after laparoscopic repair of giant paraesophageal hernia; in their study, 90% of patients showed high degree of satisfaction on a GERD health-related quality of life scale, which was the same as in our study.We also assessed patient satisfaction with the treatment results. 12 months after the procedure, 81.8% (45/55) HHR-LNF patients and 47.2% (25/53) LNF patients were fully or partially satisfied with the symptom relief following surgery. Luketich 28. The risk of treatment-associated complications did not appear to be increased by the addition of fundoplication to HHR. Although dysphagia, abdominal bloating and abdominal pain occurred with higher frequency in the HHR-LNF group, both perioperatively and at 1 months postoperatively. After symptomatic treatment, there was no difference between the two groups in the incidence of the complications at 6 months. In this study population, only 11.4% (13/114) of patients had postoperative complications in the two groups, this was consistent with the 8%-28% complication rates that had been reported by others30.Postoperative complications mainly occurred in the mean duration of postoperative hospital stay in HHR group, such as dysphagia, abdominal bloating and abdominal pain. These symptoms were due to the occurrence of postoperative esophageal hiatus narrow, poor gastric motility. The esophageal elongation associated with surgery also is a factor. Leaving 2 cm or more of the abdominal esophagus to preserve the lower esophageal sphincter is important factors of the anti-gastroesophageal reflux32. In our study, a recurrent HH was revealed in only 1.7% (1/58) HHR-LNF group and in 5.4% (3/56) HHR group (P > 0.05). The overall recurrence rates of 3.5% (4/114) was lower than reported in the above literature. Therefore, an concomitant fundoplication maybe could not increase the risk of hernia recurrence when a HHR is performed. Moreover, up to 37.7% (20/53) HHR patients were rehospitalized for LNF, only 3.6% (2/55) HHR-LNF patients were rehospitalized for laparoscopic revision . HHR patients had a significantly higher reoperation rate than HHR-LNF patient.Recurrent HH has been reported to occur following antireflux surgery in 50%-70% of patients with large paraesophageal hernias34. Although both HHR and HHR-LNF patients had significantly improved LESP during the normal value (13–43 mmHg) at 6 months after surgery, patients with HHR-LNF reported significantly fewer reflux symptoms than did HHR patients. Conversely, the procedure of only HHR did not produce improvement in mid-term or long-term reflux symptoms. Therefore, HH just is an important factor in aggravating gastroesophageal reflux; Effective control of reflux mechanisms is the key factors for the successful management of GERD-related symptoms35. Fundoplication should be routinely performed in HH patients to reduce the risk of postoperative persistence of GERD or of new-onset GERD.With regard to the mechanism of GERD, the lower esophageal sphincter, or valvular mechanism, no matter which is destroyed and it cannot prevent the occurrence of reflux. HH may predict higher risk for GERD due to its negative impact on the esophagusThis study has several limitations. For example, designed methods, excellent randomization and relevant parameters of measurement may be less valuable.In summary, we found HH patients undergoing HHR-LNF had better midterm outcomes than HHR alone. These data might indicate that early assessment of HH patients, and opting for routine adjunct of fundoplication in HHR, could significantly improve clinical outcomes.


To evaluate the optimal zinc supplementation strategy for improving growth and hematologic and micronutrient status in young Laotian children.In total, 3407 children aged 6-23 months were randomized to receive either daily preventive zinc tablets (7 mg/d), high-zinc, low-iron micronutrient powder , therapeutic zinc supplementation for diarrhea (20 mg/d for 10 days per episode), or daily placebo powder; all were followed for ~9 months. Anthropometry, hemoglobin, zinc, and iron status were assessed at baseline and endline. Analyses were by intention-to-treat, using linear and modified Poisson regression.P < .001), with no impact on stunting . The micronutrient powder reduced iron deficiency by 44%-55% compared with other groups (P < .001), with no overall impact on anemia (P = .14). Micronutrient powder tended to reduce anemia by 11%-16% among children who were anemic at baseline (P = .06).At baseline, mean (±SD) age was 14.2 ± 5.1 months and stunting and anemia prevalence were 37.9% and 55.6%, respectively. At endline, zinc deficiency in the preventive zinc (50.7%) and micronutrient powder (59.1%) groups were significantly lower than in the therapeutic zinc (79.2%) and control groups (78.6%; Despite improving zinc status, preventive zinc and micronutrient powder had no impact on growth. The micronutrient powder improved iron status and tended to reduce anemia among the subset of previously anemic children.ClinicalTrials.govNCT02428647. Growth faltering and anemia are common in low- and middle-income countries, likely due in part to the coexistence of multiple micronutrient deficiencies, including zinc and iron deficiencies.Often, zinc deficiency does not occur in isolation. Supplementation with a multiple micronutrient powder offers a low-cost option to prevent multiple micronutrient deficiencies simultaneously. The World Health Organization (WHO) and United Nations Children's Fund currently promote a micronutrient powder formulation containing 12.5 mg of iron, 4.1-5 mg of zinc, and 13 other micronutrients.Therapeutic zinc supplementation (20 mg per day for 10-14 days) during episodes of diarrhea is recommended by the WHO and the United Nations Children's Fund along with oral rehydration salts (ORS) to reduce disease severity.The goal of the present study was to compare the effects of preventive zinc supplementation, provided either as a single micronutrient or as part of new micronutrient powder formulation containing more zinc (10 mg) and less iron (6 mg) than current formulations and therapeutic zinc supplementation, on physical growth, anemia, and biomarkers of zinc and iron status in rural Laotian children.https://clinicaltrials.gov).This trial and the consent procedure were approved by the National Ethics Committee for Health Research, Ministry of Health, Lao PDR, and the institutional review board of the University of California, Davis, California. The trial is registered as the Lao Zinc Study, NCT02428647  included 300 rural villages from 5 districts . All villages in these districts were invited for enrollment, except in Nongbok. Because the prevalence of stunting in Nongbok was lower than in the other districts, only villages belonging to the catchment area of health centers with a mean stunting prevalence ≥25% were invited to participate.The study area  provided written informed consent. Children were excluded from participation if they demonstrated any of the following health conditions: severe anemia (hemoglobin <70 g/L), severe wasting (defined as weight-for-height z score <–3 with respect to WHO 2006 standards),A statistician at University of California Davis randomly assigned the study ID numbers to the 4 study arms, using a block randomization scheme with block lengths of 4 or 8. In the event that multiple siblings in the target age range resided in the same household, only the youngest was enrolled. In the case of twins, both twins were assigned to the same group and received all study-related interventions and follow-up, but only one was selected randomly for inclusion in the data analyses.www.jpeds.com): (1) the preventive zinc supplementation group, who received a daily preventive zinc supplement tablet containing 7 mg of zinc and placebo therapeutic tablets for diarrhea; (2) the micronutrient powder group, who received a daily preventive micronutrient powder containing 10 mg of zinc, 6 mg of iron and 13 other micronutrients and placebo therapeutic tablets for diarrhea; (3) the therapeutic zinc supplementation group, who received a daily placebo preventive supplement tablet and therapeutic zinc tablets containing 20 mg for 10 days for diarrhea treatment; or (4) the placebo control group, who received daily placebo preventive powder and placebo therapeutic tablets for diarrhea.Children were individually randomized into 1 of 4 groups . The preventive tablets were distributed in blister packages of 8 tablets and the therapeutic tablets in blister packages containing 10 tablets per strip. The micronutrient powder and placebo powder sachets were produced by DSM Fortitech Asia Pacific . Standard quality control procedures were used by the manufactures to confirm the nutrient concentrations. The supplements were prelabeled (by the manufacturer) with 4 different numerical codes. In the field, the 4 numerical codes were assigned specific colors (one color per intervention group) to ensure correct delivery to children in the respective study groups. Thus, each color represented one type of preventive intervention (to be taken daily) and the corresponding therapeutic tablets for diarrhea management. In addition, caretakers for each child, irrespective of the study group, were given low-osmolarity ORS sachets for diarrhea management. Supplements and ORS were replenished during weekly home visits, as needed. Caregivers were instructed to begin diarrhea treatment with the therapeutic zinc (or placebo) tablets whenever a child had 3 or more liquid stools per day and to continue the treatment for 10 days, until the blister pack was empty. In addition, caregivers also were instructed to give ORS on any day that the child had 3 or more liquid stools per day.At enrollment, caregivers were instructed on how to administer the study products to their child, and field workers repeated these instructions every month during one of the weekly home visits. For both the preventive and therapeutic dispersible zinc and placebo tablets, caregivers were instructed to dissolve 1 tablet with clean water or breastmilk and feed the resulting suspension to the child at least 30 minutes before or after a meal. Caregivers were instructed to mix the entire contents of a micronutrient powder or placebo powder sachet into a small amount of semisolid food that the child could easily consume. Each child was visited weekly for 36 weeks , unless lost to follow-up. During the weekly visits, caregivers were interviewed regarding consumption of the intervention products. In addition, used and unused blister packages and sachets were collected to assess adherence.On the day of enrollment, caregivers attended an information session, first in groups and subsequently on individual basis, during which details of the trial, including the overall goals of the study, the study interventions, duration, voluntary participation, and inclusion and exclusion criteria, were explained. After the information session, caregivers who showed interest to participate in the study were asked to sign (or fingerprint) the consent statement in the presence of an independent witness. Children with written, informed parental consent were subsequently screened for eligibility.All data were recorded electronically via a customized CommCare-HQ  application deployed on portable Samsung tablets . At baseline, duplicate anthropometric assessments were recorded by trained anthropometric teams, using standardized procedures. Measurements included weight to the nearest 0.02 kg , recumbent length to the nearest 0.1 cm , and mid-upper arm circumference (MUAC) to the nearest 0.1 cm . In the event that the duplicate measurements differed by >0.1 kg for weight, or by >0.5 cm for recumbent length and MUAC, a third independent measurement was taken.Means were computed using the 2 measures with the lowest absolute differences. Children who were severely wasted (WLZ <–3) at baseline were excluded from participation and referred to the nearest health center or hospital. The anthropometric assessments were repeated after 18 weeks and at endline (32-40 weeks). At baseline, maternal weights were measured to 0.05-kg precision (SECA 874) and maternal heights to 0.1 cm precision (SECA 213). Anthropometric teams were systematically standardized.g (3100 rpm) for 10 minutes and the plasma was aliquoted into clear or amber microcentrifuge tubes (0.2-1.5 mL per tube), depending on the preplanned analyses. Plasma samples intended for nutritional biomarker assays were stored in the field laboratories at –20°C and later shipped on dry ice to a permanent laboratory at the University of California, Davis, California, where the samples were transferred to another –20°C freezer.Anemia status, based on capillary hemoglobin concentrations, was assessed for all children at baseline and endline using a Hemocue Hb 301 System . The Hemocue devices was checked weekly against a commercial quality control sample . For the purpose of evaluating biomarkers of nutritional status and health, ~7 mL samples of venous blood were collected by trained nurses from a subsample of 760 children, using evacuated, trace element-free, 7.5 mL of lithium–heparin tubes . The heparinized samples were maintained at 4-8°C in portable cooler boxes until transported to field laboratories for plasma processing within ~3 hours. In the field laboratory, the blood samples were centrifuged  at 1097g. Plasma samples were analyzed in duplicate in the same run. In the event that concentrations of the duplicates differed by 10%, the analyses were repeated. Each batch of samples was analyzed along with a reference sample . Iron status markers (ferritin and sTfR), RBP, and inflammatory markers (CRP and AGP) were measured using a sandwiched enzyme-linked immunosorbent assay technique at the VitMin Lab .Plasma zinc was analyzed by inductively coupled plasma optical emission spectrophotometry  at the Children's Hospital of Oakland Research Institute . Details of the plasma zinc assay by inductively coupled plasma optical emission spectrophotometry have been described previously.A statistical analysis plan was developed and published before analysesBoth absolute and standardized anthropometric measures were used in defining growth outcomes,P value < .05). In all cases of statistically significant pair-wise comparisons, multiple hypothesis testing adjustments were made to determine the sensitivity of estimates.All analyses were done on an intention-to-treat basis among children with available data.P < .05 for the regression coefficient). In all models, ferritin, zinc, sTfR, and RBP were log-transformed. Potential effect modification by baseline variables was explored by incorporating interaction terms in the statistical models and further investigated if marginally significant (P < .1). The full list of the specific covariates and effect modifiers is available in the published analysis plan.For anthropometric and anemia outcomes, ANCOVA or modified Poisson regression methods were used to model continuous and binary outcomes, respectively. For each outcome, treatment effect parameters were estimated using minimally adjusted models that controlled for baseline measurement of the respective outcome, age at enrollment, sex, and district as the only covariates. In the assessment of treatment effects on the nutritional biomarkers (both continuous and dichotomous), separate models were constructed using either measured concentrations, or the inflammation-adjusted concentration calculated by adapting procedures recommended by the Biomarkers Reflecting Inflammation and Nutritional Determinant of Anemia project.www.jpeds.com). This included 3407 individually randomized children and 26 children from a twin pair . Attrition during the 36 weeks of follow-up was 10% in the therapeutic zinc group, 13% in preventive zinc and control groups, and 17% in the micronutrient powder group (P = .01). The children lost to follow-up were statistically similar to those who completed the study with respect to baseline age, maternal variables, and anemia (www.jpeds.com). Similarly, baseline anthropometric indicators were comparable between those who completed the study vs those who dropped out, except for MUAC, which was slightly, but significantly, lower (P = .04) in the children who completed the study (13.8 ± 1.0 cm) compared with those lost to follow-up (13.9 ± 1.1 cm).Of the 3830 children screened, 3433 were eligible for enrollment . This amounted to a daily supplemental zinc intake of ~6.5 mg for the preventive zinc and ~9.0 mg for the micronutrient powder group over the duration of follow-up. Children received diarrhea treatment for an average of 4 days per 100 child days. Children in the therapeutic zinc group received the equivalent of 0.8 mg/d zinc over the course of the study (The average age at baseline was 14.3 ± 5.0 months . The prehe study .Table IIP = .37) or underweight . Wasting prevalence differed significantly by study group (P = .02) and was significantly lower in the preventive zinc group  compared with the therapeutic zinc group . There were no other significant pair-wise differences in wasting prevalence. A secondary analyses of anthropometry outcomes assessed at midline (~18 months) did not find any treatment effects (data not shown).At endline, the adjusted mean length (79.1 ± 4.9 cm), mean weight (9.6 ± 1.3 kg), and mean MUAC (14.0 ± 1.0 cm) did not differ by study group . LikewisP < .001), resulting in a significantly lower prevalence of zinc deficiency (P < .001) in preventive zinc (60%) and micronutrient powder (67%) groups compared with the therapeutic zinc (85%) and control (85%) groups. Ferritin concentration in the micronutrient powder group (26.0 mg/L [95% CI 23.5-28.5]) was greater (P < .001) than the other 3 groups  and micronutrient powder (62.2 µg/dL [95% CI 59.8-64.5]) groups were significantly greater than in the therapeutic zinc (56.8 µg/dL [95% CI 54.7-58.9]) and control (56.0 µg/dL [95% CI 53.9-58.1]) groups (P = .09) but not anemia prevalence (P = .14) (P = .06), such that among children who were anemic at baseline, the micronutrient powder was associated with a trend to a reduction in anemia of 6-9 percentage points  compared with the other 3 groups (www.jpeds.com). The prevalence of anemia among previously nonanemic children was 24%-28% (P = .62).There was an overall marginal effect of the study group on hemoglobin concentration (P = .14) . Specifi3 groups , whereas 24%-28%  and did P = .72) and minimum meal frequency , and consumption of animal source foods  and iron-rich foods  did not differ by group (www.jpeds.com).Complementary feeding practices did not differ over the course of the study. Based on data pooled from 9 monthly caregiver dietary recalls, the percentage of times children met the WHO recommended criteria for minimum dietary diversity  may have had constraining effects on growth. The dose and duration of preventive zinc tablet were consistent with the range of dosing regimens used in earlier studies that did find a growth response. Thus, the dosing regimen does not seem to explain the lack of observed growth impact. Some studies have shown that the absorption of zinc from micronutrient powder is lowIn settings with a high burden of infections, there is interest in finding the minimum effective iron dose necessary for reducing anemia, without increasing the risk for adverse health outcomes.In neighboring Savannakhet (south of the study area), an earlier study by Kounnavong et al reported that in children 6-52 months, daily or twice-weekly micronutrient powder containing 12.5 mg of iron was associated with a greater reduction in anemia (32-35 percentage points decrease) compared with children receiving no micronutrient powder (only 10 percentage points decrease) during 24 weeks of follow-up.Several strengths of this study include its implementation in a zinc-deficient and thus potentially responsive population, the individual randomization protocol, the frequency of follow-up visits, and the masking procedures used to ensure continued blinding throughout the field implementation and data analyses. In addition, the drop-out rate of ~13% was lower than expected, ensuring a sufficient final sample size for addressing the proposed questions. As indicated previously, the baseline characteristics of the children lost to follow-up were comparable with those who remained in the study. Moreover, among the children lost to follow-up, there were no differences in age, weight, length, and hemoglobin between the groups at baseline.A potential weakness of this trial is the nature of the comparison groups. The micronutrient powder and control preventive interventions were formulated in powder forms, whereas the preventive zinc and therapeutic zinc were formulated in tablet forms, potentially undermining the blinding procedure. However, because there were 2 powder groups, and 2 tablet groups, it was impossible to identify the exact intervention allocation for any particular group. A related problem is that micronutrient powder is designed to be taken along with food, whereas the preventive zinc is recommended to be consumed between meals. Thus, it is possible that the children's dietary patterns were affected differently, possibly confounding treatment effects. However, analyses of the dietary data found no difference in meal frequency, dietary diversity, consumption of iron-rich foods, and animal source foods  across tIn conclusion, our data suggest that preventive zinc supplements, provided alone or in combination with other micronutrients at doses of 7-10 mg zinc/d to young children in rural central Lao PDR, improved biomarkers of zinc status but had no impact on physical growth. Also, because the micronutrient powder tended to improve iron status and reduce anemia among initially anemic children, this may be the preferred strategy to deliver zinc and other micronutrients in this population. Additional research is needed to understand the optimal strategy for reducing the high burden of stunting and anemia in this population.www.jpeds.comAcknowledgments available at


Actinidia chinensis var. chinensis, which has an active-Y sex chromosome system, to study recombination rates around the sex locus, to better understand key events in the development of sex chromosomes.Angiosperm sex chromosomes, where present, are generally recently evolved. The key step in initiating the development of sex chromosomes from autosomes is the establishment of a sex-determining locus within a region of non-recombination. To better understand early sex chromosome evolution, it is important to determine the process by which recombination is suppressed around the sex determining genes. We have used the dioecious angiosperm kiwifruit A. chinensis var. chinensis, using a combination of high density genetic mapping and fluorescent in situ hybridisation (FISH) of Bacterial Artificial Chromosomes (BACs) linked to the sex markers onto pachytene chromosomes. The SDR is a subtelomeric non-recombining region adjacent to the nucleolar organiser region (NOR). A region of restricted recombination of around 6 Mbp in size in both male and female maps spans the SDR and covers around a third of chromosome 25.We have confirmed the sex-determining region (SDR) in As recombination is suppressed over a similar region between X chromosomes and between and X and Y chromosomes, we propose that recombination is suppressed in this region because of the proximity of the NOR and the centromere, with both the NOR and centromere suppressing recombination, and this predates suppressed recombination due to differences between X and Y chromosomes. Such regions of suppressed recombination in the genome provide an opportunity for the evolution of sex chromosomes, if a sex-determining locus develops there or translocates into this region.The online version of this article (10.1186/s12870-019-1766-2) contains supplementary material, which is available to authorized users. Sex chromosomes have evolved independently in a number of plants , 2. TheySilene latifolia are estimated to have evolved 5–10 Mya octane (DABCO) (Sigma), 50% (v/v) glycerol in 1X PBS pH 7.4). Following storage at 4 °C for 2–3 days before observation, chromosome spreads were observed with an Olympus Vanox AHT3 light microscope using epi-fluorescence, and images were captured with an RS Photometrics CoolSNAP digital camera. Two images were captured per cell, at excitation wavelengths of 358 nm and 550 nm. Entire images were then manipulated using Adobe® Photoshop Version 6.0. Chromosome measurements were made using the computer application MicroMeasure version 3.3 [The FISH procedure followed a previously published method , with sosion 3.3 .Additional file 1:Table S1. Primer sequences for microsatellite analysis. (XLSX 16 kb)


While associations between salivary characteristics and dental caries have been well studied, we are not aware of this being assessed in a remote Indigenous child population, where lifestyles may be different from urban children. Our aim was to assess associations between caries experience and putative biomarkers in saliva, accounting for oral hygiene and dietary habits.Children attending schools in an Indigenous community in remote north Queensland, Australia were invited to an oral examination by qualified and calibrated examiners. Salivary flow rate, pH, buffering capacity and loads of mutans streptococci (MS), lactobacilli (LB) and yeasts were determined. Also, data on tooth brushing frequency and soft drinks consumption were obtained via a questionnaire. Caries experience was recorded by the International Caries Detection and Assessment System (ICDAS-II), and quantified as decayed, missing and filled surfaces. Relationships between the salivary variables and the cumulative caries experience (dmfs+DMFS) in the deciduous and permanent dentitions were examined by multivariate analyses to control the effect of confounders.The mean cumulative decayed (DS + ds), missing (MS + ms) and filled (FS + fs) surfaces were 3.64 (SD: 4.97), 1.08 (4.38) and 0.79 (1.84) respectively. Higher salivary MS and LB counts, low tooth brushing frequency and daily soft drink consumption were significantly related to greater caries experience. Caries experience was about twice in those with ≥10^5 CFU/ml saliva counts of MS  and LB . In the fully-adjusted multivariate model, caries experience in those with higher counts of MS and LB were 51 and 52% more than those with lower counts.As with studies in other populations, childhood salivary counts of MS and LB were significantly associated with greater caries experience in this remote Indigenous community. To address the serious burden of oral disease, we are researching ways to promote a healthy oral environment by encouraging good dietary habits, and emphasising the importance of daily tooth brushing with a fluoridated toothpaste. Our ongoing longitudinal studies will indicate the success of measures employed to reduce the counts of bacteria closely associated with cariogenesis and their impact on caries increment.ANZCTR), No: ACTRN12615000693527; date of registration: 3rd July 2015.Australian New Zealand Clinical Trials Registry ( Worldwide amongst the fifty most common chronic diseases, four occur in the mouth: 1) dental caries of permanent teeth (ranked 1st), 2) chronic periodontitis ranked 6th), 3) dental caries of deciduous teeth (ranked 10th), and 4) edentulousness  . To assess saliva flow rate, the child was asked to chew on a piece of wax for 5 min, expectorating into a collection cup every 30 s or sooner if they felt more comfortable. The volume of saliva collected after 5 min was recorded in millilitres. To measure pH, the litmus strip from the GC SalivaCheck test kit was dipped into the cup and the pH recorded after 30 s. For buffering capacity, three drops of saliva were placed onto the three GC test pads using the pipette provided, left for 5 min, and the colour matched against the manufacturer’s chart, and the three recordings added. A high count of bacteria in dental biofilm correlates with salivary bacterial counts, enabling saliva as a source for quantifying cariogenic bacteria [Prior to the oral screening examination, a sample of stimulated saliva was collected over 5 min using commercially available kits for recording flow rate, pH and buffering capacity [GC Corporation, <bacteria , 23. Thehttp://www.ivoclarvivadent.com/en/p/all/products/prevention-care/caries-risk/crt-bacteria) [(acteria) . CRT agaAge, gender, tooth brushing frequency and daily soft drink consumption information were collected via a questionnaire. Specifically, children were asked the frequency of daily tooth brushing; categorised into ‘Once or less’ and ‘Twice or more’ for this analysis. They were asked if they consumed a soft drink on a typical day (Yes or No). The effect of these variables was controlled in the fully-adjusted multivariate analysis as they could serve as confounders.p-value for the Mann Whitney U test are reported. With the count data (dmfs+DMFS) widely dispersed (variance was greater than the mean), a Generalised Linear Model with negative binomial regression and log link was used to explore the association with demographic (age and gender), salivary physiology , salivary microbiology  and behavioural characteristics . In order to observe the effect of each explanatory variable separately, univariate analyses was conducted by entering one variable at a time. This was followed by a fully-adjusted multivariate model where all the variables were entered at once. Exponential estimates of the negative binomial regression analyses are presented as Prevalence Rate Ratios (PRR) with 95% confidence intervals (95% CI). For all tests conducted, a p-value of < 0.05 was considered statistically significant.SPSS 24.0  was used for conducting the statistical analyses. Descriptive statistics were performed to evaluate the mean decayed, missing and filled surfaces for deciduous and permanent dentitions together (caries experience = dmfs+DMFS). Caries experience (dmfs+DMFS) was the outcome variable in this study. As caries experience was not normally distributed, Mann Whitney U test was used to compare the caries experience in relation to gender, salivary physiology and microbiology, and a selection of behavioural characteristics. In this part of the analysis a number of continuous explanatory variables were dichotomised: salivary pH (<=6.6 and > 6.6); flow rate (<=5 ml/5 min and > 5 ml/5 min) and buffering capacity (<=9 and > 9) Table . The meaOf the 435 children who underwent the clinical examination and answered the questionnaires, 292 had a MS and LB colony count recording Fig. . A numbeThe mean age of the children was 8.79 (SD: 3.45), there were more females (55.4%) than males. A majority of children (84.4%) had salivary pH of > 6.6 Table . Many chThe mean cumulative decayed (DS + ds), missing (MS + ms) and filled (FS + fs) surfaces were 3.64 (SD: 4.97), 1.08 (4.38) and 0.79 (1.84) respectively. The overall caries experience (dmfs+DMFS) was 5.45 (SD: 7.89). More than three-quarters of children (76.5%) had caries in either of the dentitions (dmfs+DMFS> 0).Mann-Whitney U test revealed salivary MS and LB counts, tooth brushing frequency and soft drink consumption as significant variables Table . Caries In the fully-adjusted model, the expected counts of caries experience in those with salivary MS and LB counts of ≥10^5 CFU/ml saliva were 51%  and 52%  more than those with < 10^5 CFU/ml saliva Table . Caries High salivary loads of MS and LB were significantly associated with dental caries experience, even after adjusting for other salivary characteristics , tooth brushing frequency and soft drink consumption. Further, children who brushed their teeth infrequently and consumed soft drinks daily experienced greater caries. Such findings are well known from previous studies –17, but The relationship between high counts of mutans streptococci and of lactobacilli with high levels of dental caries has long been recognised, and is summarised in the National Institute of Health (NIH) Consensus Conference . SimilarReducing MS colonisation of the mouth at early age, and limiting their contribution to oral biofilms by limiting sugar intake and the practise of good oral hygiene, is important. Inhibiting growth periodically with broad spectrum antiseptics, may help to minimise subsequent dental caries, particularly in a high risk population such as this –37. MS cApproximately three quarters of the children reported brushing their teeth twice or more a day (75.7%). This is more than the national average of 68.5% in children aged 5 to 14 years and 53.5% in Indigenous children . It was A majority of children (77.3%) were consuming soft drinks on a typical day while the data from Australian National Child Oral Health Survey indicates that half of the Australian population consume a glass or more of sugar sweetened beverages in a day . HoweverBased on our findings, there is a need to undertake oral health promotion programs in this population, targeted towards behaviour change. School based oral health promotion programs, including supervised tooth brushing, might assist children in adopting positive oral health-related behaviours. A Cochrane review reported that oral health education in association with supervised tooth brushing was effective in reducing dental caries in children . HoweverIn Australia, 80% of public water supplies are fluoridated. However, this does not include many regional and rural areas , 56. TheLimitations: Conducting research in small and very remote settings comes with many challenges. Receiving consent is a complex process and communities may feel overwhelmed by the process. Even though we recruited a community member to assist with this process a significant number of parents whose children were invited to participate did not consent; and of those who consented a number could not provide a saliva specimen. A sample of almost 300 children, comprising half of all children, is a significant sample. However, because of the modest sample size, explanatory and confounding variables were dichotomised, and some had missing data, reducing the sample in the multivariate analysis. Oral health practices and soft drink consumption are self-reported, with possible over- or under-estimation a reality. The explanatory salivary characteristics variables were, however, measured objectively.Cross-sectional design does not allow causation and temporality to be assessed. Our longitudinal data will, however, permit more confident evaluation of these parameters. A significant strength is that this, to our knowledge, is the first study to report these relationships from a remote Indigenous community in Australia, and perhaps even globally. Thus, although the sample size is modest, the findings might be applicable to remote Indigenous communities worldwide.Children with higher loads of salivary MS and LB experienced a greater burden of dental caries. In addition, less frequent tooth brushing and daily consumption of soft drinks was associated with greater caries experience. These observations will inform our approach to disease prevention in this and similar communities.