Short-chain Amino Acids Research Articles

Side‐Chain Polarity of Amino Acids within the Kir7.1 Channel Pore Lining Determine Permeability and Function

Background Inwardly rectifying potassium (K+) channels (Kir) maintain membrane potential and K+ homeostasis across many tissues. Mutations in the KCNJ13 gene encoding for Kir7.1 protein, in the retinal pigmented epithelium (RPE), cause pediatric blindness. One point mutation in the KCNJ13 gene c.458C>T Threonine (T) to Isoleucine (I) at amino acid position 153 lines the inner pore of the tetrameric protein. We sought to elucidate the impact of pore region amino-acid hydrophobicity and side-chain length on the channel function. Hypothesis Amino acid side-chain polarity and length at position T153 are important determinants of Kir7.1 channel structure/function. Methods HEK293 cells in culture were transfected with GFP tagged wildtype (WT) Kir7.1, T153I, T153G, T153A, T153L, T153C, or T153S plasmid DNA. Live-cell imaging, using the Nikon-C2 confocal system, mapped the expression of GFP fused protein. Hoescht and WGA-Alexa 594 labeled the nucleus and plasma membrane, respectively. Off-line image analysis was performed with Nikon NIS Elements. Whole-cell patch-clamp electrophysiology with either extracellular Ringer's or Rb+ determined channel function. Extracellular K+-gradient was used to determine chord conductance. Data analyzed using the Clampfit program. Results Live-cell imaging indicated that Kir7.1 WT and all mutants studied are trafficked to the membrane. The IV plot for the Kir7.1 WT showed inward current measured at -150 mV with a mean amplitude of -863.67 ± 142.44 pA (n = 8) compared to Ile -68.54 ± 10.49 pA (n = 9, P = 2.79x10-5). Extracellular K+ dependent chord conductance further confirmed that T153I is nonfunctional. The current amplitude rank order for the mutant channel K+ permeability was Cys > WT > Ser > Gly > Ile > Ala> Leu. The membrane potential, measured as zero-current potential, was -57.75 ± 3.23 mV (n = 8) for the WT compared to-5.46 ± 6.01 mV (n = 9, P = 2.27x10-6) for the disease mutant with a rank order of Cys > WT > Ser > Leu > Gly > Ile > Ala. Rb+ ion, selectively permeates through Kir7.1 channel with a Rb+-current fold-change of7.28 ± 1.63 (n = 8) for WT versus 6.48 ± 2.78 (n = 9, P=0.81) for the disease mutant. The rank order of Rb+-current fold-change was Leu > Ser > WT > Ile > Cys > Gly > Ala. Conclusion Even though all the mutant channels traffick normally to the membrane, T153I was non-functional as determined by K+ ion permeability. Upon testing a more permeable ion, Rb+, it was deemed that T153I is actually dysfunctional. Based on the rank order, a substitution of polar amino acid at position 153 is required for Kir7.1 channel function. Permeability of all the mutant channels except T153A to Rb+ indicates that channel function is dependent on size of the cation. Since aa position T153 is within the inner pore-lining of the channel, a hydrophobic aa might alter protein-lipid interaction to affect channel function. Additionally, short-chain amino acids may further alter protein-protein interactions within the channel.

Genomic and Transcriptomic Investigation of the Physiological Response of the Methylotroph Bacillus methanolicus to 5-Aminovalerate.

The methylotrophic thermophile Bacillus methanolicus can utilize the non-food substrate methanol as its sole carbon and energy source. Metabolism of L-lysine, in particular its biosynthesis, has been studied to some detail, and methanol-based L-lysine production has been achieved. However, little is known about L-lysine degradation, which may proceed via 5-aminovalerate (5AVA), a non-proteinogenic ω-amino acid with applications in bioplastics. The physiological role of 5AVA and related compounds in the native methylotroph was unknown. Here, we showed that B. methanolicus exhibits low tolerance to 5AVA, but not to related short-chain (C4–C6) amino acids, diamines, and dicarboxylic acids. In order to gain insight into the physiological response of B. methanolicus to 5AVA, transcriptomic analyses by differential RNA-Seq in the presence and absence of 5AVA were performed. Besides genes of the general stress response, RNA levels of genes of histidine biosynthesis, and iron acquisition were increased in the presence of 5AVA, while an Rrf2 family transcriptional regulator gene showed reduced RNA levels. In order to test if mutations can overcome growth inhibition by 5AVA, adaptive laboratory evolution (ALE) was performed and two mutants—AVA6 and AVA10—with higher tolerance to 5AVA were selected. Genome sequencing revealed mutations in genes related to iron homeostasis, including the gene for an iron siderophore-binding protein. Overexpression of this mutant gene in the wild-type (WT) strain MGA3 improved 5AVA tolerance significantly at high Fe2+ supplementation. The combined ALE, omics, and genetics approach helped elucidate the physiological response of thermophilic B. methanolicus to 5AVA and will guide future strain development for 5AVA production from methanol.

The influence of amino acids structure on their anaerobic digestion and the strategy to enhance biotransformation of refractory ones

Protein is an important component of organic waste, and it is first hydrolyzed to amino acids (AAs) during anaerobic digestion (AD). Until now, however, the effect of AAs structure on their AD has never been documented. In this paper the influence of AAs structure on their two-phase AD and the strategy to enhance biotransformation of refractory ones were investigated. Firstly, the AD performance of nineteen water-soluble AAs was compared. The methane yield of polar AAs was higher than that of non-polar ones (162.3–308.1 verse 10.7–140.5 mL/gCODadd) except glycine (284.1 mL/gCODadd). Further studies showed that the order of methane production with polar AAs, according to their carbon number, was 4-C < 6-C < 3-C < 5-C, while that with non-polar AAs was long-chain AAs (5 to 11-C) < short-chain AAs (2 to 3-C). The methane yield of non-polar ones with long-chain, according to their functional groups, was followed: proline > Alkyl AAs > Ph AAs. The mechanism investigation revealed the relationship between the structure of AAs and the amount of VFAs excluding propionate was consistent with the influence of AAs structure on methane yield. The microbes in AD system of refractory AAs had higher cell surface hydrophobicity (CSH) and lower cell membrane permeability (CMP), which led to lower enzyme activities and amino acid degradation as well as VFAs generation, and thus less methane production. Finally, the strategy to increase the biotransformation of refractory AAs was studied, and the methane yield was increased up to 139.8% by the application of rhamnolipid to change CSH and CMP in the digestion system. These findings elucidated the relationship between AAs structure and AD performance and provided new ideas to improve the biotransformation of refractory AAs.

Metabolomic Profiling in Neuromyelitis Optica Spectrum Disorder Biomarker Discovery.

There is no specific test for diagnosing neuromyelitis optica spectrum disorder (NMOSD), a disabling autoimmune disease of the central nervous system. Instead, diagnosis relies on ruling out other related disorders with overlapping clinical symptoms. An urgency for NMOSD biomarker discovery is underscored by adverse responses to treatment following misdiagnosis and poor prognosis following the delayed onset of treatment. Pathogenic autoantibiotics that target the water channel aquaporin-4 (AQP4) and myelin oligodendrocyte glycoprotein (MOG) contribute to NMOSD pathology. The importance of early diagnosis between AQP4-Ab+ NMOSD, MOG-Ab+ NMOSD, AQP4-Ab− MOG-Ab− NMOSD, and related disorders cannot be overemphasized. Here, we provide a comprehensive data collection and analysis of the currently known metabolomic perturbations and related proteomic outcomes of NMOSD. We highlight short chain fatty acids, lipoproteins, amino acids, and lactate as candidate diagnostic biomarkers. Although the application of metabolomic profiling to individual NMOSD patient care shows promise, more research is needed.

Anhedonia induced by high-fat diet in mice depends on gut microbiota and leptin

ABSTRACT Objectives: Imbalanced nutrition and obesity are risk factors for depression, a relationship that in rodents can be modeled by depression-like behavior in response to high-fat diet (HFD). In this work, we examined the role of the intestinal microbiota and the adipocytokine leptin as potential mediators of the effects of HFD to induce anhedonia-like behavior and reduce self-care in mice. Methods: Male mice were fed a control diet or HFD (60 kJ% from fat) for a period of 4 weeks, after which behavioral tests and molecular analyses (gut microbiome composition, intestinal metabolome, fecal fatty acids, plasma hormone levels) were performed. The role of the intestinal microbiota was addressed by selective depletion of gut bacteria with a combination of non-absorbable antibiotics, while the implication of leptin was examined by the use of leptin-deficient ob/ob mice. Results: Antibiotic treatment reduced the HFD-induced weight gain and adiposity and prevented HFD-induced anhedonia-like behavior and self-care reduction. These effects were associated with a decrease in fecal fatty acids and intestinal microbiota-related metabolites including short-chain fatty acids, glucose and amino acids. Gut microbiota depletion suppressed the HFD-induced rise of plasma leptin, and the circulating leptin levels correlated with the anhedonia-like behavior and reduced self-care caused by HFD. The anhedonic effect of HFD was absent in leptin-deficient ob/ob mice although these animals gained more weight and adiposity in response to HFD than wild-type mice. Discussion: The results indicate that anhedonia-like behavior induced by HFD in mice depends on the intestinal microbiome and involves leptin as a signaling hormone.

Optical Characterization and Monitoring of Enzyme Catalyzed Short Chain Peptides in Cellular Environment

Enzyme catalysis is an emerging technique that has been employed to identify the protein of interest in a complex cellular environment. In present article, we have utilized optical techniques for the identification of enzyme mediated catalysis of fusion protein to study the short chain amino acid epitope in both aqueous solution and cellular environment. We characterized enzyme catalysis short chain peptide with electron microscopic techniques, for the study of reaction product and its morphology in aqueous solution. Furthermore, we applied MALDI-TOF-MS technique for the analysis of fusion protein, FLAG-tag peptides and enterokinase enzyme (EK), in complex solutions and cellular environment. The enzyme catalytic reactions were studied HEK-293T cells with confocal fluorescence microscopy. In spectroscopic studies two sharp prominent peaks at 310 nm and 330 nm were appeared in case of Flag-tag peptide and for fusion protein absorption peaks were found at 315 nm and 410 nm with relative increase in intensity level. Furthermore, a linear relation between response unit and binding time (ms) for both fusion protein and EK was observed. Mass spectra reveal the presence of FLAG-tag peptide epitope at mass to charge (m/z) ratio of 2190.023 and 2191.102 in cell lysation with and without enterokinase respectively. We provide here a rapid and accurate trace detection system for enzyme cleaved peptides in fusion protein by taking a snapshot of peptide to identify specific domains based on amino acids and their mass spectrum calculations.

Dysbiosis of gut microbiota in a selected population of Parkinson's patients

IntroductionIn recent years the hypothesis that gut microbiota associates with Parkinson's disease (PD) has gained importance, although it has not been possible to define a specific microbiota composition as a predictive biomarker of this disease. We have investigated dysbiosis of gut microbiota in a selected population of PD patients from Central Italy, and examined the weight of specific confounders and predictors, in order to identify potential correlations with clinical phenotypes. Methods152 fecal samples were collected from 80 patients and 72 healthy controls. Patients were enrolled according to tight inclusion criteria. Microbiota composition was studied through 16s ribosomal RNA gene amplicon sequencing analysis in combination with data on dietary/life habits. Age, loss of weight, and sex were recognized as confounding factors, whereas PD-status, age, Body Mass Index, “eat cereals”, “gain of weigth” and “physical activity” as predictors. The presence of Lactobacillaceae, Enterobacteriaceae and Enterococcaceae families was significantly higher in feces from PD patients compared to healthy controls, while Lachnospiraceae were significantly reduced. Lower levels of Lachnospiraceae and higher levels of Enterobacteriaceae families also correlated with increased disease severity and motor impairment (Hoehn & Yahr stage, MDS-UPDRS Part III). Predictive metagenomics indicated a significant variation of genes involved in the metabolism of short chain fatty acids and amino acids, and in lipopolysaccharide biosynthesis. ConclusionsPD showed a distinctive microbiota composition. Functional predictions suggest changes in pathways favoring a pro-inflammatory environment in the gastrointestinal tract, and a reduction in the biosynthesis of amino acids acting as precursors of physiological transmitters.

OPTIMIZATION OF PARAMETERS OF FERMENTOLYSIS OF PROTEINS IN THE COMPOSITION OF SERUM-PROTEIN CONCENTRATE

The food industry is a strategic industry that works quite steadily even during periods of economic crises, providing food security to any state, and is a source of raw material for other industries with a high potential for development, for example, for the production of cosmetics. The modern cosmetics market is represented by various cosmetic products, often expensive, but not always made from natural ingredients. Therefore, the search for the newest ingredients for the production of natural cosmetics on the basis of domestic raw materials is an urgent task of the present. Ingredients from milk serum can be used for the production of natural cosmetics that in large quantities is obtained in milk processing enterprises and often remains unprocessed. Whey protein concentrations can be a source of short-chain peptides and free amino acids for the production of various cosmetic products. The process of fermentolysis of serum proteins in nanofiltration concentrate KSB-65 with the content of dry matter of 20% using neutral peptidase C from the domestic producer at a temperature of 40 ºС with the duration of the process varying from 1 to 5 hours, the content of peptidase – from 0,5 to 2,0 U/g. It is established that the optimal parameters of the fermentolysis of serum proteins in KSB-65 are as follows: temperature 40 º C, neutral peptidase C content – 0.78 U/g, duration of fermentolysis – 3.17 hours. With optimal parameters of the fermentolysis process, the hydrolyzate of the nanofiltration concentrate KSB-65 contains the maximum amount of short chain peptides (57.03 mg/cm3) and a high concentration of free amino acids (54.66 μg/cm3). Recommendations for the further use of serum protein hydrolyzate obtained using the recommended optimal parameters of the enzyme production process from the nanofiltration concentrate KSB-65, in the manufacture of cosmetic products, including with anti-age effect, and hydrolyzates of proteins enriched with probiotic cultures of lactam bifidobacteria or their lysates.

Zengye decoction induces alterations to metabolically active gut microbiota in aged constipated rats

Zengye decoction (ZYD), a traditional Chinese medicinal formula, has been used in the treatment of various chronic diseases, such as constipation and skin dryness syndrome. Clinically, the specific mechanisms and targets of ZYD for treating disease remain unclear. The present study was undertaken to investigate the effects of ZYD on the gut microbiota and host metabolites in aged constipated rats and the relationship between the intestinal microbiota and the host. Rats were divided randomly into three groups, the control group (n = 10), recovery group (n = 10) and ZYD group (n = 10). First, the aged constipation model was established for the ZYD group and recovery group. Then, rats in the ZYD group were treated with ZYD. Urinary and faecal samples of each animal were collected in microcentrifuge tubes. Next, 16s rRNA gene sequencing was employed to analyse the composition of the gut microbiome in faecal samples and afterwards the metabolic function of the altered gut microbiota was predicted. Additionally, 1H NMR profiling was used to detect the alterations of host metabolites in urine and faecal samples to verify the metabolic function results obtained from sequencing. As a result, ZYD reduced the level of harmful bacteria, such as Desulfovibrio, Ruminococcus, Prevotella and Dorea, and increased the abundance of Oxalobacter, Clostridium and Roseburia. The functional prediction of changes in the gut microbiota induced by ZYD revealed that ZYD promoted energy storage, regulated amino acid metabolism, inhibited methane metabolism, strengthened the physiological function of glutathione and reduced bacterial toxin. The 1H NMR profiles revealed that ZYD regulated the carbohydrates, short chain fatty acids, amino acids and amines in the aged constipated rats. In addition, most metabolic changes observed were related to the function of intestinal microbiota. These results suggest that ZYD can regulate the intestinal microbiota of constipated rats to normal levels and change the endogenous metabolites of the host through the intestinal microbiota to achieve therapeutic effects.

Understanding the Representative Gut Microbiota Dysbiosis in Metformin-Treated Type 2 Diabetes Patients Using Genome-Scale Metabolic Modeling.

Dysbiosis in the gut microbiome composition may be promoted by therapeutic drugs such as metformin, the world’s most prescribed antidiabetic drug. Under metformin treatment, disturbances of the intestinal microbes lead to increased abundance of Escherichia spp., Akkermansia muciniphila, Subdoligranulum variabile and decreased abundance of Intestinibacter bartlettii. This alteration may potentially lead to adverse effects on the host metabolism, with the depletion of butyrate producer genus. However, an increased production of butyrate and propionate was verified in metformin-treated Type 2 diabetes (T2D) patients. The mechanisms underlying these nutritional alterations and their relation with gut microbiota dysbiosis remain unclear. Here, we used Genome-scale Metabolic Models of the representative gut bacteria Escherichia spp., I. bartlettii, A. muciniphila, and S. variabile to elucidate their bacterial metabolism and its effect on intestinal nutrient pool, including macronutrients (e.g., amino acids and short chain fatty acids), minerals and chemical elements (e.g., iron and oxygen). We applied flux balance analysis (FBA) coupled with synthetic lethality analysis interactions to identify combinations of reactions and extracellular nutrients whose absence prevents growth. Our analyses suggest that Escherichia sp. is the bacteria least vulnerable to nutrient availability. We have also examined bacterial contribution to extracellular nutrients including short chain fatty acids, amino acids, and gasses. For instance, Escherichia sp. and S. variabile may contribute to the production of important short chain fatty acids (e.g., acetate and butyrate, respectively) involved in the host physiology under aerobic and anaerobic conditions. We have also identified pathway susceptibility to nutrient availability and reaction changes among the four bacteria using both FBA and flux variability analysis. For instance, lipopolysaccharide synthesis, nucleotide sugar metabolism, and amino acid metabolism are pathways susceptible to changes in Escherichia sp. and A. muciniphila. Our observations highlight important commensal and competing behavior, and their association with cellular metabolism for prevalent gut microbes. The results of our analysis have potential important implications for development of new therapeutic approaches in T2D patients through the development of prebiotics, probiotics, or postbiotics.

Lower plasma trans-4-hydroxyproline and methionine sulfoxide levels are associated with insulin dysregulation in horses

BackgroundInsulin dysregulation in horses is a metabolic condition defined by high insulin concentrations in the blood and peripheral insulin resistance. This hyperinsulinemia is often associated with severe damage in the hooves, resulting in laminitis. However, we currently lack detailed information regarding the potential involvement of particular metabolic pathways in pathophysiological causes and consequences of equine insulin dysregulation. This study aimed to assess the dynamic metabolic responses given to an oral glucose test (OGT) in insulin-sensitive and insulin-dysregulated horses by a targeted metabolomics approach to identify novel metabolites associated with insulin dysregulation.ResultsOral glucose testing triggered alterations in serum insulin (26.28 ± 4.20 vs. 422.84 ± 88.86 μIU/mL, p < 0.001) and plasma glucose concentrations (5.00 ± 0.08 vs. 9.43 ± 0.44 mmol/L, p < 0.001) comparing basal and stimulated conditions after 180 min. Metabolome analyses indicated OGT-induced changes in short-chain acylcarnitines (6.00 ± 0.53 vs. 3.99 ± 0.23 μmol/L, p < 0.001), long-chain acylcarnitines (0.13 ± 0.004 vs. 0.11 ± 0.002 μmol/L, p < 0.001) and amino acids (2.18 ± 0.11 vs. 1.87 ± 0.08 μmol/L, p < 0.05). Kynurenine concentrations increased (2.88 ± 0.18 vs. 3.50 ± 0.19 μmol/L, p < 0.01), whereas spermidine concentrations decreased during OGT (0.09 ± 0.004 vs. 0.08 ± 0.002 μmol/L, p < 0.01), indicating proinflammatory conditions after oral glucose load. Insulin dysregulation was associated with lower concentrations of trans-4-hydroxyproline (4.41 ± 0.29 vs. 6.37 ± 0.71 μmol/L, p < 0.05) and methionine sulfoxide (0.40 ± 0.06 vs. 0.87 ± 0.13 μmol/L, p < 0.01; mean ± SEM in insulin-dysregulated vs. insulin-sensitive basal samples, respectively), two metabolites which are related to antioxidant defense mechanisms.ConclusionOral glucose application during OGT resulted in profound metabolic and proinflammatory changes in horses. Furthermore, insulin dysregulation was predicted in basal samples (without OGT) by pathways associated with trans-4-hydroxyproline and methionine sulfoxide, suggesting that oxidative stress and oxidant–antioxidant disequilibrium are contributing factors to insulin dysregulation. The present findings provide new hypotheses for future research to better understand the underlying pathophysiology of insulin dysregulation in horses.

SCREENING POTENTIAL ANTIOXIDANT AND ANTIBACTERIAL ACTIVITIES OF PROTEIN HYDROLYSATES DERIVED FROM GERMINATED LABLAB BEAN, PIGEON PEA AND KIDNEY BEAN

Abstract Protein hydrolysate contains a mixture of various lengths of short peptides chain and free amino acids that may&#x0D; excert biological activities. This research aims to screen potential antioxidant and antibacterial activities of protein hydrolysate&#x0D; produced from three kinds of germinated beans i.e. lablab bean (Lablab purpureus), pigeon pea (Cajanus cajan (L.) Millsp) and&#x0D; kidney bean (Phaseolus vulgaris) through enzymatic hydrolysis process. The steps of research included germination process of&#x0D; the beans prior to total protein isolation, enzymatic hydrolysis of total protein isolates using pancreatin enzyme, evaluation of&#x0D; in vitro antioxidant activity of the hydrolysates protein using DPPH (1,1-diphenyl-2-picryl hydrazyl) method, and antibaterial&#x0D; activity testing towards Eschericia coli and Staphyllococcus aureus bacteria. The results revealed that pancreatine enzyme was&#x0D; able to hydrolyse germinated protein of lablab bean, pigeon pea and kidney bean at the experiment condition applied with&#x0D; degree of hydrolysis 34.12%, 27.44%, and 30,93% respectively. It was also found that protein hydrolysates of lablab bean,&#x0D; pigeon pea, and kidney bean demonstrated antioxidant activity which percentage radical DPPH scavenging activity of 84.02%,&#x0D; 68.97% and 67.89 %. On the other hand, all of those protein hydrolysates did not show any antibacterial activity towards&#x0D; Eschericia coli and Staphyllococcus aureus bacteria.

Influence of amino acids on gastric adaptive relaxation (accommodation) in rats as evaluated with a barostat.

Aim: The present study aimed to evaluate the effects of selected straight alkyl chain, hydroxylated chain and branched chain amino acids on gastric adaptive relaxation, as these have previously been shown to have differing effects on gastric emptying. Materials and Methods: Gastric adaptive relaxation was evaluated using a barostat in rats under urethane anesthesia. The pressure within the balloon, introduced from the mouth to the stomach, was changed stepwise from 1 to 8 mmHg. The increased volume just after the increase of balloon pressure was defined as distension-induced gastric adaptive relaxation (accommodation). Amino acids were administered orally or intravenously. Results: As compared with control rats administered with distilled water, those rats that were orally administered amino acids having straight alkyl chain and extra hydroxylated alkyl chain, such as glycine and l-serine, had significantly enhanced gastric adaptive relaxation, but administration of l-alanine and l-threonine did not. Branched chain amino acids, such as l-isoleucine, l-leucine and l-valine, also did not significantly influence gastric adaptive relaxation. Glycine and l-serine showed the same efficacy when administered intravenously. Conclusion: Among the amino acids evaluated in the present study, glycine and l-serine significantly enhanced gastric adaptive relaxation, suggesting that short alkyl chain amino acids may enhance gastric adaptive relaxation as compared with the other amino acids. These findings may suggest that glycine and l-serine would be useful in the therapy of functional dyspepsia, especially for early satiety, because the dysfunction of adaptive relaxation is one of the causes of early satiety.

Vaginal microbiome and metabolome highlight specific signatures of bacterial vaginosis.

In this study, we sought to find novel bacterial and metabolic hallmarks for bacterial vaginosis (BV). We studied the vaginal microbiome and metabolome of vaginal fluids from BV-affected patients (n = 43) and healthy controls (n = 37) by means of an integrated approach based on quantitative polymerase chain reaction (qPCR) and proton nuclear magnetic resonance ((1)H-NMR). The correlations between the clinical condition and vaginal bacterial communities were investigated by principal component analysis (PCA). To define the metabolomics signatures of BV, 100 discriminant analysis by projection on latent structure (PLS-DA) models were calculated. Bacterial signatures distinguishing the health condition and BV were identified by qPCR. Lactobacillus crispatus strongly featured the healthy vagina, while increased concentrations of Prevotella, Atopobium and Mycoplasma hominis specifically marked the infection. (1)H-NMR analysis has led to the identification and quantification of 17 previously unreported molecules. BV was associated with changes in the concentration of metabolites belonging to the families of amines, organic acids, short chain fatty acids, amino acids, nitrogenous bases and monosaccharides. In particular, maltose, kynurenine and NAD(+) primarily characterised the healthy status, while nicotinate, malonate and acetate were the best metabolic hallmarks of BV. This study helps to better understand the role of the vaginal microbiota and metabolome in the development of BV infection. We propose a molecular approach for the diagnosis of BV based on quantitative detection in the vaginal fluids of Atopobium, Prevotella and M. hominis, and nicotinate, malonate and acetate by combining qPCR and (1)H-NMR.

Antimicrobial activity of silk treated with Aloe-Vera

Natural Silk in its native form is made up of the filament fibroin, coated with sericin - gummy matter which is antimicrobial in nature. However, sericin is removed during the pretreatment process. Silk being a natural and hygroscopic, fiber gets attacked by microbes easily. Hence antimicrobial treatment can add value to the wear and care of silk textiles. Synthetic antimicrobial agents may be toxic and even carcinogenic and hence the natural and eco-friendly antimicrobial agents are good substitutes for imparting the desired properties. In the present paper, ready for dyeing (RDF) silk fabric was treated with Aloe-Vera using 1,2,3,4-butanetetracarboxylic acid (BTCA) as crosslinking agent and sodium hypophosphite (SHP) as catalyst. The treated fabric with a concentration of 15 % of Aloe-Vera showed excellent antimicrobial properties. Since BTCA was used as a crosslinking agent, crease recovery angle, improved with minimal loss in breaking and tearing strength. The mechanism of treatment of Aloe-Vera is found to be chemical binding with silk and not simply of coating or impregnation. FTIR studies showed that the carboxyl side groups and short chain amino acids side groups act as sites for BTCA crosslinking interalia chemical binding of Aloe-Vera. SEM studies revealed that no coating or tangible impregnation on the surface of the fiber is visible substantiating the chemical binding phenomenon. This is further substantiated by the durability of the finish to dry cleaning of treated silk. Since Aloe-Vera is a natural product and BTCA is an eco-friendly resin, the treatment of silk with Aloe-Vera is eco-friendly in nature.

Gut Microbiota Composition Modifies Fecal Metabolic Profiles in Mice

The gut microbiome is known to be extensively involved in human health and disease. In order to reveal the metabolic relationship between host and microbiome, we monitored recovery of the gut microbiota composition and fecal profiles of mice after gentamicin and/or ceftriaxone treatments. This was performed by employing (1)H nuclear magnetic resonance (NMR)-based metabonomics and denaturing gradient gel electrophoresis (DGGE) fingerprint of gut microbiota. The common features of fecal metabolites postantibiotic treatment include decreased levels of short chain fatty acids (SCFAs), amino acids and primary bile acids and increased oligosaccharides, d-pinitol, choline and secondary bile acids (deoxycholic acid). This suggests suppressed bacterial fermentation, protein degradation and enhanced gut microbial modification of bile acids. Barnesiella, Prevotella, and Alistipes levels were shown to decrease as a result of the antibiotic treatment, whereas levels of Bacteroides, Enterococcus and Erysipelotrichaceae incertae sedis, and Mycoplasma increased after gentamicin and ceftriaxone treatment. In addition, there was a strong correlation between fecal profiles and levels of Bacteroides, Barnesiella, Alistipes and Prevotella. The integration of metabonomics and gut microbiota profiling provides important information on the changes of gut microbiota and their impact on fecal profiles during the recovery after antibiotic treatment. The correlation between gut microbiota and fecal metabolites provides important information on the function of bacteria, which in turn could be important in optimizing therapeutic strategies, and developing potential microbiota-based disease preventions and therapeutic interventions.

Metabolomics of Fecal Extracts Detects Altered Metabolic Activity of Gut Microbiota in Ulcerative Colitis and Irritable Bowel Syndrome

(1)H NMR spectroscopy of aqueous fecal extracts has been used to investigate differences in metabolic activity of gut microbiota in patients with ulcerative colitis (UC) (n = 13), irritable bowel syndrome (IBS) (n = 10), and healthy controls (C) (n = 22). Up to four samples per individual were collected over 2 years giving a total of 124 samples. Multivariate discriminant analysis, based on NMR data from all three groups, was able to predict UC and C group membership with good sensitivity and specificity; classification of IBS samples was less successful and could not be used for diagnosis. Trends were detected toward increased taurine and cadaverine levels in UC with increased bile acid and decreased branched chain fatty acids in IBS relative to controls; changes in short chain fatty acids and amino acids were not significant. Previous PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of the same fecal material had shown alterations of the gut microbiota when comparing UC and IBS groups with controls. Hierarchical cluster analysis showed that DGGE profiles from the same individual were stable over time, but NMR spectra were more variable; canonical correlation analysis of NMR and DGGE data partly separated the three groups and revealed a correlation between the gut microbiota profile and metabolite composition.

Promoter characterization and role of CRE in the basal transcription of the rat SNAT2 gene

Small neutral amino acid transporter 2 (SNAT2) is the most abundant and ubiquitous transporter for zwitterionic short-chain amino acids. The activity of this amino acid transporter is stimulated in vivo or in vitro by glucagon or cAMP analogs. However, it is not known whether the increase in activity at the protein level is due to an increase in SNAT2 gene transcription. Thus, the aim of the present work was to study whether cAMP was able to stimulate SNAT2 gene expression and to localize and characterize the presence of cAMP response elements (CRE) in the promoter that controls the expression of the rat SNAT2 gene. We found that consumption of a high-protein diet that increased serum glucagon concentration or the administration of glucagon or incubation of hepatocytes with forskolin increased the SNAT2 mRNA level. We then isolated the 5' regulatory region of the SNAT2 gene and determined that the transcriptional start site was located 970 bp upstream of the translation start codon. We identified two potential CRE sites located at -354 and -48 bp. Our results, using deletion analysis of the 5' regulatory region of the SNAT2 gene, revealed that the CRE site located at -48 bp was fully responsible for SNAT2 regulation by cAMP. This evidence was strongly supported by mutation of the CRE site and EMSA and ChIP analysis. Alignment of rat, mouse, and human sequences revealed that this CRE site is highly conserved among species, indicating its essential role in the regulation of SNAT2 gene expression.

Grafting short-chain amino acids onto membrane surfaces to resist protein fouling

The suppression of nonspecific protein adsorption to synthetic surfaces, especially membrane surfaces, represents a severe challenge in biotechnological, biomedical and biosensor community. In this work, we utilize short-chain molecules rather than long-chain polymers as modifiers for antibiofouling membrane surface construction. Three types of natural amino acids, including lysine, glycine, and serine, were chemically grafted onto membranes via carbodiimide chemistry to render highly hydrophilic surfaces, which displayed the intrinsic advantages in high surface coverage, three-dimensional modification, and unsacrificed permeability. Although hydrophilicity of membranes modified with these three types of amino acids was quite close, only lysine modified membrane surface showed superior protein fouling resistance under both static exposure and dynamic filtration conditions. Molecular dynamics simulation study was performed to tentatively interpret the underlying differences.

Insights from the structure of a smallpox virus topoisomerase-DNA transition state mimic.

Poxviruses encode their own type IB topoisomerases (TopIBs), which release superhelical tension generated by replication and transcription of their genomes. To investigate the reaction catalyzed by viral TopIBs, we have determined the structure of a variola virus topoisomerase-DNA complex trapped as a vanadate transition state mimic. The structure reveals how the viral TopIB enzymes are likely to position the DNA duplex for ligation following relaxation of supercoils and identifies the sources of friction observed in single-molecule experiments that argue against free rotation. The structure also identifies a conformational change in the leaving group sugar that must occur prior to cleavage and reveals a mechanism for promoting ligation following relaxation of supercoils that involves an Asp-minor groove interaction. Overall, the new structural data support a common catalytic mechanism for the TopIB superfamily but indicate distinct methods for controlling duplex rotation in the small versus large enzyme subfamilies.