Hypertension alters the function and expression profile of the peptide cotransporters PEPT1 and PEPT2 in the rodent renal proximal tubule.

Hypertension is a major risk factor for kidney and cardiovascular disease. The treatment of hypertensive individuals by selected ACE inhibitors and certain di-and tripeptides halts the progression of renal deterioration and extends life-span. Renal reabsorption of these low molecular weight substrates are mediated by the PEPT1 and PEPT2 cotransporters. This study aims to investigate whether hypertension and ageing affects renal PEPT cotransporters at gene, protein expression and distribution as well as function in the superficial cortex and the outer medulla of the kidney. Membrane vesicles from the brush border (BBMV) and outer medulla (OMMV) were isolated from the kidneys of young Wistar Kyoto (Y-WKY), young spontaneously hypertensive (Y-SHR), and middle aged SHR (M-SHR) rats. Transport activity was measured using the substrate, ß-Ala-Lys (AMCA). Gene expression levels of PEPT genes were assessed with qRT-PCR while renal localisation of PEPT cotransporters was examined by immunohistochemistry with Western Blot validation. The Km and Vmax of renal PEPT1 were decreased significantly in SHR compared to WKY BBMV, whilst the Vmax of PEPT2 showed differences between SHR and WKY. By contrast to the reported cortical distribution of PEPT1, PEPT1-staining was detected in the outer medulla, whilst PEPT2 was expressed primarily in the cortex of all SHR; PEPT1 was significantly upregulated in the cortex of Y-SHR. These outcomes are indicative of a redistribution of PEPT1 and PEPT2 in the kidney proximal tubule under hypertensive conditions that has potential repercussions for nutrient handling and the therapeutic use of ACE inhibitors in hypertensive individuals.

Molecular changes to the rat renal cotransporters PEPT1 and PEPT2 due to ageing.

Renal PEPT1 and PEPT2 cotransporters play an important role in the balance of circulating body oligopeptides and selected peptidomimetic drugs. We aim to comprehensively characterise age-related changes of the renal PEPT cotransporters at the gene, protein, and functional level. Brush border membrane vesicles (BBMV) and outer medulla membrane vesicles (OMMV) were isolated from the kidneys of young, middle-aged and old rats. The protein expression of PEPT1 was not only increased in BBMV from old rats, but PEPT1 also appeared in OMMV from middle-aged and old rats. SLC15A1 gene expression in the renal cortex increased in middle-aged group. PEPT2 protein expression was not only increased with ageing, but PEPT2 also was found in BBMV from middle-aged and old groups. SLC15A2 gene expression in the renal outer medulla increased in the old group. These changes in the expressions and localisations of PEPT1 and PEPT2 could explain the changes to transport activity in BBMV and OMMV. These findings provide novel insights that would be useful for maintaining protein nutrition and optimising the delivery of some peptidomimetic drugs in elderly individuals.

A new use of ß-Ala-Lys (AMCA) as a transport reporter for PEPT1 and PEPT2 in renal brush border membrane vesicles from the outer cortex and outer medulla.

Integral membrane proteins PEPT1 and PEPT2 are essential for reabsorbing almost all hydrolysed or filtered di- and tripeptides alongside a wide range of peptidomimetic drugs in the kidney. The aim of this study was to investigate the potential use of the fluorophore-conjugated dipeptide ß-Ala-Lys (AMCA) as a biosensor for measuring peptide transport activity in brush border membrane vesicles isolated from the outer cortex (BBMV-OC) and outer medulla (BBMV-OM) (representing PEPT1 and PEPT2 respectively). The vesicles were isolated using a dual magnesium precipitation and centrifugation technique. Intravesicular fluorescence accumulation was measured after incubating extra-vesicular media at pH6.6 and different concentrations of ß-Ala-Lys (AMCA) with vesicles pre-equilibrated at pH7.4. Both BBMV-OC and BMMV-OM showed accumulation of an intravesicular fluorescence signal after 20min incubation. Changing the extra-vesicular pH to 7.4 caused a significant reduction in the ß-Ala-Lys (AMCA) uptake into BBMV-OC at concentrations >100µM. When different concentrations of dipeptide, Gly-Gln was added, there was a significant inhibition of 100µM ß-Ala-Lys (AMCA) uptake into BBMV-OC and BMMV-OM, reaching 69% and 80%, respectively. Kinetic analysis of ß-Ala-Lys (AMCA) at 20min showed that the Km and Vmax were 783.7±115.7µM and 2191.2±133.9ΔF/min/mg for BBMV-OC, while BMMV-OM showed significantly higher affinity, but lower capacity at Km=93.6±21.9µM and Vmax=935.8±50.2ΔF/min/mg. These findings demonstrate the applicability of ß-Ala-Lys (AMCA) as a biosensor to measure the transport activity of the renal-type PEPT1 and PEPT2 in BBMV-OC and BMMV-OM respectively.

Kinetic Measurements of Di- and Tripeptide and Peptidomimetic Drug Transport in Different Kidney Regions Using the Fluorescent Membrane Potential-Sensitive Dye, DiS-C3-(3).

Tri- and dipeptides are transported in the kidney by PEPT1 and PEPT2 isoforms. The aim of this study was to investigate differences in transport kinetics between renal brush border (BBMV) and outer medulla (OMMV) membrane vesicles (where PEPT1 and PEPT2 are sequentially available) for a range of di- and tripeptides and peptidomimetic drugs. This was accomplished through the use of the potential-sensitive fluorescent dye 3,3'-dipropylthiacarbocyanine iodide [DiS-C3-(3)]. BBMV and OMMV were prepared from the rat kidney using standard techniques. The presence of PEPT1 in BBMV and PEPT2 in OMMV was confirmed using Western blotting. Fluorescence changes were measured when extravesicular medium at pH 6.6 containing 0-1 mM substrates was added to a cuvette containing vesicles pre-equilibrated at pH 7.4 and 2.71 µM DiS-C3-(3). An increase in fluorescence intensity occurred upon substrate addition reflecting the expected positive change in membrane potential difference. Of the range of substrates studied, OMMV manifested the highest affinity to cefadroxil and valacyclovir (K m 4.3 ± 1.2 and 11.7 ± 3.2 µM, respectively) compared to other substrates, whilst the BBMV showed a higher affinity to Gly-His (K m 15.4 ± 3.1 µM) compared to other substrates. In addition, OMMV showed higher affinity and capacity to Gly-Gln (K m 47.1 ± 9.8 µM, 55.5 ± 2.8 ΔF/s/mg protein) than BBMV (K m 78.1 ± 13.3 µM and 35.5 ± 1.7 ΔF/s/mg protein, respectively). In conclusion, this study successfully separated the expression of PEPT1 and PEPT2 into different vesicle preparations inferring their activity in different regions of the renal proximal tubule.

L-Glycyl-L-glutamine provides the isolated and perfused young and middle-aged rat heart protection against ischaemia-reperfusion injury.

The amino acids glycine and glutamine have been implicated in myocardial protection of the much studied young adult heart. This study aimed to determine whether such protection could be enhanced using the dipeptide, L-glycyl-L-glutamine (gly-gln) in both young hearts and in middle-aged hearts representative of a more clinically relevant age group. Hearts from 8-week-old and 36-week-old rats were perfused in the Langendorff mode for 20 min, before 40 min global normothermic ischaemia and 30 min reperfusion. Where present, 0.5, 2, or 5 mM gly-gln was added 10 min into baseline perfusion, was present throughout ischaemia and was washed out after 10 min reperfusion. Reperfusion damage was assessed from the release of lactate dehydrogenase. Metabolic fitness was assessed from the time to ischaemic contracture and the accumulation of lactate and thiobarbituric acid reactive substances during ischaemia. The presence of 5 mM gly-gln significantly improved the post-ischaemic rate pressure product (RPP) and decreased reperfusion damage in both the 8 (RPP in control on reperfusion 5527 ± 957 vs. 10,320 ± 795 mmHg beat min(-1) in 5 mM gly-gln, n = 6 ± SE, p < 0.05) and 36-week-old (RPP in control on reperfusion 1964.33 ± 876.3 vs. 4008 ± 675 mmHg beat min(-1), n = 6 ± SE, p < 0.01) hearts. Five mM gly-gln also increased the time to ischaemic contracture and was able to protect against the rise in lactate that occurred in the controls during ischaemia. These results suggest that gly-gln has good potential as a combatant against ischaemia-reperfusion injury in both the young adult and middle-aged populations.

Design, synthesis and biological evaluation of new 1,ω-Bis-(5-alkyl-3-tosyl-1,3,4,2-triazaphospholino)alkanes as in vitro α-amylase and lipase inhibitors.

A series of new 1,ω-bis-(5-alkyl-3-tosyl-1,3,4,2-triazaphospholino)alkanes 2 and 3 were obtained in excellent yields by the condensation of 1,ω-bis-(1-tosylamidrazone)alkanes 1 with two equivalent molars of Lawesson's Reagent (LR) and trisdimethylaminophosphine, respectively. All synthesized compounds were characterized by various spectroscopic techniques including IR, 1H NMR, 13C NMR and 31P NMR and elemental analysis. The newly synthesized compounds were evaluated against key enzymes related to diabetes and obesity such as α-amylase and lipase. This study showed that the compounds 3a and 2b are an excellent inhibitor of α-amylase (with IC50 = 18.8 mM) and lipase (with IC50 = 19 mM) respectively, as compared with standard, orlistat (IC50 = 22 mM). Among this series, compounds 3a and 2b with the CH3 or C2H5 group at position 6 were identified as the most potent inhibitors against α-amylase, and lipase enzymes. The remaining compounds were found to be moderately active. Further, molecular docking simulation studies were done to identify the interactions and binding mode of synthesized analogs at binding site of α-amylase and lipase enzymes.

Bacillus Species as Direct-Fed Microbial Antibiotic Alternatives for Monogastric Production.

Antibiotic growth promoters have been utilized for long time at subtherapeutic levels as feed supplements in monogastric animal rations. Because of their side-effects such as antibiotic resistance, reduction of beneficial bacteria in the gut, and dysbiosis, it is necessary to look for non-therapeutic alternatives. Probiotics play an important role as the key substitutes to antibacterial agents due to their many beneficial effects on the monogastric animal host. For instance, enhancement of the gut microbiota balance can contribute to improvement of feed utilization efficiency, nutrients absorption, growth rate, and economic profitability of livestock. Probiotics are defined as "live microorganisms that, when administered in adequate amounts, confer a health benefit on the host." They are available in diverse forms for use as feed supplements. Their utilization as feed additives assists in good digestion of feed ingredients and hence, making the nutrients available for promoting growth. Immunity can also be enhanced by supplementing probiotics to monogastrics diets. Moreover, probiotics can help in improving major meat quality traits and countering a variety of monogastric animals infectious diseases. A proper selection of the probiotic strains is required in order to confer optimal beneficial effects. The present review focuses on the general functional, safety, and technological screening criteria for selection of ideal Bacillus probiotics as feed supplements as well as their mechanism of action and beneficial effects on monogastric animals for improving production performance and health status.

Effects of Dietary Supplementation with Bacillus amyloliquefaciens US573 on Intestinal Morphology and Gut Microbiota of European Sea Bass.

Probiotics or direct-fed microbials (DFM) have proven strong potential for improving aquaculture sustainability. This study aims to evaluate the effects of dietary supplementation with the DFM Bacillus amyloliquefaciens US573 on growth performance, intestinal morphology, and gut microbiota (GM) of European sea bass. For this purpose, healthy fish were divided into two feeding trials in triplicate of 25 fish in each tank. The fish were fed with a control basal diet or a DFM-supplemented diet for 42 days. Results showed that, while no significant effects on growth performance were observed, the length and abundance of villi were higher in the DFM-fed group. The benefic effects of DFM supplementation included also the absence of cysts formation and the increase in number of goblet cells playing essential role in immune response. Through DNA metabarcoding analysis of GM, 5 phyla and 14 major genera were identified. At day 42, the main microbiome changes in response to B. amyloliquefaciens US573 addition included the significant decrease in abundance of Actinobacteria phylum that perfectly correlates with a decrease in Nocardia genus representatives which represent serious threat in marine and freshwater fish. On the contrary, an obvious dominance of Betaproteobacteria associated with the abundance in Variovorax genus members, known for their ability to metabolize numerous substrates, was recorded. Interestingly, Firmicutes, particularly species affiliated to the genus Sporosarcina with recent promising probiotic potential, were identified as the most abundant. These results suggest that B. amyloliquefaciens US573 can be effectively recommended as health-promoting DFM in European sea bass farming.

In Vitro Antioxidant and In Vivo Hepatoprotective Properties of Wissadula periplocifolia Extract.

Wissadula periplocifolia (L.) Thwaites is a traditional medicinal plant belonging to the family Malvaceae, used in folk medicine for inflamed snake bites and bee stings. The current study was designed to investigate the in vitro antioxidant and in vivo anti-inflammatory and hepatoprotective activities of 80% ethanol extract of W. periplocifolia and its different fractions. The crude ethanolic extract (CEE) was then serially fractionated with petroleum ether fraction (PEF), chloroform fraction (CHF), and aqueous fraction (AQF). The antioxidant activity was assessed using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical assay, anti-inflammatory activity was determined in the xylene-induced ear edema model, and hepatoprotective activity was measured in the paracetamol-induced hepatic injury model. PEF showed a significant scavenging effect with an IC50 value of 33.5 µg/mL, followed by CEE (IC50 = 42.2 µg/mL), CHF (IC50 = 77 µg/mL), and AQF (IC50 = 80 µg/mL), compared to standard butylated hydroxytoluene (IC50 = 14.8 µg/mL). Both doses of CEE (250 and 500 mg/kg) could reduce ear edema by 41.3 and 50%, respectively, compared to standard diclofenac sodium (76.09%). Moreover, CEE significantly reduces the elevated liver enzymes (ALT, AST, and ALP), compared to control. Nevertheless, it elevated blood protein and reduced the blood bilirubin level (p < 0.01), compared to control. Histopathological studies also indicated significant protection of the liver from paracetamol-induced liver damage. In conclusion, W. periplocifolia could be a good source of antioxidant and hepatoprotective phytochemicals; meanwhile, toxicological and pharmacokinetic studies are recommended.

Ephedra alata subsp. alenda (Ephedraceae) leaf extracts: phytochemical screening, anti-diabetic, anti-obesity and anti-toxic activities on diabetic-induced liver-kidney-testes toxicities and inhibition of α-amylase and lipase enzymes.

The study evaluated the phytochemical composition of Ephedra alata and its effects on α-amylase and lipase enzymes and diabetic-induced liver-kidney-testes toxicities to determine the anti-diabetic, anti-obesity, and anti-toxic potentials of the plant. Obesity was induced by a high-fat and fructose diet (HFFD). Various compounds were identified and quantified: cafeic acid, apigenin 7-O-glucoside, apigenin, rutin, luteolin 7-O-glucoside, p-Coumaric acid and others in EA aqueous extract (EAWE). In vitro, this study showed that EAWE strongly inhibited lipase activity as compared to EA methanol (EAME) and ethyl acetate EA extracts (EAEE). In obese rats, the supplementation of EAWE inhibited significantly (P < 0.01) intestinal and pancreatic lipase activity by 35 and 36% respectively. This decrease in lipid digestive enzyme activity caused a significant (P < 0.05) reduce in the weight gain by 12.7% and significant (P < 0.05) decrease in the serum lipid rate as total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C). Moreover, the supplementation of EAWE to obese rats reduced the activity of α-amylase in the small intestine and pancreas by 26 and 31% respectively (P < 0.01) and consequently decreases in serum glucose level by 20.8% (P < 0.05). In addition, administration of EAWE in type 2 diabetes protected from obesity induced liver, kidney and testes alterations. The potent protective effect EAWE may be influenced by the diversity of phenolic compounds. therefore, this study showed in the first time that EAWE are efficient for the prevention and the amelioration of obesity, hyperglycemia, and various organs toxicities.

Deep learning model for classification and bioactivity prediction of essential oil-producing plants from Egypt.

Reliance on deep learning techniques has become an important trend in several science domains including biological science, due to its proven efficiency in manipulating big data that are often characterized by their non-linear processes and complicated relationships. In this study, Convolutional Neural Networks (CNN) has been recruited, as one of the deep learning techniques, to be used in classifying and predicting the biological activities of the essential oil-producing plant/s through their chemical compositions. The model is established based on the available chemical composition's information of a set of endemic Egyptian plants and their biological activities. Another type of machine learning algorithms, Multiclass Neural Network (MNN), has been applied on the same Essential Oils (EO) dataset. This aims to fairly evaluate the performance of the proposed CNN model. The recorded accuracy in the testing process for both CNN and MNN is 98.13% and 81.88%, respectively. Finally, the CNN technique has been adopted as a reliable model for classifying and predicting the bioactivities of the Egyptian EO-containing plants. The overall accuracy for the final prediction process is reported as approximately 97%. Hereby, the proposed deep learning model could be utilized as an efficient model in predicting the bioactivities of, at least Egyptian, EOs-producing plants.

Towards understanding the antagonistic activity of phytic acid against common foodborne bacterial pathogens using a general linear model.

The increasing challenge of antibiotic resistance requires not only the discovery of new antibiotics, but also the development of new alternative approaches. Therefore, in the present study, we investigated for the first time the antibacterial potential of phytic acid (myo-inositol hexakisphosphate, IP6), a natural molecule that is 'generally recognized as safe' (FDA classification), against the proliferation of common foodborne bacterial pathogens such as Listeria monocytogenes, Staphylococcus aureus and Salmonella Typhimurium. Interestingly, compared to citric acid, IP6 was found to exhibit significantly greater inhibitory activity (P<0.05) against these pathogenic bacteria. The minimum inhibitory concentration of IP6 varied from 0.488 to 0.97 mg/ml for the Gram-positive bacteria that were tested, and was 0.244 mg/ml for the Gram-negative bacteria. Linear and general models were used to further explore the antibacterial effects of IP6. The developed models were validated using experimental growth data for L. monocytogenes, S. aureus and S. Typhimurium. Overall, the models were able to accurately predict the growth of L. monocytogenes, S. aureus, and S. Typhimuriumin Polymyxin acriflavine lithium chloride ceftazidime aesculin mannitol (PALCAM), Chapman broth, and xylose lysine xeoxycholate (XLD) broth, respectively. Remarkably, the early logarithmic growth phase of S. Typhimurium showed a rapid and severe decrease in a period of less than one hour, illustrating the bactericidal effect of IP6. These results suggest that IP6 is an efficient antibacterial agent and can be used to control the proliferation of foodborne pathogens. It has promising potential for environmentally friendly applications in the food industry, such as for food preservation, food safety, and for prolonging shelf life.

Optimization of ß-mannanase production by Bacillus subtilis US191 using economical agricultural substrates.

The Bacillus subtilis US191 strain producing highly thermostable ß-mannanase was previously selected as potential probiotic candidate for application as feed supplement in poultry industry. Initially, the level of extracellular ß-mannanase production by this strain was 1.48 U ml-1 . To improve this enzyme titer, the present study was undertaken to optimize the fermentation conditions through experimental designs and valorization of agro-industrial byproducts. Using the Plackett-Burman design, in submerged fermentation, a set of 14 culture variables was evaluated in terms of their effects on ß-mannanase production. Locust bean gum (LBG), soymeal, temperature, and inoculum size were subsequently optimized by response surface methodology using Box-Behnken design. Under optimized conditions (1 g L-1 LBG, 8 g L-1 soymeal, temperature of 30°C and inoculum size of 1010 CFU ml-1 ), a 2.59-fold enhancement in ß-mannanase titer was achieved. Next, to decrease the enzyme production cost, the effect of partial substitution of LBG (1 g L-1 ) by agro-industrial byproducts was investigated, and a Taguchi design was applied. This allowed the attaining of a ß-mannanase production level of 8.75 U ml-1 in presence of 0.25 g L-1 LBG, 5 g L-1 of coffee residue powder, 5 g L-1 of date seeds powder, and 5 g L-1 of prickly pear seeds powder as mannans sources. Overall, a 5.91-fold improvement in ß-mannanase production by B. subtilis US191 was achieved.