Two intestinal microbiota-derived metabolites, deoxycholic acid and butyrate, synergize to enhance host defense peptide synthesis and alleviate necrotic enteritis

Background Necrotic enteritis(NE)is a major enteric disease in poultry,yet effective mitigation strategies remain elusive.Deoxycholic acid(DCA)and butyrate,two major metabolites derived from the intestinal microbiota,have independently been shown to induce host defense peptide(HDP)synthesis.However,the potential synergy between these two compounds remains unexplored.Methods To investigate the possible synergistic effect between DCA and butyrate in regulating HDP synthesis and barrier function,we treated chicken HD11 macrophage cells and jejunal explants with DCA and sodium butyrate(NaB),either individually or in combination,for 24 h.Subsequently,we performed RNA isolation and reverse transcrip-tion-quantitative PCR to analyze HDP genes as well as the major genes associated with barrier function.To further determine the synergy between DCA and NaB in enhancing NE resistance,we conducted two independent trials with Cobb broiler chicks.In each trial,the diet was supplemented with DCA or NaB on the day-of-hatch,followed by NE induction through sequential challenges with Eimeria maxima and Clostridium perfringens on d 10 and 14,respectively.We recorded animal mortality after infection and assessed intestinal lesions on d 17.The impact of DCA and NaB on the microbiota in the ileum and cecum was evaluated through bacterial 16S rRNA gene sequencing.Results We found that the combination of DCA and NaB synergistically induced multiple HDP genes in both chicken HD11 cells and jejunal explants.Additionally,the gene for claudin-1,a major tight junction protein,also exhibited synergistic induction in response to DCA and NaB.Furthermore,dietary supplementation with a combination of 0.75 g/kg DCA and 1 g/kg NaB led to a significant improvement in animal survival and a reduction in intestinal lesions compared to either compound alone in a chicken model of NE.Notably,the cecal microbiota of NE-infected chickens showed a marked decrease in SCFA-producing bacteria such as Bacteroides,Faecalibacterium,and Cuneatibacter,with lactobacilli becoming the most dominant species.However,supplementation with DCA and NaB largely restored the intestinal microbiota to healthy levels.Conclusions DCA synergizes with NaB to induce HDP and claudin-1 expression and enhance NE resistance,with potential for further development as cost-effective antibiotic alternatives.

Andrias davidianus bone peptides alleviates hyperuricemia-induced kidney damage in vitro and in vivo

Hyperuricemia(HUA)is a vital risk factor for chronic kidney diseases(CKD)and development of functional foods capable of protecting CKD is of importance.This paper aimed to explore the amelioration effects and mechanism of Andrias davidianus bone peptides(ADBP)on HUA-induced kidney damage.In the present study,we generated the standard ADBP which contained high hydrophobic amino acid and low molecular peptide contents.In vitro results found that ADBP protected uric acid(UA)-induced HK-2 cells from damage by modulating urate transporters and antioxidant defense.In vivo results indicated that ADBP effectively ameliorated renal injury in HUA-induced CKD mice,evidenced by a remarkable decrease in serum UA,creatinine and blood urea nitrogen,improving kidney UA excretion,antioxidant defense and histological kidney deterioration.Metabolomic analysis highlighted 14 metabolites that could be selected as potential biomarkers and attributed to the amelioration effects of ADBP on CKD mice kidney dysfunction.Intriguingly,ADBP restored the gut microbiome homeostasis in CKD mice,especially with respect to the elevated helpful microbial abundance,and the decreased harmful bacterial abundance.This study demonstrated that ADBP displayed great nephroprotective effects,and has great promise as a food or functional food ingredient for the prevention and treatment of HUA-induced CKD.

Cell Adhesion Inhibition by RGD Peptides Linked with a Photoisomerizable Nonnatural Amino Acid

RGD peptides linked with a nonnatural amino acid, phenylazophenyl alaninine (azoAla), were synthesized and applied to cell adhesion inhibitors. The RGD peptides linked with azoAla at C terminal showed potent binding to the integrin on the surface of HeLa cells. Photoisomerization effect of the azobenzene side chain of synthesized peptides on the cell adhesion inhibition was further investigated. It was demonstrated that the cis form of azoAla linked RGD peptides revealed a little weak cell adhesion inhibition effect as compared with trans form of azoAla linked RGD peptide.

Apoptosis Induced by Bcl-2 Antisense Peptide Acid in HL60 Cells

Objective To study the differences and similarities of the antisense drugs with different structures on the biological functions of HL60 cells. Methods Cytotoxic effects were measured by cell viability assay. The expression levels of protein were assayed by immunofluorescence using fluoresce isothiocyanate label. The morphological changes in apoptotic cells were observed. Flow cytometric analysis of DNA fragmentation was also performed. Results Antisense peptide nucleic acid (PNA) targeting the coding region of the Bcl-2 mRNA could effectively inhibit the growth of HL60 cells, down-regulate the synthesis of Bcl-2 protein and induce apoptosis. After HL60 cells were treated with 10 μmol/L Bcl-2 antisense PNA or antisense oligonucleotide for 72 h respectively, apoptotic rates of HL60 cells were 17.80±1.53 and 13.17±1.12, respectively( P <0.05). Conclusion Antisense PNA targeting the coding region of Bcl-2 mRNA may have stronger antisense effects than the antisense oligonucleotides and could induce apoptosis of HL60 cells.

A Study on Anti-oxidative Activity of Soybean Peptideswith Linoleic Acid Peroxidation Systems

Soybean bioactive peptides（SBPs） were prepared from the isolated soybean protein by proteolysis with an alkaline protease, alcalase, at 50℃ and pH = 8.0. The dependence of hydrolysis time on hydrolysis degree and molecular weight distribution were examined. The hydrolysate was fractionated on a Sephadex G-25 column and the anti-oxidative activities of the fractions were detected by the method of pyrogallol auto-oxidation. The average chain length of soybean peptides that have anti-oxidative activity was estimated to be about 7. The anti-oxidative properties of the soy- bean peptide were also studied by using linoleic acid peroxidation systems. The optimal condition of the peroxidation system was set up, Vc/Cu^2 ＋ as the inducer at pH = 7.4 and 25 ℃. In addition, soybean peptides show higher antioxidative activity compared with GSH.

SERUM CONCENTRATIONS OF HYALURONIC ACID, PROCOLLAGEN TYPE III NH_2-TERMINAL PEPTIDE, AND LAMININ IN PATIENTS WITH CHRONIC CONGESTIVE HEART FAILURE

Objective To explore the role of serum fibrotic indices including hyaluronic acid （HA）, procollagen type Ⅲ NH2-terminal peptide （PCIIIP）, and laminin （LN） in assessing the severity of myocardial fibrosis in chronic congestive heart failure （CHF）. Methods Serum levels of HA, PCIIIP, and LN in 39 patients with CHF E [14 with New York Heart Association （NYHA） functional class II, 21 with class Ⅲ, 4 with class Ⅳ] and in 46 patients with NYHA functional class I were assessed by radioimmunoassay. Results The serum concentrations of HA, PCMP, and LN were 359.75 ± 84.59 μg/L, 77.88 ± 24. 67 μg/L, 86. 73 ± 23.90 μg/L in CHF group, and 211.60 ±54. 80 μg/L, 64.82 ±23.99 μg/L, 82. 26 ±23.98 μg/L in NYHA functional class Ⅰ group, respectively. The HA level was significantly higher in CHF patients as compared with NYHA functional class Ⅰ group （ P 〈 0.05 ）. However, no difference was found in the levels of PCIIIP and LN between CHF group and NYHA functional class Ⅰ group. The serum HA concentration was negatively correlated with left ventricular ejection fraction （ r = - 0.71, P 〈 0.05 ）. Conclusion Serum HA level may act as an indicator for myocardial fibrosis.

A novel artificial nerve graft for repairing longdistance sciatic nerve defects:a self-assembling peptide nanofiber scaffold-containing poly (lactic-co-glycolic acid) conduit

In this study, we developed a novel artificial nerve graft termed self-assembling peptide nanofiber scaffold （SAPNS）-containing poly（lactic-co-glycolic acid） （PLGA） conduit （SPC） and used it to bridge a 10-mm-long sciatic nerve defect in the rat. Retrograde tracing, behavioral testing and histomorphometric analyses showed that compared with the empty PLGA conduit implantation group, the SPC implantation group had a larger number of growing and extending axons, a markedly increased diameter of regenerated axons and a greater thickness of the myelin sheath in the conduit. Furthermore, there was an increase in the size of the neuromuscular junction and myofiber diameter in the target muscle. These findings suggest that the novel artificial SPC nerve graft can promote axonal regeneration and remyelination in the transected peripheral nerve and can be used for repairing peripheral nerve injury.

Expression Pattern of Peptide and Amino Acid Genes in Digestive Tract of Transporter Juvenile Turbot(Scophthalmus maximus L.)

Turbot(Scophthalmus maximus L.),a carnivorous fish species with high dietary protein requirement,was chosen to examine the expression pattern of peptide and amino acid transporter genes along its digestive tract which was divided into six segments including stomach,pyloric caeca,rectum,and three equal parts of the remainder of the intestine.The results showed that the expression of two peptide and eleven amino acid transporters genes exhibited distinct patterns.Peptide transporter 1(Pep T1) was rich in proximal intestine while peptide transporter 2(PepT2) was abundant in distal intestine.A number of neutral and cationic amino acid transporters expressed richly in whole intestine including B^0-type amino acid transporter 1(B^0AT1),L-type amino acid transporter 2(LAT2),T-type amino acid transporter 1(TAT1),proton-coupled amino acid transporter 1(PAT1),y^+L-type amino acid transporter 1(y^+LAT1),and cationic amino acid transporter 2(CAT2) while ASC amino acid transporter 2(ASCT2),sodium-coupled neutral amino acid transporter 2(SNAT2),and y^+L-type amino acid transporter 2(y^+LAT2) abundantly expressed in stomach.In addition,system b^(0,+) transporters(rBAT and b^(0,+)AT) existed richly in distal intestine.These findings comprehensively characterized the distribution of solute carrier family proteins,which revealed the relative importance of peptide and amino acid absorption through luminal membrane.Our findings are helpful to understand the mechanism of the utilization of dietary protein in fish with a short digestive tract.

Peptide nucleic acid (PNA) binding-mediated gene regulation

Peptide nucleic acids (PNAs) are synthetic oligonucleotides with chemically modified backbones. PNAs can bind to both DNA and RNA targets in a sequence-specific manner to form PNA/DNA and PNA/RNA duplex structures. When bound to double-stranded DNA (dsDNA) targets, the PNA molecule replaces one DNA strand in the duplex by strand invasion to form a PNA/DNA/PNA [or (PNA)2/DNA] triplex structure and the displaced DNA strand exists as a singlestranded D-loop. PNA has been used in many studies as research tools for gene regulation and gene targeting. The Dloops generated from the PNA binding have also been demonstrated for its potential in initiating transcription and inducing gene expression. PNA provides a powerful tool to study the mechanism of transcription and an innovative strategy to regulate target gene expression. An understanding of the PNA-mediated gene regulation will have important clinical implications in treatment of many human diseases including genetic, cancerous, and age-related diseases.

Ganoderma lucidum polysaccharide peptide alleviates hepatoteatosis via modulating bile acid metabolism dependent on FXR-SHP/FGF

OBJECTIVE Ganoderma lucidum polysaccharide peptide(GLPP)is a group of extract from Ganoderma lucidum with a molecular mass of approximately 5×10^5,which ratio of polysaccharide to peptide is approximately 95%/5%.The purpose of this study was to determine whether GLPP has therapeutic effect on Non-alcoholic fatty liver disease(NAFLD).METHODS Ob/ob mouse model and ApoC3 transgenic mouse model were used for exploring the effect of GLPP on NAFLD.Key metabolic pathways and enzymes were identified by metabolomics combining with KEGG and PIUmet analyses and key enzymes were detected by Western blotting.Hepatosteatosis models of HepG2 cells and primary hepatocytes were used to further confirm the therapeutic effect of GLPP on NAFLD.RESULTS GLPP administrated for a month alleviated hepatosteatosis,dyslipidemia,liver dysfunction and liver insulin resistance.Pathways of glycerophos⁃pholipid metabolism,fatty acid metabolism and primary bile acid biosynthesis were involved in the therapeutic effect of GLPP on NAFLD.Detection of key enzymes revealed that GLPP reversed low expression of CYP7A1,CYP8B1,FXR,SHP and high expression of FGFR4 in ob/ob mice and ApoC3 mice.Besides,GLPP inhibited fatty acid synthesis by reducing the expression of SREBP1c,FAS and ACC via a FXR-SHP dependent mechanism.Additionally,GLPP reduced the accumulation of lipid droplets and the content of TG in HepG2 cells and primary hepatocytes induced by oleic acid and palmitic acid.CONCLUSION GLPP significantly improves NAFLD via regulating bile acid synthesis dependent on FXR-SHP/FGF pathway,which finally inhibits fatty acid synthesis,indicating that GLPP might be developed as a ther⁃apeutic drug for NAFLD.

Synthesis and photo activity of phenylazonaphthalene peptide nucleic acid monomers

Phenylazonaphthalene peptide nucleic acid （PNA） monomers were successfully synthesized, and their photoisomerization was examined. The new PNA monomers showed reversible trans-cis isomerization with UVand visible light irradiation, which might be the foundation of photo-regulating the hybridization between PNA containing phenylazonaphthalene unit and DNA. Simultaneously, the fluorescence of the new PNA monomers might make them especially useful as structural probes.

Study on Free Amino Acid and Short Peptide Fertilizer Production by Solid State Fermented Castor Bean Meal

Using microbial fermentation to increase the content of free amino acids and short peptides in the organic fertilizer for castor bean meal can effectively promote plant growth and improve fruit quality.Using free amino acid and short peptide content as an indicator,through single factor and response surface optimization experiments,the process parameters(moisture content,fermentation time and inoculum quantity)of castor meal solid-state fermentation were optimized.The best process parameters for the solid-state fermentation were:the moisture content 62%,the fermentation time 20 d,and the inoculum quantity 0.23%.The moisture content had the greatest impact on the conversion rate of free amino acids and short peptides,and the protein conversion rate reached 65.6%.The scale-up experiment under the optimal conditions showed that the solid-state fermentation using the inoculum had a significant beneficial effect compared with other fermentation methods.The fermentation of castor cake fertilizer provides a theoretical and practical basis for production feasibility,and has important guiding significance for the effective utilization of castor bean meal.

Effect of Duodenal Soybean Small Peptides Infusion on Mammary Uptake of Amino Acid and Expression of APN in Lactating Goat

The aim of this study was to investigate the effect of duodenal infusion of soybean small peptides(SSP) on mammary uptake of amino acids(AA).Eight Chinese Saanen goats with duodenal fistulae and catheters were used in a crossover design trial.Goats were infused with 0,60,120,180 g/d SSP.The experimental period lasted for 14 d.The results showed:1) milk protein yield and content were increased,with the increment in milk protein yield being significant(P【0.05).Milk fat yield and content were decreased with the increased amount of SSP infused(P【0.05).2) Mammary plasma flow was not changed(P】0.05) by SSP infusion though the average was slightly higher.Mammary plasma flow/milk yield was decreased by SSP infusion and there was significant difference between the 120 g/d treatment and the control(P【0.05).3) Compared with the control treatment,uptakes of most free amino acids were increased in the 60 and 120 g/d treatments,but decreased by the 180 g/d treatment.The uptakes of all peptide-bound essential amino acids(PB-EAA) were increased except for PB-Ile.Uptake of PB-Val,PB-Leu, PB-Phe,PB-Thr,PB-Met and PB-Lys was highest in the 120 g/d treatment.Among the peptide-bound nonessential amino acids(PB-NEAA),uptake of PB-Ser,PB-Tyr,and PB-Pro was increased(P【0.05) while that of PB-Gly was decreased(P】0.05).4) With the exception of Lys,secretion of all essential AA(EAA) was increased by SSP infusion from 0-120 g/d(P【0.05),while in the 180 g/d treatment the increase was not significant and was lower than that in the 120 g/d treatment.5) The expression of APN was increased by SSP;in the 60,120,180 g/d treatments expression was 13.55-,18.69-,and 10.01-fold over that of the controls.Conclusion:SSP might be used as substrates for milk protein synthesis by mammary gland,and can increase synthesis and secretion of milk protein.There was reason to believe that uptake and use of AA was either saturated or inhibited at high levels of infusion of SSP.Increased APN expression with infusion of SSP related to mammary uptake of PBAA by mammary gland,which suggested that APN was one of the peptidases regulating use of AA from small peptides in mammary tissue.

Effects of Three Humic Acid Foliar Fertilizers on Quality and Photosynthetic Characteristics of Hawthorn

[Objectives]To investigate the effects of three foliar fertilizers on photosynthetic characteristics,fruit quality and yield of hawthorn.[Methods]Taking hawthorn foliar fertilization as the reference,the photosynthetic rate,transpiration rate,intercellular CO 2 concentration,stomatal conductance and single fruit weight of hawthorn leaves were measured under different concentrations of foliar fertilizer.[Results]The results showed that the photosynthetic rate,transpiration rate and stomatal conductance of hawthorn leaves increased significantly,while the intercellular CO 2 concentration decreased.Specifically,the fish protein peptide foliar fertilizer performed best,with net photosynthetic rate and transpiration rate increased by 57.22%and 57.51%,respectively.All the three foliar fertilizers significantly reduced the intercellular CO 2 concentration.In addition,fertilization significantly increased the single fruit weight of hawthorn,and the effect of fermented fulvic acid foliar fertilizer was the most significant,with the highest growth rate of 68.49%.[Conclusions]Spraying foliar fertilizer significantly increased the content of Vc,titratable acid,anthocyanin and soluble solids of hawthorn fruit,among which fermented fulvic acid foliar fertilizer had the optimal effect.

Synthesis of Two New Chiral Blocks for the Construction of Peptide Nucleic Acid (PNA)

Protected (L) and (D)-lysine were used respectively as starting materials to synthesize two new types of chiral blocks for the construction of PNA. Nucleobase was linked to alpha-NH2 of lysine via -CH2C (O)- spacer in type I, and -C (O)- was used in type II. The corresponding oligomers were constructed in solution.

A Study of Bioactivity of Corn Peptides with Low Molecular Weight Ⅱ: Effect on Plasma Free Amino Acid Concentrations in Rats

The effects of the ingestion of corn peptides with a low molecular weight(LMCP) prepared from zein on some plasma free amino acid concentrations in rats that had taken ethanol were investigated. LMCP(1 0 g/kg body weight) in 15% ethanol(10 mL/kg body weight) was given to Wister rats by intragastrical administration. The amino acid analysis showed that the concentrations of alanine, leucine, and proline in the plasma reached their maximum levels at 30 min for the LMCP-intake group. They are 582.39, 99.60 and 272.51 μg/L , respectively. But in the control group, the plasma free amino acid levels were not changed obviously. Therefore, LMCP could cause an increase in concentration of some free amino acids such as alanine, leucine and proline etc . in plasma of the rats that have taken ethanol.

Effects of Peptide Nucleic Acids against Ki-67 Gene on the Proliferation and Apoptosis of Human Renal Carcinoma Cell Line

To investigate the effects of anti-sense peptide nucleic acids （PNAs） targeting Ki-67 gene on modulation of the proliferation and apoptosis of human renal carcinoma cell lines, human renal carcinoma cell line 786-0 cells were treated with anti-sense PNAs at different concentrations （1.0 μmol/L, 2.0 μmol/L, 10.0 μmol/L）. The Ki-67 expression of 786-0 cells was detected by immunohistochemical technique and Western blot method respectively. The proliferation of 786-0 cells was studied by cell growth curves and ^3H-thymidine incorporation. The apoptosis of 786-0 cells was detected by TUNEL assay. The control groups were treated with anti-sense oligonucleotide （ASODNs） targeting Ki-67 gene. Our results showed that the Ki-67 expression of 786-0 cells treated with anti-sense PNAs （16.9±0.7） was significantly inhibited as compared with that of the control groups （28.6±0.4） （P〈0.01）. The Ki-67 protein rate of 786-0 cells treated with anti-sense PNAs （42.1 ±2.2） was significantly reduced when compared with that of the control groups （83.6± 1.4） （P〈0.01）. Proliferation of 786-0 cells treated with anti-sense PNAs （20.7 ± 1.5） was significantly inhibited as compared with that of the control groups （58.6± 1.4） （P〈0.01）. The apoptosis rate of 786-0 cells treated with anti-sense PNAs （28.7 ± 2.3） was significantly increased higher compared with that of the control groups （13.8 ±1.0） （P〈0.01）. From these finds we are led to conclude that anti-sense PNAs targeting Ki-67 gene have stronger effects on the inhibition of the proliferation and induction of apoptosis of human renal carcinoma cells than ASODNs targeting Ki-67 gene. The strategies using anti-sense PNAs targeting Ki-67 gene may be a promising approach for the treatment of renal cell carcinoma.

Structural, thermal and enzymatic analysis of naturally occurring and D-amino acid substituted peptides

Designing of new peptide materials for biomedical and protein engineering applications are important. In the present work an attempt has been made to study the effect of D-Leu in collagen like tetra peptide on the structure and stability of peptide against enzymes and results are compared with its chiral counterpart L-form. Effect of replacement of L-Leu in Leu-Gly-Pro-Ala tetra peptide with D-Leu on structure has been studied using circular dichroic spectroscopy (CD). Our findings suggest that, D-Leu substitution leads to conformational changes in Leu-Gly-Pro-Ala secondary structure from β-sheet to turns. L → D-Leu Configurational changes in Leu-Gly-Pro-Ala owes to enhanced thermal stability which has been substantiated through CD and differential scanning calorimetry. Change in chirality of the leucine inhibits collagenolytic activity, which enables to design selective inhibition of proteases with greater specificity.

Acid-base and lipophilic properties of peptide nucleic acid derivatives

The first combined experimental and theoretical study on the ionization and lipophilic properties of peptide nucleic acid(PNA)derivatives,including eleven PNA monomers and two PNA decamers,is described.The acidity constants(pKa)of individual acidic and basic centers of PNA monomers were measured by automated potentiometric pH titrations in water/methanol solution,and these values were found to be in agreement with those obtained by MoKa software.These results indicate that single nucleobases do not change their pKa values when included in PNA monomers and oligomers.In addition,immobilized artificial membrane chromatography was employed to evaluate the lipophilic properties of PNA monomers and oligomers,which showed the PNA derivatives had poor affinity towards membrane phospholipids,and confirmed their scarce cell penetrating ability.Overall,our study not only is of potential relevance to evaluate the pharmacokinetic properties of PNA,but also constitutes a reliable basis to properly modify PNA to obtain mimics with enhanced cell penetration properties.

Electrochemical investigation of DNA hybridization with ferrocene-labelled peptide nucleic acid on gold electrodes

An electrochemical DNA sensor based on ferrocene-labelled peptide nucleic acid （PNA-Fc） was prepared. The hybridization between PNA-Fc and DNA immobilized on a gold electrode was examined by cyclic voltammetry （CV） and differential pulse voltammetry （DPV）. PNA-Fc shows a good electrochemically activity and has a redox potential of 170 mV verus Ag/AgCl electrode after hybridization, representing the characteristic of ferrocene/ferrocenium （Fc/Fc＋） transformation. The results illustrate that PNA-Fe can be used as an effective electrochemical DNA probe sensor.