Arginine alleviates Clostridium perfringens α toxin-induced intestinal injury in vivo and in vitro via the SLC38A9/mTORC1 pathway.

Introduction: Clostridium perfringens α toxin is a main virulence factor responsible for gut damage in animals. Arginine is a functional amino acid exhibiting significant immunoregulatory activities. However, the effects and immunoregulatory mechanisms of arginine supplementation on α toxin-induced intestinal injury remain unclear. Methods: In vivo, 256 male Arbor Acres chickens were randomly assigned to a 2×2 factorial arrangement, involving diet treatments (with or without 0.3% arginine supplementation) and immunological stress (with or without α toxin challenge). In vitro, IEC-6 cells were treated with or without arginine in the presence or absence of α toxin. Moreover, IEC-6 cells were transfected with siRNA targeting mTOR and SLC38A9 to explore the underlying mechanisms. Results and discussion: The results showed that in vivo, arginine supplementation significantly alleviated the α toxin-induced growth performance impairment, decreases in serum immunoglobulin (Ig)A and IgG levels, and intestinal morphology damage. Arginine supplementation also significantly reduced the α toxin-induced increase in jejunal proinflammatory cytokines interleukin (IL)-1ß, IL-6 and IL-17 mRNA expression. Clostridium perfringens α toxin significantly decreased jejunal mechanistic target of rapamycin (mTOR) and solute carrier family 38 member 9 (SLC38A9) mRNA expression, while arginine supplementation significantly increased mTOR and SLC38A9 mRNA expression. In vitro, arginine pretreatment mitigated the α toxin-induced decrease in cell viability and the increase in cytotoxicity and apoptosis. Arginine pretreatment also alleviated the α toxin-induced upregulation of mRNA expression of inflammation-related cytokines IL-6, C-X-C motif chemokine ligand (CXCL)10, CXCL11 and transforming growth factor-ß (TGF-ß), as well as apoptosis-related genes B-cell lymphoma-2 associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-extra large (Bcl-XL) and cysteinyl aspartate specific proteinase 3 (Caspase-3) and the ratio of Bax to Bcl-2. Arginine pretreatment significantly increased the α toxin-induced decrease in mTOR, SLC38A9, eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1) and ribosomal protein S6 kinase (S6K) mRNA expression. Knockdown SLC38A9 and mTOR largely abrogated the positive effects of arginine pretreatment on α toxin-induced intracellular changes. Furthermore, SLC38A9 silencing abolished the increased mTOR mRNA expression caused by arginine pretreatment. In conclusion, arginine administration attenuated α toxin-induced intestinal injury in vivo and in vitro, which could be associated with the downregulation of inflammation via regulating SLC38A9/mTORC1 pathway.

Alleviation of albumin glycation-induced diabetic cardiomyopathy by L-Arginine: Insights into Nrf-2 signaling.

In hyperglycemia, accelerated glycation and oxidative stress give rise to many diabetic complications, such as diabetic cardiomyopathy (DCM). Glycated human serum albumin (GHSA) has disturbed structural integrity and hampered functional capabilities. When GHSA accumulates around cardiac cells, Nrf-2 is dysregulated, aiding oxidative stress. L-Arginine (L-Arg) is prescribed to patients with diabetes and cardiovascular diseases. This research contributes to the mechanistic insights on antiglycation and antioxidant potential of L-Arg in alleviating DCM. HSA was glycated with methylglyoxal in the presence of L-Arg (20-640 mM). Structural and functional modifications of HSA were studied. L-Arg and HSA, GHSA interactions, and thermodynamics were determined by steady-state fluorescence. H9c2 cardiomyocytes were given treatments of GHSA-L-Arg along with the inhibitor of the receptor of AGEs. Cellular antioxidant levels, detoxification enzyme activities were measured. Gene, protein expressions, and immunofluorescence data examined the activation and nuclear translocation of Nrf-2 during glycation and oxidative stress. L-Arg protected HSA from glycation-induced structural and functional modifications. The binding affinity of L-Arg was more towards HSA (104 M-1). L-Arg, specifically at lower concentration (20 mM), upregulated Nrf-2 gene, protein expressions and facilitated its nuclear translocation by activating Nrf-2 signaling. The study concluded that L-Arg can be of therapeutic advantage in glycation-induced DCM and associated oxidative stress.

The antibacterial properties of branched peptides based on poly(l-arginine): In vitro antibacterial evaluation and molecular dynamic simulations.

The emergence of bacterial strains resistant to antibiotics is a major issue in the medical field. Antimicrobial peptides are widely studied as they do not generate as much resistant bacterial strains as conventional antibiotics and present a broad range of activity. Among them, the homopolypeptide poly(l-arginine) presents promising antibacterial properties, especially in the perspective of its use in biomaterials. Linear poly(l-arginine) has been extensively studied but the impact of its 3D structure remains unknown. In this study, the antibacterial properties of newly synthesized branched poly(l-arginine) peptides, belonging to the family of multiple antigenic peptides, are evaluated. First, in vitro activities of the peptides shows that branched poly(l-arginine) is more efficient than linear poly(l-arginine) containing the same number of arginine residues. Surprisingly, peptides with more arms and more residues are not the most effective. To better understand these unexpected results, interactions between these peptides and the membranes of Gram positive and Gram negative bacteria are simulated thanks to molecular dynamic. It is observed that the bacterial membrane is more distorted by the branched structure than by the linear one and by peptides containing smaller arms. This mechanism of action is in full agreement with in vitro results and suggest that our simulations form a robust model to evaluate peptide efficiency towards pathogenic bacteria.

Synergistic bactericidal effect and mechanism of ultrasound combined with Lauroyl Arginate Ethyl against Salmonella Typhimurium and its application in the preservation of onions.

In the present study, the synergistic bactericidal effect and mechanism of ultrasound (US) combined with Lauroyl Arginate Ethyl (LAE) against Salmonella Typhimurium were investigated. On this basis, the effect of US+LAE treatment on the washing of S. Typhimurium on the surface of onions and on the physical and chemical properties of onion during fresh-cutting and storage were studied. The results showed that treatment with US+LAE could significantly (P < 0.05) reduce the number of S. Typhimurium compared to US and LAE treatments alone, especially the treatment of US+LAE (230 W/cm2, 8 min, 71 µM) reduced S. Typhimurium by 8.82 log CFU/mL. Confocal laser scanning microscopy (CLSM), flow cytometry (FCM), protein and nucleic acid release and N-phenyl-l-naphthylamine (NPN) assays demonstrated that US+LAE disrupted the integrity and permeability of S. Typhimurium cell membranes. Reactive oxygen species (ROS) and malondialdehyde (MDA) assays indicated that US+LAE exacerbated oxidative stress and lipid peroxidation in cell membranes. Field emission scanning electron microscopy (FESEM) demonstrated that US+LAE treatment caused loss of cellular contents and led to cell crumpling and even lost the original cell morphology. US+LAE treatment caused a significant (P < 0.05) decrease in the number of S. Typhimurium on onions, but there was no significant (P > 0.05) effect on the color, hardness, weight and ascorbic acid content of onions. This study elucidated the synergistic antibacterial mechanism of US+LAE and verified the feasibility of bactericidal effect on the surface of onions, providing a theoretical basis for improving the safety of fresh produce in the food industry and to propose a new way to achieve the desired results.

Arginine-Nanoenzyme with Timely Angiogenesis for Promoting Diabetic Wound Healing.

The successful treatment of diabetic wounds requires strategies that promote anti-inflammation, angiogenesis, and re-epithelialization of the wound. Excessive oxidative stress in diabetic ulcers (DUs) inhibits cell proliferation and hinders timely vascular formation and macrophage polarization from pro-inflammatory M1 to anti-inflammatory M2, resulting in a persistent inflammatory environment and a nonhealing wound. We designed arginine-nanoenzyme (FTA) with mimic-catalase and arginine-loading. 2,3,4-trihydroxy benzaldehyde and arginine (Arg) were connected by a Schiff base bond, and the nanoassembly of Arg to FTA was driven by the coordination force between a ferric ion and polyphenol and noncovalent bond force such as a hydrogen bond. FTA could remove excess reactive oxygen species at the wound site in situ and convert it to oxygen to improve hypoxia. Meanwhile, Arg was released and catalytically metabolized by NO synthase in M1 to promote vascular repair in the early phase. In the late phase, the metabolite of Arg catalyzed by arginase in M2 was mainly ornithine, which played a vital role in promoting tissue repair, which implemented angiogenesis timely and prevented hypertrophic scars. Mechanistically, FTA activated the cAMP signaling pathway combined with reducing inflammation and ameliorating angiogenesis, which resulted in excellent therapeutic effects on a DU mice model.

Antiproliferative and Antimigratory Activity of Poly-gallic Acid in Cancer Cell Lines.

BACKGROUND/AIM: Enzyme-mediated grafting of poly (gallic acid) (PGAL) and L-arginine and a-L-lysine onto PGAL produces reactive oxygen species (ROS)-suppressor multiradical molecules with low cytotoxicity, high thermostability and water solubility with cancer treatment potential. This study examined the anticancer effects of these molecules in hepatic (HepG2, ATCC HB-8065), breast (MCF7, ATCC HTB-22), and prostate (PC-3, ATCC CRL-1435 and DU 145, ATCC HTB-81) cancer cell lines, as well as in fibroblasts from healthy human skin as control cells. MATERIALS AND METHODS: PGAL was synthesized by the oxidative polymerization of the naturally abundant GA using laccase from Trametes versicolor. Insertions of amino acids L-arginine and α-L-lysine on the PGAL chain were carried out by microwave. The cells of dermal fibroblast (Fb) were obtained from primary skin cultures and isolated from skin biopsies. The cancer cells lines of hepatic (HepG2), breast (MCF7), and prostate (PC-3, DU 145) were obtained from ATCC. The viability of the cancer cells and the primary culture was obtained by the MTT assay. Proliferation was demonstrated by crystal violet assay. Cell migration was determined by Wound healing assay. Finally, cell cycle analysis was carried out with cells. RESULTS: The results show that 200 µg/ml of PGAL cultured in vitro with prostate cancer cells decreased viability, proliferation, and migration, as well as arrested cells in the G1 and S phases of the cell cycle. In contrast, the dermal fibroblasts and the hepatic line remained unaffected. The random grafting of L-Arg and a-L-Lys onto the PGAL chain also decreased the viability of prostate cancer cells. CONCLUSION: PGAL and PGAL-grafted amino acids are potential adjuvants for prostate cancer treatment, with improved physicochemical characteristics compared to GA.

Asymmetric Dimethylarginine Enables Depolarizing Spikes and Vasospasm in Mesenteric and Coronary Resistance Arteries.

BACKGROUND: Increased vasoreactivity due to reduced endothelial NO bioavailability is an underlying feature of cardiovascular disease, including hypertension. In small resistance arteries, declining NO enhances vascular smooth muscle (VSM) reactivity partly by enabling rapid depolarizing Ca2+-based spikes that underlie vasospasm. The endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) is metabolized by DDAH1 (dimethylarginine dimethylaminohydrolase 1) and elevated in cardiovascular disease. We hypothesized ADMA might enable VSM spikes and vasospasm by reducing NO bioavailability, which is opposed by DDAH1 activity and L-arginine. METHODS: Rat isolated small mesenteric arteries and myogenic rat-isolated intraseptal coronary arteries (RCA) were studied using myography, VSM intracellular recording, Ca2+ imaging, and DDAH1 immunolabeling. Exogenous ADMA was used to inhibit NO synthase and a selective DDAH1 inhibitor, NG-(2-methoxyethyl) arginine, to assess the functional impact of ADMA metabolism. RESULTS: ADMA enhanced rat-isolated small mesenteric arteries vasoreactivity to the α1-adrenoceptor agonist, phenylephrine by enabling T-type voltage-gated calcium channel-dependent depolarizing spikes. However, some endothelium-dependent NO-vasorelaxation remained, which was sensitive to DDAH1-inhibition with NG-(2-methoxyethyl) arginine. In myogenically active RCA, ADMA alone stimulated depolarizing Ca2+ spikes and marked vasoconstriction, while NO vasorelaxation was abolished. DDAH1 expression was greater in rat-isolated small mesenteric arteries endothelium compared with RCA, but low in VSM of both arteries. L-arginine prevented depolarizing spikes and protected NO-vasorelaxation in rat-isolated small mesenteric artery and RCA. CONCLUSIONS: ADMA increases VSM electrical excitability enhancing vasoreactivity. Endothelial DDAH1 reduces this effect, and low levels of DDAH1 in RCAs may render them susceptible to endothelial dysfunction contributing to vasospasm, changes opposed by L-arginine.

Co-release of nitric oxide and L-arginine from poly (ß-amino ester)-based adhesive reprogram macrophages for accelerated wound healing and angiogenesis in vitro and in vivo.

Recently, insufficient angiogenesis and prolonged inflammation are crucial challenges of chronic skin wound healing. The sustained release of L-Arginine (L-Arg) and nitric oxide (NO) production can control immune responses, improve angiogenesis, enhance re-epithelialization, and accelerate wound healing. Here, we aim to improve wound healing via the controlled release of NO and L-Arg from poly (ß-amino ester) (PßAE). In this regard, PßAE is functionalized with methacrylate poly-L-Arg (PAMA), and the role of PAMA content (50, 66, and 75 wt%) on the adhesive properties, L-Arg, and NO release, as well as collagen deposition, inflammatory responses, and angiogenesis, is investigated in vitro and in vivo. Results show that the PAMA/ PßAE could provide suitable adhesive strength (~25 kPa) for wound healing application. In addition, increasing the PAMA content from 50 to 75 wt% results in an increased release of L-Arg (approximately 1.4-1.7 times) and enhanced NO production (approximately 2 times), promoting skin cell proliferation and migration. The in vitro studies also show that compared to PßAE hydrogel, incorporation of 66 wt% PAMA (PAMA 66 sample) reveals superior collagen I synthesis (~ 3-4 times) of fibroblasts, controlled pro-inflammatory and improved anti-inflammatory cytokines secretion of macrophages, and accelerated angiogenesis (~1.5-2 times). In vivo studies in a rat model with a full-thickness skin defect also demonstrate the PAMA66 sample could accelerate wound healing (~98 %) and angiogenesis, compared to control (untreated wound) and Tegaderm™ commercial wound dressing. In summary, the engineered multifunctional PAMA functionalized PßAE hydrogel with desired NO and L-Arg release, and adhesive properties can potentially reprogram macrophages and accelerate skin healing for chronic wound healing.

A DNA-inspired injectable adhesive hydrogel with dual nitric oxide donors to promote angiogenesis for enhanced wound healing.

Chronic diabetic wounds are a severe complication of diabetes, often leading to high treatment costs and high amputation rates. Numerous studies have revealed that nitric oxide (NO) therapy is a promising option because it favours wound revascularization. Here, base-paired injectable adhesive hydrogels (CAT) were prepared using adenine- and thymine-modified chitosan (CSA and CST). By further introducing S-nitrosoglutathione (GSNO) and binary l-arginine (bArg), we obtained a NO sustained-release hydrogel (CAT/bArg/GSON) that was more suitable for the treatment of chronic wounds. The results showed that the expression of HIF-1α and VEGF was upregulated in the CAT/bArg/GSON group, and improved blood vessel regeneration was observed, indicating an important role of NO. In addition, the research findings revealed that following treatment with the CAT/bArg/GSON hydrogel, the viability of Staphylococcus aureus and Escherichia coli decreased to 14 ± 2 % and 6 ± 1 %, respectively. Moreover, the wound microenvironment was improved, as evidenced by a 60 ± 1 % clearance of DPPH. In particular, histological examination and immunohistochemical staining results showed that wounds treated with CAT/bArg/GSNO exhibited denser neovascularization, faster epithelial tissue regeneration, and thicker collagen deposition. Overall, this study proposes an effective strategy to prepare injectable hydrogel dressings with dual NO donors. The functionality of CAT/bArg/GSON has been thoroughly demonstrated in research on chronic wound vascular regeneration, indicating that CAT/bArg/GSON could be a potential option for promoting chronic wound healing. STATEMENT OF SIGNIFICANCE: This article prepares a chitosan hydrogel utilizing the principle of complementary base pairing, which offers several advantages, including good adhesion, biocompatibility, and flow properties, making it a good material for wound dressings. Loaded GSNO and bArg can steadily release NO and l-arginine through the degradation of the gel. Then, the released l-arginine not only possesses antioxidant properties but can also continue to generate a small amount of NO under the action of NOS. This design achieves a sustained and stable supply of NO at the wound site, maximizing the angiogenesis-promoting and antibacterial effects of NO. More neovascularization and abundant collagen were observed in the regenerated tissues. This study provides an effective repair hydrogel material for diabetic wound.

Effects of X-ray cranial irradiation on metabolomics and intestinal flora in mice.

Cranial radiotherapy is an important treatment for intracranial and head and neck tumors. To investigate the effects of cranial irradiation (C-irradiation) on gut microbiota and metabolomic profile, the feces, plasma and cerebral cortex were isolated after exposing mice to cranial X-ray irradiation at a dose rate of 2.33 Gy/min (5 Gy/d for 4 d consecutively). The gut microorganisms and metabolites were detected by 16 S rRNA gene sequencing method and LC-MS method, respectively. We found that compared with sham group, the gut microbiota composition changed at 2 W and 4 W after C-irradiation at the genus level. The fecal metabolomics showed that compared with Sham group, 44 and 66 differential metabolites were found to be annotated into metabolism pathways at 2 W and 4 W after C-irradiation, which were significantly enriched in the arginine and proline metabolism. Metabolome analysis of serum and cerebral cortex showed that, at 4 W after C-irradiation, the expression pattern of metabolites in serum samples of mice was similar to that of sham group, and the cerebral cortex metabolites of the two groups were completely separated. KEGG functional analysis showed that serum and brain tissue differential metabolites were respectively enriched in tryptophan metabolism, and arginine proline metabolism. The correlation analysis showed that the changes of gut microbiota genera were significantly correlated with the changes of metabolism, especially Helicobacter, which was significantly correlated with many different metabolites at 4 W after C-irradiation. These data suggested that C-irradiation could affect the gut microbiota and metabolism profile, even at relatively long times after C-irradiation.

Oregano (Origanum vulgare) Essential Oil and Its Constituents Prevent Rat Kidney Tissue Injury and Inflammation Induced by a High Dose of L-Arginine.

This study aimed to evaluate the protective action of oregano (Origanum vulgare) essential oil and its monoterpene constituents (thymol and carvacrol) in L-arginine-induced kidney damage by studying inflammatory and tissue damage parameters. The determination of biochemical markers that reflect kidney function, i.e., serum levels of urea and creatinine, tissue levels of neutrophil-gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM-1), as well as a panel of oxidative-stress-related and inflammatory biomarkers, was performed. Furthermore, histopathological and immunohistochemical analyses of kidneys obtained from different experimental groups were conducted. Pre-treatment with the investigated compounds prevented an L-arginine-induced increase in serum and tissue kidney damage markers and, additionally, decreased the levels of inflammation-related parameters (TNF-α and nitric oxide concentrations and myeloperoxidase activity). Micromorphological kidney tissue changes correlate with the alterations observed in the biochemical parameters, as well as the expression of CD95 in tubule cells and CD68 in inflammatory infiltrate cells. The present results revealed that oregano essential oil, thymol, and carvacrol exert nephroprotective activity, which could be, to a great extent, associated with their anti-inflammatory, antiradical scavenging, and antiapoptotic action and, above all, due to their ability to lessen the disturbances arising from acute pancreatic damage. Further in-depth studies are needed in order to provide more detailed explanations of the observed activities.

Mimicking Charged Host-Defense Peptides to Tune the Antifungal Activity and Biocompatibility of Amphiphilic Polymers.

Invasive fungal infections impose a substantial global health burden. They cause more than 1.5 million deaths annually and are insufficiently met by the currently approved antifungal drugs. Antifungal peptides are a promising alternative to existing antifungal drugs; however, they can be challenging to synthesize, and are often susceptible to proteases in vivo. Synthetic polymers which mimic the properties of natural antifungal peptides can circumvent these limitations. In this study, we developed a library of 29 amphiphilic polyacrylamides with different charged units, namely, amines, guanidinium, imidazole, and carboxylic acid groups, representative of the natural amino acids lysine, arginine, histidine, and glutamic acid. Ternary polymers incorporating primary ammonium (lysine-like) or imidazole (histidine-like) groups demonstrated superior activity against Candida albicans and biocompatibility with mammalian cells compared to the polymers containing the other charged groups. Furthermore, a combination of primary ammonium, imidazole, and guanidinium (arginine-like) within the same polymer outperformed the antifungal drug amphotericin B in terms of therapeutic index and exhibited fast C. albicans-killing activity. The most promising polymer compositions showed synergistic effects in combination with caspofungin and fluconazole against C. albicans and additionally demonstrated activity against other clinically relevant fungi. Collectively, these results indicate the strong potential of these easily producible polymers to be used as antifungals.

Inclusion of guanidinoacetic acid in a low metabolizable energy diet improves broilers growth performance by elevating energy utilization efficiency through modulation serum metabolite profile.

This study was aimed to explore the elevating energy utilization efficiency mechanism for the potentially ameliorative effect of guanidinoacetic acid (GAA) addition on growth performance of broilers fed a low metabolizable energy (LME) diet. A total of 576 d old broilers were randomly allocated to one of the six treatments: a basal diet (normal ME, positive control, PC), or an LME diet (50 kcal/kg reduction in ME, negative control, NC) supplemented with 0.02%, 0.04%, 0.06%, and 0.08% GAA from 1 to 42 d of age, respectively. The GAA fortification in LME diet linearly or quadratically dropped (P < 0.05) the feed conversion ratio (FCR) from 22 to 42 and 1 to 42 d of age, abdominal fat rate on day 42, serum alanine aminotransferase (ALT) on day 21, and serum creatinine (CREAN) on days 21 and 42, elevated (P < 0.05) breast muscle rate and leg muscle rate on day 42, serum creatine kinase (CK) on days 21 and 42, as well as alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) on day 21. The dietary optimal GAA levels were 0.03%-0.08% based on the best-fitted quadratic models (P < 0.03) of the above parameters. Thus, the PC, LME, and 0.04% GAA-LME groups were selected for further analysis. Serum essential amino acids (EAA) tryptophan, histidine and arginine, non-essential amino acids (NEEA) serine, glutamine and aspartic acid were significantly decreased (P < 0.05), compared to PC diet by LME or 0.04% GAA-LME diet. 0.04% GAA-LME group reversed (P < 0.05) the reduction of arginine, 3-methyhistidine, and 1-methylhistidine by LME diet. Besides, six birds at 28 d of age from LME and 0.04% GAA-LME groups were selected for energy utilization observation in calorimetry chambers. The results demonstrated that 0.04% GAA-LME group significantly improved (P < 0.05) the ME intake (MEI) and net energy (NE) compared to the LME diet. Overall, these findings suggest that 0.04% GAA is the ideal dose of broilers fed the LME diet, which can significantly improve the growth performance and carcass characteristics by modulation of creatine metabolism through elevating serum CK activity and arginine concentration.

Guanidinoacetic acid (GAA) has been found to elevate energy utilization efficiency in broilers; however, the underlying mechanisms remain unclear. We investigated the effects of GAA addition in low metabolizable energy (LME) diet on growth performance, carcass characteristics and serum biochemical indices of broilers, and found that GAA addition linearly or quadratically dropped the feed conversion ratio from 22 to 42 and 1 to 42 d of age, abdominal fat rate on day 42, serum alanine aminotransferase on day 21, and serum creatinine on days 21 and 42, elevated breast muscle and leg muscle rate on day 42, serum creatine kinase, alkaline phosphatase, as well as lactate dehydrogenase on days 21 or 22. The dietary optimal GAA levels were 0.03%-0.08% based on the best-fitted quadratic models of the above parameters. Thus, further analysis was conducted and found that 0.04% GAA reversed the reduction of arginine, 3-methyhistidine, and 1-methylhistidine and improved the ME intake and net energy compared to the LME diet. These findings suggested that 0.04% GAA is the ideal dose for enhancing the energy utilization of broilers fed the LME diet, GAA addition can significantly improve the growth performance by elevating energy utilization efficiency through modulation serum metabolite profile.

L-arginine metabolism inhibits arthritis and inflammatory bone loss.

OBJECTIVES: To investigate the effect of the L-arginine metabolism on arthritis and inflammation-mediated bone loss. METHODS: L-arginine was applied to three arthritis models (collagen-induced arthritis, serum-induced arthritis and human TNF transgenic mice). Inflammation was assessed clinically and histologically, while bone changes were quantified by µCT and histomorphometry. In vitro, effects of L-arginine on osteoclast differentiation were analysed by RNA-seq and mass spectrometry (MS). Seahorse, Single Cell ENergetIc metabolism by profilIng Translation inHibition and transmission electron microscopy were used for detecting metabolic changes in osteoclasts. Moreover, arginine-associated metabolites were measured in the serum of rheumatoid arthritis (RA) and pre-RA patients. RESULTS: L-arginine inhibited arthritis and bone loss in all three models and directly blocked TNFα-induced murine and human osteoclastogenesis. RNA-seq and MS analyses indicated that L-arginine switched glycolysis to oxidative phosphorylation in inflammatory osteoclasts leading to increased ATP production, purine metabolism and elevated inosine and hypoxanthine levels. Adenosine deaminase inhibitors blocking inosine and hypoxanthine production abolished the inhibition of L-arginine on osteoclastogenesis in vitro and in vivo. Altered arginine levels were also found in RA and pre-RA patients. CONCLUSION: Our study demonstrated that L-arginine ameliorates arthritis and bone erosion through metabolic reprogramming and perturbation of purine metabolism in osteoclasts.

The impact of inter- and intra-species spore density on germination of the food spoilage fungus Aspergillus niger.

Aspergilli can be used to produce food but can spoil it as well. Both food production and spoilage are initiated by germination of the conidia of these fungi that have been introduced by inoculation and contamination, respectively. Germination of these spores includes activation, swelling, establishment of cell polarity, and formation of a germ tube. So far, only quantitative single-species germination studies of fungal spores have been performed. Here, spore germination of the food spoilage fungus Aspergillus niger was studied quantitatively in mono-culture or when mixed with other food-relevant aspergilli (Aspergillus nidulans, Aspergillus terreus, Aspergillus clavatus, and Aspergillus oryzae). In the presence of the germination inducing amino acids proline or alanine, but not in the case of the lowly inducing amino acid arginine, the incidence of swelling and germ tube formation was reduced when 35,000 extra conidia of Aspergillus niger were added to wells containing 5000 of these spores. Adding 35,000 spores of one of the other aspergilli also did not have an effect on germination in the presence of arginine, but the germination inhibition was stronger when compared to the extra A. niger spores in the case of alanine. A similar effect was obtained with proline. Together, results show that the germination of A. niger conidia is impacted by the density of its own spores and that of other aspergilli under favorable nutritional conditions. These results increase our understanding of food spoilage by fungi and can be used to optimize food production with fungi.

Arginine-Enhanced Antimicrobial Activity of Nanozymes against Gram-Negative Bacteria.

The continuous reduction of clinically available antibiotics has made it imperative to exploit more effective antimicrobial therapies, especially for difficult-to-treat Gram-negative pathogens. Herein, it is shown that the combination of an antimicrobial nanozyme with the clinically compatible basic amino acid L-arginine affords a potent treatment for infections with Gram-negative pathogens. In particular, the antimicrobial activity of the antimicrobial nanozyme is dramatically increased by ≈1000-fold after L-arginine stimulation. Specifically, the combination therapy enhances bacterial outer and inner membrane permeability and promotes intracellular reactive oxygen species (ROS) generation. Moreover, the metabolomic and transcriptomic results reveal that combination treatment leads to the increased ROS-mediated damage by inhibiting the tricarboxylic acid cycle and oxidative phosphorylation, thereby inducing an imbalance of the antioxidant and oxidant systems. Importantly, L-arginine dramatically significantly accelerates the healing of infected wounds in mouse models of multidrug-resistant peritonitis-sepsis and skin wound infection. Overall, this work demonstrates a novel synergistic antibacterial strategy by combining the antimicrobial nanozymes with L-arginine, which substantively facilitates the nanozyme-mediated killing of pathogens by promoting ROS production.

Pylorus ligation-induced hyperacidity: synergistic prophylactic effects of linagliptin and L-arginine via up-regulation of EP4 receptor subtype and improvement of vascular endothelial damage.

Gastric hyperacidity and ulceration are chronic diseases characterized by repeated healing followed by re-exacerbation. The study aims to protect against gastric hyperacidity without interfering with gastric acid secretion. Pylorus ligation-induced hyperacidity is commonly utilized in the induction of gastric ulcers.Forty-two rats were distributed into seven groups (n = 6). Group I comprised sham-operated group. Group II served as pylorus-ligation group. Groups III-VII were given oral Linagliptin (LN; 3 and 6 mg/kg), L-arginine (LA; 150 and 300 mg/kg) and their combination (LN 3 + LA 150 mg/kg), respectively for 7 days. On the 8th day, groups II-VII were subjected to pylorus-ligation.Treatment of pylorus-ligated rats with LN, LA and their combination improved the gastric hyperacidity as exhibited by a marked reduction in the gastric juice volume, total and free acidities and pepsin contents with a noticeable increase in pH. Pre-treatment with LN, LA and their combination showed a marked alleviation in the gastric inflammatory indicators evidenced by reduction in the gastric levels of MCP-1and Il-1ß as well as elevation of eNOS levels versus the sham-operated group. A marked up-regulation in the gastric gene expression of PGE, EP4 and VEGF accompanied by an improvement of the histopathologic pictures/scores, and TNF-α and caspase-3 immuno-staining were also recorded.By estimating the combination-index, it can be concluded that combining LN with LA exhibited prophylactic synergistic effects in ameliorating pylorus ligated-induced hyperacidity, mainly via up-regulation of EP4 receptor and improvement of vascular endothelial damage through VEGF expression in gastric mucosa.

Guanidinylated bioactive chitosan-based injectable hydrogels with pro-angiogenic and mechanical properties for accelerated wound closure.

Wound healing is a complex process involving the concerted action of many genes and signaling pathways, with angiogenesis being crucial for expediting wound closure. Dressings that possess pro-angiogenic properties are increasingly recognized as attractive candidates for wound care. Drawing inspiration from the active closure of wounds in embryos, we have developed a thermo-responsive hydrogel with mechanoactive properties, combining vascular regeneration and skin wound contraction to accelerate healing. The significant improvement in vascular reconstruction is attributed to the synergistic effect of arginine and deferoxamine (DFO) released from the hydrogels. Additionally, the contraction force of the hydrogel actively promotes skin closure in wounds. Remarkably, groups treated with hydroxybutyl chitosan methacrylate combined with arginine (HBC_m_Arg/DFO) exhibited increased vascularization, and greater wound maturity, leading to enhanced healing. These results highlight the synergistic impact of pro-angiogenic and mechanical properties of the HBC_m_Arg/DFO hydrogel in accelerating wound healing in rats.

Interaction with lipopolysaccharide is key to efficacy of tryptophan- and arginine-rich α-melanocyte-stimulating hormone analogs against Gram-negative bacteria.

Aim: In order to search for novel antibacterial therapeutics against Gram-negative bacteria, the antibacterial efficacies and mechanism of action of tryptophan- and arginine-rich α-melanocyte-stimulating hormone analogs were investigated. Materials & methods: We performed a killing assay to determine their efficacy; fluorescence, microscopic studies were used to understand their mechanism and peptide-lipopolysaccharide interaction. A checkerboard assay was used to find the effective combination of peptide and antibiotics. Results: Ana-peptides displayed good killing activity against Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Their strong interaction with lipopolysaccharide damaged the bacterial membranes and led to their subsequent death. Ana-5, the highest cationic and hydrophobic analog, emerged as the most potent peptide, showing synergistic action with rifampicin and erythromycin. Conclusion: Ana-5 can be presented as an important therapeutic candidate against bacterial infections.

Bacteria can cause infections. These infections are becoming harder to treat, because excessive use of antibiotics can cause these bacteria to become less susceptible to medicine. In hospitals, these bacteria can cause infections in the lungs, urinary tract, blood, or on the skin. Our bodies make small molecules called antimicrobial peptides (AMPs) to fight against bacteria. AMPs can weaken or quickly destroy bacteria by attaching to their surfaces and breaking them down. Our laboratory has made an AMP called Ana-5. Using Ana-5 with regular medicine is better at killing bacteria. Ana-5 is not only good at fighting these bacteria, but may also help to prevent future infections.

Glucose microenvironment sensitive degradation of arginine modified calcium sulfate reinforced poly(lactide-co-glycolide) composite scaffolds.

Poly(lactide-co-glycolide) (PLGA) and calcium sulfate composites are promising biodegradable biomaterials but are still challenging to use in people with high levels of blood glucose or diabetes. To date, the influence of glucose on their degradation has not yet been elucidated and thus calls for more research attention. Herein, a novel calcium sulfate whisker with L-arginine was used to effectively tune its crystal morphology and was employed as a reinforced phase to construct the PLGA-based composite scaffolds (ArgCSH/PLGA) with a sleeve porous structure. ArgCSH/PLGA showed excellent elastic modulus and strength in the compression and bending models. Moreover, an in vitro immersion test showed that ArgCSH/PLGA possessed degradation and redeposition behaviors sensitive to glucose concentration, and the adsorbed Arg played a crucial role in the degradation process. The subsequent cell functional evaluation showed that ArgCSH could effectively protect cells from damage caused by AGEs and promote osteogenic differentiation. The corresponding degradation products of ArgCSH/PLGA displayed the ability to regulate osteoblast bone differentiation and accelerate matrix mineralization. These findings provide new insights into the interaction between biomaterials and the physiological environment, which may be useful in expanding the targeted choice of efficient bone graft biodegradable materials for diabetic osteoporosis.