l-arginine and l-lysine supplementation to NaCl tenderizes porcine meat by promoting myosin extraction and actomyosin dissociation.

This study investigated the individual and combined effects of l-arginine, l-lysine, and NaCl on the ultrastructure of porcine myofibrils to uncover the mechanism underlying meat tenderization. Arg or Lys alone shortened A-bands and damaged M-lines, while NaCl alone destroyed M- and Z-lines. Overall, Arg and Lys cooperated with NaCl to destroy the myofibrillar ultrastructure. Moreover, these two amino acids conjoined with NaCl to increase myosin solubility, actin band intensity, and the protein concentration of the actomyosin supernatant. However, they decreased the turbidity and particle size of both myosin and actomyosin solutions, and the remaining activities of Ca2+- and Mg2+-ATPase. The current results revealed that Arg/Lys combined with NaCl to extract myosin and dissociate actomyosin, thereby aggravating the destruction of the myofibrillar ultrastructure. The present results provide a good explanation for the previous phenomenon that Arg and Lys cooperated with NaCl to improve meat tenderness.

An investigation of excipients for a stable Orf viral vector formulation.

The Orf virus (ORFV) is a promising candidate for vector vaccines as well as for immunomodulatory and oncolytic therapies. However, few publications are available on its infectivity degradation or on suitable additives for prolonging its viral stability. In this study, the non-supplemented ORFV itself showed a very high stability at storage temperatures up to 28 °C, with a linear titer loss of 0.10 log infectious particles per day at 4 °C over a period of five weeks. To prolong this inherent stability, thirty additives, i.e., detergents, sugars, proteins, salts, and buffers as well as amino acids, were tested for their time- and temperature-dependent influence on the ORFV infectivity. A stabilizing effect on the infectivity was identified for the addition of all tested proteins, i.e., gelatine, bovine serum albumin, and recombinant human serum albumin (rHSA), of several sugars, i.e., mannitol, galactose, sucrose, and trehalose, of amino acids, i.e., arginine and proline, of the detergent Pluronic F68, and of the salt Na2SO4. The infectivity preservation was especially pronounced for proteins in liquid and frozen formulations, sugars in frozen state, and arginine und Pluronic in liquid formulations at high storage temperatures (37 °C). The addition of 1% rHSA with and without 5% sucrose was evaluated as a very stable formulation with a high safety profile and economic validity at storage temperatures up to 28 °C. At increased temperatures, the supplementation with 200 mM arginine performed better than with rHSA. In summary, this comprehensive data provides different options for a stable ORFV formulation, considering temperature, storage time, economic aspects, and downstream processing integrity.

A pan-influenza antibody inhibiting neuraminidase via receptor mimicry.

Rapidly evolving influenza A viruses (IAVs) and influenza B viruses (IBVs) are major causes of recurrent lower respiratory tract infections. Current influenza vaccines elicit antibodies predominantly to the highly variable head region of haemagglutinin and their effectiveness is limited by viral drift1 and suboptimal immune responses2. Here we describe a neuraminidase-targeting monoclonal antibody, FNI9, that potently inhibits the enzymatic activity of all group 1 and group 2 IAVs, as well as Victoria/2/87-like, Yamagata/16/88-like and ancestral IBVs. FNI9 broadly neutralizes seasonal IAVs and IBVs, including the immune-evading H3N2 strains bearing an N-glycan at position 245, and shows synergistic activity when combined with anti-haemagglutinin stem-directed antibodies. Structural analysis reveals that D107 in the FNI9 heavy chain complementarity-determinant region 3 mimics the interaction of the sialic acid carboxyl group with the three highly conserved arginine residues (R118, R292 and R371) of the neuraminidase catalytic site. FNI9 demonstrates potent prophylactic activity against lethal IAV and IBV infections in mice. The unprecedented breadth and potency of the FNI9 monoclonal antibody supports its development for the prevention of influenza illness by seasonal and pandemic viruses.

GAP positions catalytic H-Ras residue Q61 for GTP hydrolysis in molecular dynamics simulations, complicating chemical rescue of Ras deactivation.

Functional interaction of Ras signaling proteins with upstream, negative regulatory GTPase activating proteins (GAPs) represents a crucial step in cellular decision making related to growth and survival. Key components of the catalytic transition state for Ras deactivation by GAP-accelerated hydrolysis of Ras-bound guanosine triphosphate (GTP) are thought to include an arginine residue from the GAP (the arginine finger), a glutamine residue from Ras (Q61), and a water molecule that is likely coordinated by Q61 to engage in nucleophilic attack on GTP. Here, we use in-vitro fluorescence experiments to show that 0.1-100 mM concentrations of free arginine, imidazole, and other small nitrogenous molecule fail to accelerate GTP hydrolysis, even in the presence of the catalytic domain of a mutant GAP lacking its arginine finger (R1276A NF1). This result is surprising given that imidazole can chemically rescue enzyme activity in arginine-to-alanine mutant protein tyrosine kinases (PTKs) that share many active site components with Ras/GAP complexes. Complementary all-atom molecular dynamics (MD) simulations reveal that an arginine finger GAP mutant still functions to enhance Ras Q61-GTP interaction, though less extensively than wild-type GAP. This increased Q61-GTP proximity may promote more frequent fluctuations into configurations that enable GTP hydrolysis as a component of the mechanism by which GAPs accelerate Ras deactivation in the face of arginine finger mutations. The failure of small molecule analogs of arginine to chemically rescue catalytic deactivation of Ras is consistent with the idea that the influence of the GAP goes beyond the simple provision of its arginine finger. However, the failure of chemical rescue in the presence of R1276A NF1 suggests that the GAPs arginine finger is either unsusceptible to rescue due to exquisite positioning or that it is involved in complex multivalent interactions. Therefore, in the context of oncogenic Ras proteins with mutations at codons 12 or 13 that inhibit arginine finger penetration toward GTP, drug-based chemical rescue of GTP hydrolysis may have bifunctional chemical/geometric requirements that are more difficult to satisfy than those that result from arginine-to-alanine mutations in other enzymes for which chemical rescue has been demonstrated.

Near infrared light triggered ternary synergistic cancer therapy via L-arginine-loaded nanovesicles with modification of PEGylated indocyanine green.

L-arginine (L-Arg) is an important nitric oxide (NO) donor, and its exploration in NO gas therapy has received widespread attention. Application of nano-platforms that can efficiently deliver L-Arg and induce its rapid conversion to NO becomes a predominant strategy to achieve promising therapeutic effects in tumor treatment. Herein, an enhanced nano-vesicular system of ternary synergistic treatment combining NO therapy, photodynamic therapy (PDT) along with mild photothermal therapy (MPTT) was developed for cancer therapy. We integrated photosensitizer PEGylated indocyanine green (mPEG-ICG) into polyphosphazene PEP nano-vesicles through co-assembly and simultaneously encapsulated NO donor L-Arg into the vesicle center chambers to form mPEG-ICG/L-Arg co-loaded system IA-PEP. The unique nanostructure of vesicle provided considerable loading capacity for mPEG-ICG and L-Arg with 15.9% and 17.95% loading content, respectively, and efficiently prevented mPEG-ICG and L-Arg from leaking. Significantly, the reactive oxygen species (ROS) was produced by IA-PEP under 808 nm laser irradiation to perform PDT against tumors, which concurrently reacted with L-Arg to release NO and arouse gas therapy effectively. Moreover, the mild heat produced by IA-PEP could exhibit cooperative anti-tumor effect with minimal damage. As a consequence, in vivo antitumor investigation on nude mice bearing xenograft MCF-7 tumors verified the potent anti-tumor efficacy of IA-PEP under 808 nm laser irradiation with complete tumor elimination. Taken together, the IA-PEP nano-vesicle system designed in this work may provide a promising treatment paradigm for synergistic cancer treatment. STATEMENT OF SIGNIFICANCE: Nitric oxide (NO) gas therapy has drawn widespread attention due to its "green" treatment paradigm with negligible side effects. L-arginine (L-Arg) is an important NO donor. However, how to efficiently deliver L-Arg and induce NO generation remains a big challenge since L-Arg is a water-soluble small molecule. Herein, we developed a nano-vesicle system IA-PEP to integrate photosensitizer PEGylated indocyanine green and L-Arg with high loading content and to produce a ternary synergistic treatment combining NO therapy, photodynamic therapy (PDT) along with mild-temperature photothermal therapy (MPTT) under 808 nm laser irradiation. The in vivo investigation on nude mice bearing xenograft MCF-7 tumors verified its potent anti-tumor efficacy with complete tumor elimination.

Concentration-Dependent Multi-Potentiality of L-Arginine: Antimicrobial Effect, Hydroxyapatite Stability, and MMPs Inhibition.

This study's objective was to examine L-arginine (L-arg) supplementation's effect on mono-species biofilm (Streptococcus mutans/Streptococcus sanguinis) growth and underlying enamel substrates. The experimental groups were 1%, 2%, and 4% arg, and 0.9% NaCl was used as the vehicle control. Sterilised enamel blocks were subjected to 7-day treatment with test solutions and S. mutans/S. sanguinis inoculum in BHI. Post-treatment, the treated biofilms stained for live/dead bacterial cells were analysed using confocal microscopy. The enamel specimens were analysed using X-ray diffraction crystallography (XRD), Raman spectroscopy (RS), and transmission electron microscopy (TEM). The molecular interactions between arg and MMP-2/MMP-9 were determined by computational molecular docking and MMP assays. With increasing arg concentrations, bacterial survival significantly decreased (p < 0.05). The XRD peak intensity with 1%/2% arg was significantly higher than with 4% arg and the control (p < 0.05). The bands associated with the mineral phase by RS were significantly accentuated in the 1%/2% arg specimens compared to in other groups (p < 0.05). The TEM analysis revealed that 4% arg exhibited an ill-defined shape of enamel crystals. Docking of arg molecules to MMPs appears feasible, with arg inhibiting MMP-2/MMP-9 (p < 0.05). L-arginine supplementation has an antimicrobial effect on mono-species biofilm. L-arginine treatment at lower (1%/2%) concentrations exhibits enamel hydroxyapatite stability, while the molecule has the potential to inhibit MMP-2/MMP-9.

Structural determination of arginine-linked cisplatin complexes via IRMPD action spectroscopy: arginine binds to platinum via NO- binding mode.

Cisplatin, (NH3)2PtCl2, has been known as a successful metal-based anticancer drug for more than half a century. Its analogue, Argplatin, arginine-linked cisplatin, (Arg)PtCl2, is being investigated because it exhibits reactivity towards DNA and RNA that differs from that of cisplatin. In order to understand the basis for its altered reactivity, the deprotonated and sodium cationized forms of Argplatin, [(Arg-H)PtCl2]- and [(Arg)PtCl2 + Na]+, are examined by infrared multiple photon dissociation (IRMPD) action spectroscopy in the IR fingerprint and hydrogen-stretching regions. Complementary electronic structure calculations are performed using density functional theory approaches to characterize the stable structures of these complexes and to predict their infrared spectra. Comparison of the theoretical IR spectra predicted for various stable conformations of these Argplatin complexes to their measured IRMPD spectra enables determination of the binding mode(s) of Arg to the Pt metal center to be identified. Arginine is found to bind to Pt in a bidentate fashion to the backbone amino nitrogen and carboxylate oxygen atoms in both the [(Arg-H)PtCl2]- and [(Arg)PtCl2 + Na]+ complexes, the NO- binding mode. The neutral side chain of Arg also interacts with the Pt center to achieve additional stabilization in the [(Arg-H)PtCl2]- complex. In contrast, Na+ binds to both chlorido ligands in the [(Arg)PtCl2 + Na]+ complex and the protonated side chain of Arg is stabilized via hydrogen-bonding interactions with the carboxylate moiety. These findings are consistent with condensed-phase results, indicating that the NO- binding mode of arginine to Pt is preserved in the electrospray ionization process even under variable pH and ionic strength.

Mesoporous polydopamine-coated hydroxyapatite nano-composites for ROS-triggered nitric oxide-enhanced photothermal therapy of osteosarcoma.

In this paper, MPDA@hydroxyapatite nanocomposites (MPHA NCs) were prepared and applied to develop a novel reactive oxygen species (ROS)-triggered nitric oxide (NO)-enhanced photothermal therapy nanocomposite system composed of indocyanine green (ICG)/L-arginine-MPDA@HAp (AI-MPHA NCs) for displaying both NO gas therapy and photothermal osteosarcoma treatment. The nanosystem exhibited a mesoporous and core-shell structure and high ICG loading efficiency (about 90%). Under near infrared (NIR) irradiation, the AI-MPHA NCs could not only produce heat but also generate reactive oxygen species (ROS), inducing the catalysis of L-Arg to obtain NO. Under NIR irradiation, the AI-MPHA NCs achieved osteosarcoma ablation by a synergistic combination of photothermal therapy and NO-gas therapy. Additionally, the cell viability of MG-63 cells decreased to 23.6% (co-incubated with AI-MPHA NCs) under irradiation with a power density at 1.0 W cm-2 for 10 min. The study proposed a novel nano-platform for NO-enhanced photothermal therapy of osteosarcoma.

Advances and impact of arginine-based materials in wound healing.

In studies on wound-dressing materials, bioactive materials have been developed rapidly to accelerate wound healing. In recent years, scientists have studied arginine as a bioactive component due to its excellent biosafety, antimicrobial properties and therapeutic effects on wound healing. Surprisingly, arginine therapy is also used under specific pathological conditions, such as diabetes and trauma/hemorrhagic shock. Due to the broad utilization of arginine-assisted therapy, we present the unique properties of arginine for healing lesions of damaged tissue and examined multiple arginine-based systems for the application of wound healing. This review shows that arginine-based therapy can be separated in two categories: direct supplemental approaches of free arginine, and indirect approaches based on arginine derivatives in which modified arginine can be released after biodegradation. Using these two pathways, arginine-based therapy may prove to be a promising strategy in the development of wound curative treatments.

Immune Modulatory Activities of Arginyl-Fructose (AF) and AF-Enriched Natural Products in In-Vitro and In-Vivo Animal Models.

The immune system plays an important role in maintaining body homeostasis. Recent studies on the immune-enhancing effects of ginseng saponins have revealed more diverse mechanisms of action. Maillard reaction that occurs during the manufacturing processes of red ginseng produces a large amount of Amadori rearrangement compounds (ARCs), such as arginyl-fructose (AF). The antioxidant and anti-hyperglycemic effects of AF have been reported. However, the possible immune enhancing effects of non-saponin ginseng compounds, such as AF, have not been investigated. In this study the effects of AF and AF-enriched natural product (Ginofos, GF) on proliferation of normal mouse splenocytes were evaluated in vitro and male BALB/c mice models. The proliferation of splenocytes treated with mitogens (concanavalin A, lipopolysaccharide) were further increased by addition of AF (p < 0.01) or GF (p < 0.01), in a dose dependent manner. After the 10 days of oral administration of compounds, changes in weights of spleen and thymus, serum immunoglobulin, and expression of cytokines were measured as biomarkers of immune-enhancing potential in male BALB/c mice model. The AF or GF treated groups had higher weights of the thymus (0.94 ± 0.25 and 0.86 ± 0.18, p < 0.05, respectively) than that of cyclophosphamide treated group (0.59 ± 0.18). This result indicates that AF or AF-enriched extract (GF) increased humoral immunity against CY-induced immunosuppression. In addition, immunoglobulin contents and expression of cytokines including IgM (p < 0.01), IgG (p < 0.05), IL-2 (p < 0.01), IL-4 (p < 0.01), IL-6 (p < 0.01), and IFN-γ (p < 0.05) were also significantly increased by supplementation of AF or GF. These results indicate that AF has immune enhancing effects by activation of adaptive immunity via increase of expression of immunoglobulins and cytokines such as IgM, IgG, IL-2, IL-4, IL-6 and thereby proliferating the weight of thymus. Our findings provide a pharmacological rationale for AF-enriched natural products such as ginseng and red ginseng that can possibly have immune-enhancement potential and should be further evaluated.

How arginine derivatives alter the stability of lipid membranes: dissecting the roles of side chains, backbone and termini.

Arginine (R)-rich peptides constitute the most relevant class of cell-penetrating peptides and other membrane-active peptides that can translocate across the cell membrane or generate defects in lipid bilayers such as water-filled pores. The mode of action of R-rich peptides remains a topic of controversy, mainly because a quantitative and energetic understanding of arginine effects on membrane stability is lacking. Here, we explore the ability of several oligo-arginines R[Formula: see text] and of an arginine side chain mimic R[Formula: see text] to induce pore formation in lipid bilayers employing MD simulations, free-energy calculations, breakthrough force spectroscopy and leakage assays. Our experiments reveal that R[Formula: see text] but not R[Formula: see text] reduces the line tension of a membrane with anionic lipids. While R[Formula: see text] peptides form a layer on top of a partly negatively charged lipid bilayer, R[Formula: see text] leads to its disintegration. Complementary, our simulations show R[Formula: see text] causes membrane thinning and area per lipid increase beside lowering the pore nucleation free energy. Model polyarginine R[Formula: see text] similarly promoted pore formation in simulations, but without overall bilayer destabilization. We conclude that while the guanidine moiety is intrinsically membrane-disruptive, poly-arginines favor pore formation in negatively charged membranes via a different mechanism. Pore formation by R-rich peptides seems to be counteracted by lipids with PC headgroups. We found that long R[Formula: see text] and R[Formula: see text] but not short R[Formula: see text] reduce the free energy of nucleating a pore. In short R[Formula: see text], the substantial effect of the charged termini prevent their membrane activity, rationalizing why only longer [Formula: see text] are membrane-active.

ICG/l-Arginine Encapsulated PLGA Nanoparticle-Thermosensitive Hydrogel Hybrid Delivery System for Cascade Cancer Photodynamic-NO Therapy with Promoted Collagen Depletion in Tumor Tissues.

Photodynamic therapy (PDT) is promising for clinical cancer therapy; however, the efficacy was limited as an individual treatment regimen. Here, an approach synergistically combining PDT and nitric oxide (NO) gas therapy along with destruction of the tumor extracellular matrix (ECM) was presented to eliminate cancer. Specifically, the NO donor l-arginine (l-Arg) and the photosensitizer indocyanine green (ICG) were co-encapsulated in poly(lactic-glycolic acid) (PLGA) nanoparticles and then loaded into the poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCL-PEG-PCL) hydrogel to develop an injectable, thermosensitive dual drug delivery system (PLGA@ICG@l-Arg/Gel). Significantly, reactive oxygen species (ROS) produced by PLGA@ICG@l-Arg/Gel under near-infrared (NIR) light irradiation could not only result in the apoptosis of cancer cells but also oxidize l-Arg to generate NO, which could suppress the proliferation of cancer cells. Moreover, ROS could further oxidize NO to generate peroxynitrite anions (ONOO-). ONOO- could activate matrix metalloproteinases (MMPs), which notably degraded collagen in ECM so as to damage the tumor microenvironment. PLGA@ICG@l-Arg/Gel significantly increased the antitumor efficacy against highly malignant 4T1 tumors in mice. Taken together, PLGA@ICG@l-Arg/Gel is a multifunctional platform that provides a novel strategy for cancer treatment with cascade amplification of the ROS oxidation effect, which holds great potential in clinical translation.

Determinants of adenine-mutagenesis in diversity-generating retroelements.

Diversity-generating retroelements (DGRs) vary protein sequences to the greatest extent known in the natural world. These elements are encoded by constituents of the human microbiome and the microbial 'dark matter'. Variation occurs through adenine-mutagenesis, in which genetic information in RNA is reverse transcribed faithfully to cDNA for all template bases but adenine. We investigated the determinants of adenine-mutagenesis in the prototypical Bordetella bacteriophage DGR through an in vitro system composed of the reverse transcriptase bRT, Avd protein, and a specific RNA. We found that the catalytic efficiency for correct incorporation during reverse transcription by the bRT-Avd complex was strikingly low for all template bases, with the lowest occurring for adenine. Misincorporation across a template adenine was only somewhat lower in efficiency than correct incorporation. We found that the C6, but not the N1 or C2, purine substituent was a key determinant of adenine-mutagenesis. bRT-Avd was insensitive to the C6 amine of adenine but recognized the C6 carbonyl of guanine. We also identified two bRT amino acids predicted to nonspecifically contact incoming dNTPs, R74 and I181, as promoters of adenine-mutagenesis. Our results suggest that the overall low catalytic efficiency of bRT-Avd is intimately tied to its ability to carry out adenine-mutagenesis.

Arginine Forks Are a Widespread Motif to Recognize Phosphate Backbones and Guanine Nucleobases in the RNA Major Groove.

RNA recognition by proteins is central to biology. Here we demonstrate the existence of a recurrent structural motif, the "arginine fork", that codifies arginine readout of cognate backbone and guanine nucleobase interactions in a variety of protein-RNA complexes derived from viruses, metabolic enzymes, and ribosomes. Nearly 30 years ago, a theoretical arginine fork model was posited to account for the specificity between the HIV-1 Tat protein and TAR RNA. This model predicted that a single arginine should form four complementary contacts with nearby phosphates, yielding a two-pronged backbone readout. Recent high-resolution structures of TAR-protein complexes have unveiled new details, including (i) arginine interactions with the phosphate backbone and the major-groove edge of guanine and (ii) simultaneous cation-π contacts between the guanidinium group and flanking nucleobases. These findings prompted us to search for arginine forks within experimental protein-RNA structures retrieved from the Protein Data Bank. The results revealed four distinct classes of arginine forks that we have defined using a rigorous but flexible nomenclature. Examples are presented in the context of ribosomal and nonribosomal interfaces with analysis of arginine dihedral angles and structural (suite) classification of RNA targets. When arginine fork chemical recognition principles were applied to existing structures with unusual arginine-guanine recognition, we found that the arginine fork geometry was more consistent with the experimental data, suggesting the utility of fork classifications to improve structural models. Software to analyze arginine-RNA interactions has been made available to the community.

Highly Enhanced Antitumor Immunity by a Three-Barreled Strategy of the l-Arginine-Promoted Nanovaccine and Gene-Mediated PD-L1 Blockade.

Weak T cell responses and immune checkpoints within tumors could be two key factors for limiting antitumor efficacy in the field of cancer immunotherapy. Thus, the combined strategy of tumor vaccines and immune checkpoint blockade has been widely studied and expected to boost antitumor immune responses. Herein, we first developed a two-barreled strategy to combine the nanovaccine with a gene-mediated PD-L1 blockade. On the one hand, polyethyleneimine (PEI) worked as a vaccine carrier to codeliver the antigen ovalbumin (OVA) and the adjuvant unmethylated cytosine-phosphate-guanine (CpG) to formulate the PEI/OVA/CpG nanovaccine through electrostatic binding, which realized both dendritic cell activation and antigen cross-presentation enhancement. On the other hand, the PD-L1 silence gene was loaded by PEI to form PEI/pshPD-L1 complexes, which were further in situ shielded by aldehyde-modified polyethylene glycol (OHC-PEG-CHO) via pH-responsive Schiff base bonds. The formed pshPD-L1@NPs could decrease PD-L1 expression on the tumor cells. However, such a combined two-barreled strategy improved feebly for tumor inhibition in comparison with monotherapy, exhibiting the antagonistic effect, which might be due to the limited T cell response enhancement in the tumor microenvironment. To solve this problem, we have further developed a three-barreled strategy to combine oral administration of l-arginine, which worked as an amplifier to induce robust T cell response enhancement, without causing the upregulation of other negative immune regulators. Superior antitumor behavior and tumor rechallenge protection were realized by the three-barreled strategy in B16F10-OVA (B16-OVA)-bearing mice. The unique three-barreled strategy we developed might offer a novel clinical therapeutic treatment.

Translational Detection of Nonproteinogenic Amino Acids Using an Engineered Complementary Cell-Free Protein Synthesis Assay.

We developed a simple and rapid method for analyzing nonproteinogenic amino acids that does not require conventional chromatographic equipment. In this technique, nonproteinogenic amino acids were first converted to a proteinogenic amino acid through in vitro metabolism in a cell extract. The proteinogenic amino acid generated from the nonproteinogenic precursors were then incorporated into a reporter protein using a cell-free protein synthesis system. The titers of the nonproteinogenic amino acids could be readily quantified by measuring the activity of reporter proteins. This method, which combines the enzymatic conversion of target amino acids with translational analysis, makes amino acid analysis more accessible while minimizing the cost and time requirements. We anticipate that the same strategy could be extended to the detection of diverse biochemical molecules with clinical and industrial implications.

IFN-γ Activates the TLR4-CCL5 Signaling Through Reducing Arginine Level, Leading to Enhanced Susceptibility of Bovine Mammary Epithelial Cells to Staphylococcus aureus.

Dairy cow mastitis is a common bacterial infectious disease which seriously threatens the development of the dairy cow industry. Previous studies have found that increased IFN-γ expression in dairy cows makes dairy cows more susceptible to mastitis, but the underlying mechanism is still not known. In this study, we utilized the in vitro bovine mammary epithelial cells (BMECs) model to explore the molecular mechanism via transcriptome sequencing technology, immunofluorescence, and Western blotting. It was found that IFN-γ promoted the adhesion and invasion of Staphylococcus aureus to BMECs through increasing the expression of TLR4-mediated CCL5 in BMECs. IFN-γ increased the activity of arginase II and reduced the level of arginine in cells, while the addition of arginine inhibited the expression of TLR4 and CCL5. An invasion experiment in mice further validated that IFN-γ treatment significantly increased the bacterial load in mammary glands and blood. However, the colonization and diffusion of S. aureus were interestingly decreased after Arg supplement. These data reveal that increased IFN-γ reduces arginine levels and activates TLR4-CCL5 signaling, leading to enhanced susceptibility of BMECs to S. aureus. Our findings are helpful to understand the pathogenesis of dairy cow mastitis and provide a theoretical basis for improvement of mastitis resistance in dairy cows.

Methanol Extract of Dicranopteris linearis Leaves Attenuate Pain via the Modulation of Opioid/NO-Mediated Pathway.

Dicranopteris linearis leaf has been reported to exert antinociceptive activity. The present study elucidates the possible mechanisms of antinociception modulated by the methanol extract of D. linearis leaves (MEDL) using various mouse models. The extract (25, 150, and 300 mg/kg) was administered orally to mice for 30 min priot to subjection to the acetic acid-induced writhing-, hot plate- or formalin-test to establish the antinociceptive profile of MEDL. The most effective dose was then used in the elucidation of possible mechanisms of action stage. The extract was also subjected to the phytochemical analyses. The results confirmed that MEDL exerted significant (p < 0.05) antinociceptive activity in those pain models as well as the capsaicin-, glutamate-, bradykinin- and phorbol 12-myristate 13-acetate (PMA)-induced paw licking model. Pretreatment with naloxone (a non-selective opioid antagonist) significantly (p < 0.05) reversed MEDL effect on thermal nociception. Only l-arginine (a nitric oxide (NO) donor) but not N(ω)-nitro-l-arginine methyl ester (l-NAME; a NO inhibitor) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; a specific soluble guanylyl cyclase inhibitor) significantly (p < 0.05) modified MEDL effect on the writhing test. Several polyphenolics and volatile antinociceptive compounds were detected in MEDL. In conclusion, MEDL exerted the opioid/NO-mediated antinociceptive activity, thus, justify D. linearis as a potential source for new analgesic agents development.

Near-Infrared Light-Triggered Nitric-Oxide-Enhanced Photodynamic Therapy and Low-Temperature Photothermal Therapy for Biofilm Elimination.

Photothermal treatment (PTT) involving a combination of therapeutic modalities recently emerged as an efficient alternative for combating biofilm. However, PTT-related local high temperature may destroy the surrounding healthy tissues. Herein, we present an all-in-one phototherapeutic nanoplatform consisting of l-arginine (l-Arg), indocyanine green (ICG), and mesoporous polydopamine (MPDA), namely, AI-MPDA, to eliminate the already-formed biofilm. The fabrication process included surface modification of MPDA with l-Arg and further adsorption of ICG via π-π stacking. Under near-infrared (NIR) exposure, AI-MPDA not only generated heat but also produced reactive oxygen species, causing a cascade catalysis of l-Arg to release nitric oxide (NO). Under NIR irradiation, biofilm elimination was attributed to the NO-enhanced photodynamic therapy and low-temperature PTT (≤45 °C). Notably, the NIR-triggered all-in-one strategy resulted in severe destruction of bacterial membranes. The phototherapeutic AI-MPDA also displayed good cytocompatibility. NIR-irradiated AI-MPDA nanoparticles not only prevented bacterial colonization but also realized a rapid recovery of infected wounds. More importantly, the all-in-one phototherapeutic platform displayed effective biofilm elimination with an efficiency of around 100% in a abscess formation model. Overall, this low-temperature phototherapeutic platform provides a reliable tool for combating already-formed biofilms in clinical applications.

Homocysteine and Asymmetrical Dimethylarginine in Diabetic Rats Treated with Docosahexaenoic Acid-Loaded Zinc Oxide Nanoparticles.

Hyperglycemia, the hallmark of diabetes mellitus, is considered one of the endothelial dysfunction risk factors, the main reason of vascular complication. In this study, we aimed to evaluate homocysteine (Hcy) and asymmetrical dimethylarginine (ADMA) levels in diabetic rats and the possibility to attenuate the elevation of these two parameters by supplementation of docosahexaenoic acid (DHA) alone or loaded zinc oxide nanoparticles (ZnONPs) to improve endothelial dysfunction in streptozotocin (STZ)-induced diabetic rats. Forty male albino rats weighing 180-200 g were classified as control, diabetic, diabetic treated with DHA, and diabetic treated with DHA-loaded zinc oxide nanoparticles (DHA/ZnONPs) groups. Fasting blood glucose, insulin, ADMA, Hcy, and nitric oxide (NO) were estimated. Fatty acids (linoleic acid (LA), arachidonic acid (AA), DHA, α-linolenic acid (ALA), and oleic acid (OA)) were also evaluated by reversed phase HPLC using a UV detector. The results showed that fasting blood sugar, insulin resistance, LA, AA, OA, ADMA, and Hcy increased significantly in diabetic rats compared with control while fasting insulin, DHA, ALA, and NO decreased significantly in diabetic rats. In both treated groups, fasting blood sugar, insulin resistance, LA, AA, OA, ADMA, and Hcy significantly decreased as compared with the diabetic group while fasting insulin, DHA, ALA, and NO were significantly increased. In conclusion, DHA and DHA/ZnONP supplementation protect against diabetic complications and improve endothelial dysfunction as well as hyperhomocysteinemia in diabetes. DHA/ZnONP-treated group appeared more efficient than DHA alone.