Exploring the predictive power of jejunal microbiome composition in clinical and subclinical necrotic enteritis caused by Clostridium perfringens: insights from a broiler chicken model.

BACKGROUND: Necrotic enteritis (NE) is a severe intestinal infection that affects both humans and poultry. It is caused by the bacterium Clostridium perfringens (CP), but the precise mechanisms underlying the disease pathogenesis remain elusive. This study aims to develop an NE broiler chicken model, explore the impact of the microbiome on NE pathogenesis, and study the virulence of CP isolates with different toxin gene combinations. METHODS: This study established an animal disease model for NE in broiler chickens. The methodology encompassed inducing abrupt protein changes and immunosuppression in the first experiment, and in the second, challenging chickens with CP isolates containing various toxin genes. NE was evaluated through gross and histopathological scoring of the jejunum. Subsequently, jejunal contents were collected from these birds for microbiome analysis via 16S rRNA amplicon sequencing, followed by sequence analysis to investigate microbial diversity and abundance, employing different bioinformatic approaches. RESULTS: Our findings reveal that CP infection, combined with an abrupt increase in dietary protein concentration and/or infection with the immunosuppressive variant infectious bursal disease virus (vIBDV), predisposed birds to NE development. We observed a significant decrease (p < 0.0001) in the abundance of Lactobacillus and Romboutsia genera in the jejunum, accompanied by a notable increase (p < 0.0001) in Clostridium and Escherichia. Jejunal microbial dysbiosis and severe NE lesions were particularly evident in birds infected with CP isolates containing cpa, netB, tpeL, and cpb2 toxin genes, compared to CP isolates with other toxin gene combinations. Notably, birds that did not develop clinical or subclinical NE following CP infection exhibited a significantly higher (p < 0.0001) level of Romboutsia. These findings shed light on the complex interplay between CP infection, the gut microbiome, and NE pathogenesis in broiler chickens. CONCLUSION: Our study establishes that dysbiosis within the jejunal microbiome serves as a reliable biomarker for detecting subclinical and clinical NE in broiler chicken models. Additionally, we identify the potential of the genera Romboutsia and Lactobacillus as promising candidates for probiotic development, offering effective alternatives to antibiotics in NE prevention and control.

Development of a multivalent adjuvanted inactivated vaccine against variant arthrotropic avian reoviruses.

Variant avian reoviruses (ARVs) are economically important emerging pathogens of poultry, which mainly affect young broiler chickens and cause significant production losses. Currently, there are no effective commercial vaccines available for control and prevention of emerging variant ARVs. In this study, monovalent inactivated adjuvated (20% Emulsigen D) broiler breeder vaccines containing antigens from ARV genotype cluster (C) group -2, -4, -5, or -6, and a multivalent vaccine containing antigens from all the four indicated genotypic cluster groups were developed and evaluated for their efficacy in protecting broiler progenies against homologous or heterologous ARV challenge. The use of monovalent or multivalent inactivated vaccines in a prime-boost immunization strategy induced the production of ARV specific antibodies in broiler breeders. The maternal antibodies were effectively transferred to broiler progenies. Broiler progenies obtained from immunized breeders demonstrated milder clinical symptoms and reduced gross and histopathological lesions after homologous ARV challenge. More severe gross and histological lesions were observed in challenged progenies from unvaccinated broiler breeders. However, cross protection was not observed when either of the monovalent-vaccine groups were challenged with a heterologous virus. In addition, the progenies from the unvaccinated ARV challenged control or heterologous ARV challenged vaccinated groups had significantly reduced body weight gain (p < 0.01) than the unchallenged-control, challenged-multivalent, or homologous ARV-challenged monovalent vaccine groups. However, homologous ARV challenged progenies in the multivalent or monovalent vaccine groups had similar body weight gain as the control unchallenged group with significantly reduced viral load (p < 0.01) in the gastrocnemius tendon tissue. This study indicates that broad-spectrum protection of broiler progenies from variant ARV infections is feasible through the development of multivalent vaccines after proper characterization, selection and incorporation of multiple antigens based on circulating ARV genotypes in targeted regions.

pH-controlled histone acetylation amplifies melanocyte differentiation downstream of MITF.

Tanning response and melanocyte differentiation are mediated by the central transcription factor MITF. This involves the rapid and selective induction of melanocyte maturation genes, while concomitantly the expression of other effector genes is maintained. In this study, using cell-based and zebrafish model systems, we report on a pH-mediated feed-forward mechanism of epigenetic regulation that enables selective amplification of the melanocyte maturation program. We demonstrate that MITF activation directly elevates the expression of the enzyme carbonic anhydrase 14 (CA14). Nuclear localization of CA14 leads to an increase of the intracellular pH, resulting in the activation of the histone acetyl transferase p300/CBP. In turn, enhanced H3K27 histone acetylation at selected differentiation genes facilitates their amplified expression via MITF. CRISPR-mediated targeted missense mutation of CA14 in zebrafish results in the formation of immature acidic melanocytes with decreased pigmentation, establishing a central role for this mechanism during melanocyte differentiation in vivo. Thus, we describe an epigenetic control system via pH modulation that reinforces cell fate determination by altering chromatin dynamics.

Non-invasive Oil-Based Method to Increase Topical Delivery of Nucleic Acids to Skin.

Topical delivery of nucleic acids to skin has huge prospects in developing therapeutic interventions for cutaneous disorders. In spite of initial success, clinical translation is vastly impeded by the constraints of bioavailability as well as stability in metabolically active environment of skin. Various physical and chemical methods used to overcome these limitations involve invasive procedures or compounds that compromise skin integrity. Hence, there is an increasing demand for developing safe skin penetration enhancers for efficient nucleic acid delivery to skin. Here, we demonstrate that pretreatment of skin with silicone oil can increase the transfection efficiency of non-covalently associated peptide-plasmid DNA nanocomplexes in skin ex vivo and in vivo. The method does not compromise skin integrity, as indicated by microscopic evaluation of cellular differentiation, tissue architecture, enzyme activity assessment, dye penetration tests using Franz assay, and cytotoxicity and immunogenicity analyses. Stability of nanocomplexes is not hampered on pretreatment, thereby avoiding nuclease-mediated degradation. The mechanistic insights through Fourier transform infrared (FTIR) spectroscopy reveal some alterations in the skin hydration status owing to possible occlusion effects of the enhancer. Overall, we describe a topical, non-invasive, efficient, and safe method that can be used to increase the penetration and delivery of plasmid DNA to skin for possible therapeutic applications.

Bioinspired Functionalized Melanin Nanovariants with a Range of Properties Provide Effective Color Matched Photoprotection in Skin.

Melanin and related polydopamine hold great promise; however, restricted fine-tunabilility limits their usefulness in biocompatible applications. In the present study, by taking a biomimetic approach, we synthesize peptide-derived melanin with a range of physicochemical properties. Characterization of these melanin polymers indicates that they exist as nanorange materials with distinct size distribution, shapes, and surface charges. These variants demonstrate similar absorption spectra but have different optical properties that correlate with particle size. Our approach enables incorporation of chemical groups to create functionalized polyvalent organic nanomaterials and enables customization of melanin. Further, we establish that these synthetic variants are efficiently taken up by the skin keratinocytes, display appreciable photoprotection with minimal cytotoxicity, and thereby function as effective color matched photoprotective agents. In effect we demonstrate that an array of functionalized melanins with distinct properties could be synthesized using bioinspired green chemistry, and these are of immense utility in generating customized melanin/polydopamine like materials.

A novel coumarin derivative, 8-methoxy chromen-2-one alleviates collagen induced arthritis by down regulating nitric oxide, NFκB and proinflammatory cytokines.

Ruta graveolens (Rue) is a well-known medicinal plant having anti-inflammatory and other healing properties. This contains many active phytochemicals such as coumarins which possess anti-inflammatory and anti-cancer activities. The present study was carried out to evaluate the therapeutic potential of a newly isolated coumarin derivative from rue plant, 8-methoxy-chromen-2-one (MCO) in the collagen induced arthritic (CIA) rat model. MCO showed inhibition of cytokines and NF-κB in LPS stimulated J774 cells which prompted its possible use in animal. In CIA, arthritic index and arthritic score reduced markedly within 15days of MCO treatment at doses of 2mg and 20mg per kg body weight. Alleviation of joint damage in CIA animals on treatment with MCO was evident from radiographic and histological data. Behavioral studies by open field tests also showed convalescence in the MCO treated CIA rats. Further, escalated plasma levels of pro-inflammatory cytokines TNF-α, IL-1ß and IL-6, and also nitric oxide reduced significantly with the treatment. All these results indicate the therapeutic efficacy of MCO and its possible use as an anti-arthritic drug.

N-terminally modified linear and branched spermine backbone dipeptidomimetics against planktonic and sessile methicillin-resistant Staphylococcus aureus.

Toward the discovery of useful therapeutic molecules, we report the design and synthesis of a focused library of new ultrashort N-terminally modified dipeptidomimetics, with or without modifications in the spermine backbone leading to linear (series 1) or branched (series 2) tryptophans, as antimicrobial agents. Eight peptidomimetics in the library showed good antibacterial activity (MICs of 1.77 to 14.2 µg/ml) against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis bacterial strains. Tryptophan fluorescence measurements on artificial bacterial or mammalian mimic membranes and assessment of the MRSA potential depolarization ability of the designed compounds revealed membrane interactions dependent on tryptophan positioning and N-terminal tagging. Among active peptidomimetics, compounds 1c and 1d were found to be nonhemolytic, displaying rapid bactericidal activity (at 4× MIC) against exponentially growing MRSA. Further, scanning electron microscopy of peptidomimetic 1c- and 1d-treated MRSA showed morphological changes with damage to cell walls, defining a membrane-active mode of action. Moreover, peptidomimetics 1c and 1d did not induce significant drug resistance in MRSA even after 17 passages. We also investigated the activity of these molecules against MRSA biofilms. At sub-MIC levels (∼2 to 4 µg/ml), both peptidomimetics inhibited biofilm formation. At concentrations higher than the MIC (35 to 140 µg/ml), peptidomimetics 1c and 1d significantly reduced the metabolic activity and biomass of mature (24-h) MRSA biofilms. These results were corroborated by confocal laser scanning microscopy (live/dead assay). The in vitro protease stability and lower cytotoxicity of peptidomimetics against peripheral blood mononuclear cells (PBMCs) support them being novel staphylocidal peptidomimetics. In conclusion, this study provides two peptidomimetics as potential leads for treatment of staphylococcal infections under planktonic and sessile conditions.

Proteomic analysis of global changes in protein expression during exposure of gamma radiation in Bacillus sp. HKG 112 isolated from saline soil.

A Gram-positive bacterium was isolated from the saline soils of Jangpura (U.P.), India, and showed high-level of radiation-resistant property and survived upto 12.5 kGy dose of gamma radiation. The 16S rDNA sequence of this strain was examined, identified as Bacillus sp. strain HKG 112, and was submitted to the NCBI GenBank (Accession No. GQ925432). The mechanism of radiation resistance and gene level expression were examined by proteomic analysis of whole-cell extract. Two proteins, 38 kDa and 86.5 kDa excised from SDS-PAGE, which showed more significant changes after radiation exposure, were identified by MALDI-TOF as being flagellin and S-layer protein, respectively. Twenty selected 2-DE protein spots from the crude extracts of Bacillus sp. HKG 112, excised from 2- DE, were identified by liquid chromatography mass spectrometry (LC-MS) out of which 16 spots showed significant changes after radiation exposure and might be responsible for the radiation resistance property. Our results suggest that the different responses of some genes under radiation for the expression of radiation-dependent proteins could contribute to a physiological advantage and would be a significant initial step towards a full-system understanding of the radiation stress protection mechanisms of bacteria in different environments.

Characterization of LPS mutants of peanut specific Bradyrhizobium (GN17).

Two mutants of Bradyrhizobium sp. (Arachis) strain GN17 having altered lipopolysaccharide (LPS) composition were isolated upon random Tn5 mutagenesis to study their binding with peanut root lectin (PRA II). These mutant strains designated as GN17M1 and GN17M2 produced rough colonies and showed autoagglutination. Flow cytometric analyses indicated that strain GN17M1 bind to PRA II with highest efficiency. Both the mutants synthesized only high molecular weight lipopolysaccharides as observed by silver staining of polyacrylamide gel. The LPSs from both the mutants cross-reacted with anti-GN17 LPS, however, GN17M1 LPS showed 3 times higher cross-reactivity as detected by ELISA. Carbohydrate analysis by high performance anion exchange chromatography (HPAEC) showed that glucose was the major constituent of the purified LPS from the parent strain whereas mannose appeared as major component in the GN17M2 LPS. Equivalent amount of glucose and galactosamine with significant amount of mannose and galactose was the characteristics of the GN17M1 LPS. Purified LPS from GN17M1 and GN17M2 were respectively 17 and 10 times more potent inhibitors of PRA II activity than that of parent strain GN17. Similar binding efficiencies of the mutant LPS towards PRA II was also observed by ELISA. The results of this study indicate that the composition and the arrangement of the LPS are crucial for lectin binding.