In silico design of a novel hybrid epitope-based antigen harboring highly exposed immunogenic peptides of BamA, OmpA, and Omp34 against Acinetobacter baumannii.

Acinetobacter baumannii, is among the highest priority bacteria according to the WHO categorization which necessitate the exploration of alternative strategies such as vaccination. OmpA, BamA, and Omp34 are assigned as appropriate antigens to serve in vaccine development against this pathogen. Experimentally validated exposed epitopes of OmpA and Omp34 along with selected exposed epitopes predicted by an integrative in silico approach were represented by the barrel domain of BamA as a scaffold. Among the 8 external loops of BamA, 5 loops were replaced with selected loops of OmpA and Omp34. The designed antigen was analyzed regarding the physicochemical properties, antigenicity, epitope retrieval, topology, structure, and safety. BamA is a two-domain OMP with a 16-stranded barrel in which L4, L6, and L7 were the longest loops of BamA in order. The designed antigen consisted of 478 amino acids with antigen probability of 0.7793. The novel antigen was a 16-stranded barrel. No identical 8-meric peptides were found in the human proteome against the designed antigen sequence. The designed construct was safe regarding the allergenicity, toxicity, and human proteome reactivity. The designed antigen could develop higher protection against A. baumannii in comparison to either OmpA, BamA, or Omp34 alone.

Leptospira interrogans encodes a canonical BamA and three novel noNterm Omp85 outer membrane protein paralogs.

The Omp85 family of outer membrane proteins are ubiquitously distributed among diderm bacteria and play essential roles in outer membrane (OM) biogenesis. The majority of Omp85 orthologs are bipartite and consist of a conserved OM-embedded 16-stranded beta-barrel and variable periplasmic functional domains. Here, we demonstrate that Leptospira interrogans encodes four distinct Omp85 proteins. The presumptive leptospiral BamA, LIC11623, contains a noncanonical POTRA4 periplasmic domain that is conserved across Leptospiraceae. The remaining three leptospiral Omp85 proteins, LIC12252, LIC12254 and LIC12258, contain conserved beta-barrels but lack periplasmic domains. Two of the three 'noNterm' Omp85-like proteins were upregulated by leptospires in urine from infected mice compared to in vitro and/or following cultivation within rat peritoneal cavities. Mice infected with a L. interrogans lic11254 transposon mutant shed tenfold fewer leptospires in their urine compared to mice infected with the wild-type parent. Analyses of pathogenic and saprophytic Leptospira spp. identified five groups of noNterm Omp85 paralogs, including one pathogen- and two saprophyte-specific groups. Expanding our analysis beyond Leptospira spp., we identified additional noNterm Omp85 orthologs in bacteria isolated from diverse environments, suggesting a potential role for these previously unrecognized noNterm Omp85 proteins in physiological adaptation to harsh conditions.

Targeting BamA, the essential component of the Acinetobacter baumannii ß-barrel assembly machinery, hinders its ability to adhere to and invade human alveolar basal epithelial cell line.

The lungs are commonly targeted by Acinetobacter baumannii. The human alveolar basal epithelial cell line, A549, serves as a valuable in vitro model for probing pathogen-cell dynamics. This study examined two Acinetobacter strains, ATCC 19606 and the clinical isolate 58ST, investigating their adherence, internalization, and cytotoxicity within the A549 cell line to illuminate pathogenic mechanisms. Anti-BamA antibodies were expressed, purified, and detected via indirect ELISA. The toxicity of BamA was assessed across BALB/c mice. Both A. baumannii strains were used to infect A549 cells to scrutinize cell invasion diversity. Serum resistance, biofilm creation and inhibition, adhesion, internalization, and intracellular proliferation of live and inactivated A. baumannii were probed with and without anti-BamA sera. A549 cell viability was evaluated in the presence of live A. baumannii and anti-BamA sera-exposed bacteria. Cytoskeleton inhibitor tests were conducted on epithelial cells. A. baumannii strains displayed differing cell invasion aptitudes, with the clinical variant manifesting the highest invasion capability. During internalization, A. baumannii cells localized within vacuoles and migrated towards the nucleus using a zipper-like invasion mechanism. Bacterial division inside host cells culminated in cell demise. Pre-treatment with anti-BamA antibodies substantially impeded A. baumannii's adherence and invasion in epithelial cells. Microscopic imaging validated the intracellular presence of A. baumannii in A549 cells, verifying their invasive potential and residency. These findings substantiate A. baumannii's capacity to proliferate in epithelial cells, with BamA pivotal role against A. baumannii-epithelial cell interplay. This study augments our insight into A. baumannii pathogenesis, facilitating the development of efficacious strategies against A. baumannii infections.

Molecular characterization, tissue expression, and antiviral activities of Bama minipig interferon-α subtypes.

Interferons play a major role in innate immunity and disease resistance. Porcine interferon alpha has 17 subtypes, and their gene sequences, tissue expression profiles, and antiviral activities have been primarily studied in domestic pigs but not in minipigs. Bama minipigs are genetically stable disease-resistant and making them as laboratory animal models for bioscience studies. To define the potential mechanism for disease resistance, in this study, we cloned 17 subtypes of Porcine interferon alpha genes in Bama minipigs using high fidelity polymerase chain reaction and subsequent sequencing. Sequence alignment showed that the 17 porcine interferon alpha subtypes were 98%-100 % homologous in those of domestic pigs. However, significantly different tissue expression profiles of PoIFN-α subtypes were found in the two pig species using real-time quantitative RT-PCR. Among the 10 different Bama minipig tissues tested, significant expression of multi-subtype porcine interferon alpha was detected in the lymph nodes and spleen, whereas no or low expression of fewer subtypes was detected in the heart, lung, brain, and small intestine. Sequence analysis revealed that the porcine interferon alpha promoters were almost similar between the two pig species. A cytopathic effect inhibition assay showed that the recombinant 17 porcine interferon alpha subtypes purified from mammalian cells had significantly different antiviral profile against vesicular stomatitis virus, porcine pseudorabies virus and porcine reproductive and respiratory syndrome virus compared with those in domestic pigs. Our findings provide evidence that porcine interferon alpha subtypes are highly conserved between Bama minipigs and domestic pigs but show varied tissue expression pattern and antiviral capabilities, which may contribute to their differences in disease resistance.

Isolation of Limosilactobacillus mucosae G01 with inhibitory effects on porcine epidemic diarrhea virus in vitro from Bama pig gastroenteritis.

Porcine epidemic diarrhea virus (PEDV) is responsible for causing fatal watery diarrhea in piglets, resulting in significant economic losses within the pig farming industry. Although vaccination is currently employed as a preventive measure, certain vaccines do not provide complete protection against PEDV field strains. Probiotics present a promising alternative due to their ability to regulate intestinal flora, enhance host immunity, and improve resistance against pathogenic microorganisms. We isolated six lactic acid bacteria (LAB) from the fecal microorganisms of Bama pigs, compared to Limosilactobacillus mucosae DSM13345 of the same genus in which Limosilactobacillus mucosae G01 (L. mucosae G01) proved to have a potent anti-PEDV effect. In a comprehensive manner, L. mucosae G01 significantly augmented the phosphorylation of IRF3 in IPEC-J2 cells, resulting in the induction of interferons (IFN α, IFN ß, IFN λ1, and IFN λ3) and subsequent upregulation of interferon-stimulated genes (ISGs) (MX1, MX2, OAS1, and ZAP) in a dose-dependent fashion, consequently leading to the mitigation of PEDV replication. These findings underscore the promising prospects of L. mucosae G01 as a naturally derived substitute for combating PEDV and other enteric coronavirus infections.

Microbiome-gut-brain axis contributes to patients and Bama miniature pigs with acute large ischemic stroke.

Acute large hemispheric infarction (ALHI) is an overwhelming emergency with a great challenge of gastrointestinal dysfunction clinically. Here, we initially proposed delayed bowel movements as the clinical phenotype of strike to gut-brain axis (GBA) in ALHI patients by epidemiological analysis of 499 acute ischemic stroke (AIS) patients. 1H NMR-based metabolomics revealed that AIS markedly altered plasma global metabolic profiling of patients compared with healthy controls. Risk factors of strike on GBA were the National Institutes of Health Stroke Scale (NIHSS) score ≥ 5 and stroke onset time ≤ 24 h. As a result, first defecating time after admission to the hospital ≥2 days could be considered as a potential risk factor for strike on GBA. Subsequently, the ALHI Bama miniature (BM) pig model with acute symptomatic seizure was successfully established by ligation of the left ascending pharyngeal artery combined with local air injection. Clinical phenotypes of brain necrosis such as hemiplegia were examined with brain diffusion-weighted imaging (DWI) and pathological diagnosis. In addition to global brain injury and inflammation, we also found that ALHI induced marked alterations of intestinal barrier integrity, the gut microbial community, and microbiota-derived metabolites including serotonin and neurotransmitters in both plasma and multiple brain tissues of BM pigs. These findings revealed that microbiota-gut-brain axis highly contributed to the occurrence and development of ALHI.

Designing of a multi-epitopes based vaccine against Haemophilius parainfluenzae and its validation through integrated computational approaches.

Haemophilus parainfluenzae is a Gram-negative opportunist pathogen within the mucus of the nose and mouth without significant symptoms and has an ability to cause various infections ranging from ear, eye, and sinus to pneumonia. A concerning development is the increasing resistance of H. parainfluenzae to beta-lactam antibiotics, with the potential to cause dental infections or abscesses. The principal objective of this investigation is to utilize bioinformatics and immuno-informatic methodologies in the development of a candidate multi-epitope Vaccine. The investigation focuses on identifying potential epitopes for both B cells (B lymphocytes) and T cells (helper T lymphocytes and cytotoxic T lymphocytes) based on high non-toxic and non-allergenic characteristics. The selection process involves identifying human leukocyte antigen alleles demonstrating strong associations with recognized antigenic and overlapping epitopes. Notably, the chosen alleles aim to provide coverage for 90% of the global population. Multi-epitope constructs were designed by using suitable linker sequences. To enhance the immunological potential, an adjuvant sequence was incorporated using the EAAAK linker. The final vaccine construct, comprising 344 amino acids, was achieved after the addition of adjuvants and linkers. This multi-epitope Vaccine demonstrates notable antigenicity and possesses favorable physiochemical characteristics. The three-dimensional conformation underwent modeling and refinement, validated through in-silico methods. Additionally, a protein-protein molecular docking analysis was conducted to predict effective binding poses between the multi-epitope Vaccine and the Toll-like receptor 4 protein. The Molecular Dynamics (MD) investigation of the docked TLR4-vaccine complex demonstrated consistent stability over the simulation period, primarily attributed to electrostatic energy. The docked complex displayed minimal deformation and enhanced rigidity in the motion of residues during the dynamic simulation. Furthermore, codon translational optimization and computational cloning was performed to ensure the reliability and proper expression of the multi-Epitope Vaccine. It is crucial to emphasize that despite these computational validations, experimental research in the laboratory is imperative to demonstrate the immunogenicity and protective efficacy of the developed vaccine. This would involve practical assessments to ascertain the real-world effectiveness of the multi-epitope Vaccine.

miR-423-5p Regulates Skeletal Muscle Growth and Development by Negatively Inhibiting Target Gene SRF.

The process of muscle growth directly affects the yield and quality of pork food products. Muscle fibers are created during the embryonic stage, grow following birth, and regenerate during adulthood; these are all considered to be phases of muscle development. A multilevel network of transcriptional, post-transcriptional, and pathway levels controls this process. An integrated toolbox of genetics and genomics as well as the use of genomics techniques has been used in the past to attempt to understand the molecular processes behind skeletal muscle growth and development in pigs under divergent selection processes. A class of endogenous noncoding RNAs have a major regulatory function in myogenesis. But the precise function of miRNA-423-5p in muscle development and the related molecular pathways remain largely unknown. Using target prediction software, initially, the potential target genes of miR-423-5p in the Guangxi Bama miniature pig line were identified using various selection criteria for skeletal muscle growth and development. The serum response factor (SRF) was found to be one of the potential target genes, and the two are negatively correlated, suggesting that there may be targeted interactions. In addition to being strongly expressed in swine skeletal muscle, miR-423-5p was also up-regulated during C2C12 cell development. Furthermore, real-time PCR analysis showed that the overexpression of miR-423-5p significantly reduced the expression of myogenin and the myogenic differentiation antigen (p < 0.05). Moreover, the results of the enzyme-linked immunosorbent assay (ELISA) demonstrated that the overexpression of miR-423-5p led to a significant reduction in SRF expression (p < 0.05). Furthermore, miR-423-5p down-regulated the luciferase activities of report vectors carrying the 3' UTR of porcine SRF, confirming that SRF is a target gene of miR-423-5p. Taken together, miR-423-5p's involvement in skeletal muscle differentiation may be through the regulation of SRF.

Dietary probiotic and synbiotic supplementation starting from maternal gestation improves muscular lipid metabolism in offspring piglets by reshaping colonic microbiota and metabolites.

Probiotics and synbiotics have been intensively used in animal husbandry due to their advantageous roles in animals' health. However, there is a paucity of research on probiotic and synbiotic supplementation from maternal gestation to the postnatal growing phases of offspring piglets. Thus, we assessed the effects of dietary supplementation of these two additives to sows and offspring piglets on skeletal muscle and body metabolism, colonic microbiota composition, and metabolite profiles of offspring piglets. Pregnant Bama mini-pigs and their offspring piglets (after weaning) were fed either a basal diet or a basal diet supplemented with antibiotics, probiotics, or synbiotics. At 65, 95, and 125 days old, eight pigs per group were euthanized and sampled for analyses. Probiotics increased the intramuscular fat content in the psoas major muscle (PMM) at 95 days old, polyunsaturated fatty acid (PUFA) and n-3 PUFA levels in the longissimus dorsi muscle (LDM) at 65 days old, C16:1 level in the LDM at 125 days old, and upregulated ATGL, CPT-1, and HSL expressions in the PMM at 65 days old. Synbiotics increased the plasma HDL-C level at 65 days old and TC level at 65 and 125 days old and upregulated the CPT-1 expression in the PMM at 125 days old. In addition, probiotics and synbiotics increased the plasma levels of HDL-C at 65 days old, CHE at 95 days old, and LDL-C at 125 days old, while decreasing the C18:1n9t level in the PMM at 65 days old and the plasma levels of GLU, LDH, and TG at 95 days old. Microbiome analysis showed that probiotic and synbiotic supplementation increased colonic Actinobacteria, Firmicutes, Verrucomicrobia, Faecalibacterium, Pseudobutyrivibrio, and Turicibacter abundances. However, antibiotic supplementation decreased colonic Actinobacteria, Bacteroidetes, Prevotella, and Unclassified_Lachnospiraceae abundances. Furthermore, probiotic and synbiotic supplementation was associated with alterations in 8, 7, and 10 differential metabolites at three different age stages. Both microbiome and metabolome analyses showed that the differential metabolic pathways were associated with carbohydrate, amino acid, and lipid metabolism. However, antibiotic supplementation increased the C18:1n9t level in the PMM at 65 days old and xenobiotic biodegradation and metabolism at 125 days old. In conclusion, sow-offspring's diets supplemented with these two additives showed conducive effects on meat flavor, nutritional composition of skeletal muscles, and body metabolism, which may be associated with the reshaping of colonic microbiota and metabolites. However, antibiotic supplementation has negative effects on colonic microbiota composition and fatty acid composition in the PMM. IMPORTANCE: The integral sow-offspring probiotic and synbiotic supplementation improves the meat flavor and the fatty acid composition of the LDM to some extent. Sow-offspring probiotic and synbiotic supplementation increases the colonic beneficial bacteria (including Firmicutes, Verrucomicrobia, Actinobacteria, Faecalibacterium, Turicibacter, and Pseudobutyrivibrio) and alters the colonic metabolite profiles, such as guanidoacetic acid, beta-sitosterol, inosine, cellobiose, indole, and polyamine. Antibiotic supplementation in sow-offspring's diets decreases several beneficial bacteria (including Bacteroidetes, Actinobacteria, Unclassified_Lachnospiraceae, and Prevotella) and has a favorable effect on improving the fatty acid composition of the LDM to some extent, while presenting the opposite effect on the PMM.

Integrated Transcriptomic and Metabolomic Analysis Reveal the Dynamic Process of Bama Hemp Seed Development and the Accumulation Mechanism of α-Linolenic Acid and Linoleic Acid.

Bama County is a world-famous longevity county in the Guangxi Province, China. Bama hemp is a traditional seed used in hemp cultivation in the Bama County. The seeds contain abundant unsaturated fatty acids, particularly linoleic acid (LA) and linolenic acid in the golden ratio. These two substances have been proven to be related to human health and the prevention of various diseases. However, the seed development and seed oil accumulation mechanisms remain unclear. This study employed a combined analysis of physiological, transcriptomic, and metabolomic parameters to elucidate the fatty acid formation patterns in Bama hemp seeds throughout development. We found that seed oil accumulated at a late stage in embryo development, with seed oil accumulation following an "S″-shaped growth curve, and positively correlated with seed size, sugar content, protein content, and starch content. Transcriptome analysis identified genes related to the metabolism of LA, α-linolenic acid (ALA), and jasmonic acid (JA). We found that the FAD2 gene was upregulated 165.26 folds and the FAD3 gene was downregulated 6.15 folds at day 21. Metabolomic changes in LA, ALA, and JA compounds suggested a competitive relationship among these substances. Our findings indicate that the peak period of substance accumulation and nutrient accumulation in Bama hemp seeds occurs during the midstage of seed development (day 21) rather than in the late stage (day 40). The results of this research will provide a theoretical basis for local cultivation and deep processing of Bama hemp.

Performance of ATT and UDFF in the diagnosis of non-alcoholic fatty liver: An animal experiment.

Objective: To establish a Bama minipigs model with Non-Alcoholic Fatty Liver (NAFL) induced by a high-fat diet and investigate the application of attenuation coefficient (ATT) and ultrasound-derived fat fraction (UDFF) in the diagnosis of NAFL. Methods: Six-month-old male Bama minipigs were randomly divided into normal control and high-fat groups (n = 3 pigs per group), and fed with a control diet and high-fat diet for 32 weeks. Weight and body length were measured every four weeks, followed by quantitative ultrasound imaging (ATT and UDFF), blood biochemical markers, and liver biopsies on the same day. Using the Non-Alcoholic Fatty Liver Disease (NAFLD) Activity Score (NAS) as a reference, we analyzed the correlation between ATT, UDFF, and their score results. Results: Compared with the normal control group, the body weight, body mass index (BMI), and serum levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in the High-fat group were significantly different at Week 12 (P < 0.05). Spearman correlation analysis showed that the ATT value was significantly correlated with NAS score (r = 0.76, P < 0.001), and the UDFF value was significantly correlated with NAS score (r = 0.80, P < 0.001). The optimal cut-off value of ATT and UDFF were 0.59 dB/cm/MHz and 5.5%, respectively. These values are optimal for diagnosis of NAFL in Bama minipig model. Conclusion: ATT and UDFF have a high correlation with steatosis, and can be used as a non-invasive method for early screening of hepatic steatosis, which can dynamically monitor the change of disease course.

Effects of Succinate on Growth Performance, Meat Quality and Lipid Synthesis in Bama Miniature Pigs.

Succinate, one of the intermediates of the tricarboxylic acid cycle, is now recognized to play a role in a broad range of physiological and pathophysiological settings, but its role in adipogenesis is unclear. Our study used Bama miniature pigs as a model to explore the effects of succinate on performance, meat quality, and fat formation. The results showed that adding 1% succinate significantly increased the average daily gain, feed/gain ratio, eye muscle area, and body fat content (p < 0.05), but had no effect on feed intake. Further meat quality analysis showed that succinate increased the marbling score and intramuscular fat content of longissimus dorsi muscle (LM), while decreasing the shear force and the cross-sectional area of LM (p < 0.05). Metabolomics analysis of LM revealed that succinate reshaped levels of fatty acids, triglycerides, glycerophospholipids, and sphingolipids in LM. Succinate promotes adipogenic differentiation in porcine primary preadipocytes. Finally, dietary succinate supplementation increased succinylation modification rather than acetylation modification in the adipose tissue pool. This study elucidated the effects of succinate on the growth and meat quality of pigs and its mechanism of action and provided a reference for the role of succinate in the nutrition and metabolism of pigs.

Histological Changes of Porcine Animal Skin with Micro-focused Ultrasound.

BACKGROUND: As a noninvasive alternative therapy, microfocused ultrasound (MFU) has become a research hotspot in recent years for its potential to enhance skin laxity. While several clinical studies have explored the effects of MFU on improving skin laxity, there is limited literature available on the histological changes resulting from MFU treatments. It has been established that the skin structure and composition of the Bama miniature pigs closely resembles that of humans, including collagen content, type I collagen distribution, and elastin distribution. METHODS: This study primarily focuses on examining the histological alterations in the skin tissue of Bama miniature pigs following MFU application. We also selected some typical clinical photographs of patients treated with MFU and compared the clinical effects with histological changes observed in porcine skin. The MFU device utilized in this study incorporates ultra-pulse technology and large focal area technology. RESULTS: Following the standard operating procedures provided by the manufacturer, different handles were used in different skin area of pigs. Biopsies were obtained immediately after treatment and 1 month after treatment. Significant histological changes were observed in the Bama miniature pigs skin, including collagen contraction and fragmentation, dilation and congestion of superficial dermal capillaries immediately after MFU treatment; dermal thickening, increased thickness and density of collagen fibers, elevated levels of elastin and type I collagen, as well as thickened fiber septa in the adipose layer 1 month later. These histological results corresponded to clinical findings in human, such as facial redness and swelling immediately after treatment, and improvement in facial relaxation after approximately 1 month after treatment. CONCLUSION: Collectively, these histological findings provide valuable evidence supporting the clinical application of MFU for enhancing skin laxity. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Specifically targeting antimicrobial peptides for inhibition of Candidatus Liberibacter asiaticus.

AIMS: Huanglongbing (citrus greening) is a plant disease putatively caused by the unculturable Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas), and it has caused severe damage to citrus plantations worldwide. There are no definitive treatments for this disease, and conventional disease control techniques have shown limited efficacy. This work presents an in silico evaluation of using specifically targeting anti-microbial peptides (STAMPs) consisting of a targeting segment and an antimicrobial segment to inhibit citrus greening by inhibiting the BamA protein of CLas, which is an outer membrane protein crucial for bacterial viability. METHODS AND RESULTS: Initially, a set of peptides with a high affinity toward BamA protein were screened and evaluated via molecular docking and molecular dynamics simulations and were verified in vitro via bio-layer interferometry (BLI). In silico studies and BLI experiments indicated that two peptides, HASP2 and HASP3, showed stable binding to BamA. Protein structures for STAMPs were created by fusing known anti-microbial peptides (AMPs) with the selected short peptides. The binding of STAMPs to BamA was assessed using molecular docking and binding energy calculations. The attachment of high-affinity short peptides significantly reduced the free energy of binding for AMPs, suggesting that it would make it easier for the STAMPs to bind to BamA. Efficacy testing in vitro using a closely related CLas surrogate bacterium showed that STAMPs had greater inhibitory activity than AMP alone. CONCLUSIONS: In silico and in vitro results indicate that the STAMPs can inhibit CLas surrogate Rhizobium grahamii more effectively compared to AMPs, suggesting that STAMPs can achieve better inhibition of CLas, potentially via enhancing the site specificity of AMPs.

A Novel Laryngopharyngeal Reflux Disease Model for Bama Pigs.

OBJECTIVE: To develop a novel Laryngopharyngeal Reflux Disease (LPRD) model in Bama pigs through endoscopic cricopharyngeal myotomy. METHODS: A total of eight 8-month-old Bama pigs were randomly assigned to either the control or surgery group. Prior to intervention, upper esophageal sphincter (UES) manometry and laryngopharyngeal Dx-pH monitoring were conducted to establish baseline physiological parameters for each pig. Subsequently, the surgery group underwent endoscopic cricopharyngeal myotomy, while the control group did not. Two weeks postintervention, these procedures were repeated to evaluate changes in UES contractility and the occurrence of reflux events. At week eight postsurgery, mucosal tissues from both groups were harvested for histological analysis. Hematoxylin and eosin (H&E) staining was used to assess inflammation, while transmission electron microscopy (TEM) examined alterations in intercellular spaces and desmosomes. RESULTS: The mean UES pressures in the control and surgery groups were 59 ± 9 mmHg and 68 ± 12 mmHg, respectively. In the surgery group, there was a significant decrease in UES pressure 2weeks after the operation compared to preoperative values (P = 0.005), whereas no significant change was observed in the control group (P = 0.488). Laryngopharyngeal reflux (LPR) was successfully induced in the surgery group as evidenced by reflux events with pH <5.0, which were not detected in the control group. HE staining revealed marked inflammatory cell infiltration and submucosal gland expansion in throat tissues of the surgery group Bama pigs. TEM further showed enlarged intercellular spaces and reduced desmosome numbers in the laryngopharyngeal epithelium compared to controls. CONCLUSION: Given analogous throat epithelial structures to humans, Bama pigs are an appropriate species for an LPRD animal model. Endoscopic cricopharyngeal myotomy effectively induces LPR and observable pathological changes in Bama pigs, providing a valuable platform for further research into LPRD pathophysiology.

Site-Specific Photocrosslinking to Investigate Toxin Delivery Mediated by the Bacterial ß-Barrel Assembly Machine.

Contact-dependent inhibition (CDI) is a mechanism of interbacterial competition in Gram-negative organisms that relies on a specific interaction between a CdiA protein on the surface of one cell and a ß-barrel protein on the surface of a neighboring cell. This interaction triggers the transport of a protein toxin into the neighboring cell where it exerts its lethal activity. Several classes of CdiA proteins that bind to different ß-barrel receptors have been identified, but the molecular mechanism by which they deliver their toxins across the outer membranes of their target cells is poorly understood. Here we describe the use of site-specific photocrosslinking to characterize the interaction between a CdiA protein and its receptor. We describe the method for an E. coli CdiA that utilizes BamA as its receptor. BamA's central role in assembling ß-barrel proteins in the outer membrane makes its role in CDI particularly intriguing; it suggests that these two different protein transport processes might share mechanistic features. Our in vitro photocrosslinking method is useful in elucidating early steps in the CDI mechanism, but it could be adapted to study later steps or to study other CdiA-receptor pairs.

Analysis of Transmembrane ß-Barrel Proteins by Native and Semi-native Polyacrylamide Gel Electrophoresis.

Membrane-embedded ß-barrel proteins are important regulators of the outer membrane permeability barrier of Gram-negative bacteria. ß-barrels are highly structured domains formed by a series of antiparallel ß-strands. Each ß-strand is locked in position by hydrogen bonds between its polypeptide backbone and those of the two neighbouring strands in the barrel structure. Some transmembrane ß-barrel proteins form larger homo- or hetero-multimeric complexes that accomplish specific functions. In this chapter, we describe native and semi-native polyacrylamide gel electrophoresis (PAGE) methods to characterize the organization of transmembrane ß-barrel proteins. We illustrate blue native (BN)-PAGE as an analytical method to assess the formation of protein complexes. Furthermore, we describe a heat-modifiability assay via semi-native PAGE as a rapid method to investigate the folding of transmembrane ß-barrels.

Phenotypic correlations of carpal gland diverticular number with production traits and its genome-wide association analysis in multiple pig populations.

Pig carpal glands play crucial roles in territorial recognition, reproductive behavior, and information exchange; however, their effects on production traits and underlying genetic mechanisms remain unclear. In this study, 1028 pigs from six populations were counted for the carpal gland diverticular numbers (CGDNs) on the left (CGDNL) and right (CGDNR) legs, and their carcass and meat quality traits were assessed. The CGDNs were significantly different among the populations, and Licha Black pigs had a lower CGDN than the Bama Xiang breed. It was also significantly different between sexes, with males having more diverticula than females (p ≤ 0.0391). Moreover, the number was asymmetric, with CGDNR being significantly higher than CGDNL. Notably, CGDNs was significantly correlated with each other in phenotype and genetics and with 24-h pH, 24-h meat color score, 24-h marbling score, fat content, moisture content, sodium salt content, and saturated fatty acid content in phenotype. Furthermore, genome-wide association analyses identified seven SNPs in association with CGDNs at a 5% genome-wide significance level, all of which were located in a 1.78-Mb (35.347-37.129 Mb) region on chromosome 1. CNC10010837 and CNC10010840 were the top SNPs: both had an additive effect of 0.789 ± 0.120 on CGDNR with p = 8.31E-10. These findings provide important insights into the functions and underlying genetic mechanisms of swine carpal glands.

Integrated Analysis of the Transcriptome and Microbial Diversity in the Intestine of Miniature Pig Obesity Model.

Obesity, a key contributor to metabolic disorders, necessitates an in-depth understanding of its pathogenesis and prerequisites for prevention. Guangxi Bama miniature pig (GBM) offers an apt model for obesity-related studies. In this research, we used transcriptomics and 16S rRNA gene sequencing to discern the differentially expressed genes (DEGs) within intestinal (jejunum, ileum, and colon) tissues and variations in microbial communities in intestinal contents of GBM subjected to normal diets (ND) and high-fat, high-carbohydrate diets (HFHCD). After a feeding duration of 26 weeks, the HFHCD-fed experimental group demonstrated notable increases in backfat thickness, BMI, abnormal blood glucose metabolism, and blood lipid levels alongside the escalated serum expression of pro-inflammatory factors and a marked decline in intestinal health status when compared to the ND group. Transcriptomic analysis revealed a total of 1669 DEGs, of which 27 had similar differences in three intestinal segments across different groups, including five immune related genes: COL6A6, CYP1A1, EIF2AK2, NMI, and LGALS3B. Further, we found significant changes in the microbiota composition, with a significant decrease in beneficial bacterial populations within the HFHCD group. Finally, the results of integrated analysis of microbial diversity with transcriptomics show a positive link between certain microbial abundance (Solibacillus, norank_f__Saccharimonadaceae, Candidatus_Saccharimonas, and unclassified_f__Butyricicoccaceae) and changes in gene expression (COL6A6 and NMI). Overall, HFHCD appears to co-contribute to the initiation and progression of obesity in GBM by aggravating inflammatory responses, disrupting immune homeostasis, and creating imbalances in intestinal flora.

Alternative Biological Material for Tissue Engineering of the Vagina: Porcine-Derived Acellular Vaginal Matrix.

BACKGROUND: Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is a severe congenital disorder characterized by vaginal hypoplasia caused by dysplasia of the Müllerian duct. Patients with MRKH syndrome often require nonsurgical or surgical treatment to achieve satisfactory vaginal length and sexual outcomes. The extracellular matrix has been successfully used for vaginal reconstruction. METHODS: In this study, we developed a new biological material derived from porcine vagina (acellular vaginal matrix, AVM) to reconstruct the vagina in Bama miniature pigs. The histological characteristics and efficacy of acellularization of AVM were evaluated, and AVM was subsequently transplanted into Bama miniature pigs to reconstruct the vaginas. RESULTS: Macroscopic analysis showed that the neovaginas functioned well in all Bama miniature pigs with AVM implants. Histological analysis and electrophysiological evidence indicated that morphological and functional recovery was restored in normal vaginal tissues. Scanning electron microscopy showed that the neovaginas had mucosal folds characteristics of normal vagina. No significant differences were observed in the expression of CK14, HSP47, and α-actin between the neovaginas and normal vaginal tissues. However, the expression of estrogen receptor (ER) was significantly lower in the neovaginas than in normal vaginal tissues. In addition, AVM promoted the expression of ß-catenin, c-Myc, and cyclin D1. These results suggest that AVM might promotes vaginal regeneration by activating the ß-catenin/c-Myc/cyclin D1 pathway. CONCLUSION: This study reveals that porcine-derived AVM has potential application for vaginal regeneration.