Solar radiation leads to increased collagenase gene expression (MMP1) in HERDA (Hereditary equine regional dermal asthenia) affected horses and may explain the typical dorsal distribution of their lesions.

BACKGROUND: Hereditary equine regional dermal asthenia (HERDA) is a genetic disease that alters collagen biosynthesis. Affected horses exhibit fragile, hyperextensible skin, especially over the dorsal region. Although ultraviolet (UV) radiation seems to contribute to the regional distribution of lesions and worsening of clinical signs, the molecular mechanisms involved are largely unknown. OBJECTIVES: To evaluate the effect of solar radiation on matrix metalloproteinase MMP1, MMP8 and MMP13 gene expression in the dorsal and ventral skin of HERDA-affected and HERDA-unaffected horses [wild-type (WT) horses]. ANIMALS: Six HERDA-affected and six unaffected Quarter horses (WT) were paired according to age, sex and coat colour. MATERIALS AND METHODS: Horses were submitted to 30 day sunlight restriction, followed by 15 day sunlight exposure. Dorsal and ventral skin biopsies were obtained at six sampling times over 45 days. The expression of MMP1, MMP8 and MMP13 genes was measured by quantitative PCR. RESULTS: Although solar radiation modulated MMP1, MMP8 and MMP13 expression, the effects were more pronounced on MMP1. Sun exposure for three days significantly upregulated MMP1 in the dorsal region when compared to the ventral skin in both unaffected and HERDA-affected horses. CONCLUSIONS AND CLINICAL RELEVANCE: This study shows that solar irradiation leads to upregulation of skin collagenase genes particularly MMP1 in the dorsal, sun-exposed skin of horses. Furthermore, this was more marked in HERDA-affected horses. The increased activity of collagenases on the disorganised collagen present in HERDA affected horses would explain why UV radiation leads to deterioration of clinical signs in affected individuals.

Mechanistic Insight into the Fragmentation of Type I Collagen Fibers into Peptides and Amino Acids by a Vibrio Collagenase.

Vibrio collagenases of the M9A subfamily are closely related to Vibrio pathogenesis for their role in collagen degradation during host invasion. Although some Vibrio collagenases have been characterized, the collagen degradation mechanism of Vibrio collagenase is still largely unknown. Here, an M9A collagenase, VP397, from marine Vibrio pomeroyi strain 12613 was characterized, and its fragmentation pattern on insoluble type I collagen fibers was studied. VP397 is a typical Vibrio collagenase composed of a catalytic module featuring a peptidase M9N domain and a peptidase M9 domain and two accessory bacterial prepeptidase C-terminal domains (PPC domains). It can hydrolyze various collagenous substrates, including fish collagen, mammalian collagens of types I to V, triple-helical peptide [(POG)10]3, gelatin, and 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-o-Arg (Pz-peptide). Atomic force microscopy (AFM) observation and biochemical analyses revealed that VP397 first assaults the C-telopeptide region to dismantle the compact structure of collagen and dissociate tropocollagen fragments, which are further digested into peptides and amino acids by VP397 mainly at the Y-Gly bonds in the repeating Gly-X-Y triplets. In addition, domain deletion mutagenesis showed that the catalytic module of VP397 alone is capable of hydrolyzing type I collagen fibers and that its C-terminal PPC2 domain functions as a collagen-binding domain during collagenolysis. Based on our results, a model for the collagenolytic mechanism of VP397 is proposed. This study sheds light on the mechanism of collagen degradation by Vibrio collagenase, offering a better understanding of the pathogenesis of Vibrio and helping in developing the potential applications of Vibrio collagenase in industrial and medical areas. IMPORTANCE Many Vibrio species are pathogens and cause serious diseases in humans and aquatic animals. The collagenases produced by pathogenic Vibrio species have been regarded as important virulence factors, which occasionally exhibit direct pathogenicity to the infected host or facilitate other toxins' diffusion through the digestion of host collagen. However, our knowledge concerning the collagen degradation mechanism of Vibrio collagenase is still limited. This study reveals the degradation strategy of Vibrio collagenase VP397 on type I collagen. VP397 binds on collagen fibrils via its C-terminal PPC2 domain, and its catalytic module first assaults the C-telopeptide region and then attacks the Y-Gly bonds in the dissociated tropocollagen fragments to release peptides and amino acids. This study offers new knowledge regarding the collagenolytic mechanism of Vibrio collagenase, which is helpful for better understanding the role of collagenase in Vibrio pathogenesis and for developing its industrial and medical applications.

Enzymatic vitreolysis using reengineered Vibrio mimicus-derived collagenase.

Purpose: Collagen is a key player contributing to vitreoelasticity and vitreoretinal adhesions. Molecular reorganization causes spontaneous weakening of these adhesions with age, resulting in the separation of the posterior hyaloid membrane (PHM) from the retina in what is called complete posterior vitreous detachment (PVD). Incomplete separation of the posterior hyaloid or tight adherence or both can lead to retinal detachment, vitreomacular traction syndrome, or epiretinal membrane formation, which requires surgical intervention. Pharmacological vitrectomy has the potential of avoiding surgical vitrectomy; it is also useful as an adjunct during retinal surgery to induce PVD. Previously studied enzymatic reagents, such as collagenase derived from Clostridium histolyticum, are nonspecific and potentially toxic. We studied a novel collagenase from Vibrio mimicus (VMC) which remains active (VMA), even after deletion of 51 C-terminal amino acids. To limit the activity of VMA to the vitreous cavity, a fusion construct (inhibitor of hyaluronic acid-VMA [iHA-VMA]) was made in which a 12-mer peptide (iHA, which binds to HA) was fused to the N-terminus of VMA. The construct was evaluated in the context of PVD. Methods: VMA and iHA-VMA were expressed in Escherichia coli, purified, and characterized with gelatin zymography, collagen degradation assay, fluorescamine-based assay, and cell-based assays. Two sets of experiments were performed in New Zealand albino rabbits. Group A (n = 10) received iHA-VMA, while group B (n = 5) received the equivalent dose of VMA. In both groups, saline was injected as a control in the contralateral eyes. Animals were monitored with indirect ophthalmoscopy, optical coherence tomography (OCT), and B-scan ultrasonography. Retinal toxicity was assessed with hematoxylin and eosin (H&E) staining of retinal tissue. Results: The activity of iHA-VMA and VMA was comparable and 65-fold lower than that of C. histolyticum collagenase Type IV. In the iHA-VMA group, all the rabbits (n = 10) developed PVD, with complete PVD seen in six animals. No statistically significant histomorphological changes were seen. In the VMA group, four of the five rabbits developed complete PVD; however, retinal morphological changes were seen in two animals. Conclusions: iHA-VMA displays targeted action confined to the vitreous and shows potential for safe pharmacologic vitreolysis.

MMP1 and MMP9 are potential prognostic biomarkers and targets for uveal melanoma.

BACKGROUND: Uveal melanoma (UVM) is the leading cause of eye-related mortality worldwide. This study aimed to explore the expression and prognostic value of matrix metalloproteinases (MMPs) in UVM. METHODS: Gene expression levels were obtained from the Gene Expression Omnibus (GEO) and Oncomine databases. Functional and pathway enrichment analyses were performed using the Metascape database. GeneMANIA was then applied to construct a protein-protein interaction network and identify the hub genes. Moreover, overall survival (OS) and disease-free survival (DFS) analysis for the hub genes was performed using the UALCAN and Gene Expression Profiling Interactive Analysis (GEPIA) online tool. Furthermore, TRRUST was used to predict the targets of the MMPs. RESULTS: Our results revealed that the transcriptional levels of MMP1, MMP9, MMP10, MMP11, MMP13, MMP14, and MMP17 were upregulated in UVM tissues compared to normal tissues. A protein-protein interaction (PPI) network was constructed and the top 50 hub genes were identified. The functions of MMPs and their neighboring proteins are mainly associated with ECM-receptor interaction, proteoglycans in cancer, the IL-17 signaling pathway, and microRNAs in cancer. Among the MMPs, MMP1/2/9/11/14/15/16/17/24 played significant roles in the progression of UVM from stage 3 to stage 4. We also found that the expression of MMP1, MMP2, MMP9, and MMP16 positively correlated with OS and DFS in patients with UVM. Additionally, 18 transcription factors associated with nine MMPs were identified. CONCLUSIONS: The results of this study may provide potential biomarkers and targets for UVM. However, further studies are required to confirm these results.

Understanding factors affecting human chondrocyte culturing: an experimental study.

Over the years, surgical strategies have been developed in hope of full regeneration of the injured cartilage. In our study, we aimed to develop an optimized chondrocyte culture isolation technique as an active ingredient of a standardized autologous chondrocte implantation product, which is able to maintain the phenotype along with the molecular features of the cartilage. We compared different enzymes, which suggested optimal performance with collagenase type II at 5 mg/ml concentration. Thereafter, we observed that COL2 and GAG expression is substantially reduced with passaging. There was a need to omit passaging to reach the optimal isolation method. We then tested various growth factors and media in order to maintain the natural character of chondrocytes. Our study also suggested the highest COL2 and GAG expressions with the highest recovery in the presence of Advanced DMEM. Autologous chondrocyte implantation manufacturing approval was recently received from the national competent authority, making it possible to utilize the process engineering protocol developed with this study at our Tissue and Cell Manufacturing Center as a part of the autologous chondrocyte implantation manufacturing standard operation procedure (SOP).

The Biological and Clinical Role of the Long Non-Coding RNA LOC642852 in Ovarian Carcinoma.

The objective of the present study was to analyze the biological and clinical role of the long non-coding RNA LOC642852 in ovarian carcinoma (OC). LOC642852 expression was analyzed in seven OC cell lines (OVCAR-3, OVCAR-8, OVCA 433, OVCA 429, OC 238, DOV13, ES-2) and 139 high-grade serous carcinoma (HGSC) specimens (85 effusions, 54 surgical specimens). Following LOC642852 knockout (KO) using the CRISPR/Cas9 system, OVCAR-8 HGSC cells were analyzed for spheroid formation, migration, invasion, proliferation, matrix metalloproteinase (MMP) activity, and expression of cell signaling proteins. OVCAR-8 cells with LOC642852 KO were significantly less motile and less invasive compared to controls, with no differences in spheroid formation, proliferation, or matrix metalloproteinase (MMP) activity. Total Akt and Erk levels were comparable in controls and KO cells, but their phosphorylation was significantly increased in the latter. In clinical specimens, LOC642852 was overexpressed in ovarian tumors and omental/peritoneal metastases compared to effusion specimens (p = 0.013). A non-significant trend for shorter overall (p = 0.109) and progression-free (p = 0.056) survival was observed in patients with HGSC effusions with high LOC642852 levels. Bioinformatics analysis showed potential roles for LOC642852 as part of the TLE3/miR-221-3p ceRNA network and in relation to the FGFR3 protein. In conclusion, LOC642852 inactivation via CRISPR/Cas9 affects cell signaling, motility, and invasion in HGSC cells. LOC642852 is differentially expressed in HGSC cells at different anatomical sites. Its potential role in regulating the TLE3/miR-221-3p ceRNA network and FGFR3 merits further research.

Effects of flexibility of the α2 chain of type I collagen on collagenase cleavage.

Cleavage of collagen by collagenases such as matrix metalloproteinase 1 (MMP-1) is a key step in development, tissue remodeling, and tumor proliferation. The abundant heterotrimeric type I collagen composed of two α1(I) chains and one α2(I) chain is efficiently cleaved by MMP-1 at a unique site in the triple helix, a process which may be initiated by local unfolding within the peptide chains. Atypical homotrimers of the α1(I) chain, found in embryonic and cancer tissues, are very resistant to MMP cleavage. To investigate MMP-1 cleavage, recombinant homotrimers were constructed with sequences from the MMP cleavage regions of human collagen chains inserted into a host bacterial collagen protein system. All triple-helical constructs were cleaved by MMP-1, with α2(I) homotrimers cleaved efficiently at a rate similar to that seen for α1(II) and α1(III) homotrimers, while α1(I) homotrimers were cleaved at a much slower rate. The introduction of destabilizing Gly to Ser mutations within the human collagenase susceptible region of the α2(I) chain did not interfere with MMP-1 cleavage. Molecular dynamics simulations indicated a greater degree of transient hydrogen bond breaking in α2(I) homotrimers compared with α1(I) homotrimers at the MMP-1 cleavage site, and showed an extensive disruption of hydrogen bonding in the presence of a Gly to Ser mutation, consistent with chymotrypsin digestion results. This study indicates that α2(I) homotrimers are susceptible to MMP-1, proves that the presence of an α1(I) chain is not a requirement for α2(I) cleavage, and supports the importance of local unfolding of α2(I) in collagenase cleavage.

Expression of Collagenase is Regulated by the VarS/VarA Two-Component Regulatory System in Vibrio alginolyticus.

Vibrio alginolyticus is an opportunistic pathogen in both humans and marine animals. Collagenase encoded by colA is considered to be one of the virulence factors. Expression of colA is regulated by multiple environmental factors, e.g., temperature, growth phase, and substrate. To elucidate the mechanism of regulation of colA expression, transposon mutagenesis was performed. VarS, a sensor histidine kinase of the two-component regulatory system, was demonstrated to regulate the expression of colA. VarA, a cognate response regulator of VarS, was also identified and shown to be involved in the regulation of colA expression. In vitro phosphorylation assays showed that phosphorylated VarS acted as a phosphoryl group donor to VarA. A site-directed mutagenesis study showed that the His300, Asp718 and His874 residues in VarS were essential for the phosphorylation of VarS, and the Asp54 residue in VarA was likely to receive the phosphoryl group from VarS. The results demonstrate that the VarS/VarA two-component regulatory system regulates the expression of collagenase in V. alginolyticus.

Canagliflozin attenuates the progression of atherosclerosis and inflammation process in APOE knockout mice.

BACKGROUND: Sodium glucose co-transporter2 inhibitors reduce the incidence of cardiovascular events in patients with type 2 diabetes mellitus based on the results of recent cardiovascular outcome studies. Herein, we investigated the effects of long-term treatment with canagliflozin on biochemical and immunohistochemical markers related to atherosclerosis and atherosclerosis development in the aorta of apolipoprotein E knockout (Apo-E(-/-)) mice. METHODS: At the age of 5 weeks, mice were switched from normal to a high-fat diet. After 5 weeks, Apo-E(-/-) mice were divided into control-group (6 mice) treated with 0.5% hydroxypropyl methylcellulose and Cana-group (7 mice) treated with canagliflozin (10 mg/kg per day) per os. After 5 weeks of intervention, animals were sacrificed, and heart and aorta were removed. Sections stained with hematoxylin-eosin (H&E) were used for histomorphometry whereas Masson's stained tissues were used to quantify the collagen content. Immunohistochemistry to assess MCP-1, CD68, a-smooth muscle actin, MMP-2, MMP-9, TIMP-1 and TIMP-2 expression was carried out and q-PCR experiments were performed to quantify mRNA expression. RESULTS: Canagliflozin-group mice had lower total-cholesterol, triglycerides and glucose levels (P < 0.01), while heart rate was significantly lower (P < 0.05). Histomorphometry revealed that one in seven Cana-group mice versus four in six control mice developed atheromatosis, while aortic root plaque was significantly less, and collagen was 1.6 times more intense in canagliflozin-group suggesting increased plaque stability. Immunohistochemistry revealed that MCP-1 was significantly less expressed (P < 0.05) in the aortic root of canagliflozin-group while reduced expression of a-actin and CD68 was not reaching significance (P = 0.15). VCAM-1 and MCP-1 mRNA levels were lower (P = 0.02 and P = 0.07, respectively), while TIMP-1/MMP-2 ratio expression was higher in canagliflozin-group approaching statistical significance (P = 0.07). CONCLUSIONS: Canagliflozin attenuates the progression of atherosclerosis, reducing (1) hyperlipidemia and hyperglycemia, and (2) inflammatory process, by lowering the expression of inflammatory molecules such as MCP-1 and VCAM-1. Moreover, canagliflozin was found to increase the atherosclerotic plaque stability via increasing TIMP-1/MMP-2 ratio expression.

Molecular characterization of the spike gene of the porcine epidemic diarrhea virus in Mexico, 2013-2016.

In Mexico, the first outbreaks suggestive of the circulation of the porcine epidemic diarrhea virus (PEDV) were identified at the beginning of July 2013. To identify the molecular characteristics of the PEDV Spike (S) gene in Mexico, 116 samples of the intestine and diarrhea of piglets with clinical signs of porcine epidemic diarrhea (PED) were obtained. Samples were collected from 14 farms located in six states of Mexico (Jalisco, Puebla, Sonora, Veracruz, Guanajuato, and Michoacán) from 2013 to 2016. To identify PEDV, we used real-time RT-PCR to discriminate between non-INDEL and INDEL strains. We chose samples according to state and year to characterize the S gene. After amplification of the S gene, the obtained products were sequenced and assembled. The complete amino acid sequences of the spike protein were used to perform an epitope analysis, which was used to determine null mutations in regions SS2, SS6, and 2C10 compared to the sequences of G2. A phylogenetic analysis determined the circulation of G2b and INDEL strains in Mexico. However, several mutations were recorded in the collagenase equivalent (COE) region that were related to the change in polarity and charge of the amino acid residues. The PEDV strain circulating in Jalisco in 2016 has an insertion of three amino acids (232LGL234) and one change in the antigenic site of the COE region, and strains from the years 2015 and 2016 changed the index of the surface probability, which could be related to the re-emergence of disease outbreaks.

Destructin-1 is a collagen-degrading endopeptidase secreted by Pseudogymnoascus destructans, the causative agent of white-nose syndrome.

Pseudogymnoascus destructans is the causative agent of white-nose syndrome, a disease that has caused the deaths of millions of bats in North America. This psychrophilic fungus proliferates at low temperatures and targets hibernating bats, resulting in their premature arousal from stupor with catastrophic consequences. Despite the impact of white-nose syndrome, little is known about the fungus itself or how it infects its mammalian host. P. destructans is not amenable to genetic manipulation, and therefore understanding the proteins involved in infection requires alternative approaches. Here, we identify hydrolytic enzymes secreted by P. destructans, and use a novel and unbiased substrate profiling technique to define active peptidases. These experiments revealed that endopeptidases are the major proteolytic activities secreted by P. destructans, and that collagen, the major structural protein in mammals, is actively degraded by the secretome. A serine endopeptidase, hereby-named Destructin-1, was subsequently identified, and a recombinant form overexpressed and purified. Biochemical analysis of Destructin-1 showed that it mediated collagen degradation, and a potent inhibitor of peptidase activity was identified. Treatment of P. destructans-conditioned media with this antagonist blocked collagen degradation and facilitated the detection of additional secreted proteolytic activities, including aminopeptidases and carboxypeptidases. These results provide molecular insights into the secretome of P. destructans, and identify serine endopeptidases that have the clear potential to facilitate tissue invasion and pathogenesis in the mammalian host.

Global Transcriptional Response to Organic Hydroperoxide and the Role of OhrR in the Control of Virulence Traits in Chromobacterium violaceum.

A major pathway for the detoxification of organic hydroperoxides, such as cumene hydroperoxide (CHP), involves the MarR family transcriptional regulator OhrR and the peroxidase OhrA. However, the effect of these peroxides on the global transcriptome and the contribution of the OhrA/OhrR system to bacterial virulence remain poorly explored. Here, we analyzed the transcriptome profiles of Chromobacterium violaceum exposed to CHP and after the deletion of ohrR, and we show that OhrR controls the virulence of this human opportunistic pathogen. DNA microarray and Northern blot analyses of CHP-treated cells revealed the upregulation of genes related to the detoxification of peroxides (antioxidant enzymes and thiol-reducing systems), the degradation of the aromatic moiety of CHP (oxygenases), and protection against other secondary stresses (DNA repair, heat shock, iron limitation, and nitrogen starvation responses). Furthermore, we identified two upregulated genes (ohrA and a putative diguanylate cyclase with a GGDEF domain for cyclic di-GMP [c-di-GMP] synthesis) and three downregulated genes (hemolysin, chitinase, and collagenase) in the ohrR mutant by transcriptome analysis. Importantly, we show that OhrR directly repressed the expression of the putative diguanylate cyclase. Using a mouse infection model, we demonstrate that the ohrR mutant was attenuated for virulence and showed a decreased bacterial burden in the liver. Moreover, an ohrR-diguanylate cyclase double mutant displayed the same virulence as the wild-type strain. In conclusion, we have defined the transcriptional response to CHP, identified potential virulence factors such as diguanylate cyclase as members of the OhrR regulon, and shown that C. violaceum uses the transcriptional regulator OhrR to modulate its virulence.

Honokiol improved chondrogenesis and suppressed inflammation in human umbilical cord derived mesenchymal stem cells via blocking nuclear factor-κB pathway.

BACKGROUND: Cartilage degradation is the significant pathological process in osteoarthritis (OA). Inflammatory cytokines, such as interleukin-1ß (IL-1ß), activate various downstream mediators contributing to OA pathology. Recently, stem cell-based cartilage repair emerges as a potential therapeutic strategy that being widely studied, whereas, the outcome is still far from clinical application. In this study, we focused on an anti-inflammatory agent, honokiol, which is isolated from an herb, investigated the potential effects on human umbilical cord derived mesenchymal stem cells (hUC-MSCs) in IL-1ß stimulation. METHODS: Second passage hUC-MSCs were cultured for multi-differentiation. Flow cytometry, qRT-PCR, von Kossa stain, alcian blue stain and oil red O stain were used for characterization and multi-differentiation determination. Honokiol (5, 10, 25, 50 µM) and IL-1ß (10 ng/ml) were applied in hUC-MSCs during chondrogenesis. Analysis was performed by MTT, cell apoptosis evaluation, ELISA assay, qRT-PCR and western blot. RESULTS: hUC-MSC was positive for CD73, CD90 and CD105, but lack of CD34 and CD45. Remarkable osteogenesis, chondrogenesis and adipogenesis were detected in hUC-MSCs. IL-1ß enhanced cell apoptosis and necrosis and activated the expression of caspase-3, cyclooxygenase-2 (COX-2), interleukin-6 (IL-6) and matrix metalloproteinase (MMP)-1, -9, 13 in hUC-MSCs. Moreover, the expression of SRY-related high-mobility group box 9 (SOX-9), aggrecan and col2α1 was suppressed. Honokiol relieved these negative impacts induced by IL-1ß and suppressed Nuclear factor-κB (NF-κB) pathway by downregulating expression of p-IKKα/ß, p-IκBα and p-p65 in dose-dependent and time-dependent manner. CONCLUSIONS: Honokiol improved cell survival and chondrogenesis of hUC-MSCs and inhibited IL-1ß-induced inflammatory response, which suggested that combination of anti-inflammation and stem cell can be a novel strategy for better cartilage repair.

Dynamic cyclic compression modulates the chondrogenic phenotype in human chondrocytes from late stage osteoarthritis.

Human osteoarthritic chondrocytes (hOACs) are characterized by their "dedifferentiated" and catabolic phenotype and lack the ability for restoring their inherent functions by themselves. Here we investigated whether extrinsically supplemented mechanical signal via compression loading would affect the phenotype of hOACs. Specifically, we applied cyclic compression loading on cultured hOACs-collagen constructs and measured the expression of the major chondrogenic factors, cell-matrix interaction molecules and matrix degradation enzymes. Dynamic compression loading stimulates the expression and nuclear localization of sox9 in hOACs and reduces the catabolic events via downregulated expression of collagenases. These results contribute to better understanding towards mechanoregulation of hOACs.

Chromosome-Encoded Hemolysin, Phospholipase, and Collagenase in Plasmidless Isolates of Photobacterium damselae subsp. damselae Contribute to Virulence for Fish.

Photobacterium damselae subsp. damselae is a pathogen of marine animals, including fish of importance in aquaculture. The virulence plasmid pPHDD1, characteristic of highly hemolytic isolates, encodes the hemolysins damselysin (Dly) and phobalysin (PhlyP). Strains lacking pPHDD1 constitute the vast majority of the isolates from fish outbreaks, but genetic studies to identify virulence factors in plasmidless strains are scarce. Here, we show that the chromosome I-encoded hemolysin PhlyC plays roles in virulence and cell toxicity in pPHDD1-negative isolates of this pathogen. By combining the analyses of whole genomes and of gene deletion mutants, we identified two hitherto uncharacterized chromosomal loci encoding a phospholipase (PlpV) and a collagenase (ColP). PlpV was ubiquitous in the subspecies and exerted hemolytic activity against fish erythrocytes, which was enhanced in the presence of lecithin. ColP was restricted to a fraction of the isolates and was responsible for the collagen-degrading activity in this subspecies. Consistent with the presence of signal peptides in PlpV and ColP sequences, mutants for the type II secretion system (T2SS) genes epsL and pilD exhibited impairments in phospholipase and collagenase activities. Sea bass virulence experiments and cell culture assays demonstrated major contributions of PhlyC and PlpV to virulence and toxicity.IMPORTANCE This study constitutes genetic and genomic analyses of plasmidless strains of an emerging pathogen in marine aquaculture, Photobacterium damselae subsp. damselae To date, studies on the genetic basis of virulence were restricted to the pPHDD1 plasmid-encoded toxins Dly and PhlyP. However, the vast majority of the recent isolates of this pathogen from fish farm outbreaks lack this plasmid. Here we demonstrate that the plasmidless strains produce two hitherto uncharacterized ubiquitous toxins encoded in chromosome I, namely, the hemolysin PhlyC and the phospholipase PlpV. We report the main roles of these two toxins in fish virulence and in cell toxicity. Our results constitute the basis for a better understanding of the virulence of a widespread marine pathogen.

Functional study of gene hp0169 in Helicobacter pylori pathogenesis.

Many virulence genes have been reported to play important roles in Helicobacter pylori pathogenesis. However the detailed mechanisms of many of them have not been completely clear. In this study, we found gene hp0169, encoding a putative collagenase (HpPrtC), was involved in pathogenesis of H. pylori. Recombinant HpPrtC shows activities to both native and heat-denatured collagens. This result indicated that HpPrtC may act as a virulence factor to help the bacterium colonize in their host stomach by degrading surrounding collagens. hp0169 was deleted by homologous recombination to study its function in bacterium-host cell interaction. For the pathogenic functions on the host cells, the hp0169 mutant exhibits no significant changes on inducing apoptosis of GES-1 cells. However, the viability and proliferation rate of GES-1 cells infected with mutant strain were higher than the cells infected with wild-type strain. These results indicated that except for its collagenolytic activity, HpPrtC might participate in H. pylori pathogenesis through an additional pathway. Functional studies on hp0169 involved in pathogenesis would shed light on deep understanding of the pathogenic mechanism of H. pylori.

Development of immersion vaccine for bacterial cold-water disease in ayu Plecoglossus altivelis.

Flavobacterium psychrophilum (F. psychrophilum) is the causative agent of bacterial cold-water disease (BCWD) that occurs in ayu Plecoglossus altivelis. Formalin-killed cell of F. psychrophilum has long been studied as an immersion vaccine for BCWD. In this study, we explored the possibility of F. psychrophilum collagenase (fpcol) for use as the immersion vaccine. BCWD convalescent ayu sera contained specific IgM antibodies against somatic F. psychrophilum and fpcol, meaning that fpcol is a promising antigen for the vaccine development. The recombinant fpcol was successfully expressed in Escherichia coli and Brevibacillus chosinensis (B. chosinensis). The culture supernatant of the B. chosinensis was used as an immersion vaccine solution. The vaccinated ayu were then challenged by soaking into F. psychrophilum culture. In two experimental groups, the relative percentages of survivals were 63 and 38%, respectively, suggesting that fpcol is promising as the immersion vaccine for ayu-BCWD.

Targeted Disruption of Ig-Hepta/Gpr116 Causes Emphysema-like Symptoms That Are Associated with Alveolar Macrophage Activation.

Ig-Hepta/GPR116 is a member of the G protein-coupled receptor family predominantly expressed in the alveolar type II epithelial cells of the lung. Previous studies have shown that Ig-Hepta is essential for lung surfactant homeostasis, and loss of its function results in high accumulation of surfactant lipids and proteins in the alveolar space. Ig-Hepta knock-out (Ig-Hepta(-/-)) mice also exhibit emphysema-like symptoms, including accumulation of foamy alveolar macrophages (AMs), but its pathogenic mechanism is unknown. Here, we show that the bronchoalveolar lavage fluid obtained from Ig-Hepta(-/-) mice contains high levels of inflammatory mediators, lipid hydroperoxides, and matrix metalloproteinases (MMPs), which are produced by AMs. Accumulation of reactive oxygen species was observed in the AMs of Ig-Hepta(-/-) mice in an age-dependent manner. In addition, nuclear factor-κB (NF-κB) is activated and translocated into the nuclei of the AMs of Ig-Hepta(-/-) mice. Release of MMP-2 and MMP-9 from the AMs was strongly inhibited by treatment with inhibitors of oxidants and NF-κB. We also found that the level of monocyte chemotactic protein-1 is increased in the embryonic lungs of Ig-Hepta(-/-) mice at 18.5 days postcoitum, when AMs are not accumulated and activated. These results suggest that Ig-Hepta plays an important role in regulating macrophage immune responses, and its deficiency leads to local inflammation in the lung, where AMs produce excessive amounts of reactive oxygen species and up-regulate MMPs through the NF-κB signaling pathway.

Hydrogen sulfide depletion contributes to microvascular remodeling in obesity.

Structural remodeling of the microvasculature occurs during obesity. Based on observations that impaired H2S signaling is associated with cardiovascular pathologies, the current study was designed to test the hypothesis that altered H2S homeostasis is involved in driving the remodeling process in a diet-induced mouse model of obesity. The structural and passive mechanical properties of mesenteric resistance arterioles isolated from 30-wk-old lean and obese mice were assessed using pressure myography, and vessel H2S levels were quantified using the H2S indicator sulfidefluor 7-AM. Remodeling gene expression was assessed using quantitative RT-PCR, and histological staining was used to quantify vessel collagen and elastin. Obesity was found to be associated with decreased vessel H2S concentration, inward hypertrophic remodeling, altered collagen-to-elastin ratio, and reduced vessel stiffness. In addition, mRNA levels of fibronectin, collagen types I and III, matrix metalloproteinases 2 and 9, and tissue inhibitor of metalloproteinase 1 were increased and elastin was decreased by obesity. Evidence that decreased H2S was responsible for the genetic changes was provided by experiments in which H2S levels were manipulated, either by inhibition of the H2S-generating enzyme cystathionine γ-lyase with dl-propargylglycine or by incubation with the H2S donor GYY4137. These data suggest that, during obesity, depletion of H2S is involved in orchestrating the genetic changes underpinning inward hypertrophic remodeling in the microvasculature.

Genetic polymorphisms of vein wall remodeling in chronic venous disease: a narrative and systematic review.

Chronic venous disease encompasses a spectrum of disorders caused by an abnormal venous system. They include chronic venous insufficiency, varicose veins, lipodermatosclerosis, postthrombotic syndrome, and venous ulceration. Some evidence suggests a genetic predisposition to chronic venous disease from gene polymorphisms associated mainly with vein wall remodeling. The literature exploring these polymorphisms has not been reviewed and compiled thus far. In this narrative and systematic review, we present the current evidence available on the role of polymorphisms in genes involved in vein wall remodeling and other pathways as contributors to chronic venous disease. We searched the EMBASE, Medline, and PubMed databases from inception to 2013 for basic science or clinical studies relating to genetic associations in chronic venous disease and obtained 38 relevant studies for this review. Important candidate genes/proteins include the matrix metalloproteinases (extracellular matrix degradation), vascular endothelial growth factors (angiogenesis and vessel wall integrity), FOXC2 (vascular development), hemochromatosis (involved in venous ulceration and iron absorption), and various types of collagen (contributors to vein wall strength). The data on associations between these genes/proteins and the postthrombotic syndrome are limited and additional studies are required. These associations might have future prognostic and therapeutic implications.