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.

Structure of Vibrio collagenase VhaC provides insight into the mechanism of bacterial collagenolysis.

The collagenases of Vibrio species, many of which are pathogens, have been regarded as an important virulence factor. However, there is little information on the structure and collagenolytic mechanism of Vibrio collagenase. Here, we report the crystal structure of the collagenase module (CM) of Vibrio collagenase VhaC and the conformation of VhaC in solution. Structural and biochemical analyses and molecular dynamics studies reveal that triple-helical collagen is initially recognized by the activator domain, followed by subsequent cleavage by the peptidase domain along with the closing movement of CM. This is different from the peptidolytic mode or the proposed collagenolysis of Clostridium collagenase. We propose a model for the integrated collagenolytic mechanism of VhaC, integrating the functions of VhaC accessory domains and its collagen degradation pattern. This study provides insight into the mechanism of bacterial collagenolysis and helps in structure-based drug design targeting of the Vibrio collagenase.

The Imbalance of MMP-2/TIMP-2 and MMP-9/TIMP-1 Contributes to Collagen Deposition Disorder in Diabetic Non-Injured Skin.

The importance of the early diagnosis and treatment of diabetes and its cutaneous complications has become increasingly recognized. When diabetic non-injured skin was stained with Masson's trichrome, its dermal collagen was found to be disordered, its density was variable, and it was dispersed or arranged in vague fascicles. The collagen type I sequencing results of RNA sequencing-based transcriptome analysis of three primary human skin cell types-dermal fibroblasts, dermal microvascular endothelial cells, and epidermal keratinocytes-under high glucose were analyzed. The results showed that both COL1A1 and COL1A2 mRNA expressions were reduced in human dermal fibroblasts (HDFs). The ratio of matrix metalloproteinase (MMP)-2/tissue inhibitors of metalloproteinase (TIMP)-2 and MMP-9/TIMP-1 in HDFs increased when treated with high glucose. By inhibiting MMP-2 and MMP-9 with SB-3CT, collagen deposition disorder of the skin in streptozotocin-induced diabetes mice was alleviated. The imbalance of MMP2/TIMP2 and MMP9/TIMP1 contributes to the non-injured skin disorder of collagen deposition in diabetes, suggesting a possibility for early treatment of diabetes skin complications.

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.

Studies on Vibrio mimicus derived collagenase variants providing insights into critical role(s) played by the FAXWXXT motifs in its collagen-binding domain.

Vibrio mimicus collagenase (VMC), a Class II Vibrio metalloprotease, contains an HEXXH motif in a zinc-binding catalytic domain, and two FAXWXXT motifs in its C-terminal domain, which is its collagen binding domain (CBD). To understand the functional role of the individual CBD motifs in the activity of VMC, if any, we created and characterized a series of VMC variants: i) VMA, with 51 amino acids deleted from the C-terminal end of full-length VMC; ii) VMT1, a form of VMA mutated in the first CBD motif; iii) VMT2, a form of VMA mutated in the second CBD motif; iv) DM, a form of VMA with both CBD motifs mutated; v) CT, a truncated form of VMA, lacking the entire CBD region; and vi) CBD, a construct containing the collagen binding domain alone. The activity of each variant was assessed by multiple means, in relation to VMA. We report that VMT1 and VMT2 show 1.6-fold and 10-fold reduced activity, respectively. The reduced activity of VMT2 correlates with reduced binding to insoluble collagen as well as an inability to cause structural perturbation of collagen. VMC appears to cause unwinding and structural alteration of the collagen triple helix prior to hydrolysis of the substrate (using both motifs for collagen binding), like Clostridium collagenases. In the absence of a known structure for VMC, our findings suggest that Vibrio collagenase, functions like Clostridium collagenases, although the two show very little sequence similarity. Also, VMC shows reduced activity with respect to Clostridium collagenases, making it an ideal enzyme for therapeutic 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.

Collagenase IV and clusterin-modified polycaprolactone-polyethylene glycol nanoparticles for penetrating dense tumor tissues.

Purpose: Novel collagenase IV (ColIV) and clusterin (CLU)-modified polycaprolactone-polyethylene glycol (PCL-PEG) nanoparticles that load doxorubicin (DOX) were designed and fully evaluated in vitro and in vivo. Methods: PCL-PEG-ColIV was synthesized by linking PCL-PEG and ColIV through a carbodiimide method. DOX-loaded nanoparticles (DOX-PCL-PEG-ColIV) were self-assembly prepared, followed by noncovalently adsorbing CLU on the DOX-PCL-PEG-ColIV surface to obtain DOX-PCL-PEG-ColIV /CLU nanoparticles, which can penetrate through the tumor extracellular matrix (ECM) and inhibit phagocytosis by macrophage. The physicochemical properties of nanoparticles were characterized. The cellular uptake and antiphagocytosis ability of nanoparticles in MCF-7 tumor cells and RAW264.7 cells were investigated. The penetration ability of nanoparticles was individually evaluated in the two-dimensional (2D) and three-dimensional (3D) ECM models. The tissue distribution and antitumor effect of nanoparticles were evaluated in MCF-7 cell-bearing nude mice. Results: Compared with DOX-PCL-PEG-COOH nanoparticles, DOX-PCL-PEG-ColIV/CLU nanoparticles could effectively overcome the phagocytosis by RAW264.7 and showed excellent cellular uptake in MCF-7 cells. In addition, they showed remarkable penetration ability through the 2D and 3D ECM models. DOX-PCL-PEG-ColIV/CLU nanoparticles significantly reduced the drug distribution in the liver and spleen and enhanced the drug accumulation in tumor tissue compared with DOX-PCL-PEG-COOH or DOX-PCL-PEG-ColIV nanoparticles. DOX-PCL-PEG-ColIV/CLU nanoparticles showed remarkable antitumor effect but did not cause severe pathological damages in the main tissues, including the heart, liver, spleen, lung, and kidney. Conclusion: Novel ColIV and CLU-modified PCL-PEG nanoparticles showed excellent cellular uptake, ECM penetration, antiphagocytosis, and antitumor effects both in vitro and in vivo.

A standardized Terminalia chebula fruit extract alters the expression of genes associated with skin architecture and barrier formation.

BACKGROUND: Terminalia chebula (TC) is a deciduous tree of which extracts have demonstrated efficacy for treatment of photodamage, skin aging, and wound healing. However, molecular and cellular mechanisms underlying these benefits remain unclear. OBJECTIVE: To profile dermal expression responses to a standardized tannin-enriched TC fruit extract (Synastol® TC). MATERIALS AND METHODS: Microarrays were used to evaluate gene expression in three-dimensional reconstituted human skin cultures. RESULTS: Genome-wide expression responses to TC were the opposite to those observed in cells exposed to oxidative stress, solar-simulated UV radiation, and wounding, with increased expression of genes associated with water homeostasis, skin barrier establishment, blood vessel development, and circadian rhythms. TC also increased expression of extracellular matrix components, such as collagens (COL1A1, COL1A2) and proteoglycans (PRELP, OGN), and in separate assays, we showed that TC inhibits MMP enzymes (MMP-1, MMP-2, MMP-3, MMP-9, MMP-12) and microbial activity (S. aureus, P. acnes). Unexpectedly, mRNA and protein levels of late keratinocyte (KC) differentiation markers (FLG, LOR) were increased by TC, and expression responses in skin cultures broadly resembled those that occur with KC differentiation. Consistent with these results, TC increased expression of transcription factors regulating KC differentiation (FOS, GHRL3, PPARG) and we noted enrichment of AP-1 binding sites in regions upstream of TC-increased genes. CONCLUSION: These results demonstrate that functionally important TC extract responses occur in the epidermis and are therefore not restricted to the dermal layer. Our findings thus suggest mechanisms by which TC may strengthen full-thickness skin architecture for treatment of skin aging and/or chronic wounds.

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.

Protease levels are significantly altered in pediatric burn wounds.

Burn wounds contain high levels of protease activity due to the need to remodel the damaged extracellular matrix proteins. While necessary, excessive protease activity can lead to improper wound healing and is associated with increased contraction and fibrosis. No studies to date have investigated the expression changes of all the collagenases and elastases in burn wounds. The present study compares gene expression changes and changes in collagenase and elastase activity between burn wound eschar and normal skin in a pediatric population. Deidentified pediatric tissues were used for these experiments. Burn wound tissue was excised as part of normal standard care within a week from injury; normal skin was removed during elective plastic surgery procedures. RNA-sequencing was performed and significant results were confirmed with qRT-PCR. Activity assays showed a significant increase in both collagenase and elastase activity in the burn wound tissue compared to the normal skin. Western blotting and substrate zymography of tissue homogenates evaluated the results at the protein levels. Four elastases and three collagenases were determined to be significantly upregulated in the wound tissues by both RNA-sequencing and qRT-PCR. Cathepsin V was the only protease that was significantly downregulated. All but one metalloproteinase studied was significantly upregulated. None of the serine proteases were significantly altered in the wound tissues. In conclusion, matrix metalloproteinases appear to be the most highly elevated proteases after a pediatric burn wound injury, at least within the first 3-7 days. The data warrant further investigation into the effects of MMPs on burn wound healing.

Evaluation of profibrotic gene transcription in renal tissues from cats with naturally occurring chronic kidney disease.

BACKGROUND: Increased gene transcription of hypoxia-induced mediators of fibrosis in renal tissue has been identified in experimentally induced, ischemic chronic kidney disease (CKD). OBJECTIVE: To characterize hypoxia-induced profibrotic pathways in naturally occurring CKD in cats. ANIMALS: Twelve client-owned cats with CKD and 8 healthy control cats. METHODS: In this prospective, cross-sectional study, bilateral renal tissue samples were assessed histologically for inflammation, tubular atrophy, and fibrosis, and by reverse transcription-quantitative PCR for characterization of transcript levels of hypoxia-inducible factor-1α (HIF1A), matrix metalloproteinases-2 (MMP2), -7 (MMP7), and -9 (MMP9), tissue inhibitor of metalloproteinase-1 (TIMP1), transforming growth factor-ß1 (TGFB1), and vascular endothelial growth factor-A (VEGFA). Linear mixed models were used to compare gene transcription between diseased and healthy kidneys, and to examine the association between transcript levels and serum creatinine concentration for all cats, and between transcript levels and histologic scores of diseased kidneys. RESULTS: Kidneys from cats with CKD had significantly higher transcript levels of HIF1A, MMP2, MMP7, MMP9, TIMP1, and TGFB1 (all P < .001), and lower levels of VEGFA (P = .006) than those from control cats. Transcript levels of MMP7 (P = .05) and TIMP1 (P = .005) were positively associated with serum creatinine in cats with CKD, but not in control cats. In diseased kidneys, transcript levels of MMP2 (P = .002), MMP7 (P = .02), and TIMP1 (P = .02) were positively, whereas those of VEGFA (P = .003) were negatively, associated with histologic score severity. CONCLUSION AND CLINICAL SIGNIFICANCE: Evaluation of the expression of the corresponding proteins in larger populations could identify therapeutic targets and/or biomarkers of tubulointerstitial fibrosis in cats.

The roles of histidine and tyrosine residues in the active site of collagenase in Grimontia hollisae.

Collagenase from the Grimontia hollisae strain 1706B (Ghcol) is a zinc metalloproteinase with the zinc-binding motif H492EXXH496. It exhibits higher collagen-degrading activity than the collagenase from Clostridium histolyticum, which is widely used in industry. We previously examined the pH and temperature dependencies of Ghcol activity; Glu493 was thought to contribute acidic pKa (pKe1), while no residue was assigned to contribute alkaline pKa (pKe2). In this study, we introduced nine single mutations at the His or Tyr residues in and near the active site. Our results showed that H412A, H485A, Y497A, H578A and H737A retained the activities to hydrolyze collagen and gelatin, while H426A, H492A, H496A and Y568A lacked them. Purification of active variants H412A, H485A, H578A and H737A, along with inactive variants H492A and H496A, were successful. H412A preferred (7-methoxycoumarin-4-yl)acetyl-L-Lys-L-Pro-L-Leu-Gly-L-Leu-[N3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH2 to collagen, while H485A preferred collagen to the peptide, suggesting that His412 and His485 are important for substrate specificity. Purification of the active variant Y497A and inactive variants H426A and Y568A were unsuccessful, suggesting that these three residues were important for stability. Based on the reported crystal structure of clostridial collagenase, Tyr568 of Ghcol is suggested to be involved in catalysis and may be the ionizable residue for pKe2.

Comparative genetics of Enterococcus faecalis intestinal tissue isolates before and after surgery in a rat model of colon anastomosis.

We have recently demonstrated that collagenolytic Enterococcus faecalis plays a key and causative role in the pathogenesis of anastomotic leak, an uncommon but potentially lethal complication characterized by disruption of the intestinal wound following segmental removal of the colon (resection) and its reconnection (anastomosis). Here we hypothesized that comparative genetic analysis of E. faecalis isolates present at the anastomotic wound site before and after surgery would shed insight into the mechanisms by which collagenolytic strains are selected for and predominate at sites of anastomotic disruption. Whole genome optical mapping of four pairs of isolates from rat colonic tissue obtained following surgical resection (herein named "pre-op" isolates) and then 6 days later from the anastomotic site (herein named "post-op" isolates) demonstrated that the isolates with higher collagenolytic activity formed a distinct cluster. In order to perform analysis at a deeper level, a single pair of E. faecalis isolates (16A pre-op and 16A post-op) was selected for whole genome sequencing and assembled using a hybrid assembly algorithm. Comparative genomics demonstrated absence of multiple gene clusters, notably a pathogenicity island in the post-op isolate. No differences were found in the fsr-gelE-sprE genes (EF1817-1822) responsible for regulation and production of collagenolytic activity. Analysis of unique genes among the 16A pre-op and post-op isolates revealed the predominance of transporter systems-related genes in the pre-op isolate and phage-related and hydrolytic enzyme-encoding genes in the post-op isolate. Despite genetic differences observed between pre-op and post-op isolates, the precise genetic determinants responsible for their differential expression of collagenolytic activity remains unknown.

The population structure of Clostridium tetani deduced from its pan-genome.

Clostridium tetani produces a potent neurotoxin, the tetanus neurotoxin (TeNT) that is responsible for the worldwide neurological disease tetanus, but which can be efficiently prevented by vaccination with tetanus toxoid. Until now only one type of TeNT has been characterized and very little information exists about the heterogeneity among C. tetani strains. We report here the genome sequences of 26 C. tetani strains, isolated between 1949 and 2017 and obtained from different locations. Genome analyses revealed that the C. tetani population is distributed in two phylogenetic clades, a major and a minor one, with no evidence for clade separation based on geographical origin or time of isolation. The chromosome of C. tetani is highly conserved; in contrast, the TeNT-encoding plasmid shows substantial heterogeneity. TeNT itself is highly conserved among all strains; the most relevant difference is an insertion of four amino acids in the C-terminal receptor-binding domain in four strains that might impact on receptor-binding properties. Other putative virulence factors, including tetanolysin and collagenase, are encoded in all genomes. This study highlights the population structure of C. tetani and suggests that tetanus-causing strains did not undergo extensive evolutionary diversification, as judged from the high conservation of its main virulence factors.

Epicatechin and scopoletin rich Morinda citrifolia (Noni) leaf extract supplementation, mitigated Osteoarthritis via anti-inflammatory, anti-oxidative, and anti-protease pathways.

The scopoletin (coumarin) and epicatechin (flavonoid) rich Morinda citrifolia L. (MC) Noni leaves are non-toxic (unlike the fruits) and consumed as vegetables. The anti-osteoarthritis effects of the MC leaf extract against joint cartilage degradation and inflammation were investigated through cartilage explant cultures and pre-clinical animal study. Osteoarthritis were induced by intra-articular monosodium iodoacetate injection into the right knee. The extract, scopoletin and epicatechin, suppressed glycosaminoglycan and nitric oxide release from the cartilage explant in the presence of Interleukin-1ß. After 28 days, the extract treatment reduced the in vivo serum levels and joint tissues mRNA expressions for joint cartilage degradation, aggrecanase, and collagenase biomarkers. The extract increased the bone formation marker PINP levels, besides improving the articular cartilage structure and chondrocytes cellularity. The extract improved bone formation/repair, subchondral bone structure, strength and integrity, as well as cartilage synthesis by suppressing inflammation, nitric oxide production, joint catabolism by proteases, and oxidative stress. PRACTICAL APPLICATIONS: The scopoletin (coumarin) and epicatechin (flavonoid) rich Morinda citrifolia (Noni) leaves may be used as vegetables, functional food ingredient, or dietary supplements to suppress osteoarthritis progression against joint cartilage degradation and inflammation. The extract, scopoletin, or epicatechin, suppressed glycosaminoglycan, and nitric oxide release from the cartilage. The Morinda citrifolia leaf extract suppressed inflammation, nitric oxide production, tissues catabolism by proteases and oxidative stress to help reduce joint cartilage degradation, besides improving the articular cartilage structure, chondrocytes health, subchondral bone structure, bone formation/repair, and cartilage synthesis.

Understanding the basis of Ehlers-Danlos syndrome in the era of the next-generation sequencing.

Ehlers-Danlos syndrome (EDS) is a clinically and genetically heterogeneous group of heritable connective tissue disorders (HCTDs) defined by joint laxity, skin alterations, and joint hypermobility. The latest EDS classification recognized 13 subtypes in which the clinical and genetic phenotypes are often overlapping, making the diagnosis rather difficult and strengthening the importance of the molecular diagnostic confirmation. New genetic techniques such as next-generation sequencing (NGS) gave the opportunity to identify the genetic bases of unresolved EDS types and support clinical counseling. To date, the molecular defects have been identified in 19 genes, mainly in those encoding collagen, its modifying enzymes or other constituents of the extracellular matrix (ECM). In this review we summarize the contribution of NGS technologies to the current knowledge of the genetic background in different EDS subtypes.

Matrix-Metalloprotease Resistant Mucin-16 (MUC16) Peptide Mutants Represent a Worse Lung Adenocarcinoma Outcome.

PURPOSE: The relationship of lung adenocarcinoma (LUAD)-specific proteases and the mutant profile of cytoskeletal and extracellular matrix proteins (CECMPs) are examined. EXPERIMENTAL DESIGN: Mutant CECMPs are assessed with an automated application of a protease binding, amino acid-based, scoring database. RESULTS: MUC16 (Human Genome Organization symbol for mucin-16 gene) mutants in particular are, more often than not, resistant to matrix-metalloproteases (MMPs) commonly secreted by LUAD cells, and LUAD cases representing the MUC16, MMP resistant mutants have a worse outcome. Similar results are obtained for MUC16 mutants resistant to cathepsins, also commonly secreted by LUAD cells. Analyses also show that MUC16, MMP resistant peptide mutants have greater binding affinities to HLA-A and HLA-B when compared to MUC16, MMP nonresistant peptide mutants. CONCLUSION: These results provide a potential, novel biomarker for lung cancer progression, in particular, protease resistant MUC16 peptides; and suggest a possible mechanism of immune escape entailing the reduction of mutant peptides available for HLA class I binding.

Isolation of Highly Viable Thymic Epithelial Cells for Use in In Vitro and In Vivo Experiments.

Thymic epithelial cells (TECs) play multiple essential roles in T-cell development and the establishment of immune tolerance, but their isolation can be challenging, and their low viability upon isolation complicates downstream experiments. A method that allows TECs to be isolated easily and to survive afterward will be useful for elucidating key questions in TEC biology. Here, we demonstrate a simple method to isolate highly viable TECs. Primary TECs isolated using papain together with collagenase IV and DNase I survive and proliferate in vitro. Moreover, these primary TECs functionally engraft after intrathymic transplantation into recipient mice. Thus, the methods described herein will be useful for elucidating the roles of TECs and TEC subsets in T-cell development and immune tolerance.