Matrix Protein Gene Research Articles

Anti-inflammatory and pro-anabolic effects of 5-aminosalicylic acid on human inflammatory osteoarthritis models.

Osteoarthritis (OA) is the most common degenerative joint disease, mainly affecting the elderly worldwide, for which the drug treatment remains a major challenge. Low-grade inflammation plays a pivotal role in OA onset and progression. Exploration of notable anti-inflammatory and disease-modifying drugs on human samples could facilitate the evaluation of therapeutic strategies for OA. The anti-inflammatory drug 5-aminosalicylic acid (5-ASA) is a first-line drug for ulcerative colitis (UC), however no study has explored the effects of 5-ASA on articular chondrocytes. In this work, both in vitro (chondrocyte pellets) and ex vivo (osteochondral explants) human inflammatory OA models were applied to evaluate the effects of 5-ASA. In the inflammatory pellet model, 5-ASA remarkably downregulated the gene expression of interleukin-6 (IL-6), and cyclooxygenase-2 (COX-2) while upregulating proteoglycan 4 (PRG4) and cartilage oligomeric matrix protein (COMP) gene expression. Total glycosaminoglycan (GAG) synthesis by pellets was markedly increased in 5-ASA-treated groups compared with the inflammatory group. In conditioned medium, inflammatory mediators (IL-8, nitric oxide) were markedly inhibited upon 5-ASA treatment. Moreover, histological staining showed 5-ASA retained proteoglycan content and inhibited degradation of extracellular matrix (ECM) core components, aggrecan (ACAN) and collagen type II (COL2). In the inflammatory explant model, 5-ASA mitigated signs of OA development by reducing inflammatory mediators and GAG loss. These findings suggest that 5-ASA has anti-inflammatory and pro-anabolic effects on human chondrocyte pellet and osteochondral explant inflammatory OA models. Disease-modifying OA drugs are an unmet clinical need for the treatment of OA. Our study explored and demonstrated the anti-inflammatory and protective effects of 5-ASA on in vitro and ex vivo human inflammatory OA models, showing its translational potential for OA treatment.

Crenigacestat blocking notch pathway reduces liver fibrosis in the surrounding ecosystem of intrahepatic CCA viaTGF-β inhibition

BackgroundIntrahepatic cholangiocarcinoma (iCCA) is a highly malignant tumor characterized by an intensive desmoplastic reaction due to the exaggerated presence of the extracellular (ECM) matrix components. Liver fibroblasts close to the tumor, activated by transforming growth factor (TGF)-β1 and expressing high levels of α-smooth muscle actin (α-SMA), become cancer-associated fibroblasts (CAFs). CAFs are deputed to produce and secrete ECM components and crosstalk with cancer cells favoring tumor progression and resistance to therapy. Overexpression of Notch signaling is implicated in CCA development and growth. The study aimed to determine the effectiveness of the Notch inhibitor, Crenigacestat, on the surrounding microenvironment of iCCA.MethodsWe investigated Crenigacestat’s effectiveness in a PDX model of iCCA and human primary culture of CAFs isolated from patients with iCCA.ResultsIn silico analysis of transcriptomic profiling from PDX iCCA tissues treated with Crenigacestat highlighted “liver fibrosis” as one of the most modulated pathways. In the iCCA PDX model, Crenigacestat treatment significantly (p < 0.001) reduced peritumoral liver fibrosis. Similar results were obtained in a hydrodynamic model of iCCA. Bioinformatic prediction of the upstream regulators related to liver fibrosis in the iCCA PDX treated with Crenigacestat revealed the involvement of the TGF-β1 pathway as a master regulator gene showing a robust connection between TGF-β1 and Notch pathways. Consistently, drug treatment significantly (p < 0.05) reduced TGF-β1 mRNA and protein levels in tumoral tissue. In PDX tissues, Crenigacestat remarkably inhibited TGF-β signaling and extracellular matrix protein gene expression and reduced α-SMA expression. Furthermore, Crenigacestat synergistically increased Gemcitabine effectiveness in the iCCA PDX model. In 31 iCCA patients, TGF-β1 and α-SMA were upregulated in the tumoral compared with peritumoral tissues. In freshly isolated CAFs from patients with iCCA, Crenigacestat significantly (p < 0.001) inhibited Notch signaling, TGF-β1 secretion, and Smad-2 activation. Consequently, Crenigacestat also inactivated CAFs reducing (p < 0.001) α-SMA expression. Finally, CAFs treated with Crenigacestat produced less (p < 005) ECM components such as fibronectin, collagen 1A1, and collagen 1A2.ConclusionsNotch signaling inhibition reduces the peritumoral desmoplastic reaction in iCCA, blocking the TGF-β1 canonical pathway.

No molecular evidence for influenza A virus and coronavirus in bats belonging to the families Phyllostomidae, Vespertilionidae, and Molossidae in the state of São Paulo, Brazil.

This study aimed to evaluate, by molecular methods, the presence of influenza A virus (IAV) and coronavirus in non-hematophagous bats collected in the state of São Paulo, Brazil. Samples of lung tissue and small intestine from 105 bats belonging to three families (Phyllostomidae, Vespertilionidae, and Molossidae) were collected in 22 municipalities in the state of São Paulo. Genetic identification of bats species was performed by amplification and sequencing of a fragment of 710bp of the mitochondrial COI gene. In the detection of IAV, genomes were performed by RT-PCR, aiming at the amplification of a 245-bp fragment of the IAV matrix (M) protein gene. For coronaviruses, two fragments of 602 and 440bp corresponding to segments along the gene encoding the RNA-dependent RNA polymerase (RdRp) were targeted. The detection limit for each of the PCRs was also determined. All samples analyzed here were negative for both viruses, and the lower limit of detection of the PCRs for the amplification of influenza virus A and coronavirus was estimated at 3.5 × 103 and 4.59 genomic copies per microliter, respectively. Although bats have been shown to harbor a large number of pathogens, the results of the present study support the theory that virus circulation in bats in the wild often occurs at low viral loads and that our understanding of the complex infectious dynamics of these viruses in wild conditions is still limited.

Sf9 Cells Metabolism and Viability When Coinfected with Two Monocistronic Baculoviruses to Produce Rabies Virus-like Particles.

This work aimed to describe the dynamics of the Sf9 insect cells death and primary metabolism when this host is infected simultaneously by two recombinant baculoviruses (BV) expressing rabies glycoprotein (BVG) and matrix protein (BVM) genes to produce rabies virus-like particles (VLP) at different multiplicities of infection (MOI). Schott flasks essays covering a wide range of MOI for both BV were performed. Viable cell density, cell viability, glucose, glutamine, glutamate, lactate, ammonium, and rabies proteins concentrations were monitored over the infection phase. The expression of both recombinantproteins was not limited by glucose, glutamine, and glutamate in a broad MOI (pfu/cell) range of BVG (0.15-12.5) and BVM (0.1-5.0) using SF900 III serum free culture medium. Death phase initiation and the specific death rate depend on BV MOI. The wave pattern of nutrient/metabolite profiles throughout the viral infection phase is related to the baculovirus lytic cycle. The optimal MOIs ratio between BVG (2.5-4.5) and BVM (1.0-3.0) for maximum protein expression was defined. The produced rabies VLP sizes are close to 78nm. In general, these work outputs bring a better understanding of the metabolic performance of Sf9 cells when infected by BV for producing VLP, and specifically, for progressing in a rabies VLP vaccine development.

Comparison of ligamentization potential between anterior cruciate ligament-derived cells and adipose-derived mesenchymal stem cells reseeded to acellularized tendon allograft.

To test the hypothesis that reseeded anterior cruciate ligament (ACL)-derived cells have a better ability to survive and integrate into tendon extracellular matrix (ECM) and accelerate the ligamentization process, compared to adipose-derived mesenchymal stem cells (ADMSCs). Acellularized tibialis allograft tendons were used. Tendons were randomly reseeded with ACL-derived cells or ADMSCs. ACL-derived cells were harvested and isolated from remnants of ruptured ACLs during reconstruction surgery and cultured at passage three. Cell suspensions (200 µl) containing 2 × 106 ACL-derived cells or ADMSCs were prepared for the purpose of reseeding. At days 1, 3, and 7 post-reseeding, graft composites were assessed for repopulation with histological and immunohistochemical analysis. Matrix protein contents and gene expression levels were analyzed. In the graft reseeded with ACL-derived cells, a large number of elongated cells that integrated into the matrix were evident at day 3 and day 7. However, in the graft reseeded with ADMSCs, only a small number of elongated cells were found integrated into the matrix. Immunofluorescence for Ki-67 and type I collagen confirmed the pronounced production of type I collagen by Ki-67-positive ACL-derived cells integrated into the ECM. A messenger RNA (mRNA) expression assay demonstrated significantly higher gene expression levels of types I (p = 0.013) and III (p = 0.050) collagen in the composites reseeded with ACL-derived cells than ADMSCs. ACL-derived cells, when reseeded to acellularized tendon graft, demonstrated earlier better survival and integration in the tendon ECM and resulted in higher gene expression levels of collagen, which may be essential to the normal ligamentization process compared to ADMSCs.Cite this article: Bone Joint Res2022;11(11):777-786.

Hc-transgelin is a novel matrix protein gene involved in the shell biomineralization of triangle sail mussel (Hyriopsis cumingii)

In triangle sail mussel (Hyriopsis cumingii), shell biomineralization is a complicated process that involves multiple gene products. Shell matrix proteins are involved in the formation of the organic framework and play an important role in the regulation of calcium carbonate deposition. In this study, A new shell matrix protein gene Hc-transgelin was identified in H. cumingii. The full-length cDNA of Hc-transgelin was 1200 bp, including a 501 bp open reading frame, which encoded 166 amino acids. Hc-transgelin is rich in lysine, it accounts for 11.40% of the protein. The predicted transgelin protein contained a conserved calmodulin homologous domain. A tissue-specific expression assay indicated that Hc-transgelin exhibited significantly highest expression in the mantle. Furthermore, Hc-transgelin in situ hybridization detected positive signals at the edge of the mantle outer fold, where nacre and prismatic layer biomineralization occur. An RNA interference assay showed that the shape of aragonite flakes in nacre changed and their growth was inhibited, and cracks appeared in the prismatic layer organic sheath when the expression of Hc-transgelin was suppressed. In a shell repair assay, a higher expression of Hc-transgelin appeared from day 12 to day 25 when the nacre accumulated quickly. These findings indicate that Hc-transgelin may be involved in the formation of aragonite flakes in the nacre and play a role in the formation of the organic sheath in the prismatic layer of the shell. This study provides new insights into the role of the Hc-transgelin gene and also contributes to the molecular understanding of mollusk shell formation.

Torticollis in Connection with Spine Phenotype.

Purpose: Torticollis is not of uncommon occurrence in orthopaedic departments. Various theories and studies concerning the pathogenesis of the deformity have been suggested. We aimed to highlight and discuss the underlying cervical and spine malformation complex in correlation with torticollis via radiographic and tomographic analysis and its connection with a specific syndromic entity. Methods: Torticollis has been recognised in six patients (2 boys and 4 girls with an age range of 14–18 years), in addition to a couple of parents manifested persistent backpain. A variable spine malformation complex was the main reason behind torticollis. In addition, some patients manifested plagiocephaly, facial asymmetry and scoliosis/kyphoscoliosis. In some patients, conventional radiographs were of limited value because of the overlapping anatomical structures. Three-dimensional reconstruction CT scanning was the modality of choice, which enlightens the path for the phenotypic characterisation. Results: A 16-year-old-boy presented with torticollis in correlation with pathologic aberration of the spine cartilaginous stage was analysed via 3DCT scan. Comprehensive clinical and radiological phenotypes were in favour of spondylomegepiphyseal dysplasia. The genotype showed a mutation of the NKX3-2 (BAPX1) gene compatible with the diagnosis of spondylo-meg-epiphyseal-metaphyseal dysplasia. His younger male sibling and parents were heterozygous carriers. In two patients with pseudoachondroplasia syndrome, in which odontoid hypoplasia associated with cervical spine synchondrosis causing life-threatening torticollis, Cartilage oligomeric matrix protein (COMP) gene mutation was identified. MURCS syndrome has been diagnosed in two unrelated girls. Torticollis associated with cervical kyphosis was the major presentation since early childhood. Interestingly, one girl showed omovertebral bones of the lower cervical and upper thoracic spine. Her karyotype manifested a balanced translocation of 46 XX, t (14q; 15q). Conclusion: To detect the underlying etiological diagnosis of torticollis, a skeletal survey was the primary diagnostic tool. Conventional radiographs of the craniocervical junction and spine resulted in confusing readings because of the overlapping anatomical structures. Cranio-cervical malformation complex could have serious neurological deficits, especially for children with indefinite diagnosis of torticollis. The widely used term of congenital muscular torticollis resulted in morbid or mortal consequences. Moreover, some patients received vigorous physical therapy on the bases of muscular torticollis. Sadly speaking, this resulted in grave complications. Understanding the imaging phenotype and the genotype in such patients is the baseline tool for precise and proper management. The value of this paper is to sensitise physicians and orthopaedic surgeons to the necessity of comprehensive clinical and radiological phenotypic characterisations in patients with long term skeletal pathology.

Loss of Jagged1 in mature endothelial cells causes vascular dysfunction with alterations in smooth muscle phenotypes

BackgroundNotch signaling is an evolutionarily conserved pathway that functions via direct cell-cell contact. The Notch ligand Jagged1 (Jag1) has been extensively studied in vascular development, particularly for its role in smooth muscle cell maturation. Endothelial cell-expressed Jag1 is essential for blood vessel formation by signaling to nascent vascular smooth muscle cells and promoting their differentiation. Given the established importance of Jag1 in endothelial cell/smooth muscle crosstalk during development, we sought to determine the extent of this communication in the adult vasculature for blood vessel function and homeostasis. MethodsWe conditionally deleted Jag1 in endothelial cells of adult mice and examined the phenotypic consequences on smooth muscle cells of the vasculature. ResultsOur results show that genetic loss of Jag1 in endothelial cells has a significant impact on Notch signaling and vascular smooth muscle function in mature blood vessels. Endothelial cell-specific deletion of Jag1 causes a concomitant loss of JAG1 and NOTCH3 expression in vascular smooth muscle cells, resulting in a transition to a less differentiated state. Aortic vascular smooth muscle cells isolated from the endothelial cell-specific Jag1 deficient mice retain an altered phenotype in culture with fixed changes in gene expression and reduced Notch signaling. Utilizing comparative RNA-sequence analysis, we found that Jag1 deficiency preferentially affects extracellular matrix and adhesion protein gene expression. Vasoreactivity studies revealed a reduced contractile response and impaired agonist-induced relaxation in endothelial cell Jag1-deficient aortas compared to controls. ConclusionsThese data are the first to demonstrate that Jag1 in adult endothelial cells is required for the regulation and homeostasis of smooth muscle cell function in arterial vessels partially through the autoregulation of Notch signaling and cell matrix/adhesion components in smooth muscle cells.

Inactivation of Ihh in Sp7-Expressing Cells Inhibits Osteoblast Proliferation, Differentiation, and Bone Formation, Resulting in a Dwarfism Phenotype with Severe Skeletal Dysplasia in Mice.

Indian hedgehog (Ihh) is an indispensable paracrine factor for proper tissue patterning, skeletogenesis, and cellular proliferation. Recent genetic studies have revealed critical roles of chondrocyte-derived Ihh in regulating chondrocyte proliferation, hypertrophy and cartilage ossification. However, the functions of Sp7-expressing cell-derived Ihh in osteoblast differentiation and bone formation remain unclear. Sp7 is an essential transcription factor for osteoblast differentiation. In the current study, we generated Sp7-iCre; Ihhfl/fl mice, in which the Ihh gene was specifically deleted in Sp7-expressing cells to investigate the roles of Ihh. Ihh ablation in Sp7-expressing cells resulted in a dwarfism phenotype with severe skeletal dysplasia and lethality at birth, but with normal joint segmentation. Sp7-iCre; Ihhfl/fl mice had fewer osteoblasts, almost no cortical and trabecular bones, smaller skulls, and wider cranial sutures. Additionally, the levels of osteogenesis- and angiogenesis-related genes, and of major bone matrix protein genes were significantly reduced. These results demonstrated that Ihh regulates bone formation in Sp7-expressing cells. Ihh deficiency in primary osteoblasts cultured in vitro inhibited their proliferation, differentiation, and mineralization ability, and reduced the expression of osteogenesis-related genes. Moreover, the deletion of Ihh also attenuated the Bmp2/Smad/Runx2 pathway in E18.5 tibial and primary osteoblasts. The activity of primary osteoblasts in mutant mice was rescued after treatment with rhBMP2. In summary, our data revealed that Ihh in Sp7-expressing cells plays an indispensable role in osteoblast differentiation, mineralization, and embryonic osteogenesis, further implicated that its pro-osteogenic role may be mediated through the canonical Bmp2/Smad/Runx2 pathway.

Control of spring viremia of carp in common carp using RNA interference

In this study, the control of Carp sprivivirus, commonly known as spring viremia of carp virus (SVCV), replication in Epithelioma papulosum cyprini (EPC) cells and for the first time in common carp was investigated by using the RNA interference (RNAi) technology targeting the glycoprotein (G), matrix protein (M), and RNA-dependent RNA polymerase (L) genes. Small-interfering RNA sequences (siRNAs) were selected to target three different regions on both the SVCV-G (G-102, G-1070, and G-1456) and SVCV-M gene (M-88, M-271, and M-414). Designed specific siRNAs were investigated individually or in combinations in EPC cells. In vitro results showed that the most effective siRNAs were G-102 and M-271 that significantly reduced the virus titer in EPC cells by 1.6 logs and 1.5 logs respectively. Various combinations of the G-102 and M-271 of this study and siRNA-L-602 (targets the L-gene) from our previous study were investigated in vitro. Results showed that the most effective combination in reducing the virus replication in EPC cells was the siRNA-M-271 + L-602 combination as indicated by the significant reduction in virus titer (2.9 logs). In-vivo investigation of the siRNA-M-271 + L-602 combination by single or two successive doses in common carp challenged by IP injection of SVCV showed a significant reduction in mortality rate by 77.78% and 88.89% respectively. Results were confirmed by measuring the downregulation of SVCV-M and -L gene expression in vivo.Results of this study proved that SVCV can be controlled in common carp through targeting the SVCV-M and -L genes by using the RNAi technology that results in a significant in-vivo reduction of the SVCV replication.

Whole Aspect of Runx2 Functions in Skeletal Development.

Runt-related transcription factor 2 (Runx2) is a fundamental transcription factor for bone development. In endochondral ossification, Runx2 induces chondrocyte maturation, enhances chondrocyte proliferation through Indian hedgehog (Ihh) induction, and induces the expression of vascular endothelial growth factor A (Vegfa), secreted phosphoprotein 1 (Spp1), integrin-binding sialoprotein (Ibsp), and matrix metallopeptidase 13 (Mmp13) in the terminal hypertrophic chondrocytes. Runx2 inhibits the apoptosis of the terminal hypertrophic chondrocytes and induces their transdifferentiation into osteoblasts and osteoblast progenitors. The transdifferentiation is required for trabecular bone formation during embryonic and newborn stages but is dispensable for acquiring normal bone mass in young and adult mice. Runx2 enhances the proliferation of osteoblast progenitors and induces their commitment to osteoblast lineage cells through the direct regulation of the expressions of a hedgehog, fibroblast growth factor (Fgf), Wnt, and parathyroid hormone-like hormone (Pthlh) signaling pathway genes and distal-less homeobox 5 (Dlx5), which all regulate Runx2 expression and/or protein activity. Runx2, Sp7, and Wnt signaling further induce osteoblast differentiation. In immature osteoblasts, Runx2 regulates the expression of bone matrix protein genes, including Col1a1, Col1a2, Spp1, Ibsp, and bone gamma carboxyglutamate protein (Bglap)/Bglap2, and induces osteoblast maturation. Osteocalcin (Bglap/Bglap2) is required for the alignment of apatite crystals parallel to the collagen fibers; however, it does not physiologically work as a hormone that regulates glucose metabolism, testosterone synthesis, or muscle mass. Thus, Runx2 exerts multiple functions essential for skeletal development.

Prevalence, Genetics and Evolutionary Properties of Eurasian Avian-like H1N1 Swine Influenza Viruses in Liaoning.

Swine influenza virus (SIV) is an important zoonosis pathogen. The 2009 pandemic of H1N1 influenza A virus (2009/H1N1) highlighted the importance of the role of pigs as intermediate hosts. Liaoning province, located in northeastern China, has become one of the largest pig-farming areas since 2016. However, the epidemiology and evolutionary properties of SIVs in Liaoning are largely unknown. We performed systematic epidemiological and genetic dynamics surveillance of SIVs in Liaoning province during 2020. In total, 33,195 pig nasal swabs were collected, with an SIV detection rate of 2%. Our analysis revealed that multiple subtypes of SIVs are co-circulating in the pig population in Liaoning, including H1N1, H1N2 and H3N2 SIVs. Furthermore, 24 H1N1 SIVs were confirmed to belong to the EA H1N1 lineage and divided into two genotypes. The two genotypes were both triple reassortant, and the predominant one with polymerase, nucleoprotein (NP), and matrix protein (M) genes originating from 2009/H1N1; hemagglutinin (HA) and neuraminidase (NA) genes originating from EA H1N1; and the nonstructural protein (NS) gene originating from triple reassortant H1N2 (TR H1N2) was detected in Liaoning for the first time. According to our evolutionary analysis, the EA H1N1 virus in Liaoning will undergo further genome variation.

In silico Prediction of Molecular Docking between Chicken BRD2 and Newcastle Disease Virus Matrix Protein to Elucidate Host-pathogen Interaction

Background: Newcastle disease is one of the most contagious viral diseases caused by the Newcastle disease virus (NDV) which causes a huge loss in the terms of economy as well as mortality level in the poultry sector. The viral matrix protein is one of the proteins which helps in the attachment of virus, budding and assembly of progeny virions in the host cell. Host-pathogen interaction plays a very important role in the changeover of genetic diversity in organisms. Methods: The amino acid sequences of chicken BRD2 gene and matrix protein of NDV were used for structure prediction and visualization of protein-protein interaction between BRD2 and matrix protein by using Discovery Studio software. The nucleotide sequences of both genes were used for evolutionary analysis using MEGA7 software followed by selection pressure analysis using online Datamonkey server. Result: The results indicated that the ligand was attached with the matrix protein, which acts as a receptor on the virus surface. The purifying selection has been evident for the viral matrix protein gene. The docking of viral protein with host BRD2 protein has been detected in the study which further warrants experimental validation. In conclusion, the viral matrix protein interacts at amino acid position Tyrosine-148, Valine-339, Alanine-341 with ligand present on the chicken BRD2 protein. The evolutionary analysis revealed that the matrix protein of NDV R2B strain was the closest to that of Peacock, pigeon, duck and quail however, it exhibited maximum distance with the chicken NDV from Ireland, Vietnam and Haryana state of India.

Transcriptomic, Proteomic, and Functional Assays Underline the Dual Role of Extrapallial Hemocytes in Immunity and Biomineralization in the Hard Clam Mercenaria mercenaria.

Circulating hemocytes in the hemolymph represent the backbone of innate immunity in bivalves. Hemocytes are also found in the extrapallial fluid (EPF), the space delimited between the shell and the mantle, which is the site of shell biomineralization. This study investigated the transcriptome, proteome, and function of EPF and hemolymph in the hard clam Mercenaria mercenaria. Total and differential hemocyte counts were similar between EPF and hemolymph. Overexpressed genes in the EPF were found to have domains previously identified as being part of the “biomineralization toolkit” and involved in bivalve shell formation. Biomineralization related genes included chitin-metabolism genes, carbonic anhydrase, perlucin, and insoluble shell matrix protein genes. Overexpressed genes in the EPF encoded proteins present at higher abundances in the EPF proteome, specifically those related to shell formation such as carbonic anhydrase and insoluble shell matrix proteins. Genes coding for bicarbonate and ion transporters were also overexpressed, suggesting that EPF hemocytes are involved in regulating the availability of ions critical for biomineralization. Functional assays also showed that Ca2+ content of hemocytes in the EPF were significantly higher than those in hemolymph, supporting the idea that hemocytes serve as a source of Ca2+ during biomineralization. Overexpressed genes and proteins also contained domains such as C1q that have dual functions in biomineralization and immune response. The percent of phagocytic granulocytes was not significantly different between EPF and hemolymph. Together, these findings suggest that hemocytes in EPF play a central role in both biomineralization and immunity.

Sustained delivery of the bone morphogenetic proteins BMP-2 and BMP-7 for cartilage repair and regeneration in osteoarthritis

ObjectiveAttempts to utilise growth factors (GF) such as bone morphogenic proteins (BMPs) to treat osteoarthritis (OA) in the clinic have not secured widespread adoption. However, the novel crystalline GF formulation called PODS offers new perspectives. This study investigated the hypothesis that Polyhedrin Delivery System (PODS) BMP-2 and PODS BMP-7, compared with conventional BMP-2 and BMP-7 increase capacity for cartilage repair. DesignSustained release from PODS BMP-2 and PODS BMP-7 and their effects on OA patient-derived cells as well as a chondrocyte cell line were first assessed in vitro. Here, extra cellular matrix (ECM) protein gene expression and actual ECM deposition were measured and compared to the effect achieved with conventional, soluble BMPs. Subsequently, in an established murine model of cartilage regeneration of the knee joint, changes were traced over 8 weeks and scored with two metrics, modified Pineda and Mankin. ResultsBoth crystalline PODS BMP formulations strongly induced proliferation in primary as well as immortal cell line chondrocytes, outperforming conventional soluble BMP-2 and BMP-7. Furthermore, ECM-producing genes were upregulated and the production of ECM could be demonstrated. In the murine cartilage regeneration model, both PODS BMP-2 and PODS-BMP-7 improved cartilage repair assessed with both histological scoring methods. ConclusionsThis study showed that the sustained release of GF from PODS BMPs is effective in promoting chondrogenesis in vitro. The small animal data suggests that this novel approach of delivering therapeutic proteins sustainably and locally to the knee has promise for developing future disease-modifying therapies of OA.

Effect of two different concentrations of 1α,25-dihydroxyvitamin D3 on odontogenic differentiation of stem cells from human exfoliated deciduous teeth

Background:Stem cells from human exfoliated deciduous teeth (SHEDs) can transform into odontoblasts in vitro and in vivo. The role of 1α, 25-dihydroxyvitamin D3 (1α,25 vitD3) has been reported in the mineralization of hard tissues and teeth, as well as osteoblastic differentiation. This study aimed to assess the effect of different concentrations of 1α,25 vitD3 on odontogenic differentiation of SHEDs.Materials and Methods:In this experimental study, second-passage SHEDs were exposed to odontogenic medium along with 0, 10, 50, 100, and 150 nmol concentrations of in 1α, 25 vitD3 to determine its optimal concentration for odontogenic differentiation. The methyl thiazolyl tetrazolium (MTT) assay was performed. Odontogenic differentiation was evaluated by QRT- polymerase chain reaction for dentin matrix protein (DMP1) and dentin sialophosphoprotein (DSPP) genes. Morphology of differentiated cells was studied by Scanning Electron Microscopy. Data were analyzed using the Kruskal–Wallis, Mann–Whitney, Friedman, and Chi-square test. P < 0.05 is considered statistically significant.Results:MTT test result showed the two groups of odontogenic medium + 10 nm 1α,25 vitD3 and odontogenic medium + 150 nm 1α,25 vitD3 provided the most suitable conditions for cell viability at 72 h. Expression of both genes significantly increased in the presence of 1α,25 vitD3 (P < 0.001). Expression of both genes was significantly higher at 14 days compared with 7 days (P < 0.01). At both time points, expression of both genes was significantly higher in the presence of 150 nm 1α,25 vitD3 compared with 10 nm (P < 0.01). The accumulation of cells with odontoblastic morphology, cell interactions, and calcifications were evident.Conclusion:1α,25 vitD3 upregulates DMP1 and DSPP and results in odontogenic differentiation of SHEDs in odontogenic medium. This upregulation increases with time and by an increase in concentration of 1α,25 vitD3.

Ecdysone signal pathway participates in shell formation in pearl oysters Pinctada fucata martensii

Ecdysone exists in arthropods, Mollusca and other invertebrates and plays vital roles in exoskeleton formation of Ecdysozoa. However, little is known about its functions in bivalve species. Herein, we identified ecdysone from the serum of pearl oyster Pinctada fucata martensii and obtained the coding sequence of ecdysone receptor (PmEcR) and homologue of its heterodimer protein retinoid X receptor (PmRXR). The deduced amino acid sequences of PmEcR and PmRXR contained a DNA-binding and ligand-binding domain and were very similar to the orthologs of other species. Moreover, PmEcR and PmRXR were located in the nuclei and cytoplasm of HEK-293T cells. PmEcR and PmRXR were highly expressed in early embryos and biomineralized mantle tissue. Moreover, the serum concentration of ecdysone significantly increased at 2, 4, 6, and 8 h post-shell notching. The expression of PmEcR in the mantle tissue was significantly induced at the corresponding time points, while that of PmRXR was significantly induced at 6 h. Ecdysone stimulation remarkably induced the expression of growth factors (BMP2 and BMP7), transcription factors (PmRunt and AP-1), and shell matrix protein genes (chitinase, lysine-rich matrix protein (KRMP), TYR2, and PmCOLVI), which indicated that ecdysone signaling plays important roles in shell repair. However, yeast two-hybrid assay and bimolecular fluorescence complementation showed that PmEcR and PmRXR did not form dimers, suggesting the different molecular interactions of EcR in bivalves. These findings provide insights into the function of ecdysone and its regulation pathway in bivalve species.

Rapid Emergence of the Reassortant 2.3.4.4b H5N2 Highly Pathogenic Avian Influenza Viruses in a Live Poultry Market in Xinjiang, Northwest China

Live poultry markets (LPMs) play a key role in reassorting and spreading avian influenza viruses (AIVs). In 2018, four strains of H5N2 AIVs were isolated from domestic ducks (Anas platyrhynchos) during AIV surveillance from the LPM in Urumqi, Xinjiang, China. All gene segments of the isolates were amplified by reverse transcription-PCR and sequenced; then, the viral genetic mutations, reassortant, and origin were analyzed. Higher nucleotide identities were observed among each gene of the isolates, indicating a common ancestor. The hemagglutinin (HA) genes of the isolates all classified into the clade 2.3.4.4b; the HA, matrix protein (MP), and nonstructural protein (NS) genes were all clustered together with the local H5N6 highly pathogenic AIVs (HPAIVs) identified in the same LPM of Urumqi in July 2017; the neuraminidase albumen, polymerase basic proteins 1 and 2, polymerase acidic protein, and nucleocapsid protein genes (NA, PB1, PB2, PA, and NP) all had close phylogenetic relationships with the local H9N2 AIVs identified in the same LPM from September to October 2018. Multiple basic amino acids were present at the cleavage site of the HA protein, which was associated with HPAIVs. These results indicated that the reassortant clade 2.3.4.4b H5N2 HPAIVs were rapidly generated from reassortment between the H5N6 and H9N2 AIVs in the local LPM of Urumqi in 2018.

Whole-Genome Sequencing Highlights Conservative Genomic Strategies of a Stress-Tolerant, Long-Lived Scleractinian Coral, Porites australiensis Vaughan, 1918

Massive corals of the genus Porites, common, keystone reef builders in the Indo-Pacific Ocean, are distinguished by their relative stress tolerance and longevity. In order to identify genetic bases of these attributes, we sequenced the complete genome of a massive coral, Porites australiensis. We developed a genome assembly and gene models of comparable quality to those of other coral genomes. Proteome analysis identified 60 Porites skeletal matrix protein genes, all of which show significant similarities to genes from other corals and even to those from a sea anemone, which has no skeleton. Nonetheless, 30% of its skeletal matrix proteins were unique to Porites and were not present in the skeletons of other corals. Comparative genomic analyses showed that genes widely conserved among other organisms are selectively expanded in Porites. Specifically, comparisons of transcriptomic responses of P. australiensis and Acropora digitifera, a stress-sensitive coral, reveal significant differences in regard to genes that respond to increased water temperature, and some of the genes expanded exclusively in Porites may account for the different thermal tolerances of these corals. Taken together, widely shared genes may have given rise to unique biological characteristics of Porites, massive skeletons and stress tolerance.

Nucleotide amplification and sequencing of the GC-rich region between matrix and fusion protein genes of peste des petits ruminants virus

Peste des petits ruminants virus (PPRV) causes a highly devastating disease of sheep and goats, that threatens the conservation of small wild ruminants. The development of PPRV vaccines, diagnostics and therapeutics, greatly depends on in-depth genomic data. Yet, high guanine-cytosine (GC) content between matrix (M) and fusion (F) genes of PPRV poses difficulty for both primer design and nucleotide amplification. In turn, this has led into absence or low nucleotide sequence coverage in this region. This poses a risk of missing important part of the genome that could help to infer viral evolution. Here, an overlapping long-read primer-based amplification strategy was developed to amplify the GC-rich fragments between M-F gene junction using nexus gradient polymerase chain reaction (PCR). The resulting amplicons were sequenced by dideoxynucleotide cycle sequencing and compared with other PPRV nucleotide sequences available at GenBank. Our findings indicate clear PCR amplification products with expected size of the GC-rich fragments on agarose gel electrophoresis. The sequencing results of these fragments indicate 99.5 % nucleotide identity with PPRV strain KY628761. An extremely difficult PCR target of 67.4 % GC contents was successfully amplified and sequenced using this long-read primer approach. The long-read primer set may be used in tiling multiplex PCR for complete genome sequencing of PPRV.