HOXA10 controls osteoblastogenesis by directly activating bone regulatory and phenotypic genes

HOXA10 is necessary for embryonic patterning of skeletal elements, but its function in bone formation beyond this early developmental stage is unknown. Here we show that HOXA10 contributes to osteogenic lineage determination through activation of Runx2 and directly regulates osteoblastic phenotypic genes. In response to bone morphogenic protein BMP2, Hoxa10 is rapidly induced and functions to activate the Runx2 transcription factor essential for bone formation. A functional element with the Hox core motif was characterized for the bone-related Runx2 P1 promoter. HOXA10 also activates other osteogenic genes, including the alkaline phosphatase, osteocalcin, and bone sialoprotein genes, and temporally associates with these target gene promoters during stages of osteoblast differentiation prior to the recruitment of RUNX2. Exogenous expression and small interfering RNA knockdown studies establish that HOXA10 mediates chromatin hyperacetylation and trimethyl histone K4 (H3K4) methylation of these genes, correlating to active transcription. HOXA10 therefore contributes to early expression of osteogenic genes through chromatin remodeling. Importantly, HOXA10 can induce osteoblast genes in Runx2 null cells, providing evidence for a direct role in mediating osteoblast differentiation independent of RUNX2. We propose that HOXA10 activates RUNX2 in mesenchymal cells, contributing to the onset of osteogenesis, and that HOXA10 subsequently supports bone formation by direct regulation of osteoblast phenotypic genes. <br/

Shrimp metabolism: The roles of lactate dehydrogenase (c31), glycogen phosphorylase (c34) and protein kinase (PK) as revealed by RNA interference

Abstract only.Energy metabolism is well-studied in vertebrate systems, providing insights on the genes and mechanisms involved in different pathways necessary for the survival of an organism. Yet, such studies are still lacking in invertebrate systems much more in shrimp. An earlier study has showed several contigs from the black tiger shrimp to be homologous to white spot syndrome virus (WSSV), a devastating pathogen in shrimp, including contig 31-WSSVORF82 (c31) and contig 34-WSSVORF21 (c34). This study aims to unveil the roles of three genes: c31, c34 and protein kinase (PK) in the shrimp system and its possible role in WSSV-infection. Rapid amplification of cDNA ends-polymerase chain reaction or RACE-PCR was used to obtain the full-length sequence of c31 and c34, followed by in vivo gene silencing using RNAi technology, and intramuscularly injecting dsRNA to WSSVchallenged Macrobrachium rosenbergii and Penaeus (Marsupenaeus) japonicus. Gene expression followed for healthy shrimps and dsRNA-treated shrimps. Mrc31 was revealed to be the enzyme lactase dehydrogenase (LDH), commonly released during tissue damage and is a marker for disease. The most parsimonious tree pictured Mrc31 to be sister clades to LDH of other shrimp species, Penaeus monodon and P. vannamei, supported with 100% and 72% bootstrap values, respectively. Mrc34 was highly homologous to the glycogen phosphorylase (GP) enzymes of other organisms including that of another shrimp, M. japonicus, bearing a bootstrap value of 99%. For PK, phylogenetic analysis revealed that the three open reading frames (ORFs) from P. monodon, M. rosenbergii and P. japonicus have 30% homology to WSSV-PK supported by a 98% bootstrap value. Mortality data from dsRNA-treated and WSSV-infected shrimps showed that treatment with dsRNA-LDH, GP and PK had significantly higher survival rates compared to that of the controls, Phosphate Buffered Saline (PBS) and Green Fluorescent Protein (GFP). Silencing the three genes in the shrimp has rendered some protective effect against the virus. Gene expression showed that all three genes are present in immune-related organs such as the gills, hepatopancreas and hemocyte. This study is the first to report the possible identities and functions of contigs 31, 34 and PK providing valuable data on the shrimp's genome

Population density controls on microbial pollution across the Ganga catchment

For millions of people worldwide, sewage-polluted surface waters threaten water security, food security and human health. Yet the extent of the problem and its causes are poorly understood. Given rapid widespread global urbanisation, the impact of urban versus rural populations is particularly important but unknown. Exploiting previously unpublished archival data for the Ganga (Ganges) catchment, we find a strong non-linear relationship between upstream population density and microbial pollution, and predict that these river systems would fail faecal coliform standards for irrigation waters available to 79% of the catchment’s 500 million inhabitants. Overall, this work shows that microbial pollution is conditioned by the continental-scale network structure of rivers, compounded by the location of cities whose growing populations contribute c. 100 times more microbial pollutants per capita than their rural counterparts

Alcohol exposure during late gestation: Multiple developmental outcomes in sheep

Alcohol consumption during pregnancy remains common in many countries. Exposure to even low amounts of alcohol (i.e. ethanol) in pregnancy can lead to the heterogeneous fetal alcohol spectrum disorders (FASD), while heavy alcohol consumption can result in the fetal alcohol syndrome (FAS). FAS is characterized by cerebral dysfunction, growth restriction and craniofacial malformations. However, the effects of lower doses of alcohol during pregnancy, such as those that lead to FASD, are less well understood. In this article, we discuss the findings of recent studies performed in our laboratories on the effects of fetal alcohol exposure using sheep, in which we investigated the effects of late gestational alcohol exposure on the developing brain, arteries, kidneys, heart and lungs. Our studies indicate that alcohol exposure in late gestation can (1) affect cerebral white matter development and increase the risk of hemorrhage in the fetal brain, (2) cause left ventricular hypertrophy with evidence of altered cardiomyocyte maturation, (3) lead to a decrease in nephron number in the kidney, (4) cause altered arterial wall stiffness and endothelial and smooth muscle function and (5) result in altered surfactant protein mRNA expression, surfactant phospholipid composition and pro-inflammatory cytokine mRNA expression in the lung. These findings suggest that fetal alcohol exposure in late gestation can affect multiple organs, potentially increasing the risk of disease and organ dysfunction in later life

The unconventional myosin CRINKLED and its mammalian orthologue MYO7A regulate caspases in their signalling roles

Caspases provide vital links in non-apoptotic regulatory networks controlling inflammation, compensatory proliferation, morphology and cell migration. How caspases are activated under non-apoptotic conditions and process a selective set of substrates without killing the cell remain enigmatic. Here we find that the Drosophila unconventional myosin CRINKLED (CK) selectively interacts with the initiator caspase DRONC and regulates some of its non-apoptotic functions. Loss of CK in the arista, border cells or proneural clusters of the wing imaginal discs affects DRONC-dependent patterning. Our data indicate that CK acts as substrate adaptor, recruiting SHAGGY46/GSK3-β to DRONC, thereby facilitating caspase-mediated cleavage and localized modulation of kinase activity. Similarly, the mammalian CK counterpart, MYO7A, binds to and impinges on CASPASE-8, revealing a new regulatory axis affecting receptor interacting protein kinase-1 (RIPK1)>CASPASE-8 signalling. Together, our results expose a conserved role for unconventional myosins in transducing caspase-dependent regulation of kinases, allowing them to take part in specific signalling events

A cartilage tissue engineering approach combining starch-polycaprolactone fibre mesh scaffolds with bovine articular chondrocytes

In the present work we originally tested the suitability of corn starch-polycaprolactone (SPCL) scaffolds for pursuing a cartilage tissue engineering approach. Bovine articular chondrocytes were seeded on SPCL scaffolds under dynamic conditions using spinner flasks (total of 4 scaffolds per spinner flask using cell suspensions of 0.5×106 cells/ml) and cultured under orbital agitation for a total of 6 weeks. Poly(glycolic acid) (PGA) non-woven scaffolds and bovine native articular cartilage were used as standard controls for the conducted experiments. PGA is a kind of standard in tissue engineering approaches and it was used as a control in that sense. The tissue engineered constructs were characterized at different time periods by scanning electron microscopy (SEM), hematoxylin-eosin (H&E) and toluidine blue stainings, immunolocalisation of collagen types I and II, and dimethylmethylene blue (DMB) assay for glycosaminoglycans (GAG) quantification assay. SEM results for SPCL constructs showed that the chondrocytes presented normal morphological features, with extensive cells presence at the surface of the support structures, and penetrating the scaffolds pores. These observations were further corroborated by H&E staining. Toluidine blue and immunohistochemistry exhibited extracellular matrix deposition throughout the 3D structure. Glycosaminoglycans, and collagen types I and II were detected. However, stronger staining for collagen type II was observed when compared to collagen type I. The PGA constructs presented similar features toSPCLat the end of the 6 weeks. PGA constructs exhibited higher amounts of matrix glycosaminoglycans when compared to the SPCL scaffolds. However, we also observed a lack of tissue in the central area of the PGA scaffolds. Reasons for these occurrences may include inefficient cells penetration, necrosis due to high cell densities, or necrosis related with acidic by-products degradation. Such situation was not detected in the SPCL scaffolds, indicating the much better biocompatibility of the starch based scaffolds

Detection of Taura Syndrome Virus (TSV) in Litopenaeus vannamei in the Philippines

Farming of Litopenaeus vannamei in the Philippines was seen as a measure to increase shrimp production in the country. Taura Syndrome Virus (TSV) is a viral pathogen reported to be associated with Litopenaeus vannamei and other shrimp species. TSV outbreak impedes the production of Litopenaeus vannamei population and other species susceptible to the virus. Detection of TSV in L. vannamei in the Philippines calls for proper mitigation and appropriate actions for aquaculture and environmental management towards sustainable development. This study embarked on the detection of TSV using a purposive sampling method. Samples showing morphological symptoms of TSV such as redness of the carapace and pleopods and necrotic gill tissue collected from sites in Luzon and Visayas in the Philippines were processed. Viral RNA was extracted from the gills, the central area of infiltration and replication site of the virus. Complementary DNA (cDNA) was synthesized from the RNA templates and were subjected to RT-PCR under specific conditions using TSV specific primers against a positive control. Selected sites in the Philippines were proven to be positive of infection as they showed morphological symptoms and RT-PCR result in having a band at around 200 base pairs (bp). Percentage of prevalence results were obtained: Bulacan with 5 out of 15 samples (33%), Batangas with 7 out of 15 samples (47%), Bohol with 1 out of 15 samples (7%) and Cebu with 2 out of 15 samples (13%). The Batangas samples showed the highest prevalence of 47% (7 out of 15 samples) that were positive of the infection. This study reported the presence of TSV in L. vannamei in the country using morphological and molecular techniques

Prospects of micromass culture technology in tissue engineering

Tissue engineering of bone and cartilage tissue for subsequent implantation is of growing interest in cranio- and maxillofacial surgery. Commonly it is performed by using cells coaxed with scaffolds. Recently, there is a controversy concerning the use of artificial scaffolds compared to the use of a natural matrix. Therefore, new approaches called micromass technology have been invented to overcome these problems by avoiding the need for scaffolds. Technically, cells are dissociated and the dispersed cells are then reaggregated into cellular spheres. The micromass technology approach enables investigators to follow tissue formation from single cell sources to organised spheres in a controlled environment. Thus, the inherent fundamentals of tissue engineering are better revealed. Additionally, as the newly formed tissue is devoid of an artificial material, it resembles more closely the in vivo situation. The purpose of this review is to provide an insight into the fundamentals and the technique of micromass cell culture used to study bone tissue engineering

Phytotoxicity, cytotoxicity and genotoxicity evaluation of organic and inorganic pollutants rich tannery wastewater from a Common Effluent Treatment Plant (CETP) in Unnao district, India using Vigna radiata and Allium cepa

The leather industry is a major source of environmental pollution in India. The wastewater generated by leather industries contains very high pollution parameters due to the presence of a complex mixture of organic and inorganic pollutants even after the treatment at a Common Effluent Treatment Plant (CETP) and disturbs the ecological flora and fauna. The nature, characteristics and toxicity of CETP treated wastewater is yet to be fully elucidated. Thus, this study aims to characterize and evaluate the toxicity of CETP treated tannery wastewater collected from the Unnao district of Uttar Pradesh, India. In addition to measuring the physico-chemical parameters, the residual organic pollutants was identified by GC-MS analysis and phytotoxicity, cytotoxicity and genotoxicity of the treated wastewater was evaluated using Vigna radiata L. and Allium cepa L. Results showed that the treated wastewater contained very high pollution parameters (TDS 3850mg/L, BOD 680mg/L, COD-1300mg/L). GC-MS analysis revealed the presence of various types of residual organic pollutants including benzoic acid, 3-[4,-(T-butyl) Phenyl] furan-2-5-dione, benzeneacetamide, resorcinol, dibutyl phthalate, and benzene-1,2,4-triol. Further, toxicological studies showed the phytotoxic nature of the wastewater as it inhibited seed germination in V. radiata L. and root growth of A. cepa. Genotoxicity was evidenced in the root tip cell of A. cepa where chromosomal aberrations (stickiness, chromosome loss, C-mitosis, and vagrant chromosome) and nuclear abnormalities like micronucleated and binucleated cells were observed. Thus, results suggested that it is not safe to discharge these wastewater into the environment