Craniofacial chondrogenesis in organoids from human stem cell-derived neural crest cells.

Knowledge of cell signaling pathways that drive human neural crest differentiation into craniofacial chondrocytes is incomplete, yet essential for using stem cells to regenerate craniomaxillofacial structures. To accelerate translational progress, we developed a differentiation protocol that generated self-organizing craniofacial cartilage organoids from human embryonic stem cell-derived neural crest stem cells. Histological staining of cartilage organoids revealed tissue architecture and staining typical of elastic cartilage. Protein and post-translational modification (PTM) mass spectrometry and snRNA-seq data showed that chondrocyte organoids expressed robust levels of cartilage extracellular matrix (ECM) components: many collagens, aggrecan, perlecan, proteoglycans, and elastic fibers. We identified two populations of chondroprogenitor cells, mesenchyme cells and nascent chondrocytes, and the growth factors involved in paracrine signaling between them. We show that ECM components secreted by chondrocytes not only create a structurally resilient matrix that defines cartilage, but also play a pivotal autocrine cell signaling role in determining chondrocyte fate.

PI3K-Akt pathway mediated antiviral mechanism in silkworm Antheraea mylitta.

Bombyx mori nucleopolyhedrosis virus (BmNPV) is a highly pathogenic virus to domestic silkworm Bombyx mori, often causing severe economic losses to silk industry. Interestingly, there are no documented reports of NPV infection in the wild silkworm Antheraea mylitta. Analysis of gene expression datasets and comparative genomic analysis of sequence datasets from B. mori and A. mylitta was undertaken to unravel the potential immune-related proteins and immune pathways involved. The B. mori silkworm races Sarupat and CSR-2 which are resistant and susceptible to BmNPV respectively were selected and challenged with virus to study BmNPV resistance related genes and their expression profile. The genes were filtered to isolate membrane proteins, their sequences were retrieved from UniProt and were compared against A. mylitta using BLASTp to search for similarity. Major proteins were putative defence proteins. Further, KEGG database was used to check for the presence of differentially regulated proteins in certain metabolic pathways. The analysis of the pathways was carried out using cytoscape software based on betweeness centrality and stress. The PI3K-Akt pathway was found to be the hub of all signals triggered during the course of NPV infection. We analyzed how the NPV infection modulates PI3K-Akt signaling by gene expression studies of the key regulator of the pathway i.e. Akt using real-time PCR in A. mylitta. A significant upregulation of Akt expression from 72 h post infection reaching its peak with a 2.30 fold change at 120 h pi clearly indicates an enhanced level of immune response in host towards the viral infection.

Molecular evaluation of BRAF V600 mutation and its association with clinicopathological characteristics: First findings from Indian malignant melanoma patients.

Mutations in the BRAF gene have been described to occur in two-third of melanomas. The objective of the study was to establish the frequency of BRAF V600E/K/R mutation in a series of melanomas from Indian origin and to correlate mutation status with clinicopathological features. Seventy melanoma cases were evaluated for BRAF V600 mutation by pyrosequencing. Overall, BRAF mutations were detected in 30% of the patients. All mutations observed were missense type (GTG > GAG) resulting in p.V600E, while none showed V600K/R mutation. The frequency of BRAF V600E mutations were more in patients with onset age of 50 years. BRAF mutations were significantly associated with tumor site wherein more mutations were seen in tumors from head and neck and extremities region. Acral and mucosal tumor subtype showed a mutation frequency of 31% and 20%, respectively. Epithelial cell morphology tends to harbor frequent BRAF V600E mutation (36%) than other morphological subtypes. Tumors with ulceration and necrosis showed increased BRAF mutation rate (32.5% and 33%) respectively. In conclusion, this is the first study to report a mutation frequency of 30% in this cohort. Our results demonstrated that the BRAF V600E mutation is a frequent event in Indian melanomas, and represents an important molecular target for novel therapeutic approaches.