Early insights into the role of Exoc6B associated with spondyloepimetaphyseal dysplasia with joint laxity type 3 in primary ciliogenesis and chondrogenic differentiation in vitro.

BACKGROUND: Spondyloepimetaphyseal dysplasia with joint laxity type 3 (SEMDJL3) is a rare skeletal dysplasia associated with EXOC6B, a component of the exocyst complex, involved in vesicle tethering and exocytosis at the plasma membrane. So far, EXOC6B and the pathomechanisms underlying SEMDJL3 remain obscure. METHODS AND RESULTS: Exoc6b was detected largely at the perinuclear regions and the primary cilia base in ATDC5 prechondrocytes. Its shRNA lentiviral knockdown impeded primary ciliogenesis. In Exoc6b silenced prechondrocytes, Hedgehog signaling was attenuated, including when stimulated with Smoothened agonist. Exoc6b knockdown deregulated the mRNA and protein levels of Col2a1, a marker of chondrocyte proliferation at 7- and 14-days following differentiation. It led to the upregulation of Ihh another marker of proliferative chondrocytes. The levels of Col10a1, a marker of chondrocyte hypertrophy was enhanced at 14 days of differentiation. Congruently, Axin2, a canonical Wnt pathway modulator that inhibits chondrocyte hypertrophy was repressed. The expression of Mmp13 and Adamts4 that are terminal chondrocyte hypertrophy markers involved in extracellular matrix (ECM) remodelling were downregulated at 7 and 14 days of chondrogenesis. Bglap that encodes for the most abundant non-collagenous bone matrix constituent and promotes ECM calcification was suppressed at 14 days of chondrocyte differentiation. ECM mineralization was assessed by Alizarin Red staining. Gene expression and ciliogenesis were investigated by reverse transcription quantitative real-time PCR, immunoblotting, and immunocytochemistry. CONCLUSIONS: These findings provide initial insights into the potential role of Exoc6b in primary ciliogenesis and chondrogenic differentiation, contributing towards a preliminary understanding of the molecular pathomechanisms underlying SEMDJL3.

Genome-wide DNA methylation profiling after Ayurveda intervention to bronchial asthmatics identifies differential methylation in several transcription factors with immune process related function.

BACKGROUND: The Indian traditional medicinal system, Ayurveda, describes several lifestyle practices, processes and medicines as an intervention to treat asthma. Rasayana therapy is one of them and although these treatment modules show improvement in bronchial asthma, their mechanism of action, particularly the effect on DNA methylation, is largely understudied. OBJECTIVES: Our study aimed at identifying the contribution of DNA methylation changes in modulating bronchial asthma phenotype upon Ayurveda intervention. MATERIALS AND METHODS: In this study, genome-wide methylation profiling in peripheral blood DNA of healthy controls and bronchial asthmatics before (BT) and after (AT) Ayurveda treatment was performed using array-based profiling of reference-independent methylation status (aPRIMES) coupled to microarray technique. RESULTS: We identified 4820 treatment-associated DNA methylation signatures (TADS) and 11,643 asthma-associated DNA methylation signatures (AADS), differentially methylated [FDR (≤0.1) adjusted p-values] in AT and HC groups respectively, compared to BT group. Neurotrophin TRK receptor signaling pathway was significantly enriched for differentially methylated genes in bronchial asthmatics, compared to AT and HC subjects. Additionally, we identified over 100 differentially methylated immune-related genes located in the promoter/5'-UTR regions of TADS and AADS. Various immediate-early response and immune regulatory genes with functions such as transcription factor activity (FOXD1, FOXD2, GATA6, HOXA3, HOXA5, MZF1, NFATC1, NKX2-2, NKX2-3, RUNX1, KLF11), G-protein coupled receptor activity (CXCR4, PTGER4), G-protein coupled receptor binding (UCN), DNA binding (JARID2, EBF2, SOX9), SNARE binding (CAPN10), transmembrane signaling receptor activity (GP1BB), integrin binding (ITGA6), calcium ion binding (PCDHGA12), actin binding (TRPM7, PANX1, TPM1), receptor tyrosine kinase binding (PIK3R2), receptor activity (GDNF), histone methyltransferase activity (MLL5), and catalytic activity (TSTA3) were found to show consistent methylation status between AT and HC group in microarray data. CONCLUSIONS: Our study reports the DNA methylation-regulated genes in bronchial asthmatics showing improvement in symptoms after Ayurveda intervention. DNA methylation regulation in the identified genes and pathways represents the Ayurveda intervention responsive genes and may be further explored as diagnostic, prognostic, and therapeutic biomarkers for bronchial asthma in peripheral blood.

Understanding the effects of Abhraka Bhasma on genotoxicity and its DNA repair potential in mouse model.

BACKGROUND: Metal toxicity is of major concern to human health. The metals may modulate molecular mechanisms of various pathways. Rasashastra, the branch of Ayurveda, narrates the properties, unique preparation, processing techniques, and therapeutic uses of minerals. The use of herbal metallic preparations has evoked concern for their potential to produce toxicity, interest in efficacy as therapeutic agents and safety related issues. Abhraka Bhasma, is one such incinerated herbo-metallic preparation of mica, widely used by traditional medicine practitioners. Although there are reports of Abhraka Bhasma on beneficial effects, clear evidence is lacking on the effect of Abhraka Bhasma on genotoxicity and DNA repair. OBJECTIVE: The present study aims to understand the effects of Abhraka Bhasma on geno toxicity, DNA repair, and other mechanisms in the mice test model. MATERIAL AND METHODS: The experiments were conducted in in vivo Swiss albino mice. The acute oral toxicity was performed as per the OECD guidelines. The mice were treated with Abhraka Bhasma (120 or 360 mg/kg body weight) for 7 days. They were then challenged with ethyl methanesulfonate and the DNA repair was analyzed. RESULTS: The data obtained indicated that the Abhraka Bhasma is not a genotoxic and reproductive toxic formulation. The selected higher concentration of Abhraka Bhasma showed a protective role against ethyl methanesulfonate induced chromosomal damages and enhanced constitutive DNA base excision repair in mice. CONCLUSION: The anti-oxidant, potentiation of DNA repair and hematinic properties of Abhraka Bhasma may be attributed to the synergistic actions of its bioactive components.

Advances in mitochondrial medicine and translational research.

Current knowledge of mitochondrial biology and function has provided tools and technologies that helped a better understanding of the molecular etiology of complex mitochondrial disorders. Dual genetic control of this subcellular organelle function regulates various signaling mechanisms which are essential for metabolism, bioenergetics, fatty acid biosynthesis, and DNA replication & repair. Understanding nuclear mitochondrial crosstalk through advanced genomics as well as clinical perspectives is the overall basis of mitochondrial research and medicine, also the sole objective of Society for Mitochondrial Medicine and Research (SMRM) - India. The eighth virtual international conference on 'Advances in Mitochondrial Medicine and Translational Research' was organized at the Manipal School of Life Sciences, MAHE, Manipal, India, during 6 - 7 November 2020. The aim of the virtual conference was to highlight the recent advances and future perspectives that represent comprehensive clinical and fundamental research interests in the area of mitochondrial biology of human diseases. To systematically present the various findings in mitochondrial biology, the meeting was themed with specific aspects comprising (a) mitochondrial disorders: clinical & genomic perspectives, (b) mitochondria in cancer, (c) mitochondrial metabolism & disorders, and (d) mitochondrial diseases & therapy. This report provides an overview of the recent advancements in the area of mitochondrial biology and medicine that was discussed at the conference.

Advances in Mitochondrial Medicine and Translational Research.

Current knowledge of mitochondrial biology and function has provided with tools and technologies that helped a better understanding of the molecular etiology of complex mitochondrial disorders. Dual genetic control of this subcellular organelle function regulates various signaling mechanisms which are essential for metabolism, bioenergetics, fatty acid biosynthesis, and DNA replication & repair. Understanding nuclear mitochondrial crosstalk through advanced genomics as well as clinical perspectives is the overall basis of mitochondrial research and medicine, also the sole objective of Society for Mitochondrial Medicine and Research (SMRM) - India. The eighth virtual international conference on 'Advances in Mitochondrial Medicine and Translational Research' was organized at the Manipal School of Life Sciences, MAHE, Manipal, India, during 6 - 7 November 2020. The aim of the virtual conference was to highlight the recent advances and future perspectives that represent comprehensive clinical and fundamental research interests in the area of mitochondrial biology of human diseases. To systematically present the various findings in mitochondrial biology, the meeting was themed with specific aspects comprising (a) mitochondrial disorders: clinical & genomic perspectives, (b) mitochondria in cancer, (c) mitochondrial metabolism & disorders, and (d) mitochondrial diseases & therapy. This report provides an overview of the recent advancements in the area of mitochondrial biology and medicine that was discussed at the conference.

Advances in development and application of human organoids.

Innumerable studies associated with cellular differentiation, tissue response and disease modeling have been conducted in two-dimensional (2D) culture systems or animal models. This has been invaluable in deciphering the normal and disease states in cell biology; the key shortcomings of it being suitability for translational or clinical correlations. The past decade has seen several major advances in organoid culture technologies and this has enhanced our understanding of mimicking organ reconstruction. The term organoid has generally been used to describe cellular aggregates derived from primary tissues or stem cells that can self-organize into organotypic structures. Organoids mimic the cellular microenvironment of tissues better than 2D cell culture systems and represent the tissue physiology. Human organoids of brain, thyroid, gastrointestinal, lung, cardiac, liver, pancreatic and kidney have been established from various diseases, healthy tissues and from pluripotent stem cells (PSCs). Advances in patient-derived organoid culture further provides a unique perspective from which treatment modalities can be personalized. In this review article, we have discussed the current strategies for establishing various types of organoids of ectodermal, endodermal and mesodermal origin. We have also discussed their applications in modeling human health and diseases (such as cancer, genetic, neurodegenerative and infectious diseases), applications in regenerative medicine and evolutionary studies.

ROS-dependent DNA damage and repair during germination of NaCl primed seeds.

Reactive oxygen species (ROS) generated during rehydration of seeds is a major source of cellular damage. Successful germination depends on maintaining the oxidative window and ability of the cells to repair the DNA damage accumulated during seed developmental process, maturational drying, and germination. We explored the role of DNA damage, repair, cell cycle progression and antioxidant machinery in germination of seeds of Solanum melongena L. primed with 0, 320, 640 and 1200 mM sodium chloride (NaCl). The expression of antioxidant genes such as ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase2 (CAT2), and glutathione reductase (GR) was upregulated to maintain the oxidative window required for germination in seeds treated with 320 mM NaCl. ROS generated upon treatment with 320 mM NaCl resulted in minimal DNA damage and activated non-homologous end joining (NHEJ) and mismatch repair (MMR) pathway genes such as KU70 and mutS homolog 2 (MSH2) respectively. Treatment with higher concentrations of NaCl resulted in increased DNA damage despite lower ROS, without evoking DNA repair mechanisms. Uncontrolled rehydration resulted in higher levels of ROS and DNA damage, but activation of homologous recombination (HR) pathway gene, Nijmegen breakage syndrome 1 (NBS1), and genes involved in repairing oxidized guanine, such as oxoguanine DNA glycosylase (OGG1) and proliferating cell nuclear antigen (PCNA). In summary, controlled rehydration with 320 mM NaCl decreased the DNA damage, reactivated the antioxidant and DNA repair machinery, and cell cycle progression, thereby enhancing the seed germination.

Dataset on fat body proteome of Anopheles stephensi Liston.

Fat body from Anopheles stephensi female mosquitoes were dissected and processed for proteomic analysis. Both SDS-PAGE and basic Reverse Phase Liquid Chromatography-based fractionation strategies were used to achieve a broad coverage of protein identification. The fractionated peptides were then analyzed on a high-resolution mass spectrometer. Searching the raw data against the protein database of An. stephensi resulted in identification of 4535 proteins, which is, to our knowledge, the largest catalog of fat body proteome in any mosquito vector species reported so far. Bioinformatics analysis on these fat body proteins suggested the enrichment of biological processes including carbon and lipid metabolism, amino acid metabolism, signal peptide processing and oxidation-reduction. In addition, using proteogenomic approaches, 43 novel proteins were identified, which were not listed in the annotated gene annotations of An. stephensi. The data used in the analysis are related to the article 'Integrating transcriptomic and proteomic data for accurate assembly and annotation of genomes' (Prasad et al., 2017).

AMD Genetics in India: The Missing Links.

Age related macular degeneration is a disease which occurs in aged individuals. There are various changes that occur at the cellular, molecular and physiological level with advancing age (Samiec et al., 1988; Sharma K. et al., 2014). Drusen deposition between retinal pigment epithelium (RPE) and Bruch's membrane (BM) is one of the key features in AMD patients (Mullins et al., 2000; Hageman et al., 2001) similar to Aß/tau aggregates in Alzheimer's disease (AD) patients. The primary goal of this review is to discuss whether the various candidate genes and associated biomarkers, that are known to play an independent role in progression of AMD, exert deleterious effect on phenotype, alone or in combination, in Indian AMD patients from the same ethnic group and the significance of such research. A statistical model for probable interaction between genes could be derived from such analysis. Therefore, one can use multiple modalities to identify and enrol AMD patients based on established clinical criteria and examine the risk factors to determine if these genes are associated with risk factors, biomarkers or disease by Mendelian randomization. Similarly, there are large numbers of single nucleotide polymorphisms (SNPs) identified in human population. Even non-synonymous SNPs (nsSNPs) are believed to induce deleterious effects on the functionality of various proteins. The study of such snSNPs could provide a better genetic insight for diverse phenotypes of AMD patients, predicting significant risk factors for the disease in Indian population. Therefore, the prediction of biological effect of nsSNPs in the candidate genes and the associated grant applications in the subject are highly solicited.Therefore, genotyping and levels of protein expression of various genes would provide wider canvas in genetic complexity of AMD pathology which should be evaluated by valid statistical and bioinformatics' tools. Longitudinal follow up of Indian AMD patients to evaluate the temporal effect of SNPs and biomarkers on progression of disease would provide a unique strategy in the field.

Pathway Analysis of Proteomics Profiles in Rabies Infection: Towards Future Biomarkers?

Rabies is a zoonotic viral disease that invariably leads to fatal encephalitis, which can be prevented provided post-exposure prophylaxis is initiated timely. Ante-mortem diagnostic tests are inconclusive, and rabies is nontreatable once the clinical signs appear. A large number of host factors are responsible for the altered neuronal functions observed in rabies; however their precise role remains uninvestigated. We therefore used two-dimensional electrophoresis and mass spectrometry analysis to identify differentially expressed host proteins in an experimental murine model of rabies. We identified 143 proteins corresponding to 45 differentially expressed spots (p < 0.05) in neuronal tissues of Swiss albino mice in response to infection with neurovirulent rabies strains. Time series analyses revealed that a majority of the alterations occur at 4 to 6 days post infection, in particular affecting the host's cytoskeletal architecture. Extensive pathway analysis and protein interaction studies using the bioinformatic tools such as Ingenuity Pathway Analysis and STRING revealed novel pathways and molecules (e.g., protein ubiquitination) unexplored hitherto. Further activation/inhibition studies of these pathway molecular leads would be relevant to identify novel biomarkers and mechanism-based therapeutics for rabies, a disease that continues to severely impact global health.

PAOO for faster function, aesthetics and harmony.

Periodontally accelerated osteogenic orthodontics (PAOO) is a clinical procedure that combines selective alveolar corticotomy, particulate bone grafting and the application of orthodontic forces. PAOO can play an important role in the comprehensive treatment of a patient's occlusal and esthetic needs. This technique has been shown to increase alveolar bone thickness, decrease treatment time and enhance post-treatment orthodontic stability. Contained in this review is a discussion of the different aspects of PAOO and an assessment of its role in comprehensive treatment planning for the orthodontic patient.