Role of HRTPT in kidney proximal epithelial cell regeneration: Integrative differential expression and pathway analyses using microarray and scRNA-seq

Damage to proximal tubules due to exposure to toxicants can lead to conditions such as acute kidney injury (AKI), chronic kidney disease (CKD) and ultimately end-stage renal failure (ESRF). Studies have shown that kidney proximal epithelial cells can regenerate particularly after acute injury. In the previous study, we utilized an immortalized in vitro model of human renal proximal tubule epithelial cells, RPTEC/TERT1, to isolate HRTPT cell line that co-expresses stem cell markers CD133 and CD24, and HREC24T cell line that expresses only CD24. HRTPT cells showed most of the key characteristics of stem/progenitor cells; however, HREC24T cells did not show any of these characteristics. The goal of this study was to further characterize and understand the global gene expression differences, upregulated pathways and gene interaction using scRNA-seq in HRTPT cells. Affymetrix microarray analysis identified common gene sets and pathways specific to HRTPT and HREC24T cells analysed using DAVID, Reactome and Ingenuity software. Gene sets of HRTPT cells, in comparison with publicly available data set for CD133+ infant kidney, urine-derived renal progenitor cells and human kidney-derived epithelial proximal tubule cells showed substantial similarity in organization and interactions of the apical membrane. Single-cell analysis of HRTPT cells identified unique gene clusters associated with CD133 and the 92 common gene sets from three data sets. In conclusion, the gene expression analysis identified a unique gene set for HRTPT cells and narrowed the co-expressed gene set compared with other human renal–derived cell lines expressing CD133, which may provide deeper understanding in their role as progenitor/stem cells that participate in renal repair.     

Evaluating the DNA barcodes for identification of butterflies from Western Himalayas,Uttarakhand, India

DNA barcodes in species tagging have become a popular tool for taking inventories of species from different groups worldwide. The present study aimed to generate DNA barcodes of butterfly species from the Western Himalayas in Uttarakhand, India. The Indian Western Himalayan region (IWHR) has been explored to a limited extent about butterfly species’ diversity. However, the IWHR is prone to environmental change, and slight variations in climatic conditions can influence species diversity and change butterflies' range. The mitochondrial cytochrome c oxidase I (COI) gene was first used to generate the DNA barcode for butterflies from this region on a broad scale. 28 morphologically identified species, consisting of 102 sequences, were finally grouped into 26 species, with only two species showing ambiguity in species identification. These species had < 3% sequence variations from their neighboring relatives, suggesting cryptic species diversity. Generated sequences were also compared with the GenBank data of conspecific geographical locations, which showed intraspecies variation ranging from 1.3% to 7.3%. It was also noted that butterfly species have both intra and interspecies sequence divergence. In the phylogenetic-based species identification, a total of 28 species belonging to 4 families of butterflies were successfully discriminated, and two species at the genus level, which had high intra-specific divergence (0.025), were considered. However, the high intra-species sequence divergence observed may represent the presence of hidden species.     

Stable germ line transformation of a leafy vegetable crop amaranth (Amaranthus tricolor L.) mediated by Agrobacterium tumefaciens

We have optimized a procedure for genetic transformation of a major leafy vegetable crop, Amaranthus tricolor L., using epicotyl explant co-cultivation with Agrobacterium tumefaciens. Two disarmed A. tumefaciens strains EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT harboring the neomycin phosphotransferase II gene (nptII) and the β-glucuronidase gene (gus), were evaluated as vector systems. The former displayed a higher transforming efficiency. Several key factors influencing the transformation events were optimized. The highest percentage of transformed shoots (24.24%) was achieved using hand-pricked epicotyl explants, a 10-min infection period, with 100 μM acetosyringone-pretreated Agrobacterium culture corresponding to OD₆₀₀???0.6 and diluted to 10⁹ cells ml^?1, followed by 4 d co-cultivation in the regeneration medium. Putative transformed explants capable of forming shoots were selected on medium supplemented with 75 μg?ml^?1 kanamycin, and transient as well as stable glucuronidase expression was determined by histochemical analysis. From a total of 48 selected shoot lines derived from independent transformation events with epicotyl explants co-cultivated with EHA 105, 32 showed positive PCR amplification for both the nptII and gus genes. Germ line transformation and transgene stability were evident in progeny of primary transformed plants (T₀). Among T₁ seedlings of 12 selected transgenic plant lines, kanamycin-resistant and kanamycin-sensitive seedlings segregated in a ratio typical of the Mendelian monohybrid pattern (3:1) as verified by the chi-square (χ ²) test. Southern hybridization of genomic DNA from kanamycin-resistant T₁ transgenic segregants to an nptII probe substantiated stable integration of the transgene. Neomycin phosphotransferase (NPTII) activity was detected in leaf protein extracts of selected T₁ transgenic plants, thereby confirming stable expression of the nptII gene.     

Results of testing the comparatory method: The curvature of the shell valve frontal section is inappropriate as a systematic character for the freshwater pearl mussel of the genus Margaritifera

This paper continues a discussion on the number of pearl mussel species of the genus Margaritifera in northern Europe. A biometric study of 1711 pearl mussel Margaritifera margaritifera shells from 15 rivers in Russia and Latvia (basins of the White and Baltic seas) has been conducted. All the examined samples fall into two groups: the northern group (with the shells more flattened on average, f. margaritifera) and the southern one (with more convex shells, f. elongata); the boundary between these groups is at 63° N. Analysis of intrapopulation variation has shown that the samples contain individuals that correspond to f. margaritifera, f. elongata, and f. borealis. However, any hiatus between these forms is absent in all the samples, and individuals belonging to two intermediate forms are rather frequent. The hypothesis on the species specificity of the shell valve frontal section has not been confirmed based on examination of large shell samples. The pearl mussels inhabiting rivers of Northern Europe belong to a single species, M. margaritifera.     

Delphinidin Reduces Cell Proliferation and Induces Apoptosis of Non-Small-Cell Lung Cancer Cells by Targeting EGFR/VEGFR2 Signaling Pathways

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) have emerged as two effective clinical targets for non-small-cell lung cancer (NSCLC). In the present study, we found that delphinidin, an anthocyanidin, present in pigmented fruits and vegetables, is a potent inhibitor of both EGFR and VEGFR2 in NSCLC cells that overexpress EGFR/VEGFR2. Using these cells, we next determined the effects of delphinidin on cell growth and apoptosis in vitro and on tumor growth and angiogenesis in vivo. Delphinidin (5-60 µM) treatment of NSCLC cells inhibited the activation of PI3K, and phosphorylation of AKT and MAPKs. Additionally, treatment of NSCLC cells with delphinidin resulted in inhibition of cell growth without having significant toxic effects on normal human bronchial epithelial cells. Specifically, treatment of NCI-H441 and SK-MES-1 cells with delphindin (5-60 µM) resulted in (i) cleavage of PARP protein, (ii) activation of caspase-3 and -9, (iii) downregulation of anti-apoptotic proteins (Bcl2, Bcl-xL and Mcl-1), (iv) upregulation of pro-apoptotic proteins (Bax and Bak), and (v) decreased expression of PCNA and cyclin D1. Furthermore, in athymic nude mice subcutaneously implanted with human NSCLC cells, delphinidin treatment caused a (i) significant inhibition of tumor growth, (ii) decrease in the expression of markers for cell proliferation (Ki67 and PCNA) and angiogenesis (CD31 and VEGF), and (iii) induction of apoptosis, when compared with control mice. Based on these observations, we suggest that delphinidin, alone or as an adjuvant to current therapies, could be used for the management of NSCLC, especially those that overexpress EGFR and VEGFR2.