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.     

Genomic rearrangements generate hypervariable mini-chromosomes in host-specific isolates of the blast fungus

Supernumerary mini-chromosomes–a unique type of genomic structural variation–have been implicated in the emergence of virulence traits in plant pathogenic fungi. However, the mechanisms that facilitate the emergence and maintenance of mini-chromosomes across fungi remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae), mini-chromosomes have been first described in the early 1990s but, until very recently, have been overlooked in genomic studies. Here we investigated structural variation in four isolates of the blast fungus M. oryzae from different grass hosts and analyzed the sequences of mini-chromosomes in the rice, foxtail millet and goosegrass isolates. The mini-chromosomes of these isolates turned out to be highly diverse with distinct sequence composition. They are enriched in repetitive elements and have lower gene density than core-chromosomes. We identified several virulence-related genes in the mini-chromosome of the rice isolate, including the virulence-related polyketide synthase Ace1 and two variants of the effector gene AVR-Pik. Macrosynteny analyses around these loci revealed structural rearrangements, including inter-chromosomal translocations between core- and mini-chromosomes. Our findings provide evidence that mini-chromosomes emerge from structural rearrangements and segmental duplication of core-chromosomes and might contribute to adaptive evolution of the blast fungus.     

Targeting monocarboxylate transporter by α-cyano-4-hydroxycinnamate modulates apoptosis and cisplatin resistance of Colo205 cells: implication of altered cell survival regulation

The present investigation was undertaken to study the effect of in vitro exposure of Colo205, colonadenocarcinoma cells, to monocarboxylate transporter inhibitor α-cyano-4-hydroxycinnamate (αCHC) on cell survival and evolution of resistance to chemotherapeutic drug cisplatin. αCHC-treated Colo205 cells showed inhibition of survival accompanied by an augmented induction of apoptosis. Changes in cell survival properties were associated with alterations in lactate efflux, pH homeostasis, expression of glucose transporters, glucose uptake, HIF-1α, generation of nitric oxide, expression pattern of some key cell survival regulatory molecules: Bcl2, Bax, active caspase-3 and p53. Pretreatment of Colo205 cells with αCHC also altered their susceptibility to the cytotoxicity of cisplatin accompanied by altered expression of multidrug resistance regulating MDR1 and MRP1 genes. This study for the first time deciphers some of the key molecular events underlying modulation of cell survival of cancer cells of colorectal origin by αCHC and its contribution to chemosensitization against cisplatin. Thus these findings will be of immense help in further research for optimizing the use of αCHC for improving the chemotherapeutic efficacy of anticancer drugs like cisplatin.     

Optimizing dopaminergic differentiation of pluripotent stem cells for the manufacture of dopaminergic neurons for transplantation

Background aimsWe have previously described a xeno-free scalable system to generate transplantable dopaminergic neurons from human pluripotent stem cells. However, several important questions remain to be answered about our cell therapy efforts. These include determining the exact time at which cells should be transplanted and whether cells at this stage can be frozen, shipped, thawed and injected without compromising their ability to mature and survive the transplantation procedure. We also needed to determine whether further optimization of the culture process could shorten the development time and reduce variability and whether a current Good Manufacture Practice (CGMP) facility could manufacture cells with fidelity.MethodsWe developed an optimized protocol that included modulating the sonic hedgehog homolog gradient with bone morphogenetic proteins (BMP2) and addition of activin to the culture medium, which shortened the time to generate Lmx1A and FoxA2 immunoreactive cells by 4–6 days.ResultsWe showed that cells at this stage could be safely frozen and thawed while retaining an excellent ability to continue to mature in vitro and survive transplant in vivo. Importantly, we successfully adapted this process to a CGMP facility and manufactured two lots of transplant-ready dopaminergic neurons (>250 vials) under CGMP-compatible conditions. In vitro characterization, including viability/recovery on thawing, whole genome expression as well as expression of midbrain/dopaminergic markers, showed that the cells manufactured under GMP-compatible conditions were similar to cells produced at lab scale.ConclusionsOur results suggest that this optimized protocol can be used to generate dopaminergic neurons for Investigational New Drug enabling studies.     

Myelopoietic Efficacy of Orlistat in Murine Hosts Bearing T Cell Lymphoma: Implication in Macrophage Differentiation and Activation

Orlistat, an inhibitor of fatty acid synthase (FASN), acts as an antitumor agent by blocking de novo fatty acid synthesis of tumor cells. Although, myelopoiesis also depends on de novo fatty acid synthesis, the effect of orlistat on differentiation of macrophages, which play a central role in host’s antitumor defence, remains unexplored in a tumor-bearing host. Therefore, the present investigation was undertaken to examine the effect of orlistat administration on macrophage differentiation in a T cell lymphoma bearing host. Administration of orlistat (240 mg/kg/day/mice) to tumor-bearing mice resulted in a decline of tumor load accompanied by an augmentation of bone marrow cellularity and survival of bone marrow cells (BMC). The expression of apoptosis regulatory caspase-3, Bax and Bcl2 was modulated in the BMC of orlistat-administered tumor-bearing mice. Orlistat administration also resulted in an increase in serum level of IFN-γ along with decreased TGF-β and IL-10. BMC of orlistat-administered tumor-bearing mice showed augmented differentiation into macrophages accompanied by enhanced expression of macrophage colony stimulating factor (M-CSF) and its receptor (M-CSFR). The macrophages differentiated from BMC of orlistat-administered mice showed characteristic features of M₁ macrophage phenotype confirmed by expression of CD11c, TLR-2, generation of reactive oxygen species, phagocytosis, tumor cell cytotoxicity, production of IL-1,TNF-α and nitric oxide. These novel findings indicate that orlistat could be useful to support myelopoesis in a tumor-bearing host.     

p120-catenin suppresses proliferation and tumor growth of oral squamous cell carcinoma via inhibiting nuclear phospholipase C-γ1 signaling

p120-catenin (p120) serves as a stabilizer of the calcium-dependent cadherin-catenin complex and loss of p120 expression has been observed in several types of human cancers. The p120-dependent E-cadherin-β-catenin complex has been shown to mediate calcium-induced keratinocyte differentiation via inducing activation of plasma membrane phospholipase C-γ1 (PLC-γ1). On the other hand, PLC-γ1 has been shown to interact with phosphatidylinositol 3-kinase enhancer in the nucleus and plays a critical role in epidermal growth factor-induced proliferation of oral squamous cell carcinoma (OSCC) cells. To determine whether p120 suppresses OSCC proliferation and tumor growth via inhibiting PLC-γ1, we examined effects of p120 knockdown or p120 and PLC-γ1 double knockdown on proliferation of cultured OSCC cells and tumor growth in xenograft OSCC in mice. The results showed that knockdown of p120 reduced levels of PLC-γ1 in the plasma membrane and increased levels of PLC-γ1 and its signaling in the nucleus in OSCC cells and OSCC cell proliferation as well as xenograft OSCC tumor growth. However, double knockdown of p120 and PLC-γ1 or knockdown of PLC-γ1 alone did not have any effect. Immunohistochemical analysis of OSCC tissue from patients showed a lower expression level of p120 and a higher expression level of PLC-γ1 compared with that of adjacent noncancerous tissue. These data indicate that p120 suppresses OSCC cell proliferation and tumor growth by inhibiting signaling mediated by nuclear PLC-γ1.     

A Role for Ifit2 in Restricting West Nile Virus Infection in the Brain

Previous studies have demonstrated that type I interferon (IFN-I) restricts West Nile virus (WNV) replication and pathogenesis in peripheral and central nervous system (CNS) tissues. However, the in vivo role of specific antiviral genes that are induced by IFN-I against WNV infection remains less well characterized. Here, using Ifit2^−/− mice, we defined the antiviral function of the interferon-stimulated gene (ISG) Ifit2 in limiting infection and disease in vivo by a virulent North American strain of WNV. Compared to congenic wild-type controls, Ifit2^−/− mice showed enhanced WNV infection in a tissue-restricted manner, with preferential replication in the CNS of animals lacking Ifit2. Virological analysis of cultured macrophages, dendritic cells, fibroblasts, cerebellar granule cell neurons, and cortical neurons revealed cell type-specific antiviral functions of Ifit2 against WNV. In comparison, small effects of Ifit2 were observed on the induction or magnitude of innate or adaptive immune responses. Our results suggest that Ifit2 restricts WNV infection and pathogenesis in different tissues in a cell type-specific manner.     

QTLs associated with root traits increase yield in upland rice when transferred through marker-assisted selection

Altering root morphology of rice (Oryza sativa L.) cultivars could improve yields in drought-prone upland ecosystems. Marker-assisted backcross breeding was used to introgress four QTLs for root traits into an upland rice cultivar. The QTLs had previously been identified under experimental conditions in a different genetic background. The introgressed lines and the recurrent parent were grown for 6 years by resource-poor farmers in upland sites in Eastern India and yields recorded. In combination the QTLs significantly increased yield by 1 t ha^?1 under relatively favourable field conditions. In less favourable trials, the QTL effects were not detected due to greater heterogeneity in soil–water availability in very low yielding environments and consequent yield variability. Root studies under controlled conditions showed that lines with the introgressions had longer roots throughout tillering than the recurrent parent (14 cm longer 2 weeks after sowing). Therefore, both improved roots and increased yield can be attributed to the introgression of QTLs. This is the first demonstration that marker-assisted backcross breeding (MABC) to introgress multiple root QTLs identified under controlled conditions is an effective strategy to improve farmers’ yields of upland rice. The strategy was used to breed a novel upland rice cultivar that has been released in India as Birsa Vikas Dhan 111.     

Genetic dissection of a TIR‐NB‐LRR locus from the wild North American grapevine species Muscadinia rotundifolia identifies paralogous genes conferring resistance to major fungal and oomycete pathogens in cultivated grapevine

The most economically important diseases of grapevine cultivation worldwide are caused by the fungal pathogen powdery mildew (Erysiphe necator syn. Uncinula necator) and the oomycete pathogen downy mildew (Plasmopara viticola). Currently, grapegrowers rely heavily on the use of agrochemicals to minimize the potentially devastating impact of these pathogens on grape yield and quality. The wild North American grapevine species Muscadinia rotundifolia was recognized as early as 1889 to be resistant to both powdery and downy mildew. We have now mapped resistance to these two mildew pathogens in M. rotundifolia to a single locus on chromosome 12 that contains a family of seven TIR‐NB‐LRR genes. We further demonstrate that two highly homologous (86% amino acid identity) members of this gene family confer strong resistance to these unrelated pathogens following genetic transformation into susceptible Vitis vinifera winegrape cultivars. These two genes, designated r esistance to P lasmopara v iticola (MrRPV1) are the first resistance genes to be cloned from a grapevine species. Both MrRUN1 and MrRPV1 were found to confer resistance to multiple powdery and downy mildew isolates from France, North America and Australia; however, a single powdery mildew isolate collected from the south‐eastern region of North America, to which M. rotundifolia is native, was capable of breaking MrRUN1‐mediated resistance. Comparisons of gene organization and coding sequences between M. rotundifolia and the cultivated grapevine V. vinifera at the MrRUN1/MrRPV1 locus revealed a high level of synteny, suggesting that the TIR‐NB‐LRR genes at this locus share a common ancestor.     

Synthesis and properties of thymidines with six-membered amide bridge

Artificial thymidine monomers possessing amide or N-methylamide bridges were designed, synthesized, and introduced into oligonucleotides. UV-melting experiments showed that these oligonucleotides preferred single-stranded RNA (ssRNA) to single-stranded DNA (ssDNA) in duplex formation. Both amide- and N-methylamide-modified oligonucleotides led to a significant increase in the binding affinity to ssRNA by up to +4.7 and +3.7 °C of the T_m value per modification, respectively, compared with natural oligonucleotide. In addition, their oligonucleotides showed high stability against 3′-exonuclease.