Induction of gene expression in Dictyostelium by prestarvation factor, a factor secreted by growing cells

During growth, Dictyostelium cells continuously secrete a factor, PSF, that accumulates in proportion to cell density. At sufficient concentration, it triggers the production of discoidin I and certain lysosomal enzymes. Our earlier studies demonstrated these effects of PSF on protein and enzyme levels [Clarke et al., Differentiation 34:79-87, 1987; Clarke et al., Dev Genet 9: 315-326, 1988]. In the present study, we have examined whether PSF induces increased mRNA levels. By Northern blot analysis, we have found that discoidin I mRNA accumulates in exponentially growing NC4 cells as the cells reach high density; significant levels of mRNA are detectable in cells growing either on plates or in suspension, beginning about four generations before the end of exponential growth. High levels of discoidin I mRNA are also found in low-density cells grown in the presence of buffer conditioned by high-density cells. These results indicate that PSF induces the accumulation of discoidin I mRNA. Other "early developmental" genes, pCZ22 and the early I genes (16, 18, and 111), are also expressed in exponentially growing cells at high density or in the presence of conditioned buffer. We conclude that several genes previously found to be preferentially expressed very early in development are actually induced during late exponential growth by PSF.     

Host-induced silencing of Mi-msp-1 confers resistance to root-knot nematode Meloidogyne incognita in eggplant

RNA interference (RNAi)-based host-induced gene silencing (HIGS) is emerging as a novel, efficient and target-specific tool to combat phytonematode infection in crop plants. Mi-msp-1, an effector gene expressed in the subventral pharyngeal gland cells of Meloidogyne incognita plays an important role in the parasitic process. Mi-msp-1 effector is conserved in few of the species of root-knot nematodes (RKNs) and does not share considerable homology with the other phytonematodes, thereby making it a suitable target for HIGS with minimal off-target effects. Six putative eggplant transformants harbouring a single copy RNAi transgene of Mi-msp-1 was generated. Stable expression of the transgene was detected in T₁, T₂ and T₃ transgenic lines for which a detrimental effect on RKN penetration, development and reproduction was documented upon challenge infection with nematode juveniles. The post-parasitic nematode stages extracted from the transgenic plants showed long-term RNAi effect in terms of targeted downregulation of Mi-msp-1. These findings suggest that HIGS of Mi-msp-1 enhances nematode resistance in eggplant and protect the plant against RKN parasitism at very early stage.

     

Potential of bacterial culture media in biofabrication of metal nanoparticles and the therapeutic potential of the as-synthesized nanoparticles in conjunction with artemisinin against MDA-MB-231 breast cancer cells

In the recent past, various groups have proposed diverse biocompatible methods for the synthesis of metal nanoparticles (NPs). Besides culture biomass, culture supernatants (CS) are increasingly being explored for the synthesis of NPs; however, with the ever-increasing exploration of various CS in the biofabrication of NPs, it is equally important to explore the potential of various culture media (CMs) in the synthesis of metal NPs. Considering these aspects, in the present investigation, we explore the possible applicability of various CMs in the biofabrication of metal NPs. The synthesis of NPs was primarily followed by UV/VIS spectroscopy, and, thereafter, the NPs were characterized by various physiochemical techniques, including EM, EDX, FT_IR, X-ray diffraction, and DLS measurements, and finally, their anticancer potentialities were investigated against breast cancer. In addition, the NPs were examined in conjunction with artemisinin for therapeutic benefits against aggressive and highly metastatic MDA-MB-231 breast cancer cells. Cumulatively, the results of the present study collated the potentials of various bacterial CMs in the biofabrication of metal NPs and ascertained the efficacy of the as-synthesized silver nanoparticles, especially the combinatorial entity as intriguing breast cancer therapeutics. The data of the present study plausibly assist in advancing the therapeutic applicability of the combinatorial amalgam against aggressive and highly metastatic MDA-MB-231 breast cancer cells.     

HHV-5 epitope: A potential vaccine candidate with high antigenicity and large coverage

Outbreak of Human Herpes virus-5 (HHV-5) infection in emerging countries has raised worldwide health concern owing to prevalence of congenital impairments and life threatening consequences in immunocompromised individuals. Thus, there lies an impending need to develop vaccine against HHV-5. HHV-5 enters into host cells with the help of necessary components glycoprotein B (gB) and H/L. In this study, the conformational linear B-cell and T-cell epitopes for gB of HHV-5 have been predicted using conformational approaches, for their possible collective use as vaccine candidates. We examined epitope’s interactions with major histocompatibility complexes using molecular docking and also investigated their stable binding with specific toll like receptor-2 (TLR2), present on host cells during HHV-5 infection. Predicted MHC-I epitope ‘LVAIAVVII’ with high antigenicity and large coverage of HLA alleles was found to superimpose on MHC-II epitope (Rank 1) and was also identified to be the core sequence of putative B cell epitope ‘ILVAIAVVIITYLI’. Resulting epitope was found to have consistent interaction with TLR2 during long term (100?ns) MD run. We also validated this nonamer epitope for its dissimilarity with human genome and high population coverage, suggesting it to be a potential vaccine candidate with higher coverage for both the MHC alleles of Indian population.

Communicated by Ramaswamy H. Sarma     

The Slowing Rate of CpG Depletion in SARS-CoV-2 Genomes Is Consistent with Adaptations to the Human Host

Depletion of CpG dinucleotides in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genomes has been linked to virus evolution, host-switching, virus replication, and innate immune responses. Temporal variations, if any, in the rate of CpG depletion during virus evolution in the host remain poorly understood. Here, we analyzed the CpG content of over 1.4 million full-length SARS-CoV-2 genomes representing over 170 million documented infections during the first 17 months of the pandemic. Our findings suggest that the extent of CpG depletion in SARS-CoV-2 genomes is modest. Interestingly, the rate of CpG depletion is highest during early evolution in humans and it gradually tapers off, almost reaching an equilibrium; this is consistent with adaptations to the human host. Furthermore, within the coding regions, CpG depletion occurs predominantly at codon positions 2-3 and 3-1. Loss of ZAP (Zinc-finger antiviral protein)-binding motifs in SARS-CoV-2 genomes is primarily driven by the loss of the terminal CpG within the motifs. Nonetheless, majority of the CpG depletion in SARS-CoV-2 genomes occurs outside ZAP-binding motifs. SARS-CoV-2 genomes selectively lose CpGs-motifs from a U-rich context; this may help avoid immune recognition by TLR7. SARS-CoV-2 alpha-, beta-, and delta-variants of concern have reduced CpG content compared to sequences from the beginning of the pandemic. In sum, we provide evidence that the rate of CpG depletion in virus genomes is not uniform and it greatly varies over time and during adaptations to the host. This work highlights how temporal variations in selection pressures during virus adaption may impact the rate and the extent of CpG depletion in virus genomes.     

Ischemia dysregulates DNA methyltransferases and p16INK4a methylation in human colorectal cancer cells

Epigenetic modifications are involved in the initiation and progression of cancer. Expression patterns and activity of DNA methyltransferases (DNMTs) are strictly controlled in normal cells; however, regulation of these enzymes is lost in cancer cells due to unknown reasons. Cancer therapies which target DNMTs are promising treatments of hematologic cancers, but they lack effectiveness in solid tumors. Solid tumors exhibit areas of hypoxia and hypoglycaemia due to their irregular and dysfunctional vasculature, and we previously showed that hypoxia reduces global DNA methylation. Colorectal carcinoma (CRC) cells (HCT116 and 379.2; p53^+/+ and p53^-/-, respectively) were subjected to ischemia (hypoxia and hypoglycaemia) in vitro and levels of DNMTs were assessed. We found a significant decrease in mRNA for DNMT1, DNMT3a and DNMT3b, and similar reductions in DNMT1 and DNMT3a protein levels were detected by western blotting. In addition, total activity levels of DNMTs (as measured by an ELISA-based DNMT activity assay) were reduced in cells exposed to hypoxic and hypoglycaemic conditions. Immunofluorescence of HCT116 tumor xenografts demonstrated an inverse relationship between ischemia (as revealed by carbonic anhydrase IX staining) and DNMT1 protein. Bisulfite sequencing of the proximal promoter region of p16^INK4a showed a decrease in 5-methylcytosine following in vitro exposure to ischemia. These studies provide evidence for the downregulation of DNMTs and modulation of methylation patterns by hypoxia and hypoglycaemia in human CRC cells, both in vitro and in vivo. Our findings suggest that ischemia, either intrinsic or induced through the use of anti-angiogenic drugs, may influence epigenetic patterning and hence tumor progression.Key words: DNA methylation, DNA methyltransferases, colorectal carcinoma, ischemia, p53, hypoxia, hypoglycaemia     

Informed Consent in Cross-cultural Perspective: Clinical Research in the Tibetan Autonomous Region, PRC

Procedures of Informed Consent are considered a high priority for international biomedical research. However, informed consent protocols are not necessarily transferable across cultural, national or ethnic groups. Recent debates identify the need for balancing ethical universals with practical and local conditions and paying attention to questions of cultural competence when it comes to the Informed Consent process for clinical biomedical research. This article reports on the results of a two-year effort to establish a culturally appropriate Informed Consent process for biomedical research in the Tibet Autonomous Region in the People's Republic of China. A team of Tibetan and American researchers, physicians, health professionals and medical anthropologists conducted the research. The Informed Consent was specifically for undertaking a triple-blind, double placebo-controlled randomized clinical trial of a Tibetan medicine compared with Misoprostol for reducing postpartum blood loss. The findings suggest greater need for flexibility and cooperation in establishing Informed Consent protocols across cultures and nations.