Mycobacterium leprae Transcriptome During In Vivo Growth and Ex Vivo Stationary Phases

Mycobacterium leprae, the causative agent of leprosy, is an obligate intracellular pathogen primarily residing within host macrophages and Schwann cells. Whole genome sequencing predicts a highly degraded genome with approximately one third of the coding capacity resulting in the loss of many catabolic pathways. Therefore, it can be assumed that M. leprae obtains many of the necessary metabolites for intracellular survival and growth from the host cells. In this study, global transcriptomic analyses were done on freshly harvested M. leprae growing in athymic mouse footpads for five months (MFP5) and compared to those held in axenic medium for 48 (ML48) and 96 (ML96) hours. Results show that all of the genes and pseudogenes were transcribed under both in vivo and in vitro conditions. 24% and 33% of gene transcript levels were significantly altered in ML48 and ML96 respectively, compared to MFP5. Approximately 45% (39/86) of lipid metabolism genes were significantly downregulated in ML96 compared to MFP5, majority of which are in the β-oxidation pathway. Cholesterol oxidase, acyl-CoA dehydrogenase, and coenzyme F420-dependent oxidoreductase, were significantly upregulated in both ML48 and ML96 compared to MFP5. 30% of cell wall and cell processes functional category genes had altered gene transcription at 96hr compared to MFP5. 40% of 57 genes associated with mycobacterial virulence showed significantly altered transcript levels with 52% significantly downregulated in ML96, including most of the Pro-Glu/Pro-Pro-Glu genes. All 111 hypothetical protein genes with unknown function were expressed. Adenosine triphosphate (ATP) synthesis in M. leprae appears to be significantly downregulated under ex vivo conditions. This is the first study comparing M. leprae global gene expression during in vivo growth and ex vivo stationery phase in axenic medium confirming that during the growth phase in the footpads of experimentally infected mice, M. leprae is metabolically active and its primary source of energy production is probably lipids.

Delayed processing of blood samples impairs the accuracy of mRNA-based biomarkers

Background: The transcriptome of peripheral white blood cells (PWBCs) contains valuable physiological information, thus making them a prime biological sample for investigating mRNA-based biomarkers. However, prolonged storage of whole blood samples can alter gene transcript abundance in PWBCs, compromising the results of biomarker discovery. Here, we designed an experiment to interrogate the impacts of delayed processing of whole blood samples on gene transcript abundance in PWBCs. We hypothesized that storing blood samples for 24 hours at 4°C would cause RNA degradation resulting in altered transcriptome profiles. Results: We produced RNA-sequencing data for 30 samples collected from five estrus synchronized heifers (Bos taurus). We quantified transcript abundance for 12,414 protein-coding genes in PWBCs. Analysis of parameters of RNA quality revealed no statistically significant differences (P>0.05) between samples collected from the jugular vein and coccygeal vein, as well as among samples processed after one, three, six, or eight hours. However, samples processed after 24 hours of storage had a lower RNA integrity number value (P=0.03) in comparison to those processed after one hour of storage. Next, we analyzed RNA-sequencing data between samples using those processed after one hour of storage as the baseline for comparison. Interestingly, evaluation of 3/5 prime bias revealed no differences between genes with lower transcript abundance in samples stored for 24 hours relative to one hour. In addition, sequencing coverage of transcripts was similar between samples from the 24-hour and one-hour groups. We identified four and 515 genes with differential transcript abundance in samples processed after storage for eight and 24 hours, respectively, relative to samples processed after one hour. Conclusions: The PWBCs respond to prolonged cold storage by increasing genes related to active chromatin compaction which in turn reduces gene transcription. This alteration in transcriptome profiles can impair the accuracy of mRNA-based biomarkers. Therefore, blood samples collected for mRNA-based biomarker discovery should be refrigerated immediately and processed within six hours post sampling.

Cellular Processes in Human Ovarian Follicles Are Regulated by Expression Profile of New Gene Markers—Clinical Approach

In the growing ovarian follicle, the maturing oocyte is accompanied by cumulus (CCs) and granulosa (GCs) cells. Currently, there remain many unanswered questions about the epithelial origin of these cells. Global and targeted gene transcript levels were assessed on 1, 7, 15, 30 days of culture for CCs and GCs. Detailed analysis of the genes belonging to epithelial cell-associated ontological groups allowed us to assess a total of 168 genes expressed in CCs (97 genes) and GCs (71 genes) during long-term in vitro culture. Expression changes of the analyzed genes allowed the identification of the group of genes: TGFBR3, PTGS2, PRKX, AHI1, and IL11, whose expression decreased the most and the group of ANXA3, DKK1, CCND1, STC1, CAV1, and SFRP4 genes, whose expression significantly increased. These genes’ expression indicates CCs and GCs epithelialization processes and their epithelial origin. Expression change analysis of genes involved in epithelization processes in GCs and CCs during their in vitro culture made it possible to describe the most significantly altered of the 11 genes. Detailed analysis of gene expression in these two cell populations at different time intervals confirms their ovarian surface epithelial origin. Furthermore, some gene expression profiles appear to have tumorigenic properties, suggesting that granulosa cells may play a role in cancerogenesis.

Direct RNA sequencing of Respiratory Syncytial Virus infected human cells generates a detailed overview of RSV polycistronic mRNA and transcript abundance

To characterize species of viral mRNA transcripts generated during respiratory syncytial virus (RSV) infection, human fibroblast-like MRC5 lung cells were infected with subgroup A RSV for 6, 16 and 24 hours. Total RNA was harvested and polyadenylated mRNA was enriched and sequenced by direct RNA sequencing on an Oxford nanopore device. This yielded over 150,000 direct mRNA transcript reads which were mapped to the viral genome and analysed to determine relative mRNA levels of viral genes using our in-house ORF-centric pipeline. We were also able to examine frequencies with which polycistronic readthrough mRNAs were generated and to assess the length of the polyadenylated tails for each group of transcripts. We show that there is a general but non-linear decline in gene transcript abundance across the viral genome, as predicted by the model of RSV gene transcription. However, the decline in transcript abundance is not consistent. We show that the polyadenylate tails generated by the viral polymerase are similar in length to those generated by the host cells polyadenylation machinery and broadly declined in length for most transcripts as infection progressed. Finally, we observed that the steady state abundance of transcripts with very short polyadenylate tails is much less for N, SH and G transcripts compared to NS1, NS2, P, M, F and M2 which may reflect differences in mRNA stability and/or translation rates.

Phylogenetic analysis and designing new primers for molecular identification of Drosophila suzukii

The family Drosophilidae includes over 3750 species worldwide and over 2000 of these are species of Drosophila. Spotted wing drosophila (SWD), Drosophila suzukii is one of the most dangerous species in this family. The insects live on undamaged ripening fruits, using its peculiar serrated ovipositor to break the skin of fresh ripening fruits and lay eggs in it. Drosophila species are very difficult and practically impossible to detect at larval stages. The present investigation was conducted at the All-Russian Plant Quarantine Center and Agrarian and Technological Institute of RUDN University, Moscow, Russia in 20182020. The aim of this study was to investigate the method of accurate and rapid identification of D. suzukii, and to design specific primer pairs for pest identification by Real-Time PCR method. The real-time quantitative PCR is a fast, sensitive, repeatable and accurate method for quantifying gene transcript levels. In this study, we designed specific primers (4.Dsuz.FRP) for Real-Time PCR to identify D. suzukii from other relative species. Although D. suzukii is absent in the Russian Federation and has not been reported so far, the project could be a precautionary measure.

Plasmodium berghei-Released Factor, PbTIP, Modulates the Host Innate Immune Responses

The Plasmodium parasite has to cross various immunological barriers for successful infection. Parasites have evolved mechanisms to evade host immune responses, which hugely contributes to the successful infection and transmission by parasites. One way in which a parasite evades immune surveillance is by expressing molecular mimics of the host molecules in order to manipulate the host responses. In this study, we report a Plasmodium berghei hypothetical protein, PbTIP (PbANKA_124360.0), which is a Plasmodium homolog of the human T-cell immunomodulatory protein (TIP). The latter possesses immunomodulatory activities and suppressed the host immune responses in a mouse acute graft-versus-host disease (GvHD) model. The Plasmodium berghei protein, PbTIP, is expressed on the merozoite surface and exported to the host erythrocyte surface upon infection. It is shed in the blood circulation by the activity of an uncharacterized membrane protease(s). The shed PbTIP could be detected in the host serum during infection. Our results demonstrate that the shed PbTIP exhibits binding on the surface of macrophages and reduces their inflammatory cytokine response while upregulating the anti-inflammatory cytokines such as TGF-β and IL-10. Such manipulated immune responses are observed in the later stage of malaria infection. PbTIP induced Th2-type gene transcript changes in macrophages, hinting toward its potential to regulate the host immune responses against the parasite. Therefore, this study highlights the role of a Plasmodium-released protein, PbTIP, in immune evasion using macrophages, which may represent the critical strategy of the parasite to successfully survive and thrive in its host. This study also indicates the human malaria parasite TIP as a potential diagnostic molecule that could be exploited in lateral flow-based immunochromatographic tests for malaria disease diagnosis.

An Evolving Role for the Long Non-Coding RNA H19 in Aging and Senescence

The long non-coding RNA (lncRNA) H19 is a maternally imprinted gene transcript that, in conjunction with the neighboring Igf2 gene, is critical in controlling embryonic growth. Loss of H19 results in fetal overgrowth associated with Beckwith Weidemann syndrome, while elevated H19 occurs in human cancers. In the adult, H19 functions in cancer cells where it promotes migration and is correlated with poor prognosis, and in adult stem cells where it is a key regulator of cell fate decisions during differentiation. While the function of H19 in primary somatic cells has not been defined, a reduction in the abundance of H19 has been reported during senescence in endothelial cells. Given the critical importance of H19 in cell fate decisions, it is likely that understanding the precise function of H19 in somatic cells in general and why reduced levels occur with cellular senescence will provide novel insights into both somatic cell maintenance and the senescence program. Towards this end, we examined the role of H19 in somatic cell growth using cardiac interstitial fibroblasts. Our results indicate that H19 is not only vital for somatic cell proliferation and survival, but that depletion of H19 leads to cell cycle arrest and the formation of abnormal nuclei resulting in senescent cells. We are defining both the upstream regulators of H19 and the downstream mediators of senescence following H19 depletion. Overall, these results indicate an essential role for H19 in cell cycle progression, chromatin structure, and possibly proper mitotic division.

No effect of acute exercise on the dynamic change in the expression of genes involved in epigenetic modification in professional athletes

Background:The adaptation of the organism to exercise in the context of gene expression profile is an interesting phenomenon. Exercise can change the expression of individual genes due to changes in the degree of DNA methylation, changes in miRNA expression, or through methylation or acetylation of histones.Hypothesis:Acute exercise increases the expression of genes such as HDAC1, DNMT1, and JHDM1D that can affect epigenetic modifications in PBMCs.Methods:The aim of this study was to determine whether there was a change in gene expression in the blood cells during acute exercise and after a 1-hour recovery. The transcriptions of genes involved in epigenetic modifications (HDAC1, HDAC1 and JHDM1D) were examined in 9 professional athletes at rest, during consecutive stages of a treadmill exercise until exhaustion, and following recovery.Results:No significant differences in the level of transcript were observed in the course of the experiment in the tested PBMC cells. On the other hand, a significant (p = 0.007) correlation was observed in the level of the JHDM1D gene transcript and the number of monocytes in the samples obtained after reaching peak exercise intensity, but in the initial samples this correlation was not significant (p = 0.053).Conclusion:Acute physical exercise does not rapidly alter the transcript levels of the JHDM1D, DNMT1 and HDAC1 genes in PBMCs. The observed correlation between the level of JHDM1D mRNA and the level of monocytes and HDAC1 with lymphocytes requires further investigation.

Alu insertion variants alter gene transcript levels

Alu are high copy number interspersed repeats that have accumulated near genes during primate and human evolution. They are a pervasive source of structural variation in modern humans. Impacts that Alu insertions may have on gene expression are not well understood, although some have been associated with expression quantitative trait loci (eQTLs). Here, we directly test regulatory effects of polymorphic Alu insertions in isolation of other variants on the same haplotype. To screen insertion variants for those with such effects, we used ectopic luciferase reporter assays and evaluated 110 Alu insertion variants, including more than 40 with a potential role in disease risk. We observed a continuum of effects with significant outliers that up- or down-regulate luciferase activity. Using a series of reporter constructs, which included genomic context surrounding the Alu, we can distinguish between instances in which the Alu disrupts another regulator and those in which the Alu introduces new regulatory sequence. We next focused on three polymorphic Alu loci associated with breast cancer that display significant effects in the reporter assay. We used CRISPR to modify the endogenous sequences, establishing cell lines varying in the Alu genotype. Our findings indicate that Alu genotype can alter expression of genes implicated in cancer risk, including PTHLH, RANBP9, and MYC. These data show that commonly occurring polymorphic Alu elements can alter transcript levels and potentially contribute to disease risk.

Silencing of vitellogenin gene contributes to the promise of controlling red palm weevil, Rhynchophorus ferrugineus (Olivier)

Red palm weevil [Rhynchophorus ferrugineus (Olivier)], is native to South Asia and expanding its distribution range globally. Recent invasions of red palm weevil around the world, including Saudi Arabia, has become a global constraint for the production of palm species. Although, several control measures have been tested, none of them seemed successful against this invasive species. Therefore, we focused on silencing the reproduction control gene vitellogenin (Vg) based on RNA interference (RNAi) strategy for its possible application in the management of R. ferrugineus. The Vg is a major yolk protein precursor critical for oogenesis. To do this, fat body transcriptome of R. ferrugineus female adults was sequenced, which provided partial Vg gene transcript (FPKM 5731.60). A complete RfVg gene transcript of 5504 bp encoding 1787 amino acids was then sequenced using RCAE-PCR strategy and characterized. Phylogenetic analysis suggested that RfVg has closer ancestry to the coleopteran insects. The RfVg-based RNAi significantly suppressed the expressions of Vg gene. The 15, 20 and 25 days post-injection periods suppressed Vg expressions by 95, 96.6 and 99%, respectively. The suppressed Vg expressions resulted in the dramatic failure of Vg protein expression, which caused atrophied ovaries or no oogenesis and ultimately eggs were not hatched. These results suggest that knockdown of Vg gene involved in R. ferrugineus reproduction is a promising target for RNAi-based management of R. ferrugineus.