Unraveling the genetic landscape of pulmonary arterial hypertension in Indian patients: A transcriptome study.

BACKGROUND: Pulmonary hypertension (PH) is the abnormal elevation of pressure in the pulmonary vascular system, with various underlying causes. A specific type of PH is pulmonary arterial hypertension (PAH), a severe condition characterized by high pulmonary arterial pressure resulting from structural changes in distal pulmonary vessels, altered arterial tone, and inflammation. This leads to right ventricular hypertrophy and heart failure. The molecular mechanisms behind PAH are not well understood. This manuscript aims to elucidate these mechanisms using the genetic tool, aiding in diagnosis and treatment selection. METHOD: In our present study, we have obtained blood samples from both patients with pulmonary arterial hypertension (PAH) and healthy individuals. We conducted a comparative transcriptome analysis to identify genes that are either upregulated or downregulated in PAH patients when compared to the control group. Subsequently, we carried out a validation study focusing on the log2-fold downregulated genes in PAH, employing Quantitative Real-Time PCR for confirmation. Additionally, we quantified the proteins encoded by the validated genes using the ELISA technique. RESULTS: The results of the transcriptome analysis revealed that 97 genes were significantly upregulated, and 6 genes were significantly downregulated. Among these, we chose to focus on and validate only four of the downregulated genes, as they were directly or indirectly associated with the hypertension pathway. We also conducted validation studies for the proteins encoded by these genes, and the results were consistent with those obtained in the transcriptome analysis. CONCLUSION: In conclusion, the findings of this study indicate that the four validated genes identified in the context of PAH can be further explored as potential targets for both diagnostic and therapeutic applications.

Heat shock induces alternative polyadenylation through dynamic DNA methylation-regulated chromatin looping.

Alternative cleavage and polyadenylation (APA) is a gene regulatory mechanism used by cells under stress to upregulate proteostasis-promoting transcripts, but how cells achieve this remains poorly understood. Previously, we elucidated a DNA methylation-regulated APA mechanism, in which gene body DNA methylation enhances distal poly(A) isoform expression by blocking CTCF binding and chromatin loop formation at APA control regions. We hypothesized that DNA methylation-regulated APA is one mechanism cells employ to induce proteostasis-promoting poly(A) isoforms. At the DNAJB6 co-chaperone gene locus, acute heat shock resulted in binding of stress response transcription factors HSF1, ATF6, and YY1 at the APA control region and an increase in the expression of the proximal poly(A) isoform known to prevent protein aggregation. Furthermore, TET1 was recruited to rapidly demethylate DNA, facilitating CTCF binding and chromatin loop formation, thereby reinforcing preferential proximal poly(A) isoform expression. As cells recovered, the transcription factors vacated the APA control region, and DNMT1 was recruited to remethylate the region. This process resolved chromatin looping and reset the poly(A) isoform expression pattern. Our findings unveil an epigenetic mechanism enabling cells to dynamically modulate poly(A) isoforms in response to stress while shedding light on the interplay between DNA methylation, transcription factors, and chromatin looping.

MicroRNAs from Holarrhena pubescens stems: Identification by small RNA Sequencing and their Potential Contribution to Human Gene Targets.

Holarrhena pubescens is an effective medicinal plant from the Apocynaceae family, widely distributed over the Indian subcontinent and extensively used by Ayurveda and ethno-medicine systems without apparent side effects. We postulated that miRNAs, endogenous non-coding small RNAs that regulate gene expression at the post-transcriptional level, may, after ingestion into the human body, contribute to the medicinal properties of plants of this species by inducing regulated human gene expression to modulate. However, knowledge is scarce about miRNA in Holarrhena. In addition, to test the hypothesis on the potential pharmacological properties of miRNA, we performed a high-throughput sequencing analysis using the Next Generation Sequencing Illumina platform; 42,755,236 raw reads have been generated from H. pubescens stems from a library of small RNA isolated, identifying 687 known and 50 new miRNAs led. The novel H. pubescens miRNAs were predicted to regulate specific human genes, and subsequent annotations of gene functions suggested a possible role in various biological processes and signaling pathways, such as Wnt, MAPK, PI3K-Akt, and AMPK signaling pathways and endocytosis. The association of these putative targets with many diseases, including cancer, congenital malformations, nervous system disorders, and cystic fibrosis, has been demonstrated. The top hub proteins STAT3, MDM2, GSK3B, NANOG, IGF1, PRKCA, SNAP25, SRSF1, HTT, and SNCA show their interaction with human diseases, including cancer and cystic fibrosis. To our knowledge, this is the first report of uncovering H. pubescens miRNAs based on high-throughput sequencing and bioinformatics analysis. This study has provided new insight into a potential cross-species control of human gene expression. The potential for miRNA transfer should be evaluated as one possible mechanism of action to account for the beneficial properties of this valuable species.

Small RNA sequencing and identification of Andrographis paniculata miRNAs with potential cross­kingdom human gene targets.

Cross-species post-transcriptional regulatory potential of plant derived small non-coding microRNAs (miRNAs) has been well documented by plenteous studies. MicroRNAs are transferred to host cells via oral ingestion wherein they play a decisive role in regulation of host genes; thus, miRNAs have evolved as the nascent bioactive molecules imparting pharmacological values to traditionally used medicinal plants. The present study aims to investigate small RNA profiling in order to uncover the potential regulatory role of miRNAs derived from Andrographis paniculata, one of the most widely used herb by tribal communities for liver disorders and document the pharmacological properties of A. paniculata miRNAs. In this study, high-throughput sequencing method was used to generate raw data, ~ 60 million sequences were generated from A. paniculata leaves. Using computational tools and bioinformatics approach, analyses of 3,480,097 clean reads resulted in identification of 3440 known and 51 putative novel miRNAs regulating 1365 and 192 human genes respectively. Remarkably, the identified plausible novel miRNAs apa-miR-5, apa-miR-1, apa-miR-26, and apa-miR-30 are projected to target significant host genes including CDK6, IKBKB, TRAF3, CHD4, MECP2, and ADIPOQ. Subsequent annotations revealed probable involvement of the target genes in various pathways for instance p38-MAPK, AKT, AMPK, NF-Kß, ERK, WNT signalling, MYD88 dependant cascade, and pathways in cancer. Various diseases such as human papilloma virus infection, Alzheimer's, Non-alcoholic Fatty Liver, Alcoholic liver diseases, HepatoCellular Carcinoma (HCC), and numerous other cancers were predominantly found to be linked with target genes. Our findings postulate novel interpretations regarding modulation of human transcripts by A. paniculata miRNAs and exhibit the regulation of human diseases by plant-derived miRNAs. Though our study elucidates miRNAs as novel therapeutic agents, however, experimental validations for assessment of therapeutic potential of these miRNAs are still warranted.

TbTRF suppresses the TERRA level and regulates the cell cycle-dependent TERRA foci number with a TERRA binding activity in its C-terminal Myb domain.

Telomere repeat-containing RNA (TERRA) has been identified in multiple organisms including Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis. T. brucei regularly switches its major surface antigen, VSG, to evade the host immune response. VSG is expressed exclusively from subtelomeric expression sites, and we have shown that telomere proteins play important roles in the regulation of VSG silencing and switching. In this study, we identify several unique features of TERRA and telomere biology in T. brucei. First, the number of TERRA foci is cell cycle-regulated and influenced by TbTRF, the duplex telomere DNA binding factor in T. brucei. Second, TERRA is transcribed by RNA polymerase I mainly from a single telomere downstream of the active VSG. Third, TbTRF binds TERRA through its C-terminal Myb domain, which also has the duplex DNA binding activity, in a sequence-specific manner and suppresses the TERRA level without affecting its half-life. Finally, levels of the telomeric R-loop and telomere DNA damage were increased upon TbTRF depletion. Overexpression of an ectopic allele of RNase H1 that resolves the R-loop structure in TbTRF RNAi cells can partially suppress these phenotypes, revealing an underlying mechanism of how TbTRF helps maintain telomere integrity.

DNA Methylation Regulates Alternative Polyadenylation via CTCF and the Cohesin Complex.

Dysregulation of DNA methylation and mRNA alternative cleavage and polyadenylation (APA) are both prevalent in cancer and have been studied as independent processes. We discovered a DNA methylation-regulated APA mechanism when we compared genome-wide DNA methylation and polyadenylation site usage between DNA methylation-competent and DNA methylation-deficient cells. Here, we show that removal of DNA methylation enables CTCF binding and recruitment of the cohesin complex, which, in turn, form chromatin loops that promote proximal polyadenylation site usage. In this DNA demethylated context, either deletion of the CTCF binding site or depletion of RAD21 cohesin complex protein can recover distal polyadenylation site usage. Using data from The Cancer Genome Atlas, we authenticated the relationship between DNA methylation and mRNA polyadenylation isoform expression in vivo. This DNA methylation-regulated APA mechanism demonstrates how aberrant DNA methylation impacts transcriptome diversity and highlights the potential sequelae of global DNA methylation inhibition as a cancer treatment.

Telomere and Subtelomere R-loops and Antigenic Variation in Trypanosomes.

Trypanosoma brucei is a kinetoplastid parasite that causes African trypanosomiasis, which is fatal if left untreated. T. brucei regularly switches its major surface antigen, VSG, to evade the host immune responses. VSGs are exclusively expressed from subtelomeric expression sites (ESs) where VSG genes are flanked by upstream 70 bp repeats and downstream telomeric repeats. The telomere downstream of the active VSG is transcribed into a long-noncoding RNA (TERRA), which forms RNA:DNA hybrids (R-loops) with the telomeric DNA. At an elevated level, telomere R-loops cause more telomeric and subtelomeric double-strand breaks (DSBs) and increase VSG switching rate. In addition, stabilized R-loops are observed at the 70 bp repeats and immediately downstream of ES-linked VSGs in RNase H defective cells, which also have an increased amount of subtelomeric DSBs and more frequent VSG switching. Although subtelomere plasticity is expected to be beneficial to antigenic variation, severe defects in subtelomere integrity and stability increase cell lethality. Therefore, regulation of the telomere and 70 bp repeat R-loop levels is important for the balance between antigenic variation and cell fitness in T. brucei. In addition, the high level of the active ES transcription favors accumulation of R-loops at the telomere and 70 bp repeats, providing an intrinsic mechanism for local DSB formation, which is a strong inducer of VSG switching.

Synthesis and biological evaluation of selective tubulin inhibitors as anti-trypanosomal agents.

African trypanosomiasis is still a threat to human health due to the severe side-effects of current drugs. We identified selective tubulin inhibitors that showed the promise to the treatment of this disease, which was based on the tubulin protein structural difference between mammalian and trypanosome cells. Further lead optimization was performed in the current study to improve the efficiency of the drug candidates. We used Trypanosoma brucei brucei cells as the parasite model, and human normal kidney cells and mouse macrophage cells as the host model to evaluate the compounds. One new analog showed great potency with an IC50 of 70nM to inhibit the growth of trypanosome cells and did not affect the viability of mammalian cells. Western blot analyses reveal that the compound decreased tubulin polymerization in T. brucei cells. A detailed structure activity relationship (SAR) was summarized that will be used to guide future lead optimization.

Trypanosoma brucei RAP1 maintains telomere and subtelomere integrity by suppressing TERRA and telomeric RNA:DNA hybrids.

Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, thereby evading the host's immune response. VSGs are monoallelically expressed from subtelomeric expression sites (ESs), and VSG switching exploits subtelomere plasticity. However, subtelomere integrity is essential for T. brucei viability. The telomeric transcript, TERRA, was detected in T. brucei previously. We now show that the active ES-adjacent telomere is transcribed. We find that TbRAP1, a telomere protein essential for VSG silencing, suppresses VSG gene conversion-mediated switching. Importantly, TbRAP1 depletion increases the TERRA level, which appears to result from longer read-through into the telomere downstream of the active ES. Depletion of TbRAP1 also results in more telomeric RNA:DNA hybrids and more double strand breaks (DSBs) at telomeres and subtelomeres. In TbRAP1-depleted cells, expression of excessive TbRNaseH1, which cleaves the RNA strand of the RNA:DNA hybrid, brought telomeric RNA:DNA hybrids, telomeric/subtelomeric DSBs and VSG switching frequency back to WT levels. Therefore, TbRAP1-regulated appropriate levels of TERRA and telomeric RNA:DNA hybrid are fundamental to subtelomere/telomere integrity. Our study revealed for the first time an important role of a long, non-coding RNA in antigenic variation and demonstrated a link between telomeric silencing and subtelomere/telomere integrity through TbRAP1-regulated telomere transcription.

Trypanosoma brucei TIF2 and TRF Suppress VSG Switching Using Overlapping and Independent Mechanisms.

Trypanosoma brucei causes debilitating human African trypanosomiasis and evades the host's immune response by regularly switching its major surface antigen, VSG, which is expressed exclusively from subtelomeric loci. We previously showed that two interacting telomere proteins, TbTRF and TbTIF2, are essential for cell proliferation and suppress VSG switching by inhibiting DNA recombination events involving the whole active VSG expression site. We now find that TbTIF2 stabilizes TbTRF protein levels by inhibiting their degradation by the 26S proteasome, indicating that decreased TbTRF protein levels in TbTIF2-depleted cells contribute to more frequent VSG switching and eventual cell growth arrest. Surprisingly, although TbTIF2 depletion leads to more subtelomeric DNA double strand breaks (DSBs) that are both potent VSG switching inducers and detrimental to cell viability, TbTRF depletion does not increase the amount of DSBs inside subtelomeric VSG expression sites. Furthermore, expressing an ectopic allele of F2H-TbTRF in TbTIF2 RNAi cells allowed cells to maintain normal TbTRF protein levels for a longer frame of time. This resulted in a mildly better cell growth and partially suppressed the phenotype of increased VSG switching frequency but did not suppress the phenotype of more subtelomeric DSBs in TbTIF2-depleted cells. Therefore, TbTIF2 depletion has two parallel effects: decreased TbTRF protein levels and increased subtelomeric DSBs, both resulting in an acute increased VSG switching frequency and eventual cell growth arrest.

Orally Active and Selective Tubulin Inhibitors as Anti-Trypanosome Agents.

OBJECTIVES: There is an urgent need to develop a safe, effective, orally active, and inexpensive therapy for African trypanosomiasis due to the drawbacks of current drugs. Selective tubulin inhibitors have the potential to be promising drug candidates for the treatment of this disease, which is based on the tubulin protein structural difference between mammalian and trypanosome cells. We propose to identify novel tubulin inhibitors from a compound library developed based on the lead compounds that selectively target trypanosomiasis. METHODS: We used Trypanosoma brucei brucei as the parasite model, and human normal kidney cells and mouse microphage cells as the host model. Growth rates of both trypanosomes and mammalian cells were determined as a means to screen compounds that selectively inhibit the proliferation of parasites. Furthermore, we examined the cell cycle profile of the parasite and compared tubulin polymerization dynamics before and after the treatment using identified compounds. Last, in vivo anti-parasite activities of these compounds were determined in T. brucei-infected mice. RESULTS: Three compounds were selected that are 100 fold more effective against the growth of T. brucei cells than mammalian cells. These compounds caused cell cycle progression defects in T. brucei cells. Western analyses indicated that these compounds decreased tubulin polymerization in T. brucei cells. The in vivo investigation revealed that these compounds, when admitted orally, inhibited T. brucei cell proliferation in mouse blood. However, they were not potent enough to clear up the infection completely. CONCLUSIONS: These compounds are promising lead compounds as orally active agents for drug development of anti-trypanosome agents. A more detail structure activity relationship (SAR) was summarized that will be used to guide future lead optimization to improve the selectivity and potency of the current compounds.