AMPK Activation in TET2 Downregulated Leukemia Cells Upon Glutamine Limitation.

Objective: Metabolic rewiring is a characteristic of cancer cells. Cancer cells require more nutrients for survival and proliferation. Although glutamine can be produced in cells via a series of enzymatic reactions, a group of cancer cells are dependent on extracellular glutamine for survival. TET2 plays a role in DNA demethylation and is a tumor suppressor gene. The TET2 gene is frequently mutated in various cancers, including acute myeloid leukemia (AML). Our study aimed to investigate the association between TET2-knockdown AML cell line HL-60 cells and glutamine metabolism. Methods: To evaluate the association between TET2 expression and glutamine limitation, TET2 was downregulated in HL-60 cells using shRNA plasmids. The proliferation of TET2-knockdown HL-60 cells was calculated in normal and glutamine-deficient medium. GLUL mRNA expression was investigated using quantitative reverse transcription polymerase chain reaction and protein levels were evaluated using immunoblotting. Results: The numbers and viability of TET2-knockdown HL-60 cells were decreased in low glutamine-containing medium, but the viability of TET2-knockdown HL-60 cells was higher than that of control cells. GLUL mRNA expressions were increased in TET2-knockdown cells in low glutamine. In addition, P-AMPKα protein expression was increased in TET2-knockdown HL-60 cells in low glutamine-containing medium. Conclusions: Our findings indicate that TET2-knockdown HL-60 cells may be more resistant to glutamine deprivation. In glutamine-deficient medium, the mRNA expression of glutamine synthetase is increased, which could be related to glutamine addiction in cells. In addition, low-glutamyl medium increased the P-AMPKα protein level in TET2-knockdown HL-60 cells.

The TOP-2/condensin II axis silences transcription during germline specification in C. elegans.

In C. elegans, the germline is specified via a preformation mechanism that relies on the PIE-1 protein's ability to globally silence mRNA transcription in germline precursor cells, also known as the P lineage. Recent work from our group has identified additional genome silencing events in C. elegans during oogenesis and in starved L1 larvae, and these require the condensin II complex, topoisomerase II (TOP-2), and components of the H3K9me/heterochromatin pathway. Interestingly, silencing in oocytes also requires PIE-1, but this is not the case in starved L1s. Here, we ask if additional genome silencing components besides PIE-1 are required to repress gene expression in the P lineage of early embryos, and we find that condensin II and TOP-2 are required and the H3K9me/heterochromatin pathway is not. We show that depletion of TOP-2/condensin II activates the normally suppressed RNA polymerase II to inappropriately transcribe somatic genes in the P lineage. We also present evidence that while both PIE-1 and TOP-2/condensin II are required for genome silencing in the P lineage, PIE-1 can silence transcription independently of TOP-2/condensin II when misexpressed in somatic cells. Thus, in oocytes, all three genome silencing systems (TOP-2/condensin II, H3K9me, and PIE-1) are operational while in both early embryos and starved L1s two of the three are active. Our data show that multiple, redundantly acting genome silencing mechanisms act in a mix and match manner to repress transcription at different developmental stages in the C. elegans germline.

Abf1 negatively regulates the expression of EPA1 and affects adhesion in Candida glabrata.

Introduction. Adherence is a major virulence trait in Candida glabrata that, in many strains, depends on the EPA (epithelial adhesin) genes, which confer the ability to adhere to epithelial and endothelial cells of the host. The EPA genes are generally found at subtelomeric regions, which makes them subject to subtelomeric silencing. In C. glabrata, subtelomeric silencing depends on different protein complexes, such as silent information regulator and yKu complexes, and other proteins, such as Repressor/activator protein 1 (Rap1) and Abf1. At the EPA1 locus, which encodes the main adhesin Epa1, we previously found at least two cis-acting elements, the protosilencer Sil2126 and the negative element, that contribute to the propagation of silencing from the telomere to the subtelomeric region.Hypothesis. Abf1 binds to the regulatory regions of EPA1 and other regions at the telomere E-R, thereby negatively regulating EPA1 transcription.Aim. To determine whether Abf1 and Rap1 silencing proteins bind to previously identified cis-acting elements on the right telomere of chromosome E (E-R subtelomeric region), resulting in negative regulation of EPA1 transcription and infer Abf1 and Rap1 recognition sites in C. glabrata.Methodology. We used chromatin immunoprecipitation (ChIP) followed by quantitative PCR to determine the binding sites for Abf1 and Rap1 in the intergenic regions between EPA1 and EPA2 and HYR1 and EPA1, and mutants were used to determine the silencing level of the EPA1 promoter region.Results. We found that Abf1 predominantly binds to the EPA1 promoter region, leading to negative regulation of EPA1 expression. Furthermore, the mutant abf1-43, which lacks the last 43 amino acids at its C-terminal end and is defective for subtelomeric silencing, exhibits hyperadherence to epithelial cells in vitro compared to the parental strain, suggesting that EPA1 is derepressed. We also determined the motif-binding sequences for Abf1 and Rap1 in C. glabrata using data from the ChIP assays.Conclusion. Together these data indicate that Abf1 negatively regulates EPA1 expression, leading to decreased adhesion of C. glabrata to epithelial cells.

Harnessing RNA interference for the control of Fusarium species: A critical review.

Fusarium fungi are a pervasive threat to global agricultural productivity. They cause a spectrum of plant diseases that result in significant yield losses and threaten food safety by producing mycotoxins that are harmful to human and animal health. In recent years, the exploitation of the RNA interference (RNAi) mechanism has emerged as a promising avenue for the control of Fusarium-induced diseases, providing both a mechanistic understanding of Fusarium gene function and a potential strategy for environmentally sustainable disease management. However, despite significant progress in elucidating the presence and function of the RNAi pathway in different Fusarium species, a comprehensive understanding of its individual protein components and underlying silencing mechanisms remains elusive. Accordingly, while a considerable number of RNAi-based approaches to Fusarium control have been developed and many reports of RNAi applications in Fusarium control under laboratory conditions have been published, the applicability of this knowledge in agronomic settings remains an open question, and few convincing data on RNAi-based disease control under field conditions have been published. This review aims to consolidate the current knowledge on the role of RNAi in Fusarium disease control by evaluating current research and highlighting important avenues for future investigation.

Silencing the glycerol-3-phosphate acyltransferase-1 gene in the liver of mice fed a high-fat diet, enhances insulin sensitivity and glucose metabolism by promoting fatty acid beta-oxidation.

BACKGROUND: Liver plays a central role in systemic glucose and lipid metabolism. High-fat diet (HFD) and obesity are related to hepatic lipid accumulation and insulin resistance (InsR). Diacylglycerols (DAG) play a key role in the induction of InsR, however their involvement in hepatic InsR remains debated. This study aimed to clarify and confirm the role of glycero-3-phosphate acyltransferase 1 (GPAT1), a rate-limiting enzyme in DAG synthesis, in the progression of hepatic InsR in the context of HFD-induced lipid accumulation and insulin resistance in the liver. METHODS: Liver-targeted GPAT1 silencing was performed using shRNA-mediated hydrodynamic gene delivery. Lipid species including LCA-CoA, sphingolipids, DAG and acyl-carnitines were quantified using UHPLC/MS/MS while insulin signalling was assessed at protein level by Western Blot. Hepatic glucose metabolism, including glucose-6-pasphate content and gluconeogenesis rate was evaluated using GC/MS. RESULTS: HFD-fed animals developed InsR, evidenced by increased HOMA-IR, enhanced gluconeogenesis and reduced glycogen content compared to controls. Hepatic GPAT1 silencing in HFD-fed animals resulted in a significant reduction of DAG and TAG levels, increased acyl-carnitines content and upregulated mitochondrial ß-oxidation protein expression. These changes were accompanied by improved insulin signalling, enhanced glycogen storage, and reduced gluconeogenesis. CONCLUSIONS: Silencing GPAT1, and thereby reducing glycerolipid synthesis, promotes ß-oxidation and ameliorates HFD-induced hepatic insulin resistance, confirming the enzyme's pivotal role in liver metabolic dysfunction associated with increased lipid supply.

The C5 protein of euphorbia leaf curl virus is a virulence factor and gene silencing suppressor.

The genome of a monopartite begomovirus, or the DNA-A component of a bipartite begomovirus, typically encodes six proteins: two on the viral strand (AV1/V1 and AV2/V2) and four on the complementary strand (AC1/C1, AC2/C2, AC3/C3, AC4/C4). Recent studies, however, have identified additional begomoviral proteins with various functions. This paper reports that euphorbia leaf curl virus (EuLCV), a monopartite begomovirus, encodes a seventh protein, C5. Promoter activity of the upstream fragment of the EuLCV C5 gene was shown using a GUS expression vector. EuLCV C5 also enhanced the pathogenicity and accumulation of potato virus X (PVX) in Nicotiana benthamiana. Localization studies revealed that EuLCV C5 localizes to the cytoplasm and nucleus, forming granular structures on the cell membrane. Additionally, C5 acts as a post-transcriptional gene silencing (PTGS) suppressor. A C5 deletion mutant of EuLCV (EuLCV-ΔC5) exhibited reduced pathogenicity and viral accumulation compared to wild-type EuLCV in N. benthamiana.

A novel gene silencing strategy based on tobacco rattle virus in Hibiscus mutabilis.

Background: Hibiscus mutabilis L. is a popular regional characteristic plant in China, cultivated for its attractive flower colors, extended bloom time, and medicinal properties. To enhance molecular breeding and gene function studies, we conducted transcriptome analysis and identified valuable genes in previous research. Nonetheless, the current inefficient and labor-intensive transformation techniques have hindered their applications. Virus-induced gene silencing (VIGS) provides a precise and effective strategy for post-transcriptional down-regulation of endogenous gene expression. Methods: We investigated the performance of tobacco rattle virus (TRV) as a tool for targeting and silencing the gene encoding the protein involved in chloroplast development, cloroplastos alterados 1 (altered chloroplast; CLA1), of H. mutabilis through Agrobacterium tumefaciens-mediated infiltration. Results: By effectively suppressing the CLA1 gene associated with chloroplast development in H. mutabilis via the TRV-VIGS system, we have illustrated the inaugural implementation of VIGS in this species. Quantitative RT-PCR proved that HmCLA1 expression in agro-infiltrated plants was lower than in the mock-infiltrated (mock) and the control (CK) plants. Phenotypic observations corroborated the albino phenotype in leaves following successful HmCLA1 silencing. Conclusions: Our study showcases TRV-VIGS as a potential gene silencing tool for H. mutabilis, facilitating functional genomics studies and molecular breeding efforts in this species.

Representation of non-coding RNA-mediated regulation of gene expression using the Gene Ontology.

Regulatory non-coding RNAs (ncRNAs) are increasingly recognized as integral to the control of biological processes. This is often through the targeted regulation of mRNA expression, but this is by no means the only mechanism through which regulatory ncRNAs act. The Gene Ontology (GO) has long been used for the systematic annotation of protein-coding and ncRNA gene function, but rapid progress in the understanding of ncRNAs meant that the ontology needed to be revised to accurately reflect current knowledge. Here, a targeted effort to revise GO terms used for the annotation of regulatory ncRNAs is described, focusing on microRNAs (miRNAs), long non-coding RNAs (lncRNAs), small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs). This paper provides guidance to biocurators annotating ncRNA-mediated processes using the GO and serves as background for researchers wishing to make use of the GO in their studies of ncRNAs and the biological processes they regulate.

Splice-switching antisense oligonucleotide controlling tumor suppressor REST is a novel therapeutic medicine for neuroendocrine cancer.

RNA splicing regulation has revolutionized the treatment of challenging diseases. Neuroendocrine cancers, including small cell lung cancer (SCLC) and neuroendocrine prostate cancer (PCa), are highly aggressive, with metastatic neuroendocrine phenotypes, leading to poor patient outcomes. We investigated amido-bridged nucleic acid (AmNA)-based splice-switching oligonucleotides (SSOs) targeting RE1-silencing transcription factor (REST) splicing as a novel therapy. We designed AmNA-based SSOs to alter REST splicing. Tumor xenografts were generated by subcutaneously implanting SCLC or PCa cells into mice. SSOs or saline were intraperitoneally administered and tumor growth was monitored. Blood samples were collected from mice after SSO administration, and serum alanine aminotransferase and aspartate aminotransferase levels were measured to assess hepatotoxicity using a biochemical analyser. In vitro, REST_SSO reduced cancer cell viability. In a tumor xenograft model, it exhibited significant antitumor effects. It repressed REST-controlled RE1-harboring genes and upregulated miR-4516, an SCLC biomarker. Our findings suggest that REST_SSO suppresses tumorigenesis in neuroendocrine cancers by restoring REST function. This novel therapeutic approach holds promise for intractable neuroendocrine cancers such as SCLC and neuroendocrine PCa.

Target cleavage and gene silencing by Argonautes with cityRNAs.

TinyRNAs (tyRNAs) are ≤17-nt guide RNAs associated with Argonaute proteins (AGOs), and certain 14-nt cleavage-inducing tyRNAs (cityRNAs) catalytically activate human Argonaute3 (AGO3). We present the crystal structure of AGO3 in complex with a cityRNA, 14-nt miR-20a, and its complementary target, revealing a different trajectory for the guide-target duplex from that of its ∼22-nt microRNA-associated AGO counterpart. cityRNA-loaded Argonaute2 (AGO2) and AGO3 enhance their endonuclease activity when the immediate 5' upstream region of the tyRNA target site (UTy) includes sequences with low affinity for AGO. We propose a model where cityRNA-loaded AGO2 and AGO3 efficiently cleave fully complementary tyRNA target sites unless they directly recognize the UTy. To investigate their gene silencing, we devised systems for loading endogenous AGOs with specific tyRNAs and demonstrated that, unlike microRNAs, cityRNA-mediated silencing heavily relies on target cleavage. Our study uncovered that AGO exploits cityRNAs for target recognition differently from microRNAs and alters gene silencing.

The biogenesis, regulation and functions of transitive siRNA in plants.

Small RNA (sRNA)-mediated RNA interference (RNAi) is a sequence-specific gene silencing mechanism that modulates gene expression in eukaryotes. As core molecules of RNAi, various sRNAs are encoded in the plant genome or derived from invading RNA molecules, and their biogenesis depends on distinct genetic pathways. Transitive small interfering RNAs (siRNAs), which are sRNAs produced from double-strand RNA (dsRNA) in a process that depends on RNA-dependent RNA polymerases (RDRs), can amplify and spread silencing signals to additional transcripts, thereby enabling a phenomenon termed "transitive RNAi". Members of this class of siRNAs function in various biological processes ranging from development to stress adaptation. In Arabidopsis thaliana, two RDRs participate in the generation of transitive siRNAs, acting cooperatively with various siRNA generation-related factors, such as the RNA-induced silencing complex (RISC) and aberrant RNAs. Transitive siRNAs are produced in diverse subcellular locations and structures under the control of various mechanisms, highlighting the intricacies of their biogenesis and functions. In this review, we discuss recent advances in understanding the molecular events of transitive siRNA biogenesis and its regulation, with a particular focus on factors involved in RDR recruitment. We aim to provide a comprehensive description of the generalized mechanism governing the biogenesis of transitive siRNAs. Additionally, we present an overview of the diverse biological functions of these siRNAs and raise some pressing questions in this area for further investigation.

Cy-1, a major QTL for tomato leaf curl New Delhi virus resistance, harbors a gene encoding a DFDGD-Class RNA-dependent RNA polymerase in cucumber (Cucumis sativus).

BACKGROUND: Tomato leaf curl New Delhi virus (ToLCNDV) (family Geminiviridae, genus Begomovirus) is a significant threat to cucumber (Cucumis sativus) production in many regions. Previous studies have reported the genetic mapping of loci related to ToLCNDV resistance, but no resistance genes have been identified. RESULTS: We conducted map-based cloning of the ToLCNDV resistance gene in cucumber accession No.44. Agroinfiltration and graft-inoculation analyses confirmed the resistance of No.44 to ToLCNDV isolates from the Mediterranean and Asian countries. Initial mapping involving two rounds of phenotyping with two independent F2 populations generated by crossing the begomovirus-susceptible cultivar SHF and No.44 consistently detected major quantitative trait loci (QTLs) on chromosomes 1 and 2 that confer resistance to ToLCNDV. Fine-mapping of Cy-1, the dominant QTL on chromosome 1, using F3 populations narrowed the candidate region to a 209-kb genomic segment harboring 24 predicted genes. Among these genes, DFDGD-class RNA-dependent RNA polymerase (CsRDR3), an ortholog of Ty-1/Ty-3 of tomato and Pepy-2 of capsicum, was found to be a strong candidate conferring ToLCNDV resistance. The CsRDR3 sequence of No.44 contained multiple amino acid substitutions; the promoter region of CsRDR3 in No.44 had a large deletion; and the CsRDR3 transcript levels were greater in No.44 than in SHF. Virus-induced gene silencing (VIGS) of CsRDR3 using two chromosome segment substitution lines harboring chromosome 1 segments derived from No.44 compromised resistance to ToLCNDV. CONCLUSIONS: Forward and reverse genetic approaches identified CsRDR3, which encodes a DFDGD-class RNA-dependent RNA polymerase, as the gene responsible for ToLCNDV resistance at the major QTL Cy-1 on chromosome 1 in cucumber. Marker-assisted breeding of ToLCNDV resistance in cucumber will be expedited by using No.44 and the DNA markers developed in this study.

Hydrogen Sulfide-Triggered Artificial DNAzyme Switches for Precise Manipulation of Cellular Functions.

The development of synthetic molecular tools responsive to biological cues is crucial for advancing targeted cellular regulation. A significant challenge is the regulation of cellular processes in response to gaseous signaling molecules such as hydrogen sulfide (H2S). To address this, we present the design of Gas signaling molecule-Responsive Artificial DNAzyme-based Switches (GRAS) to manipulate cellular functions via H2S-sensitive synthetic DNAzymes. By incorporating stimuli-responsive moieties to the phosphorothioate backbone, DNAzymes are strategically designed with H2S-responsive azide groups at cofactor binding locations within the catalytic core region. These modifications enable their activation through H2S-reducing decaging, thereby initiating substrate cleavage activity. Our approach allows for the flexible customization of various DNAzymes to regulate distinct cellular processes in diverse scenarios. Intracellularly, the enzymatic activity of GRAS promotes H2S-induced cleavage of specific mRNA sequences, enabling targeted gene silencing and inducing apoptosis in cancer cells. Moreover, integrating GRAS with dynamic DNA assembly allows for grafting these functional switches onto cell surface receptors, facilitating H2S-triggered receptor dimerization. This extracellular activation transmits signals intracellularly to regulate cellular behaviors such as migration and proliferation. Collectively, synthetic switches are capable of rewiring cellular functions in response to gaseous cues, offering a promising avenue for advanced targeted cellular engineering.

LinQURE: A novel AAV gene silencing platform that supports multi-transcript targeting for complex disorders.

Given that numerous genetic disorders, driven by diverse pathogenic mechanisms, may be amenable to recombinant adeno-associated virus (rAAV)-delivered gene therapies, the sustained innovation of rAAV-based therapeutic modalities is crucial. The progression and severity of genetic diseases can be reduced by targeting the toxic transcripts of a defective gene using microRNA (miRNA)-based miQURE technology delivered within an AAV vector. By adapting the delivered cassette, it may be possible to simultaneously regulate the expression profile of multiple genes involved in the pathogenesis of complex genetic diseases. The established miQURE gene silencing strategy was expanded by concatenating several miQURE molecules in a single construct, resulting in the novel linQURE platform. Here, a proof of mechanism is established by demonstrating that linQURE technology enables the concomitant expression of two synthetic miRNAs in vitro and in vivo, allowing more efficient downregulation of their disease-causing mRNA targets. This approach supports the development of multi-targeting therapeutic strategies, enabling gene therapy products to adapt to more complex multigenic indications, thus expanding the toolbox of readily available gene therapies.

Suppression of Thrips palmi population by spray-on application of dsRNA targeting V-ATPase-B.

The RNA interference (RNAi)-based gene silencing technique has enormous potential as a non-chemical and eco-friendly alternative to hazardous pesticides. This study reports a spray-induced gene silencing (SIGS) approach for managing Thrips palmi by lowering survival and offspring development. Vacuolar ATP synthases (V-ATPases) are responsible for survival, egg-laying, and viability of eggs in insects. In the current study, T. palmi V-ATPase-B was targeted to suppress the pest population by spray-on application of double-stranded RNA (dsRNA). Silencing of V-ATPase-B was first validated by oral administration of dsV-ATPase-B. The expression of V-ATPase-B was reduced by 5.40-fold post-dsRNA feeding leading to increased mortality (57.03 %) and reduced reproductive fitness (67.73 %). Spray-on application of naked dsV-ATPase-B at concentrations of 3.0 µg/mL and 5.0 µg/mL effectively suppressed the population by 30.00 % and 43.33 %, respectively. The expression of the target gene was downregulated by up to 4.24-fold. Two consecutive sprays at a concentration of 5.0 µg/mL provided substantial protection against the fresh release of T. palmi for up to 10 days. The spray-on application of dsV-ATPase-B would be an eco-friendly alternative for managing T. palmi populations thereby reducing crop damage and limiting the spread of orthotospoviruses.

Influence of the P6 effector protein of Cauliflower mosaic virus (CaMV) on the sustained expression and subcellular localization of the CaMV movement protein.

The P6 protein of cauliflower mosaic virus (CaMV) is a multifunctional protein that forms the electron dense, amorphous inclusion bodies that accumulate in the cytoplasm and has been shown to physically interact with all other CaMV proteins, including the CaMV movement protein (P1). In this study, we have investigated the subcellular localization of the P6 and P1 proteins in transient expression assays in Nicotiana benthamiana, as well as the influence of P6 on the expression and subcellular localization of P1. A version of P6 tagged with RFP was shown to envelop the endoplasmic reticulum (ER), whereas P1 tagged with RFP was shown to induce the fragmentation of the ER. Co-expression of P6 with P1 led to an enhancement of the spatial and temporal expression of P1, with a shift from expression through the plasma membrane and interior of the cell to punctate spots associated with the cell wall.

A novel RAV transcription factor from pear interacts with viral RNA-silencing suppressors to inhibit viral infection.

In plants, RNA silencing constitutes a strong defense against viral infection, which viruses counteract with RNA-silencing suppressors (RSSs). Understanding the interactions between viral RSSs and host factors is crucial for elucidating the molecular arms race between viruses and host plants. We report that the helicase motif (Hel) of the replicase encoded by apple stem grooving virus (ASGV)-the main virus affecting pear trees in China-is an RSS that can inhibit both local and systemic RNA silencing, possibly by binding double-stranded (ds) siRNA. The transcription factor related to ABSCISIC ACID INSENSITIVE3/VIVIPAROUS1 from pear (PbRAV1) enters the cytoplasm and binds Hel through its C terminus, thereby attenuating its RSS activity by reducing its binding affinity to 21- and 24-nt ds siRNA, and suppressing ASGV infection. PbRAV1 can also target p24, an RSS encoded by grapevine leafroll-associated virus 2 (GLRaV-2), with similar negative effects on p24's suppressive function and inhibition of GLRaV-2 infection. Moreover, like the positive role of the PbRAV1 homolog from grapevine (VvRAV1) in p24's previously reported RSS activity, ASGV Hel can also hijack VvRAV1 and employ the protein to sequester 21-nt ds siRNA, thereby enhancing its own RSS activity and promoting ASGV infection. Furthermore, PbRAV1 neither interacts with CP, an RSS encoded by grapevine inner necrosis virus, nor has any obvious effect on CP's RSS activity. Our results identify an RSS encoded by ASGV and demonstrate that PbRAV1, representing a novel type of RAV transcription factor, plays a defensive role against viral infection by targeting viral RSSs.

Macrophage Activation Syndrome in Coinciding Pandemics of Obesity and COVID-19: Worse than Bad.

Epigenetic changes have long-lasting impacts, which influence the epigenome and are maintained during cell division. Thus, human genome changes have required a very long timescale to become a major contributor to the current obesity pandemic. Whereas bidirectional effects of coronavirus disease 2019 (COVID-19) and obesity pandemics have given the opportunity to explore, how the viral microribonucleic acids (miRNAs) use the human's transcriptional machinery that regulate gene expression at a posttranscriptional level. Obesity and its related comorbidity, type 2 diabetes (T2D), and new-onset diabetes due to severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) are additional risk factors, which increase the severity of COVID-19 and its related mortality. The higher mortality rate of these patients is dependent on severe cytokine storm, which is the sum of the additional cytokine production by concomitant comorbidities and own cytokine synthesis of COVID-19. Patients with obesity facilitate the SARS-CoV-2 entry to host cell via increasing the host's cell receptor expression and modifying the host cell proteases. After entering the host cells, the SARS-CoV-2 genome directly functions as a messenger ribonucleic acid (mRNA) and encodes a set of nonstructural proteins via processing by the own proteases, main protease (Mpro), and papain-like protease (PLpro) to initiate viral genome replication and transcription. Following viral invasion, SARS-CoV-2 infection reduces insulin secretion via either inducing ß-cell apoptosis or reducing intensity of angiotensin-converting enzyme 2 (ACE2) receptors and leads to new-onset diabetes. Since both T2D and severity of COVID-19 are associated with the increased serum levels of pro-inflammatory cytokines, high glucose levels in T2D aggravate SARS-CoV-2 infection. Elevated neopterin (NPT) value due to persistent interferon gamma (IFN-γ)-mediated monocyte-macrophage activation is an indicator of hyperactivated pro-inflammatory phenotype M1 macrophages. Thus, NPT could be a reliable biomarker for the simultaneously occurring COVID-19-, obesity- and T2D-induced cytokine storm. While host miRNAs attack viral RNAs, viral miRNAs target host transcripts. Eventually, the expression rate and type of miRNAs also are different in COVID-19 patients with different viral loads. It is concluded that specific miRNA signatures in macrophage activation phase may provide an opportunity to become aware of the severity of COVID-19 in patients with obesity and obesity-related T2D.

Sustained intra-cellular siRNA release from poly(L-arginine) multilayered nanoparticles for prolonged gene silencing.

BACKGROUND: Sustained siRNA release from nanocarriers is difficult to achieve inside the cell after entry: typically, all nanocarriers exhibit burst release of the cargo into the cytoplasm. RESEARCH DESIGN AND METHODS: Layer-by-layer (LbL) nanoparticles (NPs) can be constructed so that they escape endosomes intact, and subsequently exhibit sustained release of the cargo. Our work quantifies intra-cellular siRNA release from multilayered NPs, evaluates mechanism behind the sustained release, and optimizes the duration of release. RESULTS: Intra-cellular studies showed that NPs developed with four layers of poly-L-arginine, alternated with three layers of siRNA layers, were able to elicit effective and prolonged SPARC knockdown activity over 21 days with a single-dose treatment. For the first time, we have quantified the amounts of released siRNA in the cytoplasm and the amount of siRNA remaining inside the NPs at each timepoint. Furthermore, we have correlated the amount of released siRNA within cells by LbL NPs to the cellular knockdown efficiency of multilayered delivery system. CONCLUSIONS: This methodology may provide an excellent screening tool for assessing the duration of gene silencing by various nanocarrier formulations.

Molecular approaches for the management of papaya ringspot virus infecting papaya: a comprehensive review.

Papaya ringspot virus (PRSV) is a catastrophic disease that causes huge yield losses in papaya cultivation around the world. Yield losses in severely infected plants can be upto 100%. Because of this disease, papaya cultivation has been shifted to other crops in some areas of the world. Many conventional methods and breeding approaches are used against this disease, which turns out to be less effective. Considering the yield loss caused by PRSV in papaya, it is high time to focus on alternative control methods. To implement effective management strategies, molecular approaches such as Marker Assisted Breeding (MAS) or transgenic methods involving post-transcriptional gene silencing targeting the genome viz., coat protein, replicase gene, or HC Pro can be pursued. However, the public's reluctance to widely accept the transgenic approach due to health and environmental concerns necessitates a consideration of non-transgenic alternatives. Prioritizing safety and ensuring efficient virus control, non-transgenic approaches which encompass cross-protection, genome editing, and topical applications of dsRNA to induce gene silencing within the host, can be adopted. This review aims to provide comprehensive insights of various molecular tools used in managing PRSV which in turn will help in sustainable agriculture.