Gene Silencing Research Articles

Arrayed CRISPR libraries for the genome-wide activation, deletion and silencing of human protein-coding genes.

Arrayed CRISPR libraries extend the scope of gene-perturbation screens to non-selectable cell phenotypes. However, library generation requires assembling thousands of vectors expressing single-guide RNAs (sgRNAs). Here, by leveraging massively parallel plasmid-cloning methodology, we show that arrayed libraries can be constructed for the genome-wide ablation (19,936 plasmids) of human protein-coding genes and for their activation and epigenetic silencing (22,442 plasmids), with each plasmid encoding an array of four non-overlapping sgRNAs designed to tolerate most human DNA polymorphisms. The quadruple-sgRNA libraries yielded high perturbation efficacies in deletion (75-99%) and silencing (76-92%) experiments and substantial fold changes in activation experiments. Moreover, an arrayed activation screen of 1,634 human transcription factors uncovered 11 novel regulators of the cellular prion protein PrPC, screening with a pooled version of the ablation library led to the identification of 5 novel modifiers of autophagy that otherwise went undetected, and 'post-pooling' individually produced lentiviruses eliminated template-switching artefacts and enhanced the performance of pooled screens for epigenetic silencing. Quadruple-sgRNA arrayed libraries are a powerful and versatile resource for targeted genome-wide perturbations.

Arsenic Trioxide (ATOIII) Induces NAD(P)H Quinone Oxidoreductase 1 (NQO1) Expression in Hepatic and Extrahepatic Tissues of C57BL/6 Mice.

Arsenic trioxide (ATOIII) has emerged as a potent therapeutic agent for acute promyelocytic leukemia (APL), yet its clinical application is often limited by significant adverse effects. This study investigates the molecular mechanisms underlying ATOIII's impact on cellular detoxification pathways, focusing on the regulation of NAD(P)H/quinone oxidoreductase (NQO1), a crucial enzyme in maintaining cellular homeostasis and cancer prevention. We explored ATOIII's effects on NQO1 expression in C57BL/6 mice and Hepa-1c1c7 cells, both independently and in combination with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a known NQO1 inducer. Our findings revealed that ATOIII significantly increased NQO1 expression in hepatic and extrahepatic tissues, as well as in Hepa-1c1c7 cells, at mRNA, protein, and activity levels. This upregulation occurred both in the presence and absence of TCDD. Mechanistically, we demonstrated that ATOIII promotes the nuclear translocation of both nuclear factor erythroid 2-related factor-2 (NRF2) and aryl hydrocarbon receptor (AHR) transcription factors. Furthermore, ATOIII exposure increased antioxidant response element (ARE)-driven reporter gene activity, indicating a transcriptional mechanism of NQO1 induction. Notably, gene silencing experiments confirmed the critical roles of both NRF2 and AHR in mediating ATOIII-induced NQO1 expression. In conclusion, ATOIII exposure is found to upregulate the NQO1 enzyme through a transcriptional mechanism via AHR- and NRF2- dependent mechanisms, offering valuable insights into its therapeutic mechanisms.

Silencing CaPIP5K4-1 leads to decreased male fertility in Capsicum annuum L.

Phosphatidylinositol 4-phosphate 5-kinase gene CaPIP5K4-1 is highly expressed in the pepper anthers. Virus-induced gene silencing of CaPIP5K4-1 leads to reduced male fertility in pepper. The phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is a pivotal enzyme in the phosphatidylinositol signaling pathway, and its crucial involvement in both plant development and stress response has been established. Here, we found that the expression of CaPIP5K4-1 in pepper was significantly higher in the fertile flower buds compared to sterile flower buds. Furthermore, its expression was validated in anthers and pollens by qRT-PCR and RNA-ISH assays, respectively. Its GFP fusion protein was mainly located on the plasma membrane. Silencing CaPIP5K4-1 in fertile pepper accessions resulted in wrinkled pollen grain cell walls, decreased pollen germination efficiency, and inhibited pollen tube growth. The transcription levels of multiple genes in the phosphatidylinositol signaling pathway were also assessed. Five phospholipase C (PLC) genes were downregulated in silenced plants. On the contrary, inositol phosphatase SAC and phosphatase and tensin homolog (PTEN) were upregulated. This study reported the role of CaPIP5K4-1 in pepper male fertility and provided insights into the regulatory mechanisms of PI signaling in pepper.

Virus-Induced Gene Silencing (VIGS) in Hydrangea macrophylla and Functional Analysis of HmF3′5′H

Hydrangea macrophylla, renowned for its large inflorescences and a diverse range of colors, highlights a significant limitation in current gene function research, which is the lack of effective molecular genetic tools. This study utilized a tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) system to investigate gene function through posttranscriptional gene silencing in H. macrophylla for the first time. The ortholog of phytoene desaturase (PDS) in H. macrophylla, termed HmPDS, was identified. Infection of tissue-cultured seedlings with TRV-HmPDS led to photobleaching of the leaves. Additionally, infection with TRV containing the HmCHS1 fragment in the flowers resulted in decreased anthocyanin production in sepals and a lightening of sepal coloration in the infected flowers. The phenomena and RT-qPCR results proved that the PDS and CHS genes of hydrangea were successfully silenced via the vacuum infiltration method. Furthermore, the introduction of TRV-HmF3′5′H revealed a decrease in delphinidin-3-glucoside content in sepals and caused a color change in the sepals from blue to pink. This study demonstrated that the TRV-VIGS system was successfully established in H. macrophylla and effectively applied to the function analysis of HmF3′5′H.

Epigenetic Modifiers: Exploring the Roles of Histone Methyltransferases and Demethylases in Cancer and Neurodegeneration

Histone methyltransferases (HMTs) and histone demethylases (HDMs) are critical enzymes that regulate chromatin dynamics and gene expression through the addition and removal of methyl groups on histone proteins. HMTs, such as PRC2 and SETD2, are involved in the trimethylation of histone H3 at lysine 27 and lysine 36, influencing gene silencing and activation. Dysregulation of these enzymes often leads to abnormal gene expression and contributes to tumorigenesis. In contrast, HDMs including KDM7A and KDM2A reverse these methylation marks, and their dysfunction can drive disease progression. In cancer, the aberrant activity of specific HMTs and HDMs can lead to the silencing of tumor suppressor genes or the activation of oncogenes, facilitating tumor progression and resistance to therapy. Conversely, in neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD), disruptions in histone methylation dynamics are associated with neuronal loss, altered gene expression, and disease progression. We aimed to comprehend the odd activity of HMTs and HDMs and how they contribute to disease pathogenesis, highlighting their potential as therapeutic targets. By advancing our understanding of these epigenetic regulators, this review provides new insights into their roles in cancer and neurodegenerative diseases, offering a foundation for future research.

Polyethylene glycol-phospholipid functionalized single-walled carbon nanotubes for enhanced siRNA systemic delivery

Small interfering RNAs (siRNA) technology has emerged as a promising therapeutic tool for human health conditions like cancer due to its ability to regulate gene silencing. Despite FDA-approved, their delivery remains localized and limiting their systemic use. This study used single-walled carbon nanotubes (SWNTs) functionalized with polyethylene glycolated (PEGylated) phospholipids (PL-PEG) derivatives for systemic siRNA delivery. We developed an siRNA systemic delivery vehicle (SWNT-siRNA) by conjugating SWNT functionalized with PL-PEG containing either amine (PA) or maleimide (MA). The functionalized SWNT with a lower molecular weight of PA produced the SWNT-siRNA conjugate system with the highest stability and high siRNA loading quantity. The system delivered siRNA to a panel of tumour cell lines of different organs (i.e. HeLa, H1299 and MCF-7) and a non-cancerous human embryonic kidney 293 cells (HEK293T) with high biocompatibility and low toxicity. The cellular uptake of SWNT-siRNA conjugates by epithelial cells was found to be energy dependent. Importantly, the presence of P-glycoprotein, a marker for drug resistance, did not inhibit SWNT-mediated siRNA delivery. Mouse xenograft model further confirmed the potential of SWNT-siRNA conjugates with a significant gene knock-down without signs of acute toxicity. These findings pave the way for potential gene therapy applications using SWNTs as delivery vehicles.

ArMYB89 and ArCOP1 interaction modulates anthocyanin biosynthesis in Acer rubrum leaves under low-temperature conditions.

Acer rubrum, a famous ornamental tree, produces bright red-coloured leaves because of the temperature decline from summer to autumn. This process's molecular mechanism is elusive, so we have investigated how anthocyanin biosynthesis is induced in A. rubrum leaves under low temperatures. The results of low-temperature treatment under light and dark conditions showed that the low-temperature promoted anthocyanin accumulation in A. rubrum is light-dependent. The transcriptome analysis showed that ArMYB89 was significantly highly expressed in leaves of A. rubrum growing under low temperatures with light conditions. The findings from the Dap-seq analysis, yeast one hybridisation, electrophoretic mobility shift assay and luciferase reporter assay indicated that the ArMYB89 transcription factor binds directly to the promoter of ArUGT52 and stimulates its transcription. The co-expression of ArUGT52 with ArMYB89 significantly induced anthocyanin levels under low temperatures with light conditions. Enzyme activity analysis showed that ArUGT52 could convert Cyanidins and Pelargonidins into Cyanidin-3-O-glucoside and Pelargonidin 3-glucoside, which are considered the main anthocyanins in red colour leaves of A. rubrum. The results of yeast two hybridisation, pulldown assay and bimolecular fluorescence complementation experiment showed an interaction between COP1 and ArMYB89, while in vivo and in vitro protein ubiquitination assay demonstrated that ArCOP1 ubiquitinates ArMYB89. Notably, co-expression of ArCOP1 with ArMYB89 significantly reduced anthocyanin levels, while the virus-induced gene silencing of ArCOP1 significantly induced anthocyanin levels under low temperatures with light conditions. In conclusion, this work revealed the molecular mechanism regulating anthocyanin accumulation in the A. rubrum leaves under low temperatures.

Genome-Wide Identification of the GbUBC Gene Family in Sea-Island Cotton (Gossypium barbadense) and the Active Regulation of Drought Resistance in Cotton by GbUBC23

Cotton is an economically critical crop worldwide, and drought stress strongly affects its growth and development. Ubiquitination modifies protein activity and is crucial in numerous biological processes. Ubiquitin-conjugating enzymes serve as intermediaries in the protein ubiquitination process and play important roles in plant responses to abiotic stress. However, the impact of ubiquitination on the response of cotton to abiotic stress is not fully understood. Bioinformatic methods were employed in this study to analyze the physiochemical characteristics, gene structure, collinearity, expression patterns, and evolutionary relationships of GbUBC gene family members in sea-island cotton. In sea-island cotton, a minimum of 125 GbUBC genes are irregularly distributed across the 26 chromosomes, with multiple instances of gene duplication observed among the members. Phylogenetic analysis categorized the GbUBC gene family into 15 ubiquitin-conjugating enzyme (E2) subgroups, one ubiquitin E2 enzyme variant (UEV) subgroup, and one COP10 subgroup. GbUBC gene expression pattern analyses revealed that most GbUBC genes responded differently to cold, heat, NaCl, and polyethylene glycol (PEG) treatments, with certain GbUBC genes exhibiting high expression levels in specific fiber development period and organs. Furthermore, molecular biology methods were employed to elucidate the biological functions of GbUBC23. The GbUBC23 gene was highly expressed in the cotyledons of sea-island cotton and was activated by PEG treatment. GbUBC23 is localized to the nucleus and cytomembrane. The silencing of the GbUBC23 gene under drought conditions led to decreased drought tolerance and survival rates in sea-island cotton. Compared with those in the control plants, the activity of proline and superoxide dismutase and the expression levels of the drought-induced genes GbNCED3, GbRD22, GbRD26 were significantly lower, but the levels of malondialdehyde and hydrogen peroxide were significantly higher. Our findings revealed 125 members of the GbUBC gene family in sea-island cotton, with the GbUBC23 gene critically contributing to the abiotic stress response. These findings indicate that the GbUBC gene family may play a crucial role in the drought stress response in sea-island cotton.

High-Pressure Processing Influences Antibiotic Resistance Gene Transfer in Listeria monocytogenes Isolated from Food and Processing Environments

The study aimed to assess the high-pressure processing (HPP) impact on antibiotic resistance gene transfer in L. monocytogenes from food and food processing environments, both in vitro (in microbiological medium) and in situ (in carrot juice), using the membrane filter method. Survival, recovery, and frequency of antibiotic resistance gene transfer analyses were performed by treating samples with HPP at different pressures (200 MPa and 400 MPa). The results showed that the higher pressure (400 MPa) had a significant effect on increasing the transfer frequency of genes such as fosX, encoding fosfomycin resistance, and tet_A1, tet_A3, tetC, responsible for tetracycline resistance, both in vitro and in situ. In contrast, the Lde gene (the gene encoding ciprofloxacin resistance) was not transferred under any conditions. In the food matrix (carrot juice), greater variability in results was observed, suggesting that food matrices may have a protective effect on bacteria and modify HPP efficacy. In general, an increase in MIC values for antibiotics was noted in transconjugants compared to donors. Genotypic analysis of transconjugants showed differences in genetic structure, especially after exposure to 400 MPa pressure, indicating genotypic changes induced by pressure stress. The study confirms the possibility of antibiotic resistance genes transfer in the food environment, even from strains showing initial susceptibility to antibiotics carrying so-called silent antibiotic resistance genes, highlighting the public health risk of the potential spread of antibiotic-resistant strains through the food chain. The findings suggest that high-pressure processing can increase and decrease the frequency of resistance gene transfer depending on the strain, antibiotic combination, and processing conditions.

Repressor MrERF4 and Activator MrERF34 Synergistically Regulate High Flavonol Accumulation Under UV-B Irradiation in Morella rubra Leaves.

Flavonols are important plant photoprotectants to defence UV-B irradiation, however, the underlying transcriptional regulatory mechanism of rapid flavonol accumulation in response to UV-B remains unknown. In this study, content of flavonols was significantly induced from 0.11 to 3.80 mg/g fresh weight by UV-B irradiation in leaves of Morella rubra seedlings. MrERF34 was identified as an activator that can regulate the expression of MrFLS2, and promoted flavonol biosynthesis with activator MrMYB12 under UV-B treatment. Transient overexpression of MrERF34 resulted in higher flavonol accumulation, while virus-induced gene silencing of MrERF34 reduced the content of flavonols in bayberry leaves. We further demonstrated that a repressor MrERF4 inhibited the expression of MrERF34 and MrMYB12 as well as MrFLS2 via ERF-associated-amphiphilic repression motif. Exposure to UV-B reduced the promoter activity and transcription of MrERF4, which weakened the inhibitory effect of MrERF4 on MrERF34, MrMYB12, and MrFLS2, leading to a tremendous accumulation of flavonols. Such inhibitory roles of MrERF4 in regulation of flavonol biosynthesis were further validated by transient overexpression in leaves of Nicotiana benthamiana and M. rubra. These findings enrich the synergistical regulatory mechanisms between repressor and activators in flavonol biosynthesis, and provide new insights into photoprotectants biosynthesis to mitigate UV-B stress in plants.

ARRDC3, a novel α-arrestin, modulates WSSV replication and AHPND pathogenesis in Litopeneaus vannamei

Although shrimp are a valuable protein source, shrimp aquaculture has numerous challenges from various infectious diseases and understanding molecular mechanisms of disease pathogenesis is crucial for disease management. In this study, a gene-to-gene correlation network generated from a transcriptomic database of the stomach of shrimp infected with acute hepatopancreatic necrosis disease (AHPND) was used to identify a new α-arrestin, termed arrestin domain containing-3 gene (LvARRDC3), with crucial roles in development of both AHPND and white spot disease (WSD). Double stranded RNA-mediated silencing or plasmid-mediated overexpression of LvARRDC3 gene significantly decreased expression of WSSV genes (IE1, VP28, and ICP11) and viral genome copy numbers. Nevertheless, in AHPND, silencing the LvARRDC3 gene increased the AHPND-associated plasmid and Pir toxins copy numbers, whereas overexpression of LvARRDC3 had the opposite effect. An in vitro pathogen binding assay with recombinant LvARRDC3 protein produced robust binding to WSSV virions and AHPND-causing V. parahaemolyticus. Moreover, based on immunofluorescence, LvARRDC3 was localized in the cytoplasm of Spodoptera frugiperda (Sf9) insect cells. Therefore, we inferred that LvARRDC3 has a role in pathogen internalization, making it a valuable target for addressing AHPND and WSD and also a biomarker for marker-associated shrimp breeding.

GhLCYε-3 characterized as a lycopene cyclase gene responding to drought stress in cotton

Drought stress significantly affects crop productivity. Carotenoids are essential photosynthetic pigment for plants, bacteria, and algae, with signaling and antioxidant functions. Lutein is a crucial branch product in the carotenoid synthesis pathway, which effectively improves the stress tolerance of higher plants. lycopene cyclase, a central enzyme for lutein synthesis, holds great significance in regulating lutein production. This research establishes a correlation between lutein content and stress resistance by measuring the drought resistance and lutein content of various cotton materials. To identify which crucial genes are associated with lutein, the lycopene cyclase family (LCYs) was analyzed. The research found that LCYs form a highly conserved family divided into two subfamilies, LCY-ε (lycopene ε-cyclase) and LCY-β (lycopene β-cyclase). Most members of the LCY family contain photoresponsive elements and abscisic acid elements. qRT-PCR demonstrates showed that most genes responded positively to drought stress, and GhLCYε-3 was expressed significantly differently under drought stress. Virus-induced gene silencing (VIGS) assay showed that the content of GhLCYε-3 was significantly increased with MDA and PRO, and the contents of chlorophyll and lutein were significantly decreased in pYL156 plants. The decrease in GhLCYε-3 expression is speculated to lead to reduced lutein content in vivo, resulting in the accumulation of reactive oxygen species (ROS) and decreased drought tolerance. This research enriched the understanding of LCY gene family and lutein function, and provided a new reference for cotton planting in arid areas. SynopsisLycopene cyclase plays an important role in enhancing the ability of scavenging ROS and drought resistance of plants.

A Computational Approach for Designing and Validating Small Interfering RNA against SARS-CoV-2 Variants.

The aim of this study is to develop a novel antiviral strategy capable of efficiently targeting a broad set of SARS-CoV-2 variants. Since the first emergence of SARS-CoV-2, it has rapidly transformed into a global pandemic, posing an unprecedented threat to public health. SARS-CoV-2 is prone to mutation and continues to evolve, leading to the emergence of new variants capable of escaping immune protection achieved due to previous SARS-CoV-2 infections or by vaccination. RNA interference (RNAi) is a remarkable biological mechanism that can induce gene silencing by targeting complementary mRNA and inhibiting its translation. In this study, using the computational approach, we predicted the most efficient siRNA capable of inhibiting SARS-CoV-2 variants of concern (VoCs). The presented siRNA was characterized and evaluated for its thermodynamic properties, offsite-target hits, and in silico validation by molecular docking and molecular dynamics simulations (MD) with Human AGO2 protein. The study contributes to the possibility of designing and developing an effective response strategy against existing variants of concerns and preventing further.

BSA-seq and transcriptome analysis reveal GhDSA05 as a candidate gene responsible for sensitivity to defoliants in Gossypium hirsutum L.

With the development of machine-harvested cotton, the application of defoliants is a key technology that can promote defoliation and reduce impurities in cotton fibers. Therefore, sensitivity to defoliants is a very important trait in machine-harvested cotton. In this study, to uncover the genetic basis of defoliant sensitivity in cotton, bulked segregant analysis by sequencing was used with a F2 population derived from Xinluzao 57 (XLZ57, defoliant sensitive) and Nan 2 (N2, defoliant insensitive) to mapped the quantitative trait locus (QTL) to a 3.66-Mb interval at chromosome A05. Then, 284 F2 individuals were used to validate and narrow the QTL to a 1.6-Mb interval and was named qSD-A05. To identify the candidate genes, dynamic transcriptome analysis of the abscission zone (AZ) in XLZ57 and N2 after defoliant treatment was performed. A total of 132 and 142 common differentially expressed genes (DEGs) were identified at all time points in N2 and XLZ57, respectively, and were enriched in the defense response pathway and abscisic acid-activated signaling pathway. More importantly, 15 DEGs were identified in the qSD-A05 interval, among which, Ghi_A05G23601 was significantly upregulated after defoliant treatment. There were multiple variants between N2 and XLZ57, including missense and frameshift mutations. Therefore, Ghi_A05G23601 was regarded as the key candidate gene and was named GhDSA05, which encodes a Facilitator superfamily transporter protein. To verify the function of GhDSA05, virus-induced gene silencing was performed, which showed that the GhDSA05-silenced plants exhibited decreased defoliant sensitivity. Additionally, the expression of organ abscission-related genes was downregulated in the GhDSA05-silenced plants. In summary, GhDSA05 positively regulates the formation of AZ under defoliant treatment and promotes leaf abscission in cotton. The results of this study not only enhance our knowledge of the defoliation mechanism of cotton but also provide a target for the genetic improvement of defoliant sensitivity.

Surfactant Protein-C Regulates Alveolar Type 2 Epithelial Cell Lineages via the CD74 Receptor.

Deficiency of surfactant protein-C (SPC) increases susceptibility to lung infections and injury, and suppressed expression of SPC has been associated with the severity of acute respiratory distress syndrome (ARDS). Alveolar type 2 epithelial cells (AT2) are critical for maintenance and repair of the lung. However, the role of the SPC in the regulation of AT2 cell lineage and the underlying mechanisms are not completely understood. This study aimed to investigate the mechanisms by which SPC regulates AT2 lineages. Sftpc-/- mice were used to model the SPC deficiency in ARDS patients. We utilized three-dimensional (3D) organoids to compare AT2 lineage characteristics between wild type (WT) and Sftpc-/- mice by analyzing AT2 proliferation, alveolar type 1 cells (AT1) differentiation and CD74 expression, using colony-formation assay, immunofluorescence, flow cytometry, and immunoblots. The results showed that Sftpc-/- mice demonstrated a reduced AT2 cell population. Influenza A virus subtype H1N1 (H1N1) infected Sftpc-/- mice demonstrated reduced AT2 proliferation and AT1 differentiation. Western blot indicated elevated levels of CD74 protein in AT2 cells of Sftpc-/- mice. Colony-forming efficiency was significantly attenuated in AT2 cells isolated from Sftpc-/- mice compared to the WT controls. Podoplanin (PDPN, a marker of AT1 cells) expression and transient cell count significantly increased in Sftpc-/- organoids. Moreover, siRNA-mediated gene silencing of CD74 in AT2 cells significantly increased AT2 proliferation and AT1 differentiation in Sftpc-/- organoids. This study suggests that SPC regulates AT2 lineage in vitro and in vivo. The SPC might influence AT2 lineage during the lung epithelium repair by activating signaling mechanism involving CD74 receptor.

In Vivo Glucose Transporter-2 Regulation of Dorsomedial Versus Ventrolateral VMN Astrocyte Metabolic Sensor and Glycogen Metabolic Enzyme Gene Expression in Female Rat.

Astrocyte glycogenolysis shapes ventromedial hypothalamic nucleus (VMN) regulation of glucostasis in vivo. Glucose transporter-2 (GLUT2), a plasma membrane glucose sensor, controls hypothalamic primary astrocyte culture glycogen metabolism in vitro. In vivo gene silencing tools and single-cell laser-catapult-microdissection/multiplex qPCR techniques were used here to examine whether GLUT2 governs dorsomedial (VMNdm) and/or ventrolateral (VMNvl) VMN astrocyte metabolic sensor and glycogen metabolic enzyme gene profiles. GLUT2 gene knockdown diminished astrocyte GLUT2 mRNA in both VMN divisions. Hypoglycemia caused GLUT2 siRNA-reversible up-regulation of this gene profile in the VMNdm, but down-regulated VMNvl astrocyte GLUT2 transcription. GLUT2 augmented baseline VMNdm and VMNvl astrocyte glucokinase (GCK) gene expression, but increased (VMNdm) or reduced (VMNvl) GCK transcription during hypoglycemia. GLUT2 imposed opposite control, namely stimulation versus inhibition of VMNdm or VMNvl astrocyte 5'-AMP-activated protein kinase-alpha 1 and -alpha 2 gene expression, respectively. GLUT2 stimulated astrocyte glycogen synthase (GS) gene expression in each VMN division. GLUT2 inhibited transcription of the AMP-sensitive glycogen phosphorylase (GP) isoform GP-brain type (GPbb) in each site, yet diminished (VMNdm) or augmented (VMNvl) astrocyte GP-muscle type (GPmm) mRNA. GLUT2 enhanced VMNdm and VMNvl glycogen accumulation during euglycemia, and curbed hypoglycemia-associated VMNdm glycogen depletion. Results show that VMN astrocytes exhibit opposite, division-specific GLUT2 transcriptional responsiveness to hypoglycemia. Data document divergent GLUT2 control of GCK, AMPK catalytic subunit, and GPmm gene profiles in VMNdm versus VMNvl astrocytes. Ongoing studies seek to determine how differential GLUT2 regulation of glucose and energy sensor function and glycogenolysis in each VMN location may affect local neuron responses to hypoglycemia.

Ex vivo delivery of dsRNA targeting ryanodine receptors for control of Tuta absoluta.

RNA interference (RNAi) is an endogenous eukaryote viral defence mechanism representing a unique form of post-transcriptional gene silencing. Owing to its high specificity, this technology is being developed for use in dsRNA-based biopesticides for control of pest insects. Whilst many lepidopteran species are recalcitrant to RNAi, Tuta absoluta, a polyphagous insect responsible for extensive crop damage, is sensitive. Ryanodine receptors (RyRs) are intracellular calcium channels regulating calcium ion (Ca2+) release. The chemical pesticide class of diamides functions agonistically against lepidopteran RyR, resulting in uncontrolled Ca2+ release, feeding cessation and death. Resistance to diamides has emerged in T. absoluta, derived from RyR point mutations. RNAi was used to target RyR transcripts of T. absoluta. Data presented here demonstrate the systemic use of exogenous T. absoluta RyR-specific (TaRy) dsRNA in tomato plants (Solanum lycopersicum) to significantly downregulate expression of the target gene, resulting in significant insect mortality and reduced leaf damage. Using a leaflet delivery system, daily dosing of 3 μg TaRy dsRNA for 72 h resulted in 50% downregulation of the target gene and 50% reduction in tomato leaf damage. Corrected larval mortality and adult emergence were reduced by 38% and 33%, respectively. TaRy dsRNA demonstrated stability in tomato leaves ≤72 h after dosing. This work identifies TaRy as a promising target for RNAi control of this widespread crop pest. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Enhancing Solanine Production and Antifungal Activity by Streptomyces abikoensis XH-17 through Combined Ribosome Engineering and Post-Translational Modification

Solanine, a strong poison alkaloid typically produced by staple food crops, such as sprouted potatoes, poses a potential risk to food safety, therefore, controlling solanine levels is one of the important targets for human health. Streptomyces species are ubiquitous across diverse environments and are acclaimed for their extensive production of bioactive compounds, including antibiotics, anticancer agents, and immunosuppressants. Despite their potential, many biosynthetic gene clusters (BGCs) in Streptomyces remain silent under standard laboratory conditions. The present study introduces a combination strategy that integrates ribosome engineering and post-translational modification (PTM) to activate the silent BGCs in Streptomyces XH-17, which was identified as S. abikoensis by whole gene cluster sequencing and phylogenetic analysis. The antimicrobial activity of the engineered strain increased by 3.74-fold, 1.40-fold, and 4.08-fold against Alternaria alternata, Fusarium oxysporum, and F. proliferatum, respectively. The main compounds responsible for antifungal activity were identified as α-chaconine and α-solanine by high-resolution mass spectrometry (HRMS), and their BGCs were silent in the wild type (WT). Compared to WT, the two compounds’ production increased by 5.78-fold and 4.68-fold in engineered strain XH-17rpoB+Svp, respectively, and the biosynthetic pathways of α-chaconine and α-solanine were inferred by bioinformatics analysis. This is the first to identify solanine and presumed its biosynthetic pathway from Streptomyces. Our research provides new insights into the control of solanine to protect food safety from Streptomyces contamination. The results from the current investigation provide an effective pipeline to activate silent gene clusters in Streptomyces and offer new insight into discovering new bioactive compounds within this genus.

RNA-based Therapeutics: Past, Present and Future Prospects, Challenges in Cancer Treatment.

It is becoming more and harder in today's climate to disregard the impact of cancer on social health. Even though a significant amount of money is spent annually on cancer research, it still ranks as the second leading cause of death worldwide. Additionally, only about half of the patients suffering from complex forms of cancer survive a year after receiving traditional cancer therapies. A method for silencing genes is called RNA interference (RNAi). Such a method is very effective in focusing on genes linked to cancer. Most gene products implicated in cancer have recently been used as RNA interference (RNAi) therapeutic targets. According to the findings from this research, RNAi application is necessary for today's cancer treatment to target functioning carcinogenic molecules and tumor resistance to chemotherapy and radiation. Proapoptotic and antiproliferative activity has been reported from previous research studies on cell culture systems, animal models, and clinical trials through the knockdown of gene products from RNAi technology. Numerous novel RNAi-based medications are now in the clinical trial stages thanks to the discovery of the RNAi mechanism and advancements in the area. In the future, genomic-based personalized medicines can be developed through this RNAi therapy. Hopefully, cancer sufferers will find this sort of therapy to be one of the most effective ones. Various kinds of RNA-based treatments, such as aptamers, small interfering RNAs, microRNAs, antisense oligonucleotides, and messenger RNA, are covered in broad terms in this study. We also present an overview of the RNA-based therapies that have received regulatory approval in the past or are now undergoing clinical studies.

Juvenile hormone regulates reproductive diapause through both canonical and noncanonical pathways in the bean bug Riptortus pedestris

Diapause is an adaptive developmental arrest commonly utilized by animals to cope with seasonal changes. Central to this process are hormonal events that bridge photoperiodic cues and physiological changes. In insect reproductive diapause, the absence of juvenile hormone (JH) serves as the primary endocrine event that governs key diapause traits, including ovarian developmental arrest and lipid accumulation. Conventionally, it is believed that the effects of JH are conveyed through the receptor Methoprene-tolerant (Met) and its transcriptional factor Krüppel homolog 1 (Kr-h1). However, our study with the bean bug Riptortus pedestris reveals that JH independently regulates lipid accumulation, bypassing Met and Kr-h1 pathways. R. pedestris enters reproduction under long-day (LD) conditions, while diapause occurs under short-day (SD) conditions. Treatment of SD females with the JH mimic methoprene stimulated reproductive activities, enhancing ovary development and reducing lipid accumulation. In contrast, silencing genes essential for JH biosynthesis in LD females led to pronounced diapause characteristics, including ovarian developmental arrest and substantial lipid accumulation. Interestingly, disruptions in the JH action genes, either Met or Kr-h1, solely affected ovary development, leaving lipid accumulation unchanged, indicating an independent pathway for regulating JH in lipid accumulation. This was further confirmed by RNA interference experiments in SD females, where knockdown of Met or Kr-h1 did not alter the effects of methoprene on lipid reduction. Collectively, these results suggest that JH controls ovary development through the established Met-Kr-h1 pathway, while it modulates lipid accumulation through an alternative, yet to be identified noncanonical pathway during reproductive diapause in R. pedestris.