Transcriptional Downregulation Research Articles

BRD4 Degradation Enhanced Glioma Sensitivity to Temozolomide by Regulating Notch1 via Glu-Modified GSH-Responsive Nanoparticles.

Temozolomide (TMZ) serves as the principal chemotherapeutic agent for glioma; nonetheless, its therapeutic efficacy is compromised by the rapid emergence of drug resistance, the inadequate targeting of glioma cells, and significant systemic toxicity. ARV-825 may play a role in modulating drug resistance by degrading the BRD4 protein, thereby exerting anti-glioma effects. Therefore, to surmount TMZ resistance and achieve efficient and specific drug delivery, a dual-targeted glutathione (GSH)-responsive nanoparticle system (T+A@Glu-NP) is designed and synthesized for the co-delivery of ARV-825 and TMZ. As anticipated, T+A@Glu-NPs significantly enhanced penetration of the blood-brain barrier (BBB), facilitated drug uptake by glioma cells, and exhibited efficient accumulation in brain tissue. Additionally, T+A@Glu-NPs exhibited augmented efficacy against glioma both in vitro and in vivo through the induction of apoptosis, inhibition of proliferation, and cell cycle arrest. Furthermore, mechanistic exploration revealed that T+A@Glu-NPs degraded the BRD4 protein, leading to the downregulation of Notch1 gene transcription and the inhibition of the Notch1 signaling pathway, thereby augmenting the therapeutic efficacy of glioma chemotherapy. Taken together, the findings suggest that T+A@Glu-NPs represents a novel and promising therapeutic strategy for glioma chemotherapy.

Maternal exercise represses FGF21 via SIRT1 to improve the phenotypic transformation of vascular smooth muscle in hypertensive offspring.

Maternal exercise during pregnancy is widely recognized as an effective means of promoting cardiovascular health in offspring. Few studies have explored how maternal exercise impacts vascular function and phenotypic switching in hypertensive offspring, despite the known involvement of vascular structural and functional remodeling in hypertension pathogenesis. Research indicates a significant relationship between elevated blood pressure and fibroblast growth factor 21 (FGF21) levels. It remains unclear whether maternal exercise during pregnancy can improve vascular function in hypertensive offspring by regulating FGF21 and its underlying mechanisms. In this study, pregnant spontaneously hypertensive rats and Wistar-Kyoto rats were randomly assigned to either a sedentary or exercise group. The exercise group underwent weightless swimming exercise from gestation day 1 (GD1) to GD20. The aim was to investigate the epigenetic modifications mediated by histone deacetylase sirtuin 1 (SIRT1) during the fetal period and the phenotypic changes in the mesenteric arteries (MAs) of hypertensive offspring. We found that maternal exercise significantly improved vascular remodeling in hypertensive offspring. Specifically, maternal exercise upregulated SIRT1 expression, which led to decreased H3K9ac (histone H3 lysine 9 acetylation) in the promoter region of the FGF21 gene. This epigenetic modification resulted in the transcriptional downregulation of FGF21 in the MAs of hypertensive fetuses. These results suggest that maternal exercise may lower blood pressure in hypertensive offspring by regulating deacetylation of the FGF21 gene promoter region through SIRT1, thereby reversing phenotypic switching and vascular structural remodeling.

AcsS inhibits the hemolytic activity and thermostable direct hemolysin (TDH) gene expression in Vibrio parahaemolyticus.

Vibrio parahaemolyticus produces a key virulent factor known as thermostable direct hemolysin (TDH). TDH exhibits diverse biological activities, including hemolytic activity. The β-type hemolysis observed on Wagatsuma agar due to TDH is recognized as the Kanagawa phenomenon (KP). The tdh2 gene is primarily responsible for TDH production and the associated KP. AcsS was originally identified as an activator of swimming and swarming motility in V. parahaemolyticus. However, the extent of its potential to regulate other cellular pathways remains unclear. In this study, we explored the regulatory effects of AcsS on the hemolytic activity and tdh2 expression in V. parahaemolyticus. The data showed that V. parahaemolyticus hemolytic activity and tdh2 transcription were under the negative control of AcsS. Additionally, in vitro binding assays revealed that His-AcsS could not bind to the regulatory DNA region of tdh2. However, overexpression of AcsS in an Escherichia coli strain suppressed the expression of tdh2. Collectively, these results suggested that AcsS suppresses the hemolytic activity of V. parahaemolyticus through the downregulation of tdh2 transcription. The data enhanced our understanding of the regulatory networks governing tdh2 expression and the roles of AcsS in this bacterium.

HSF-1 promotes longevity through ubiquilin-1-dependent mitochondrial network remodelling.

Increased activity of the heat shock factor, HSF-1, suppresses proteotoxicity and enhances longevity. However, the precise mechanisms by which HSF-1 promotes lifespan are unclear. Using an RNAi screen, we identify ubiquilin-1 (ubql-1) as an essential mediator of lifespan extension in worms overexpressing hsf-1. We find that hsf-1 overexpression leads to transcriptional downregulation of all components of the CDC-48-UFD-1-NPL-4 complex, which is central to both endoplasmic reticulum and mitochondria associated protein degradation, and that this is complemented by UBQL-1-dependent turnover of NPL-4.1. As a consequence, mitochondrial network dynamics are altered, leading to increased lifespan. Together, our data establish that HSF-1 mediates lifespan extension through mitochondrial network adaptations that occur in response to down-tuning of components associated with organellar protein degradation pathways.

DDDR-44. REPURPOSING SSRI/SNRI TARGETING NEUROTRANSMITTER SIGNALING TO OVERCOME RESISTANCE TO CDK9 INHIBITORS IN DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline glioma (DMG) is a subset of aggressive brain tumors with few effective therapies. Cyclin-dependent kinase 9 (CDK9i) inhibitors such as zotiraciclib (ZTR) are in clinical trials for glioma, but CDK9i treatment alone may be ineffective due to the development of resistance. To identify CDK9i resistance mechanisms, we analyzed the transcriptomes of DMG xenograft tumors after three weeks of treatment with ZTR, which revealed that transcripts encoding synaptic signaling regulators such as glutamate receptors were overexpressed in surviving cells, suggesting that altered regulation of synaptic signaling may mediate CDK9i resistance. We also performed drug synergy screens using a library of FDA-approved and bioactive compounds and found that drugs that modulate neurotransmitter signaling, such as SSRIs and SNRIs (selective serotonin and serotonin/norepinephrine reuptake inhibitors), increased DMG sensitivity to an IC25 dose of ZTR. Follow-up studies confirm that combinations of structurally distinct CDK9i (ZTR, AZD4573) and SSRI/SNRI (sertraline, duloxetine) synergistically reduce DMG growth in neurospheres by BLISS analysis and show that co-treatment with CDK9i and SSRI/SNRI strongly induces the apoptotic marker cleaved caspase 3 and reduces the CDK9 target phos-Pol2Ser2. Additional phospho-proteomic analyses identified differentially phosphorylated proteins following CDK9i+SSRI treatment, including chromatin regulators, Pol2 elongation factors, and splicing regulators. Further RNAseq studies reveal that CDK9i+SSRI treatment resulted in more robust transcriptional changes than either drug alone, including the downregulation of transcripts associated with ZTR resistance such as GRIA3/4, and SYT12. These observations suggest that CDK9i +SSRI/SNRI drug combinations may overcome some of the transcriptional changes underlying CDK9i resistance. Finally, preclinical studies show that co-treatment with low doses of AZD4573 and sertraline reduces the growth of orthotopic DMG xenografts and prolongs overall survival. Taken together, these data suggest that SSRI/SNRI and CDK9i might be used in combination to synergistically reduce DMG growth and overcome CDK9i resistance.

Efficient directional biosynthesis of isoquercitrin from quercetin by Bacillus subtilis CD-2 and its anti-inflammatory activity

Isoquercetin, as the sole product, was directionally biosynthesized from quercetin in a non-aqueous system using Bacillus subtilis CD-2 (1 g/L quercetin), and its structure was identified by LC-MS and NMR analysis. CCK8 assays showed no cytotoxicity and good cell proliferation. The anti-inflammatory experiments demonstrated strong inhibition of NO release, transcriptional downregulation of classical effective cellular factors tumour necrosis factor-α, interleukin-6, interleukin-1β, and transcriptional upregulation of interleukin-10 in LPS-induced RAW264.7 cells. Its stronger anti-inflammatory activity than quercetin provided a reference for modifying the structure of quercetin to obtain more compounds with better pharmacological activities for medical industry.

Degradome analysis to identify direct protein substrates of small-molecule degraders.

Targeted protein degradation (TPD) has emerged as a powerful strategy to selectively eliminate cellular proteins using small-molecule degraders, offering therapeutic promise for targeting proteins that are otherwise undruggable. However, a remaining challenge is to unambiguously identify primary TPD targets that are distinct from secondary downstream effects in the proteome. Here we introduce an approach for selective analysis of protein degradation by mass spectrometry (DegMS) at proteomic scale, which derives its specificity from the exclusion of confounding effects of altered transcription and translation induced by target depletion. We show that the approach efficiently operates at the timescale of TPD (hours) and we demonstrate its utility by analyzing the cyclin K degraders dCeMM2 and dCeMM4, which induce widespread transcriptional downregulation, and the GSPT1 degrader CC-885, an inhibitor of protein translation. Additionally, we apply DegMS to characterize a previously uncharacterized degrader, and identify the zinc-finger protein FIZ1 as a degraded target.

Blocking the HIF-1α/glycolysis axis inhibits allergic airway inflammation by reducing ILC2 metabolism and function.

The role of lung group 2 innate lymphoid cell (ILC2) activation in allergic asthma is increasingly established. However, the regulatory mechanisms underlying hypoxia-inducible factor-1α (HIF-1α)-mediated glycolysis in ILC2-mediated allergic airway inflammation remain unclear. To investigate the role of the HIF-1α/glycolysis axis in ILC2-mediated allergic airway inflammation. Glycolysis and HIF-1α inhibitors were used to identify their effect on the function and glucose metabolism of mouse and human ILC2s invivo and vitro. Blocking glycolysis and HIF-1α in mice under interleukin-33 (IL-33) stimulation were performed to test ILC2 responses. Conditional HIF-1α-deficient mice were used to confirm the specific role of HIF-1α in ILC2-driven airway inflammation models. Transcriptomic, metabolic, and chromatin immunoprecipitation analyses were performed to elucidate the underlying mechanism. HIF-1α is involved in ILC2 metabolism and is crucial in allergic airway inflammation. Single-cell sequencing data analysis and qPCR confirmation revealed a significant upregulation of glycolysis-related genes, particularly HIF-1α, in murine lung ILC2s after IL-33 intranasal administration or injection. Treatment with the glycolysis inhibitor 2-deoxy-D-glucose (2-DG) and the HIF-1α inhibitor 2-methoxyestradiol (2-ME) abrogated inflammation by suppressing ILC2s function. Conditional HIF-1α-deficient mice showed reduced ILC2 response and airway inflammation induced upon IL-33 or house dust mite (HDM) stimulation. Transcriptome and metabolic analyses revealed significantly impaired glycolysis in lung ILC2s in conditional HIF-1α knockout mice compared to that in their littermate controls. Chromatin immunoprecipitation results confirmed the transcriptional downregulation of glycolysis-related genes in HIF-1α-knockout and 2-DG-treated mice. Furthermore, impaired HIF-1α/glycolysis axis activation is correlated with downregulated ILC2 in patients with asthma. The HIF-1α/glycolysis axis is critical for controlling ILC2 responses in allergic airway inflammation and has potential immunotherapeutic value in asthma.

Molecular determinants of skeletal muscle force loss in response to 5days of dry immersion in human.

Astronauts in Earth's orbit experience microgravity, resulting in a decline of skeletal muscle mass and function. On Earth, models simulating microgravity have shown that the extent of the loss in muscle force is greater than the loss in muscle mass. The reasons behind this disproportionate loss of muscle force are still poorly understood. In the present study, we hypothesize that alongside the loss in skeletal muscle mass, modifications in the expression profile of genes encoding critical determinants of resting membrane potential, excitation-contraction coupling and Ca2+ handling contribute to the decline in skeletal muscle force. Healthy male volunteers (n=18) participated in a 5-day dry immersion (DI) study, an Earth-based model of simulated microgravity. Muscle force measurement and MRI analysis of the cross-sectional area of thigh muscles were performed before and after DI. Biopsies of the vastus lateralis skeletal muscle performed before and after DI were used for the determination Ca2+ properties of isolated muscle fibres, molecular and biochemical analyses. The extent of the decline in force, measured as maximal voluntary contraction of knee extensors (-11.1%, P<0.01) was higher than the decline in muscle mass (-2.5%, P<0.01). The decline in muscle mass was molecularly supported by a significant repression of the anabolic IGF-1/Akt/mTOR pathway (-19.9% and -40.9% in 4E-BP1 and RPS6 phosphorylation, respectively), a transcriptional downregulation of the autophagy-lysosome pathway and a downregulation in the mRNA levels of myofibrillar protein slow isoforms. At the single fibre level, biochemical and tension-pCa curve analyses showed that the loss in force was independent of fibre type (-11% and -12.3% in slow and fast fibres, respectively) and Ca2+ activation properties. Finally, we showed a significant remodelling in the expression of critical players of resting membrane potential (aquaporin 4: -24.9%, ATP1A2: +50.4%), excitation-contraction coupling (CHRNA1: +75.1%, CACNA2D1: -23.5%, JPH2: -24.2%, TRDN: -15.6%, S100A1: +27.2%), and Ca2+ handling (ATP2A2: -32.5%, CASQ1: -15%, ORAI1: -36.2%, ATP2B1: -19.1%). These findings provide evidence that a deregulation in the expression profile of critical molecular determinants of resting membrane potential, excitation-contraction coupling, and Ca2+ handling could be involved in the loss of muscle force induced by DI. They also provide the paradigm for the understanding of muscle force loss during prolonged bed rest periods as those encountered in intensive care unit.

A data mining approach to identify key radioresponsive genes in mouse model of radiation-induced intestinal injury

Background Radiation-mediated GI injury (RIGI) is observed in humans either due to accidental or intentional exposures. This can only be managed with supporting care and no approved countermeasures are available till now. Early detection and monitoring of RIGI is important for effective medical management and improve survival chances of exposed individuals. Objective The present study aims to identify new signatures of RIGI using data mining approach followed by validation of selected hub genes in mice. Methods Data mining study was performed using microarray datasets from Gene Expression Omnibus database. The differentially expressed genes were identified and further validated in total-body irradiated mice. Results Based on KEGG pathway analysis, lipid metabolism was found as one of the predominant pathways altered in irradiated intestine. Extensive alteration in lipid profile and lipid modification was observed in this tissue. A protein–protein interaction network revealed top 08 hub genes related to lipid metabolism, namely Fabp1, Fabp2, Fabp6, Npc1l1, Ppar-α, Abcg8, Hnf-4α, and Insig1. qRT-PCR analysis revealed significant up-regulation of Fabp6 and Hnf-4α and down-regulation of Fabp1, Fabp2 and Insig1 transcripts in irradiated intestine. Radiation dose and time kinetics study revealed that the selected 05 genes were altered differentially in response to radiation in intestine. Conclusion Finding suggests that lipid metabolism is one of the key targets of radiation and its mediators may act as biomarkers in detection and progression of RIGI.

Antiaging in a Bottle: Bioactive Competency of Plasma-Generated Nitric Oxide Water for Modulation of Aging-Related Signature in Human Dermal Cells.

Nitric oxide (NO), a potential therapeutic antiaging molecule, modulates various physiological and cellular processes. However, alterations in endogenous NO levels brought on by aging impact multiple organ systems and heighten susceptibility to age-related skin diseases. This correlation underscores the importance of investigating NO-based antiaging interventions. Nonthermal plasma-generated NO is a promising avenue for cosmetic and regenerative medicine due to its capacity to stimulate cellular growth. Herein, we examine the potential of plasma-generated nitric oxide water (NOW) as a bioactive agent in human dermal fibroblasts, emphasizing gene expression patterns linked to extracellular matrix (ECM) breakdown and cellular senescence. The findings of our study indicate that administering NOW at lower dosages enhances cell migration and proliferation. Moreover, the genetic signatures associated with ECM synthesis, antioxidant defense, and antisenescence pathways have been analyzed in NOW-exposed cells. Notably, the downregulation of ECM-degrading enzyme transcripts─collagenase, elastase, and hyaluronidase─suggests NOW's potential in mitigating the intrinsic skin aging phenomena, emphasizing the promise of NO-based interventions in advancing antiaging strategies within regenerative medicine.

Isoginkgetin and Madrasin are poor splicing inhibitors.

The production of eukaryotic mRNAs requires transcription by RNA polymerase (pol) II and co-transcriptional processing, including capping, splicing, and cleavage and polyadenylation. Pol II can positively affect co-transcriptional processing through interaction of factors with its carboxyl terminal domain (CTD), comprising 52 repeats of the heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7, and pol II elongation rate can regulate splicing. Splicing, in turn, can also affect transcriptional activity and transcription elongation defects are caused by some splicing inhibitors. Multiple small molecule inhibitors of splicing are now available, some of which specifically target SF3B1, a U2 snRNP component. SF3B1 inhibition results in a general downregulation of transcription elongation, including premature termination of transcription caused by increased use of intronic poly(A) sites. Here, we have investigated the effect of Madrasin and Isoginkgetin, two non-SF3B1 splicing inhibitors, on splicing and transcription. Surprisingly, we found that both Madrasin and Isoginkgetin affect transcription before any effect on splicing, indicating that their effect on pre-mRNA splicing is likely to be indirect. Both small molecules promote a general downregulation of transcription. Based on these and other published results, we conclude that these two small molecules should not be considered as primarily pre-mRNA splicing inhibitors.

Upregulation of microRNAs correlates with downregulation of HERV-K expression in Parkinson's disease.

Human endogenous retroviruses (HERVs) involvement in neurological diseases has been extensively documented, although the etiology of HERV reactivation remains unclear. MicroRNAs represent one of the potential regulatory mechanisms of HERV reactivation. We identified fourteen microRNAs predicted to bind the HERV-K transcript, and subsequently analyzed for their gene expression levels alongside those of HERV-K. We documented an increased expression of four microRNAs in patients with Parkinson's disease compared to healthy controls, which correlated with a downregulation of HERV-K transcripts. We hypothesize that specific microRNAs may bind to HERV-K transcripts, leading to its downregulation.

HPV16 integration regulates ferroptosis resistance via the c-Myc/miR-142-5p/HOXA5/SLC7A11 axis during cervical carcinogenesis

BackgroundFerroptosis, a newly identified form of regulated cell death triggered by small molecules or specific conditions, plays a significant role in virus-associated carcinogenesis. However, whether tumours arising after high-risk HPV integration are associated with ferroptosis is unexplored and remains enigmatic.MethodsHigh-risk HPV16 integration was analysed by high­throughput viral integration detection (HIVID). Ferroptosis was induced by erastin, and the levels of ferroptosis were assessed through the measurement of lipid-reactive oxygen species (ROS), malondialdehyde (MDA), intracellular Fe2+ level and transmission electron microscopy (TEM). Additionally, clinical cervical specimens and an in vivo xenograft model were utilized for the study.ResultsExpression of HPV16 integration hot spot c-Myc negatively correlates with ferroptosis during the progression of cervical squamous cell carcinoma (CSCC). Further investigation revealed that the upregulated oncogene miR-142-5p in HPV16-integrated CSCC cells served as a critical downstream effector of c-Myc in its target network. Inhibiting miR-142-5p significantly decreased the ferroptosis-suppressing effect mediated by c-Myc. Through a combination of computational and experimental approaches, HOXA5 was identified as a key downstream target gene of miR-142-5p. Overexpression of miR-142-5p suppressed HOXA5 expression, leading to decreased accumulation of intracellular Fe2+ and lipid peroxides (ROS and MDA). HOXA5 increased the sensitivity of CSCC cells to erastin-induced ferroptosis via transcriptional downregulation of SLC7A11, a negative regulator of ferroptosis. Importantly, c-Myc knockdown increased the anti-tumour activity of erastin by promoting ferroptosis both in vitro and in vivo.ConclusionsCollectively, these data indicate that HPV16 integration hot spot c-Myc plays a novel and indispensable role in ferroptosis resistance by regulating the miR-142-5p/HOXA5/SLC7A11 signalling axis and suggest a potential therapeutic approach for HPV16 integration-related CSCC.

Combinatorial Anti-Cancer Effect of Polypurine Reverse Hoogsteen Hairpins against KRAS and MYC Targeting in Prostate and Pancreatic Cancer Cell Lines

Introduction: KRAS and MYC are proto-oncogenes that are strictly regulated in healthy cells that have key roles in several processes such as cell growth, proliferation, differentiation, or apoptosis. These genes are tightly interconnected, and their dysregulation can lead to cancer progression. We previously individually targeted these oncogenes using Polypurine Reverse Hoogsteen (PPRH) hairpins, mostly targeting the complementary strand of G-quadruplex-forming sequences. We validated them in vitro in different cancer cell lines with deregulated KRAS and/or MYC. In this work we focused on our understanding of the cooperative dynamics between these oncogenes, by investigating the combined impact of PPRHs targeting KRAS and MYC in pancreatic and prostate cancer cells. Results: The combinations had a modulatory impact on the expression of both oncogenes, with transcriptional and translational downregulation occurring five days post-treatment. Out of the four tested PPRHs, MYC-targeting PPRHs, especially HpMYC-G4-PR-C directed against the promoter, showed a greater cytotoxic and expression modulation effect. When both KRAS- and MYC-targeting PPRHs were applied in combination, a synergistic reduction in cell viability was observed. Conclusion: The simultaneous targeting of KRAS and MYC demonstrates efficacy in gene modulation, thus in decreasing cell proliferation and viability.

Impacts of time-restricted feeding on middle-aged and old mice with obesity.

Time-restricted feeding is known to ameliorate obesity in young mice. However, evaluation of its effect in old age is still lacking. The current work aims to investigate the effects of time-restricted feeding on treating pre-existing obesity in old animals. The study utilized middle-aged and old high fat diet-induced obese mice and subjected them to 8h daily time-restricted feeding. Aged obese mice did not lose fat mass but lost lean mass after 8 weeks of treatment. In addition, time-restricted feeding reduced adiposity in brown adipose tissue, reversed excessive hepatic lipid accumulation, and improved glucose homeostasis in middle-aged and old obese mice. Mechanistic studies show that these metabolic benefits were mediated by transcriptional downregulation of essential genes responsible for hepatic adipogenesis and adipose tissue chronic inflammation. These results demonstrate that time-restricted feeding improves metabolic health and has beneficial effects in combating diet-induced obesity in aged obese mice. KEY POINTS: Contrary to in young obese mice, in old obese mice time-restricted feeding did not significantly reduce body fat but decreased lean mass. Time-restricted feeding reduced adipose tissue inflammation, reversed fatty liver, and improved glucose homeostasis in aged mice with diet-induced obesity. Time-restricted feeding is effective in improving metabolic homeostasis in aged mice, but less effective in terms of reducing obesity. Future studies should investigate the underlying mechanism of how ageing impaired intermittent fasting induced fat loss.

Seasonal environmental fluctuations alter the transcriptome dynamics of oocytes and granulosa cells in beef cows

BackgroundExamining the mechanistic cellular responses to heat stress could aid in addressing the increasing prevalence of decreased fertility due to elevated ambient temperatures. Here, we aimed to study the differential responses of oocytes and granulosa cells to thermal fluctuations due to seasonal differences. Dry beef cows (n = 10) were housed together, synchronized and subjected to a stimulation protocol to induce follicular growth before ovum pick-up (OPU). Two OPU’s were conducted (summer and winter) to collect cumulus-oocyte-complexes (COCs) and granulosa cells. In addition, rectal temperatures and circulating blood samples were collected during OPU. Oocytes were separated from the adherent cumulus cells, and granulosa cells were isolated from the collected OPU fluid. RNA was extracted from pools of oocytes and granulosa cells, followed by library preparation and RNA-sequencing. Blood samples were further processed for the isolation of plasma and leukocytes. The transcript abundance of HSP70 and HSP90 in leukocytes was evaluated using RT-qPCR, and plasma cortisol levels were evaluated by immunoassay. Environmental data were collected daily for three weeks before each OPU session. Data were analyzed using MIXED, Glimmix or GENMOD procedures of SAS, according to each variable distribution.ResultsAir temperatures (27.5 °C vs. 11.5 °C), average max air temperatures (33.7 °C vs. 16.9 °C), and temperature-humidity indexes, THI (79.16 vs. 53.39) were shown to contrast significantly comparing both the summer and winter seasons, respectively. Rectal temperatures (Summer: 39.2 ± 0.2 °C; Winter: 38.8 ± 0.2 °C) and leukocyte HSP70 transcript abundance (Summer: 4.18 ± 0.47 arbitrary units; Winter: 2.69 ± 0.66 arbitrary units) were shown to increase in the summer compared to the winter. No visual differences persisted in HSP90 transcript abundance in leukocytes and plasma cortisol concentrations during seasonal changes. Additionally, during the summer, 446 and 940 transcripts were up and downregulated in oocytes, while 1083 and 1126 transcripts were up and downregulated in the corresponding granulosa cells, respectively (Fold Change ≤ -2 or ≥ 2 and FDR ≤ 0.05). Downregulated transcripts in the oocytes were found to be involved in ECM-receptor interaction and focal adhesion pathways, while the upregulated transcripts were involved in protein digestion and absorption, ABC transporters, and oocyte meiosis pathways. Downregulated transcripts in the granulosa cells were shown to be involved in cell adhesion molecules, chemokine signaling, and cytokine-cytokine receptor interaction pathways, while those upregulated transcripts were involved in protein processing and metabolic pathways.ConclusionIn conclusion, seasonal changes dramatically alter the gene expression profiles of oocytes and granulosa cells in beef cows, which may in part explain the seasonal discrepancies in pregnancy success rates during diverging climatic weather conditions.

Developmental and Molecular Effects of C-Type Natriuretic Peptide Supplementation in In Vitro Culture of Bovine Embryos.

The use of C-type natriuretic peptide (CNP) in the interaction with the oocyte and in the temporary postponement of spontaneous meiosis resumption has already been well described. However, its action in pre-implantation developmental-stage embryos is yet to be understood. Thus, our study aimed to detect the presence of the canonical CNP receptor (natriuretic peptide receptor, NPR2) in germinal vesicle (GV)-, metaphase II (MII)-, presumptive zygote (PZ)-, morula (MO)-, and blastocyst (BL)-stage embryos and, later, to observe possible modulations on the embryos when co-cultured with CNP. In Experiment I, we detected and quantified NPR2 on the abovementioned embryo stages. Further, in Experiment II, we intended to test different concentrations (100, 200, or 400 nM of CNP) at different times of inclusion in the in vitro culture (IVC; inclusion from the beginning, i.e., day 1, or from day 5). In Experiment III, 400 nM of CNP was used on day 1 (D1) in the IVC, which was not demonstrated to be embryotoxic, and it showed potentially promising results in the blastocyst production rate when compared to the control. Thus, we analyzed the embryonic development rates of bovine embryos (D7) and hatching kinetics (D7, D8, and D9). Subsequently, morula and blastocyst were collected and evaluated for transcript abundance of their competence and quality (apoptosis, oxidative stress, proliferation, and differentiation) and lipid metabolism. Differences with probabilities less than p < 0.05, and/or fold change (FC) > 1.5, were considered significant. We demonstrate the presence of NPR2 until the blastocyst development stage, when there was a significant decrease in membrane receptors. There was no statistical difference in the production rate after co-culture with 400 nM CNP. However, when we evaluated the abundance of morula transcripts, there was an upregulated transcription in ADCY6 (p = 0.057) and downregulated transcripts in BMP15 (p = 0.013), ACAT1 (p = 0.040), and CASP3 (p = 0.082). In addition, there was a total of 12 transcriptions in morula that presented variation FC > 1.5. In blastocysts, the treatment with CNP induced upregulation in BID, CASP3, SOX2, and HSPA5 transcripts and downregulation in BDNF, NLRP5, ELOVL1, ELOVL4, IGFBP4, and FDX1 transcripts (FC > 1.5). Thus, our study identified and quantified the presence of NPR2 in bovine pre-implantation embryos. Furthermore, 400 nM of CNP in IVC, a concentration not previously described in the literature, modulated some transcripts related to embryonic metabolism, and this was not embryotoxic morphologically.

Second messenger c-di-AMP regulates multiple antibiotic sensitivity pathways in Mycobacterium smegmatis by discrete mechanisms.

One of the debilitating causes of high mortality in the case of tuberculosis and other bacterial infections is the resistance development against standard drugs. There are limited studies so far to describe how a bacterial second messenger molecule can directly participate in distinctive antibiotic tolerance characteristics of a cell in a mechanism-dependent manner. Here we show that intracellular cyclic di-AMP (c-di-AMP) concentration can modulate drug sensitivity of Mycobacterium smegmatis by interacting with an effector protein or interfering with the 5'-UTR regions in mRNA of the genes and thus causing transcriptional downregulation of important genes in the pathways. We studied four antibiotics with different mechanisms of action: rifampicin, ciprofloxacin, erythromycin, and tobramycin and subsequently found that the level of drug sensitivity of the bacteria is directly proportional to the c-di-AMP concentration inside the cell. Further, we unraveled the underlying molecular mechanisms to delineate the specific genes and pathways regulated by c-di-AMP and hence result in differential drug sensitivity in M. smegmatis.

9250 carRNA m6A methylation regulates transcription state and chromatin accessibility in human islets in normal physiology and type 2 diabetes

Abstract Disclosure: N. Shukla: None. C. Ju: None. H. Li: None. C. He: None. R. Kulkarni: Advisory Board Member; Self; Novo Nordisk, Biomea Fusion Inc, REDD Pharma. Grant Recipient; Self; Inversago. Chromatin-associated regulatory RNAs (carRNA), which include promoter- and enhancer-associated RNAs as well as several non-coding repeat elements have been established in the literature as important regulators of global gene transcription. However, the regulation of carRNAs themselves has been an enigma. Recent publications from the He Lab at UChicago have shown the role of RNA N6-methyladenosine (m6A) in regulating carRNA stability and function. m6A is among the most abundant mRNA modifications in mammals. Seminal work from our lab has reported that the “writers” of m6A (e.g. methyltransferase-like [METTL3] and METTL14) are significantly downregulated in islets of humans with type 2 diabetes (T2D). Consequently, the global m6A landscape in the islets segregates the T2D from the control group significantly better than the transcriptome (n=7 for Control and n=8 for T2D islets). Hypomethylation of key transcripts in the insulin/IGF-1-AKT-PDX1 pathway led to impaired insulin secretion and decreased β-cell proliferation. We now show this decreased m6A on RNA is not limited to the mRNA but also affects chromatin-associated regulatory RNAs (carRNA). Intriguingly, downregulation of METTL3 and METTL14 in human β-cell line EndoC-βH1 differentially regulates these regulatory versus protein-coding transcripts. We observe that while METTL14 KD causes global downregulation of transcripts belonging to pathways important for β-cell identity and function (including calcium signaling, PI3K-AKT/MAPK signaling pathway, pancreatic secretion etc.), it leads to an upregulation of pathways involved in RNA processing and global gene transcription (Transcription by RNA Pol II, metabolism and splicing of mRNA, chromatin organization and modification etc.) {false discovery rate (FDR) &amp;lt; 0.05}. Considering carRNAs have been previously reported to regulate global chromatin accessibility and transcription, our observations on their ability to get differentially regulated m6A writer proteins provide novel insights into the epigenetic regulation of human islet cells. These findings provide opportunities to develop therapeutic approaches to regulate β-cell identity and/or insulin secretion in T2D. Presentation: 6/3/2024