Expanding RNAi to Kidneys, Lungs, and Spleen via Selective ORgan Targeting (SORT) siRNA Lipid Nanoparticles.

Inhibition of disease-causing mutations using RNA interference (RNAi) has resulted in clinically approved medicines with additional candidates in late stage trials. However, targetable tissues currently in preclinical development are limited to liver following systemic intravenous (IV) administration because predictable delivery of siRNA to non-liver tissues remains an unsolved challenge. Here, evidence of durable extrahepatic gene silencing enabled by siRNA Selective ORgan Targeting lipid nanoparticles (siRNA SORT LNPs) to the kidneys, lungs, and spleen is provided. LNPs excel at dose-dependent silencing of tissue-enriched endogenous targets resulting in 60%-80% maximal knockdown after a single IV injection and up to 88% downregulation of protein expression in mouse lungs after two doses. To examine knockdown potency and unbiased organ targeting, B6.129TdTom/EGFP mice that constitutively express the TdTomato transgene across all cell types are utilized to demonstrate 58%, 45%, and 15% reduction in TdTomato fluorescence in lungs, spleen, and kidneys, respectively. Finally, physiological relevance of siRNA SORT LNP-mediated gene silencing is established via acute suppression of endogenous Tie2 which induces lung-specific phenotypic alteration of vascular endothelial barrier. Due to plethora of extrahepatic diseases that may benefit from RNAi interventions, it is anticipated that the findings will expand preclinical landscape of therapeutic targets beyond the liver.

BASELINE SUSCEPTABILITY AND FIRST OBSERVATION OF KNOCKDOWN RESISTANCE IN AEDES TAENIORHYNCHUS POPULATION OF ST. JOHNS COUNTY, FLORIDA.

The black saltmarsh mosquito, Aedes taeniorhynchus, is a prominent nuisance mosquito within St. Johns County, Florida. Due to their characteristically large outbreaks, and the elevated amount of insecticide application correlated with the outbreaks, local populations of Ae. taeniorhynchus are at an increased risk of developing insecticide resistance. This study was established to form a baseline susceptibility of Ae. taeniorhynchus against two technical grade materials, permethrin, and chlorpyrifos. Centers for Disease Control and Prevention bottle bioassays were conducted with technical-grade materials during two outbreaks in the fall of 2023. Results indicated a baseline susceptibility against the materials tested, but most notably, the phenotypic expression of knockdown resistance (kdr) was observed. Results highlight the need for continued monitoring and investigation into the resistance status and resistance level of this common Florida species.

Knockdown of type 2 orexin receptor in adult mouse testis potentiates testosterone production and germ cell proliferation.

Orexins (OXs) are neuropeptides which regulate various physiological processes. OXs exist in two different forms, mainly orexin A (OXA) and orexin B (OXB) and their effects are mediated via OX1R and OX2R. Presence of OXB and OX2R in mouse testis is also reported. However, the role of OXB/OX2R in the male gonad remains unexplored. Herein we investigated the role of OXB/OX2R system in testicular physiology under in vivo and ex vivo conditions. Adult mice were given a single dose of bilateral intratesticular injection of siRNA targeting OX2R and were sacrificed 96 h post-injection. OX2R-knockdown potentiated serum and intratesticular testosterone levels with up-regulation in the expressions of major steroidogenic proteins. Germ cell proliferation also increased in siRNA-treated mice. Results of the ex vivo experiment also supported the findings of the in vivo study. In conclusion, OX2R may regulate testosterone production and thereby control the fine-tuning between steroidogenesis and germ cell dynamics.

Knockdown of ATRX enhances radiosensitivity in glioblastoma.

BACKGROUND: Glioblastoma are highly malignant type of primary brain tumors. Treatment for glioblastoma multiforme (GBM) generally involves surgery combined with chemotherapy and radiotherapy. However, the development of tumoral chemo- and radioresistance induces complexities in clinical practice. Multiple signaling pathways are known to be involved in radiation-induced cell survival. However, the role of alpha-thalassemia X-linked mutant retardation syndrome (ATRX), a chromatin remodeling protein, in GBM radioresistance remains unclear. METHODS: In the present study, the ATRX mutation rate in patients with glioma was obtained from The Cancer Genome Atlas, while its expression analyzed using bioinformatics. Datasets were also obtained from the Gene Expression Omnibus, and ATRX expression levels following irradiation of GBM were determined. The effects of ATRX on radiosensitivity were investigated using a knockdown assays. RESULTS: The present study demonstrated that the ATRX mutation rate in patients with GBM was significantly lower than that in patients with low-grade glioma, and that patients harboring an ATRX mutation exhibited a prolonged survival, compared with to those harboring the wild-type gene. Single-cell RNA sequencing demonstrated that ATRX counts increased 2 days after irradiation, with ATRX expression levels also increasing in U-251MG radioresistant cells. Moreover, the results of in vitro irradiation assays revealed that ATRX expression was increased in U-251MG cells, while ATRX knockdown was associated with increased levels of radiosensitivity. CONCLUSIONS: High ATRX expression levels in primary GBM may contribute to high levels of radioresistance. Thus ATRX is a potential target for overcoming the radioresistance in GBM.

Molecular cloning, identification, transcriptional analysis, and silencing of enolase on the life cycle of Haemaphysalis longicornis (Acari, Ixodidae) tick.

Ticks, blood-sucking ectoparasites, spread diseases to humans and animals. Haemaphysalis longicornis is a significant vector for tick-borne diseases in medical and veterinary contexts. Identifying protective antigens in H. longicornis for an anti-tick vaccine is a key tick control strategy. Enolase, a multifunctional protein, significantly converts D-2-phosphoglycerate and phosphoenolpyruvate in glycolysis and gluconeogenesis in cell cytoplasm. This study cloned a complete open reading frame (ORF) of enolase from the H. longicornis tick and characterized its transcriptional and silencing effect. We amplified the full-length cDNA of the enolase gene using rapid amplification of cDNA ends. The complete cDNA, with an ORF of 1,297 nucleotides, encoded a 432-amino acid polypeptide. Enolase of the Jeju strain H. longicornis exhibited the highest sequence similarity with H. flava (98%), followed by Dermacentor silvarum (82%). The enolase motifs identified included N-terminal and C-terminal regions, magnesium binding sites, and several phosphorylation sites. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that enolase mRNA transcripts were expressed across all developmental stages of ticks and organs such as salivary gland and midgut. RT-PCR showed higher transcript levels in syn-ganglia, suggesting that synganglion nerves influence enolase,s role in tick salivary glands. We injected enolase double-stranded RNA into adult unfed female ticks, after which they were subsequently fed with normal unfed males until they spontaneously dropped off. RNA interference significantly (P<0.05) reduced feeding and reproduction, along with abnormalities in eggs (no embryos) and hatching. These findings suggest enolase is a promising target for future tick control strategies.

Species identification and pyrethroid resistance genotyping of recently resurgent Cimex lectularius and Cimex hemipterus in Korea.

The global resurgence of bed bug infestations, exacerbated by increasing international travel, trade, and insecticide resistance, has significantly impacted Korea. This study identified the bed bug species and performed pyrethroid resistance genotyping of recently resurgent bed bugs in Korea. Thirty-one regional bed bug samples were collected from 5 administrative regions: Gyeonggi-do (n=14), Seoul (n=13), Busan (n=2), Jeonllanam-do (n=1), and Chungcheongbuk-do (n=1). The samples underwent morphological and molecular identification. Twenty-four regional samples (77.4%) were identified as the tropical bed bug, Cimex hemipterus, and the remaining 7 regional samples (22.6%) were identified as the common bed bug, Cimex lectularius. The C. hemipterus regional samples carried at least three mutations associated with knockdown resistance (kdr), including 2 super-kdr mutations. The 7 C. lectularius regional samples possessed at least one of the 3 kdr-related mutations associated with pyrethroid resistance. This study confirms that the prevalent bed bug species recently in Korea is C. hemipterus, replacing the previously endemic C. lectularius. Additionally, the rise in bed bug populations with pyrethroid resistance underscores the necessity of introducing alternative insecticides.

Post-GWAS functional analyses of CNTNAP5 suggests its role in glaucomatous neurodegeneration.

Primary angle closure glaucoma (PACG) affects more than 20 million people worldwide, with an increased prevalence in south-east Asia. In a prior haplotype-based GWAS, we identified a novel CNTNAP5 genic region, significantly associated with PACG. In the current study, we have extended our perception of CNTNAP5 involvement in glaucomatous neurodegeneration in a zebrafish model, through investigating phenotypic consequences pertinent to retinal degeneration upon knockdown of cntnap5 by translation-blocking morpholinos. While cntnap5 knockdown was successfully validated using an antibody, immunofluorescence followed by western blot analyses in cntnap5-morphant (MO) zebrafish revealed increased expression of acetylated tubulin indicative of perturbed cytoarchitecture of retinal layers. Moreover, significant loss of Nissl substance is observed in the neuro-retinal layers of cntnap5-MO zebrafish eye, indicating neurodegeneration. Additionally, in spontaneous movement behavioural analysis, cntnap5-MO zebrafish have a significantly lower average distance traversed in light phase compared to mismatch-controls, whereas no significant difference was observed in the dark phase, corroborating with vision loss in the cntnap5-MO zebrafish. This study provides the first direct functional evidence of a putative role of CNTNAP5 in visual neurodegeneration.

Anticipating change: The impact of simulated seasonal heterogeneity on heat tolerances along a latitudinal cline.

An understanding of thermal limits and variation across geographic regions is central to predicting how any population may respond to global change. Latitudinal clines, in particular, have been used to demonstrate that populations can be locally adapted to their own thermal environment and, as a result, not all populations will be equally impacted by an increase in temperature. But how robust are these signals of thermal adaptation to the other ecological challenges that animals commonly face in the wild? Seasonal changes in population density, food availability, or photoperiod are common ecological challenges that could disrupt patterns of thermal tolerance along a cline if each population differentially used these signals to anticipate future temperatures and adjust their thermal tolerances accordingly. In this study, we aimed to test the robustness of a cline in thermal tolerance to simulated signals of seasonal heterogeneity. Experimental animals were derived from clones of the Australian water flea, Daphnia carinata, sampled from nine distinct populations along a latitudinal transect in Eastern Australia. We then factorially combined summer (18 h light, 6 h dark) and winter (6 h light, 18 h dark) photoperiods with high (5 million algal cells individual-1 day-1) and low (1 million algal cells individual-1 day-1) food availabilities, before performing static heat shock assays to measure thermal tolerance. We found that the thermal tolerances of the clonal populations were sensitive to both measures of seasonal change. In general, higher food availability led to an increase in thermal tolerances, with the magnitude of the increase varying by clone. In contrast, a switch in photoperiod led to rank-order changes in thermal tolerances, with heat resistance increasing for some clones, and decreasing for others. Heat resistance, however, still declined with increasing latitude, irrespective of the manipulation of seasonal signals, with clones from northern populations always showing greater thermal resistance, most likely driven by adaptation to winter thermal conditions. While photoperiod and food availability can clearly shape thermal tolerances for specific populations, they are unlikely to overwhelm overarching signals of thermal adaptation, and thus, observed clines in heat resistance will likely have remained robust to these forms of seasonal heterogeneity.

Development of a rapamycin-inducible protein-knockdown system in the unicellular red alga Cyanidioschyzon merolae.

An inducible protein-knockdown system is highly effective for investigating the functions of proteins and mechanisms essential for the survival and growth of organisms. However, this technique is not available in photosynthetic eukaryotes. The unicellular red alga Cyanidioschyzon merolae possesses a very simple cellular and genomic architecture and is genetically tractable but lacks RNA interference machinery. In this study, we developed a protein-knockdown system in this alga. The constitutive system utilizes the destabilizing activity of the FRB domain of human target of rapamycin (TOR) kinase or its derivatives to knock down target proteins. In the inducible system, rapamycin treatment induces the heterodimerization of the human FKBP12-rapamycin binding (FRB) domain fused to the target proteins with the human FK506-binding protein 12 (FKBP) fused to S-phase kinase associated protein 1 (SKP1) or Cullin 1 (CUL1), subunits of the SCF E3 ubiquitin ligase. This results in the rapid degradation of the target proteins through the ubiquitin-proteasome pathway. With this system, we successfully degraded endogenous essential proteins such as the chloroplast division protein Dynamin related protein 5B (DRP5B) and E2 transcription factor (E2F), a regulator of the G1/S transition, within 2-3 hours after rapamycin administration, enabling the assessment of resulting phenotypes. This rapamycin-inducible protein-knockdown system contributes to the functional analysis of genes whose disruption leads to lethality.

Effect of Silencing subolesin and enolase impairs gene expression, engorgement and reproduction in Haemaphysalis longicornis (Acari: Ixodidae) ticks.

IMPORTANCE: Haemaphysalis longicornis is an obligate blood-sucking ectoparasite that has gained attention due its role of transmitting medically and veterinary significant pathogens and it is the most common tick species in Republic of Korea. The preferred strategy for controlling ticks is a multi-antigenic vaccination. Testing the efficiency of a combination antigen is a promising method for creating a tick vaccine. OBJECTIVE: The aim of the current research was to analyze the role of subolesin and enolase in feeding and reproduction of H. longicornis by gene silencing. METHODS: In this study, we used RNA interference to silence salivary enolase and subolesin in H. longicornis. Unfed female ticks injected with double-stranded RNA targeting subolesin and enolase were attached and fed normally on the rabbit's ear. Real-time polymerase chain reaction was used to confirm the extent of knockdown. RESULTS: Ticks in the subolesin or enolase dsRNA groups showed knockdown rates of 80% and 60% respectively. Ticks in the combination dsRNA (subolesin and enolase) group showed an 80% knockdown. Knockdown of subolesin and enolase resulted in significant depletion in feeding, blood engorgement weight, attachment rate, and egg laying. Silencing of both resulted in a significant (p < 0.05) reduction in tick engorgement, egg laying, egg hatching (15%), and reproduction. CONCLUSIONS AND RELEVANCE: Our results suggest that subolesin and enolase are an exciting target for future tick control strategies.

RNAi-mediated pest control targeting the Troponin I (wupA) gene in sweet potato weevil, Cylas formicarius.

The sweet potato weevil (Cylas formicarius) is a critical pest producing enormous global losses in sweet potato crops. Traditional pest management approaches for sweet potato weevil, primarily using chemical pesticides, causes pollution, food safety issues, and harming natural enemies. While RNA interference (RNAi) is a promising environmentally friendly approach to pest control, its efficacy in controlling the sweet potato weevil has not been extensively studied. In this study, we selected a potential target for controlling C. formicarius, the Troponin I gene (wupA), which is essential for musculature composition and crucial for fundamental life activities. We determined that wupA is abundantly expressed throughout all developmental stages of the sweet potato weevil. We evaluated the efficiency of double-stranded RNAs in silencing the wupA gene via microinjection and oral feeding of sweet potato weevil larvae at different ages. Our findings demonstrate that both approaches significantly reduced the expression of wupA and produced high mortality. Moreover, the 1st instar larvae administered dswupA exhibited significant growth inhibition. We assessed the toxicity of dswupA on the no-target insect silkworm and assessed its safety. Our study indicates that wupA knockdown can inhibit the growth and development of C. formicarius and offer a potential target gene for environmentally friendly control.

Long non-coding RNA TUG1 is downregulated in Friedreich's ataxia.

Friedreich's ataxia is a neurodegenerative disorder caused by reduced frataxin levels. It leads to motor and sensory impairments and has a median life expectancy of around 35 years. As the most common inherited form of ataxia, Friedreich's ataxia lacks reliable, non-invasive biomarkers, prolonging and inflating the cost of clinical trials. This study proposes TUG1, a long non-coding RNA, as a promising blood-based biomarker for Friedreich's ataxia, which is known to regulate various cellular processes. In a previous study using a frataxin knockdown mouse model, we observed several hallmark Friedreich's ataxia symptoms. Building on this, we hypothesized that a dual-source approach-comparing the data from peripheral blood samples from Friedreich's ataxia patients with tissue samples from affected areas in Friedreich's ataxia knockdown mice, tissues usually unattainable from patients-would effectively identify robust biomarkers. A comprehensive reanalysis was conducted on gene expression data from 183 age- and sex-matched peripheral blood samples of Friedreich's ataxia patients, carriers and controls and 192 tissue data sets from Friedreich's ataxia knockdown mice. Blood and tissue samples underwent RNA isolation and quantitative reverse transcription polymerase chain reaction, and frataxin knockdown was confirmed through enzyme-linked immunosorbent assays. Tug1 RNA interaction was explored via RNA pull-down assays. Validation was performed in serum samples on an independent set of 45 controls and 45 Friedreich's ataxia patients and in blood samples from 66 heterozygous carriers and 72 Friedreich's ataxia patients. Tug1 and Slc40a1 emerged as potential blood-based biomarkers, confirmed in the Friedreich's ataxia knockdown mouse model (one-way ANOVA, P ≤ 0.05). Tug1 was consistently downregulated after Fxn knockdown and correlated strongly with Fxn levels (R 2 = 0.71 during depletion, R 2 = 0.74 during rescue). Slc40a1 showed a similar but tissue-specific pattern. Further validation of Tug1's downstream targets strengthened its biomarker candidacy. In additional human samples, TUG1 levels were significantly downregulated in both whole blood and serum of Friedreich's ataxia patients compared with controls (Wilcoxon signed-rank test, P < 0.05). Regression analyses revealed a negative correlation between TUG1 fold-change and disease onset (P < 0.0037) and positive correlations with disease duration and functional disability stage score (P < 0.04). This suggests that elevated TUG1 levels correlate with earlier onset and more severe cases. This study identifies TUG1 as a potential blood-based biomarker for Friedreich's ataxia, showing consistent expression variance in human and mouse tissues related to disease severity and key Friedreich's ataxia pathways. It correlates with frataxin levels, indicating its promise as an early, non-invasive marker. TUG1 holds potential for Friedreich's ataxia monitoring and therapeutic development, meriting additional research.

Acclimation to warmer temperatures can protect host populations from both further heat stress and the potential invasion of pathogens.

Thermal acclimation can provide an essential buffer against heat stress for host populations, while acting simultaneously on various life-history traits that determine population growth. In turn, the ability of a pathogen to invade a host population is intimately linked to these changes via the supply of new susceptible hosts, as well as the impact of warming on its immediate infection dynamics. Acclimation therefore has consequences for hosts and pathogens that extend beyond simply coping with heat stress-governing both population growth trajectories and, as a result, an inherent propensity for a disease outbreak to occur. The impact of thermal acclimation on heat tolerances, however, is rarely considered simultaneously with metrics of both host and pathogen population growth, and ultimately fitness. Using the host Daphnia magna and its bacterial pathogen, we investigated how thermal acclimation impacts host and pathogen performance at both the individual and population scales. We first tested the effect of maternal and direct thermal acclimation on the life-history traits of infected and uninfected individuals, such as heat tolerance, fecundity, and lifespan, as well as pathogen infection success and spore production. We then predicted the effects of each acclimation treatment on rates of host and pathogen population increase by deriving a host's intrinsic growth rate (rm) and a pathogen's basic reproductive number (R0). We found that direct acclimation to warming enhanced a host's heat tolerance and rate of population growth, despite a decline in life-history traits such as lifetime fecundity and lifespan. In contrast, pathogen performance was consistently worse under warming, with within-host pathogen success, and ultimately the potential for disease spread, severely hampered at higher temperatures. Our results suggest that hosts could benefit more from warming than their pathogens, but only by linking multiple individual traits to population processes can the full impact of higher temperatures on host and pathogen population dynamics be realised.

Endoplasmic reticulum stress-dependent regulation of the expression of serine hydroxymethyltransferase 2 in glioblastoma cells.

Objective. Serine hydroxymethyltransferase (SHMT2) plays a multifunctional role in mitochondria (folate-dependent tRNA methylation, translation, and thymidylate synthesis). The endoplasmic reticulum stress, hypoxia, and glucose and glutamine supply are significant factors of malignant tumor growth including glioblastoma. Previous studies have shown that the knockdown of the endoplasmic reticulum to nucleus signaling 1 (ERN1) pathway of endoplasmic reticulum stress strongly suppressed glioblastoma cell proliferation and modified the sensitivity of these cells to hypoxia and glucose or glutamine deprivations. The present study aimed to investigate the regulation of the SHMT2 gene in U87MG glioblastoma cells by ERN1 knockdown, hypoxia, and glucose or glutamine deprivations with the intent to reveal the role of ERN1 signaling in sensitivity of this gene expression to hypoxia and nutrient supply. Methods. The control U87MG glioblastoma cells (transfected by an empty vector) and ERN1 knockdown cells with inhibited ERN1 endoribonuclease and protein kinase (dnERN1) or only ERN1 endoribonuclease (dnrERN1) were used. Hypoxia was introduced by dimethyloxalylglycine (500 ng/ml for 4 h). For glucose and glutamine deprivations, cells were exposed in DMEM without glucose and glutamine, respectively for 16 h. RNA was extracted from cells and reverse transcribed. The expression level of the SHMT2 gene was studied by real-time qPCR and normalized to ACTB. Results. It was found that inhibition of ERN1 endoribonuclease and protein kinase in glioblastoma cells led to a down-regulation of SHMT2 gene expression in U87MG cells. At the same time, the expression of this gene did not significantly change in cells with inhibited ERN1 endoribonuclease, but tunicamycin strongly increased its expression. Moreover, the expression of the SHMT2 gene was not affected in U87MG cells after silencing of XBP1. Hypoxia up-regulated the expression level of the SHMT2 gene in both control and ERN1 knockdown U87MG cells. The expression of this gene was significantly up-regulated in glioblastoma cells under glucose and glutamine deprivations and ERN1 knockdown significantly increased the sensitivity of the SHMT2 gene to these nutrient deprivation conditions. Conclusion. The results of the present study demonstrate that the expression of the SHMT2 gene responsible for serine metabolism and formation of folate one-carbon is controlled by ERN1 protein kinase and induced by hypoxia as well as glutamine and glucose deprivation conditions in glioblastoma cells and reflects the ERN1-mediated reprogramming of sensitivity this gene expression to nutrient deprivation.

The role of Sirtuin 1 in regulation of fibrotic genes expression in pre-adipocytes.

Purpose: Considering inhibition of pre-adipocyte cells differentiation in adipose tissue fibrosis, we aimed to explore whether Sirt1 and Hif-1α in pre-adipocytes have a significant effect on fibrotic gene expression. Methods: 3T3-L1 pre-adipocytes were transfected with SIRT1-specific siRNA, confirmed by real-time polymerase chain reaction (RT-PCR) and western blotting. Additionally, cells were treated with varying concentrations of resveratrol and sirtinol as the activator and inhibitor of Sirt1, respectively. Involvement of Hif-1α was evaluated by treatment with echinomycin. Subsequently, we assessed the gene and protein expressions related to fibrosis in the extracellular matrix of adipose tissue, including collagen VI (Col VI), lysyl oxidase (Lox), matrix metalloproteinase-2 (Mmp-2), Mmp-9, and osteopontin (Opn) in pre-adipocytes through RT-PCR and western blot. Results: The current study demonstrated that Sirt1 knockdown and reduced enzyme activity significantly increased the expression of Col VI, Lox, Mmp-2, Mmp-9, and Opn genes in the treated 3T3-L1 cells compared to the control group. Interestingly, resveratrol significantly decreased the gene expression related to the fibrosis pathway. Inhibition of Hif-1α by echinomycin led to a significant reduction in Col VI, Mmp-2, and Mmp-9 gene expression in the treated group compared to the control. Conclusion: This study highlights that down-regulation of Sirt1 might be a predisposing factor in the emergence of adipose tissue fibrosis by enhancing the expression of extracellular matrix (ECM) components. Activation of Sirt1, similar to suppressing of Hif-1α in pre-adipocytes may be a beneficial approach for attenuating fibrotic gene expression. Supplementary Information: The online version contains supplementary material available at 10.1007/s40200-024-01389-4.

Characterization of bovine long non-coding RNAs, OOSNCR1, OOSNCR2 and OOSNCR3, and their roles in oocyte maturation and early embryonic development.

In mammals, early embryogenesis relies heavily on the regulation of maternal transcripts including protein-coding and non-coding RNAs stored in oocytes. In this study, the expression of three bovine oocyte expressed long non-coding RNAs (lncRNAs), OOSNCR1, OOSNCR2, and OOSNCR3, was characterized in somatic tissues, the ovarian follicle, and throughout early embryonic development. Moreover, the functional requirement of each transcript during oocyte maturation and early embryonic development was investigated using a siRNA-mediated knockdown approach. Tissue distribution analysis revealed that OOSNCR1, OOSNCR2 and OOSNCR3 are predominantly expressed in fetal ovaries. Follicular cell expression analysis revealed that these lncRNAs are highly expressed in the oocytes, with minor expression detected in the cumulus cells (CCs) and mural granulosa cells (mGCs). The expression for all three genes was highest during oocyte maturation, decreased at fertilization, and ceased altogether by the 16-cell stage. Knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 in immature oocytes was achieved by microinjection of the cumulus-enclosed germinal vesicle (GV) oocytes with siRNAs targeting these lncRNAs. Knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 did not affect cumulus expansion, but oocyte survival at 12 h post-insemination was significantly reduced. In addition, knockdown of OOSNCR1, OOSNCR2 and OOSNCR3 in immature oocytes resulted in a decreased rate of blastocyst development, and reduced expression of genes associated with oocyte competency such as nucleoplasmin 2 (NPM2), growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), and JY-1 in MII oocytes. The data herein suggest a functional requirement of OOSNCR1, OOSNCR2, and OOSNCR3 during bovine oocyte maturation and early embryogenesis.

Poor Decision Making and Sociability Impairment Following Central Serotonin Reduction in Inducible TPH2-Knockdown Rats.

Serotonin is an essential neuromodulator for mental health and animals' socio-cognitive abilities. However, we previously found that a constitutive depletion of central serotonin did not impair rat cognitive abilities in stand-alone tests. Here, we investigated how a mild and acute decrease in brain serotonin would affect rats' cognitive abilities. Using a novel rat model of inducible serotonin depletion via the genetic knockdown of tryptophan hydroxylase 2 (TPH2), we achieved a 20% decrease in serotonin levels in the hypothalamus after three weeks of non-invasive oral doxycycline administration. Decision making, cognitive flexibility, and social recognition memory were tested in low-serotonin (Tph2-kd) and control rats. Our results showed that the Tph2-kd rats were more prone to choose disadvantageously in the long term (poor decision making) in the Rat Gambling Task and that only the low-serotonin poor decision makers were more sensitive to probabilistic discounting and had poorer social recognition memory than other low-serotonin and control individuals. Flexibility was unaffected by the acute brain serotonin reduction. Poor social recognition memory was the most central characteristic of the behavioral network of low-serotonin poor decision makers, suggesting a key role of social recognition in the expression of their profile. The acute decrease in brain serotonin appeared to specifically amplify the cognitive impairments of the subgroup of individuals also identified as poor decision makers in the population. This study highlights the great opportunity the Tph2-kd rat model offers to study inter-individual susceptibilities to develop cognitive impairment following mild variations of brain serotonin in otherwise healthy individuals. These transgenic and differential approaches together could be critical for the identification of translational markers and vulnerabilities in the development of mental disorders.

Quercetin Induces Mitochondrial Apoptosis and Downregulates Ganglioside GD3 Expression in Melanoma Cells.

Malignant melanoma represents a form of skin cancer characterized by a bleak prognosis and heightened resistance to traditional therapies. Quercetin has demonstrated notable anti-carcinogenic, anti-inflammatory, anti-oxidant, and pharmacological effects across various cancer types. However, the intricate relationship between quercetin's anti-cancer properties and ganglioside expression in melanoma remains incompletely understood. In this study, quercetin manifests specific anti-proliferative, anti-migratory, and cell-cycle arrest effects, inducing mitochondrial dysfunction and apoptosis in two melanoma cancer cell lines. This positions quercetin as a promising candidate for treating malignant melanoma. Moreover, our investigation indicates that quercetin significantly reduces the expression levels of ganglioside GD3 and its synthetic enzyme. Notably, this reduction is achieved through the inhibition of the FAK/paxillin/Akt signaling pathway, which plays a crucial role in cancer development. Taken together, our findings suggest that quercetin may be a potent anti-cancer drug candidate for the treatment of malignant melanoma.

Knockdown of SCN5A alters metabolic-associated genes and aggravates hypertrophy in the cardiomyoblast.

SCN5A mutations have been reported to cause various cardiomyopathies in humans. Most of the SCN5A mutations causes loss of function and thereby, alters the overall cellular function. Therefore, to understand the loss of SCN5A function in cardiomyocytes, we have knocked down the SCN5A gene (SCN5A-KD) in H9c2 cells and explored the cell phenotype and molecular behaviors in the presence and absence of isoproterenol (ISO), an adrenergic receptor agonist that induces cardiac hypertrophy. Expression of several genes related to hypertrophy, inflammation, fibrosis, and energy metabolism pathways were evaluated. It was found that the mRNA expression of hypertrophy-related gene, brain (B-type) natriuretic peptide (BNP) was significantly increased in SCN5A-KD cells as compared to 'control' H9c2 cells. There was a further increase in the mRNA expressions of BNP and ßMHC in SCN5A-KD cells after ISO treatment compared to their respective controls. Pro-inflammatory cytokine, tumor necrosis factor-alpha expression was significantly increased in 'SCN5A-KD' H9c2 cells. Further, metabolism-related genes like glucose transporter type 4, cluster of differentiation 36, peroxisome proliferator-activated receptor alpha, and peroxisome proliferator-activated receptor-gamma were significantly elevated in the SCN5A-KD cells as compared to the control cells. Upregulation of these metabolic genes is associated with increased ATP production. The study revealed that SCN5A knock-down causes alteration of gene expression related to cardiac hypertrophy, inflammation, and energy metabolism pathways, which may promote cardiac remodelling and cardiomyopathy.

Regulatory impacts of PPARGC1A gene expression on milk production and cellular metabolism in buffalo mammary epithelial cells.

The PPARGC1A gene plays a fundamental role in regulating cellular energy metabolism, including adaptive thermogenesis, mitochondrial biogenesis, adipogenesis, gluconeogenesis, and glucose/fatty acid metabolism. In a previous study, our group investigated seven SNPs in Mediterranean buffalo associated with milk production traits, and the current study builds on this research by exploring the regulatory influences of the PPARGC1A gene in buffalo mammary epithelial cells (BuMECs). Our findings revealed that knockdown of PPARGC1A gene expression significantly affected the growth of BuMECs, including proliferation, cell cycle, and apoptosis. Additionally, we observed downregulated triglyceride secretion after PPARGC1A knockdown. Furthermore, the critical genes related to milk production, including the STATS, BAD, P53, SREBF1, and XDH genes were upregulated after RNAi, while the FABP3 gene, was downregulated. Moreover, Silencing the PPARGC1A gene led to a significant downregulation of ß-casein synthesis in BuMECs. Our study provides evidence of the importance of the PPARGC1A gene in regulating cell growth, lipid, and protein metabolism in the buffalo mammary gland. In light of our previous research, the current study underscores the potential of this gene for improving milk production efficiency and overall dairy productivity in buffalo populations.