Dose-dependent Increase Research Articles

Impact of fluoxetine exposure on Lymnaea stagnalis and its developing eggs: integrating untargeted lipidomics, targeted metabolomics, and classical risk assessment

Pharmaceuticals such as selective serotonin reuptake inhibitors (SSRIs), are increasingly detected in aquatic environments, posing potential risks to non-target organisms, because many of those substances are widely shared neuromodulator. In this study, we investigated the effects of SSRI antidepressant, namely, fluoxetine, exposure on the freshwater snail L. stagnalis, focusing on egg development, neurochemical pathways, and lipid metabolism. Snails were exposed to a range of 51–434 µg fluoxetine L⁻1 for 7 days, followed by analysis of survival, feeding behaviour, reproduction, and metabolomic changes in the central nervous system (CNS), albumen gland, and eggs. Although no significant effects were observed on survival or fecundity, fluoxetine exposure significantly impaired egg development in a dose-dependent manner, reducing hatching rates with an EC50 of 126 µg fluoxetine L⁻1. Removal of eggs from the contaminated environment partially reversed these developmental effects, suggesting potential recovery if fluoxetine levels decrease. Molecular analysis revealed several neurochemical and lipidomic alterations. In the CNS, elevated levels of catecholamines, phosphatidylcholines (PC), and ceramides were linked to disruptions in neurotransmission, membrane integrity, and impaired embryo development. In the albumen gland, we detected a decrease of key lipid classes, including sphingomyelins and fatty acids, which can be linked with impaired egg quality. Additionally, a decrease in histamine in both the albumen gland and eggs suggested further disruption of egg development, potentially affecting metamorphosis success. Moreover, the dose-dependent increase in choline, along with PC and oxidized PC, indicated oxidative stress and lipid peroxidation in the CNS and exposed eggs of Lymnaea stagnalis. Our findings highlight the benefits of combining behavioral assessments with metabolomic profiling to better understand the mechanistic pathways underlying fluoxetine’s adverse effects.

Exposure to polystyrene nanoplastics induces lysosomal enlargement and lipid droplet accumulation in KGN human ovarian granulosa cells.

Given the ubiquitous presence of plastic products in daily life, human exposure to nanoplastics (NPs) is inevitable. Previous studies have suggested that exposure to polystyrene nanoplastics (PSNPs) may contribute to reproductive disorders; however, the underlying mechanism remains elusive. The goal of this study was to investigate the impact of PSNPs on KGN human ovarian granulosa cells. KGN cells were exposed to varying concentrations of PSNPs (0-400 μg/mL) for 48 h; alterations in cell survival and morphology were assessed to elucidate potential toxic effects. PSNPs were shown to enter KGN cells. Exposure to PSNPs did not induce significant changes in cytotoxicity, Calcein intensity, or active mitochondria levels in KGN cells. However, PSNP exposure did induce a dose-dependent increase in cytoplasmic vacuoles and an increase in total lysosome area and in the numbers of lipid droplets in KGN cells. Our findings provide compelling evidence that PSNPs can penetrate cell cytoplasm and induce toxicity, resulting in an elevation in the numbers of lysosomes and lipid droplets. This may represent one mechanism by which PSNPs exert damage on the reproductive system.

Proteomic analysis and effects on osteogenic differentiation of exosomes from patients with ossification of the spinal ligament

Abstract Ossification of the spinal ligament (OSL), including ossification of the posterior longitudinal ligament (OPLL) and ossification of the ligamentum flavum (OLF), is a multifactorial disease that includes genetic predisposition. The association between the rate of ossification in the spinal canal and the severity of myelopathy symptoms is well known, but the degree of progression varies widely among patients. Although many candidate genes and biomarkers have been reported, there are no definitive and quantitative conclusions to date, probably because of low reproducibility due to individual differences. In this study, we focused on exosomes secreted by ossified spinal ligament cells. Exosomes are crucial for intercellular communication during development and progression of disease. In a co-culture study of non-OLF cells with OLF cells, there was increased osteogenic differentiation, including Runx2 and Wnt3a expression, with use of exosome-penetrating filters (1.2 μm) compared to exosome-non-penetrating filters (0.03 μm). Dose-dependent increases in alkaline phosphatase activity and mineral deposition were observed in non-OLF cells treated with OLF-derived exosomes. These results support the hypothesis that OLF-derived exosomes are involved in regulation of osteogenic differentiation. In comparative proteomics analysis, 32 factors were increased and 40 were decreased in OLF-derived exosomes compared to non-OLF-derived exosomes. Molecular network analysis of these 72 factors indicated 10 significant pathways, including the MMP signaling, mTOR signaling, Wnt signaling and VDR-associated pathways. Among the upregulated exosomal membrane proteins in OLF samples, COL IV, FMNL3, mTORC2, and PIP4K showed increased expression with greater ossification, suggesting they may serve as biomarkers of disease activity and therapeutic targets. These factors are involved in the PI3K/Akt/mTOR signaling pathway, and particularly mTOR is known to regulate osteogenic and chondrogenic differentiation. In contrast, FABP5, several KRT family proteins, S100A8, SERPINB3, and TGM, were significantly downregulated in OLF-derived exosomes. These findings provide novel insights into the molecular mechanisms underlying OSL pathogenesis.

Overview of preclinical and phase II clinical studies on Pegmolesatide’s long-term erythropoiesis stimulating effect via EPOR-mediated signal transduction

IntroductionAnemia is a prevalent complication of chronic kidney disease (CKD), primarily due to insufficient erythropoietin (EPO). Pegmolesatide (by Hansoh Pharma) is currently the only marketed long-acting EPO mimetic peptide (EMP) for the treatment of anemia in both dialysis and non-dialysis CKD patients. This paper aimed to explore the long-acting erythropoiesis stimulating molecular mechanism of Pegmolesatide.MethodsIn vitro assays were utilized to assess Pegmolesatide erythropoietin receptor (EPOR) affinity, competitive binding, cell proliferation/survival, apoptosis, cell surface receptor expression, and signal transduction. Pharmacokinetics (PK) and Pharmacodynamics (PD) parameters were evaluated in BALB/c mice following single administration. Furthermore, two Phase II clinical trials in dialysis and non-dialysis chronic kidney disease (CKD) patients with anemia, respectively CTR20140533 and CTR20140539, assessed PK-PD and safety following repeated administration.ResultsIn vitro Pegmolesatide demonstrated enhanced binding stability and prolonged residency at EPOR, surpassing erythropoiesis-stimulating agents (ESAs) rHuEPO and Darbepoetin. This sustained EPOR binding facilitated heightened endogenous EPOR expression post-drug withdrawal, maintaining downstream signal transduction pathways (JAK2/STAT5, ERK1/2 MAPK) for erythropoiesis. Pegmolesatide promoted UT-7 cell proliferation & survival and suppressed apoptosis. Following a single 0.08 mg/kg dose of Pegmolesatide in BALB/c mice, reticulocyte count, red blood cells, hemoglobin, and hematocrit persisted at elevated levels 4-6 days after administration. In the two clinical Phase II studies dose-dependent increases in hemoglobin and prolonged response duration were independently observed. Pegmolesatide showed significant PK-PD dual prolongation effects and was well tolerated. Adverse events were mild and manageable, with no reports of severe anaphylaxis.DiscussionPreclinical and clinical evidence signifies that Pegmolesatide is a unique, potent PEGylated EPO-memetic peptide (EMP) with a prolonged PD efficacy and PK half-life and a good safety-tolerability profile. To elucidate further, future studies will address the endocytosis, intracellular degradation, and ligand release of EPOR subsequent to Pegmolesatide binding, thereby supplementing our understanding of the molecular mechanism at play.Trial registration: Phase IIa clinical study of Pegmolesatide on renal anemia, CTR20140533. Initial Public Notice 14 Jan 2015, http://www.chinadrugtrials.org.cn/clinicaltrials.searchlistdetail.dhtml. Phase II clinical study of Pegmolesatide on renal anemia, CTR20140539. Initial Public Notice 26 Jan 2015, http://www.chinadrugtrials.org.cn/clinicaltrials.searchlistdetail.dhtml.

Phenyl salicylate induces neurotoxicity and early Alzheimer's disease-like symptoms through ndrg1-regulated myelin damage, increasing bace1 in zebrafish.

Phenyl salicylate, an important industrial raw material, is widely used in plastics, cosmetics, and pharmaceuticals. However, little is known about its neurotoxicity on wildlife. Here, we exposed zebrafish embryos at 4 hours post-fertilization (hpf) to 0.025, 0.05, 0.1, 0.25, 0.5, and 1.0mg/L of phenyl salicylate up to 144hpf and found its developmental- and neuro-toxicity. Specifically, a dose-dependent increase in mortality and malformation in zebrafish were revealed. Phenyl salicylate also adversely affected the development of monoaminergic neurons, cerebral blood vessels, and the blood-brain barrier (BBB), as well as induced cerebral hemorrhages and locomotion change. RNA-sequencing results combined with verification data showed that phenyl salicylate downregulated the expression of the N-myc downstream regulated gene-1 (ndrg1), caused myelin damage in zebrafish, and then increased expression of beta-secretase 1 (bace1), which ultimately led to early Alzheimer's disease (AD)-like symptoms, including BBB leakage, bleeding in the brain, and upregulation of the glial fibrillary acidic protein gene (gfap) and cholinergic system-related gene (chrna7a). In conclusion, phenyl salicylate exposure triggered developmental toxicity and neurotoxicity in zebrafish, which has a potential risk for the development of AD. Given the effects of phenyl salicylate exposure to ecosystem, the safety usage limit should be treated with caution.

Faculty of Environment and Labour Safety, Ton Duc Thang University, 19 Nguyen Huu Tho street, Tan Phong ward, District 7, Ho Chi Minh City 700000, Vietnam

This study investigates the acute and sub-chronic toxicity of the emerging antibiotic, sulfamethoxazole (SMX) on the tropical freshwater microalga Chlorella sp. under laboratory condition. An acute exposure for 96 h and a sub-chronic exposure for 10 days were conducted to explore the adverse effects, utilizing growth inhibition, pigment content, and cell diameter as endpoints. The calculated half maximal (50 %) effective concentration (EC50) values for SMX after 24 h and 96 h were estimated to be 0.56 mg/L and 0.41 mg/L, respectively. Sub-chronic exposure unveiled a pronounced inhibitory effect of SMX on algal growth, with significant and dose-dependent increases in growth inhibition observed. Across all treatments, exposure to SMX resulted in growth inhibition and a decline in chlorophyll-a (Chl-a) concentration in the test algae. Notably, at a concentration of 1 mg/L, SMX completely inhibited the growth of Chlorella sp. and reduced Chl-a content by up to 98 %. High concentrations of SMX (0.5 and 1.0 mg/L) led to a decrease in cell diameter in the tested algae. This study contributes valuable insights into the acute and sub-chronic toxic effects of environmentally relevant concentrations of antibiotics on tropical freshwater microalgae.

GCMS Analysis of Bioactive Compounds and Evaluation of Anti-parkinson Activity of Leaf Extract of Curculigo orchioides in Experimental Animals

Background: Curculigo orchioides Gaertn, a plant belonging to the Hypoxidaceae family, is well-known for its traditional uses of all plant parts. This plant is commonly known as golden eye grass, black musli, Kali musli, or Kali Musali. Parkinson's disease is a neurodegenerative disorder that is commonly observed in older adults. It is caused by a variety of mechanisms, including neuroinflammation, which results in the loss of dopamine release when triggered and causes motor symptoms. Methods: Ethanolic extract was prepared by shade-drying the leaves, making them into a coarse powder, and the extract was prepared by maceration technique with the help of ethanol. GCMS analysis and Haloperidol-induced Parkinsonism study were carried out to evaluate the antiParkinson activity in albino Wistar rats. Ethanolic extract of Curculigo orchioides was subjected to GCMS analysis. The National Institute of Standards and Technology database was used to interpret the mass spectrum of the GC-MS. The screening models were catalepsy, rotarod and a few in vitro models, namely the estimation of catalase acetylcholinesterase levels. The animals were divided into 6 groups, namely control, disease, standard (levodopa), low dose (100 mg/kg p.o.), medium dose (200 mg/kg p.o.), high dose (400 mg/kg p.o.) groups having 6 animals each were used for rotarod and catalepsy models for 14 days Results: The results obtained showed a dose-dependent increase in the anti-Parkinson activity. The evaluation of neuromuscular movements of the ethanolic leaf extract of Curculigo orchioides showed that the pre-treatment with different ranges of dose (100 mg/ml, 200 mg/kg, and 400 mg/kg) suggested a dose-dependent significant increase in fall-off time when compared with both standard and disease groups GCMS analysis revealed the presence of flavonoids, Vitamin and fatty acid esters. Conclusion: The anti-Parkinson activity of Flavonoids has been reported previously. Hence, the activity may be attributed to kaempferol-3-O-glucuronide.

Celecoxib Enhances Oxidative Muscle Fibre Formation and Improves Muscle Functions Through Prokr1 Activation in Mice.

Muscle diseases are serious challenges to human health. Prokineticin receptor 1 (PROKR1) has emerged as a potential target to improve muscle function through increasing oxidative muscle fibres, but there are no clinically applicable synthetic PROKR1 agonists. Drugs with biological properties of prokineticin 2 (PK2) were discovered through connectivity map (CMap) analysis. Their effects on PROKR1 were evaluated using molecular docking, PROKR1 signalling and competitive binding assays. Pregnant dams were fed diets containing varying celecoxib concentrations (0, 500, 1000 and 1500 ppm) from gestation day 5 through weaning. Offspring were given high-fat diets (HFD) from weaning until 20 weeks old, and body composition, insulin resistance, energy expenditure, exercise performance and histological analysis of muscle tissues were evaluated. Celecoxib, with a connectivity score of 64.19 to PK2 and a docking score of -9.0 to PROKR1, selectively activated Gs signalling at 4 μM of EC50 and increased NR4A2 protein levels by 1.6-fold (p < 0.01) in PROKR1-overexpressing cells. It competitively inhibited PK2 binding to PROKR1 and reduced cAMP accumulation. In murine and human myotubes, celecoxib increased Prokr1 protein levels by 1.8-fold (p < 0.05), pCreb by 1.5-fold (p < 0.05) and Nr4a2 by 1.3-fold (p < 0.05). It also elevated Myh7 index by 2.2-fold (p < 0.0001), mitochondrial content by 1.6-fold (p < 0.001) and fatty acid oxidation (FAO) activity by 4.1-fold (p < 0.05). Offspring exposed to celecoxib during pre- and postnatal muscle development exhibited activated Prokr1 signalling, enhanced oxidative muscle fibre formation and improved muscle phenotype despite HFD. At weaning, both male and female offspring showed dose-dependent increases in lean mass (> 9.35%, p < 0.001) and grip strength (< 18.0%, p < 0.01). At 12 weeks old, mice displayed a dose-dependent decrease in weight loss (> 13.3%, p < 0.05), increased lean mass (> 16.2%, p < 0.05), improved insulin resistance (> 70.4%, p < 0.0001), energy expenditure (> 173%, p < 0.0001) and grip strength (> 23.5%, p < 0.001). Celecoxib also increased Myh7-positive muscle fibre composition (> 10.8%, p < 0.05) and mitochondrial mass (> 32.8%, p < 0.05) in the gastrocnemius and soleus muscles, accompanied by significant Prokr1 signalling activation. These effects persisted in both male and female mice at 20 weeks old. Celecoxib shows promise as a PROKR1 agonist and clinically applicable exercise mimetic for the treatment of muscular disorders.

Preclinical and phase 1/2 data of the CHK1 inhibitor BBI-355 in development for esophageal and gastric cancers (EGC) with EGFR or FGFR2 amplifications.

TPS517 Background: High-copy number amplifications of oncogenes (e.g., EGFR , FGFR2 ) frequently occur on extrachromosomal DNA (ecDNA), highly transcribed units of circular non-chromosomal DNA. While targeted therapies have improved survival for patients with oncogene mutations, they have limited activity in oncogene amplified cancers. In Barrett’s esophagus-associated esophageal adenocarcinoma (EAC) ecDNA can be found early in the transition from high-grade dysplasia to EAC. In EAC and gastric cancer (EGC), ~7% and ~3% have EGFR and FGFR2 amplifications, respectively, with possibly &gt;50% occurring on ecDNA. Tumor cells with oncogene amplifications, particularly on ecDNA, have increased DNA replication stress and are sensitive to inactivation of checkpoint kinase 1 (CHK1). We developed BBI-355, an oral, potent, and selective small molecule inhibitor of CHK1, which is in Phase 1/2 clinical development. Methods: BBI-355-101 is a first-in-human Phase 1/2 study of BBI-355 alone or in combination with targeted therapies for advanced or metastatic solid tumors with oncogene amplification (NCT05827614). Part 1 is dose escalation of BBI-355 (PO Q2D). In Parts 2 and 3, patients with EGFR or FGFR 1-3 amplifications are treated with BBI-355 in combination with the EGFR inhibitor erlotinib (150 mg PO QD) or the FGFR inhibitor futibatinib (20 mg PO QD). While this study is enrolling all tumor types, EGC are some of the most likely to harbor ecDNA amplifications. Amplification-driven cancers rarely contain driver oncogene mutations or fusions, and so are excluded. Results: Preclinical: Tumors that harbor oncogene amplifications on ecDNA can evade targeted therapeutic pressure via ecDNA-based resistance mechanisms. BBI-355 in combination with agents targeting the amplified oncogenes ( FGFR2 or EGFR ) prevented resistance to targeted therapies in multiple ecDNA+ gastric cancer CDX and PDX models. Clinical: Safety and PK data of BBI-355 alone and in combination with erlotinib or futibatinib will be presented. The most common drug-related TEAEs, SAEs, and DLTs were hematologic (i.e., leukopenia, neutropenia, lymphopenia and thrombocytopenia) which are on target for CHK1 inhibition and generally well managed. Dose escalation is ongoing and the RP2D/MTD has not been determined. PK showed dose-dependent increase in exposure of BBI-355. Target engagement as determined by pCHK1 IHC as a PD biomarker was observed at all dose levels of BBI-355. Conclusions: The first ecDNA directed therapy, BBI-355, demonstrated significant synergistic anti-tumor activity in combination with targeted therapies in multiple ecDNA+ oncogene amplified EGC models. BBI-355 alone and in combination with erlotinib/futibatinib was well tolerated. Clinical testing in patients with EGFR and FGFR2 oncogene amplifications is ongoing with a strong rational for EGC. Clinical trial information: NCT05827614 .

Acute and chronic detrimental effects induced by short-chain chlorinated paraffins in the marine mysid Neomysis awatschensis.

To determine the potentially detrimental impacts of short-chain chlorinated paraffins (SCCPs), we conducted assessments of acute effects on 96-h survival rate and biochemical markers, as well as chronic and multigenerational impacts on growth and reproduction over three generations in the marine mysid, Neomysis awatschensis. Dose-dependent increase of mortality was measured in both juvenile and adult mysids for 96 h. Exposure to the LC10 value (derived from the 96-h acute toxicity value) significantly reduced feeding activity in juveniles, accompanied by a significant elevation in oxidative stress and a reduction in acetylcholinesterase activity. When juvenile and adult mysids were exposed to 1/10 of the NOEC and NOEC values for four weeks, mortality significantly increased in juveniles. Furthermore, mysids subjected to constant exposure to 1/10 of the NOEC and NOEC values across three generations, F0-F2, displayed more pronounced growth retardation, an extended intermolt duration, and a reduced rate of reproduction. These results collectively indicate that even sublethal concentrations of SCCPs can have harmful effects on the health status of mysid populations when they are consistently exposed.

Dose-response Association Between Alcohol Consumption and Kidney Cancer Risk Differs According to Glycemic Status: A Nationwide Cohort Study of 9.4 Million Individuals.

Previous studies suggested an association between alcohol consumption and reduced kidney cancer risk. Given a potential interaction between alcohol's insulin-sensitizing effect and hyperglycemia-related insulin resistance, we aimed to assess whether the dose-response association between alcohol intake and kidney cancer risk varies based on glycemic status. This nationwide cohort study analyzed data from 9,492,331 adults who underwent a national health screening program in 2009 and were followed until 2018. Multivariable-adjusted Cox regression models were applied to estimate the hazard ratios (aHRs) and 95% confidence intervals (CIs). Over a median follow-up period of 8.3 years, 12,381 participants were diagnosed with kidney cancer. A U-shaped relationship between alcohol consumption and kidney cancer risk was observed among individuals with normoglycemia (light-to-moderate; HR, 0.94; 95% CI, 0.89-0.99 and heavy; HR, 1.00; 95% CI, 0.91-1.09, respectively). In prediabetic individuals, alcohol consumption was not significantly associated with kidney cancer risk. In individuals with diabetes, a dose-dependent increase in kidney cancer risk was noted with higher alcohol consumption (light-to-moderate consumption: HR, 1.12; 95% CI, 1.03-1.22; heavy consumption: HR, 1.24; 95% CI, 1.09-1.42; P for trend <0.01). A modest U-shaped dose-response association between alcohol consumption and kidney cancer risk was observed exclusively in individuals with normoglycemia. Individuals with diabetes demonstrated a dose-dependent increased risk of kidney cancer with higher alcohol consumption. Tailored patient education and personalized risk assessments regarding alcohol consumption and kidney cancer risk should be emphasized over a generalized 'one-size-fits-all' approach.

Structural Analysis of Cardanol and Its Biological Activities on Human Keratinocyte Cells

Background/Objectives: Cashew nutshell liquid (CNSL) is obtained during the industrial processing of cashew nuts. It contains anacardic acid (2-hydroxy-6-n-pentadecylbenzoic acid) and cardanol (3-n-pentadecylphenol). Therefore, CNSL provides a rich source of phenolic lipids serving as natural antioxidants or precursors for industrial uses. Here, we have analyzed in detail a commercial sample of cardanol by nuclear magnetic resonance (NMR) spectroscopy and its biological activities in the human keratinocyte cell line (HaCaT cells). Methods: The cytotoxic effects, genotoxicity, cell proliferation, and healing properties on HaCaT cells were studied using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, comet assay, proliferation assay, and scratch assay, respectively. Additionally, the modulatory effect of cardanol on the cellular fatty acid profile of HaCaT cells was analyzed by gas chromatography. Results: NMR showed the structure of cardanol as a mixture of the 8′-monoene (42%), the 8′,11′-diene (22%), and the 8′,11′,14′-triene (36%) for the pentadecyl side chain with all double bonds in Z configuration. The cytotoxic effects on HaCaT cells only occurred at high concentrations of cardanol (&gt;10 µg/mL), which caused significant reductions in cell viability. Using the comet assay, a dose-dependent increase in DNA damage was found at concentrations above 10 µg/mL. Scratch assays revealed that cardanol achieved 99% wound closure of HaCaT cells treated with 1 µg/mL cardanol after 48 h. Cardanol at 1 and 0.1 µg/mL significantly enhanced HaCaT cell proliferation and promoted migration, contributing to accelerated wound healing processes. As shown by gas chromatography, 1 µg/mL cardanol increased the total amount of polyunsaturated fatty acids (PUFA), including ω-3, ω-6, and ω-9 fatty acids. Conclusions: Together, these findings suggest that concentrations of &lt;10 µg/mL cardanol are safe and exhibit beneficial biological activities, particularly wound-healing effects on HaCaT cells. Further studies are necessary to explore additional potential applications of cardanol, to refine its formulations for clinical use, and to ensure its safety and action in other target cells and species.

Inhibition of FTO expression by thiodiphenol impairs Leydig cell maturation in pubertal male rats.

This study investigated the multifaceted effects of 4,4'-thiodiphenol (TDP) on male reproductive health, focusing on its toxicity, hormonal modulation, and impact on steroidogenesis. Male rats were exposed to TDP at varying doses (0, 1, 10, and 100 mg/kg) from day 35-56 postpartum. Overt toxicity was evident as TDP significantly reduced inhibited spermatogenesis at 10 and 100 mg/kg. TDP exhibited anti-androgenic properties by inhibiting testosterone biosynthesis at 10 and 100 mg/kg, correlating with increased LH levels at 10 mg/kg and altered T/LH ratios at ≥ 1 mg/kg. Immunohistochemical analysis revealed a dose-dependent increase in Leydig cell (LC) numbers, particularly at 100 mg/kg, attributed to enhanced LC proliferation as evidenced by elevated PCNA labeling. RNA-seq and qPCR analyses demonstrated the impact of TDP on LC gene expression, notably downregulating steroidogenesis-related genes such as Lhcgr, Star, Cyp11a1, and Hsd3b1, and inducing oxidative stress pathway genes. Protein expression studies confirmed reduced levels of key steroidogenic proteins and increased oxidative stress, evidenced by elevated malondialdehyde levels and reduced SOD1 expression. In vitro studies further elucidated the inhibition of TDP on steroidogenesis and induction of reactive oxygen species (ROS), independent of cytotoxicity. Additionally, the modulation of m6A RNA methylation was explored, showing an increase in m6A levels and a reduction in FTO and ALKBH5 expression and overexpression of Fto reversed TDP-mediated suppression of testosterone and steroidogenic and antioxidative genes, suggesting a novel regulatory mechanism involving RNA modification. Collectively, these findings underscore the potential of TDP as an endocrine disruptor with significant implications for male reproductive health.

λ-cyhalothrin induced sex-specific inflammation, glia activation and GABAergic interneuron disruption in the hippocampus of rats

BackgroundGlia mediated neuroinflammation and degeneration of inhibitory GABAergic interneurons are some of the hall marks of pyrethroid neurotoxicity. Here we investigated the sex specific responses of inflammatory cytokines, microglia, astrocyte and parvalbumin positive inhibitory GABAergic interneurons to λ-cyhalothrin (LCT) exposures in rats.MethodsEqual numbers of male and female rats were given oral corn oil, 2 mg/kg.bw and 4 mg/kg.bw of LCT for fourteen days. They were euthanized on day 15, brains were excised and hippocampus (n = 5/group) isolated for interleukin 1 beta (IL-1β) and tumor necrotic factor alpha (TNF-α) analysis. The remaining brains (n = 3/group) were processed for Ionized calcium binding adaptor molecule 1 (Iba1), glial fibrillary acidic protein (GFAP) and parvalbumin (PV) distribution in the hippocampus. All quantitative data was subjected to one way analysis of variance (ANOVA).ResultsLCT caused sex and dose dependent increase in IL-1β and TNF-α concentrations, distribution of microglia (Iba1+) and astrocytes (GFAP+), and reduction of PV + GABAergic interneurons. These effects were greater in males compared to females, and dose-dependent in both sexes.ConclusionLCT specifically induced inflammation and disrupted GABAergic interneurons’ integrities via activation of microglia and reactive astrogliosis and such effects are dose-dependent and sexually dimorphic.

Positive feedback regulation between RpoS and BosR in the Lyme disease pathogen.

In Borrelia burgdorferi, the causative agent of Lyme disease, differential gene expression is primarily governed by the alternative sigma factor RpoS (σS). Understanding the regulation of RpoS is crucial for elucidating how B. burgdorferi is maintained throughout its enzootic cycle. Our recent studies have shown that the homolog of Fur/PerR repressor/activator BosR functions as an RNA-binding protein that controls the rpoS mRNA stability. However, the mechanisms regulating BosR, particularly in response to host signals and environmental cues, remain largely unclear. In this study, we uncovered a positive feedback loop between RpoS and BosR, wherein RpoS post-transcriptionally regulates BosR levels. Specifically, mutation or deletion of rpoS significantly reduced BosR levels, whereas artificial induction of rpoS resulted in a dose-dependent increase in BosR levels. Notably, RpoS does not affect bosR mRNA levels but instead modulates the turnover rate of the BosR protein. Moreover, we demonstrated that environmental cues do not directly influence bosR expression but instead induce rpoS transcription and RpoS production, thereby enhancing BosR protein levels. These findings reveal a new layer of complexity in the RpoN-RpoS regulatory pathway, challenging the existing paradigm and suggesting a need to re-evaluate the factors and signals previously implicated in regulating RpoS via BosR. This study provides new insights into the intricate regulatory networks underpinning B. burgdorferi's adaptation and survival in its enzootic cycle.IMPORTANCELyme disease is the most prevalent arthropod-borne infection in the United States. The etiological agent, Borreliella (or Borrelia) burgdorferi, is maintained in nature through an enzootic cycle involving a tick vector and a mammalian host. RpoS, the master regulator of differential gene expression, plays a crucial role in tick transmission and mammalian infection of B. burgdorferi. This study reveals a positive feedback loop between RpoS and a Fur/PerR homolog. Elucidating this regulatory network is essential for identifying potential therapeutic targets to disrupt B. burgdorferi's enzootic cycle. The findings also have broader implications for understanding the regulation of RpoS and Fur/PerR family in other bacteria.

Discovery of Highly Potent and Orally Bioavailable Histone Deacetylase 3 Inhibitors as Immunomodulators and Enhancers of DNA-Damage Response in Cancer Therapy.

Histone deacetylase 3 (HDAC3) is a well-established target for cancer therapy. Herein, we developed LSQ-28 as a novel HDAC3 inhibitor, which exhibited high HDAC3 inhibitory activity (IC50 = 42 nM, SI > 161) and displayed potent antiproliferative activity against four cancer cells and further demonstrated excellent antimigratory, anti-invasive, and antiwound healing activities. Further studies revealed that LSQ-28 induced a dose-dependent increase in Ac-H3 expression and promoted the degradation of PD-L1. Additionally, LSQ-28 enhanced the DNA damage response induced by PARP inhibitor, as evidenced by regulated expression of PARP1 and γ-H2AX. Notably, LSQ-28 also possessed favorable pharmacokinetic properties with significant oral bioavailability (F = 95.34%). Importantly, the combination of LSQ-28 with the PD-L1 inhibitor NP-19 could enhance antitumor immune response (TGI = 80%). When combined with olaparib, LSQ-28 significantly enhanced the in vivo tumor-suppression activity (TGI = 91%). Collectively, LSQ-28 represents a promising HDAC3 inhibitor for further exploration in cancer therapeutic strategies.

Combinations of approved oral nucleoside analogues confer potent suppression of alphaviruses in vitro and in vivo.

Alphaviruses such as chikungunya virus (CHIKV) pose a significant threat to global health, yet specific antiviral therapies remain unavailable. In this study, we evaluated combinations of three approved oral directly acting antiviral (DAA) drugs (sofosbuvir (SOF), molnupiravir (MPV) and favipiravir (FAV)) against CHIKV, Semliki Forest virus (SFV), Sindbis virus (SINV), and Venezuelan Equine Encephalitis virus (VEEV) in vitro and in vivo . In human skin fibroblasts, synergistic antiviral effects were observed for the drug combinations MPV + SOF and FAV + SOF against CHIKV, and for FAV + SOF against SFV. In human liver Huh7 cells, the combinations of FAV + MPV conferred additive to synergistic activity against VEEV and SINV strains, while SOF synergized with FAV against SINV strains. In a mouse model of CHIKV arthritis, MPV improved CHIKV-induced foot swelling and reduced systemic infectious virus titers. Combination treatment with suboptimal doses of MPV and SOF significantly reduced foot swelling and decreased infectious virus titers in serum as compared to single doses of each drug. Sequencing of CHIKV RNA from mouse joint tissue revealed that MPV caused dose-dependent increases in mutations in the CHIKV genome. Upon combination therapy of MPV with SOF, the number of mutations was significantly lower compared to single treatment with several higher doses of MPV. In summary, combining approved oral nucleoside analogs confers potent suppression of multiple alphaviruses in vitro and in vivo with enhanced control of viral genetic evolution in the face of antiviral drug pressure. These drug combinations may ultimately lead to the development of potent combinations of pan-family alphavirus inhibitors.

Fluoride Exposure Modulates Skeletal Development and Mineralization in Zebrafish Larvae.

The presence of high levels of fluoride (F) in groundwater is a major issue worldwide. Although F is essential for healthy teeth and bones, excessive exposure can cause fluorosis or F toxicity. This condition primarily affects the hard tissues due to their high F retention capacity. F accumulation alters bone formation and resorption mechanisms interfering with mineral homeostasis and eventually manifests as skeletal fluorosis. Albeit the numerous studies on skeletal fluorosis, the effect of F on developmental osteogenesis is inconclusive. In light of this, we studied the effect of F on osteogenic differentiation, bone development, and mineralization in zebrafish. Zebrafish embryos were subjected to a low (25 ppm NaF), and a moderately high (50 ppm NaF) dose, along with a control (E3 medium alone) until 7 days postfertilization (dpf). The F content in the larvae was quantified to reveal a dose-dependent increase in the exposed groups. Alizarin Red and alkaline phosphatase (ALP) staining suggested enhanced mineralization in the F-treated groups. Quantitative analyses of the ALP activity and hydroxyproline (Hyp) content revealed similar results. Alcian blue staining of pharyngeal cartilages showed that F exposure alters the morphology of the major cartilages, indicating a possible craniofacial defect. Moreover, gene expression analyses of the bone markers associated with osteogenic differentiation, early mineralization, and remodeling (runx2a/b, bmp4, ocn, osx, col1a1, alp, rank, rankl, and opg) showed enhanced expression in the low F group. While the 50 ppm F group showed a decline in osteogenic activity, a considerable increase in the expression of mineralization markers was observed. The expression levels of cartilage markers sox9a and sox9b, remained insignificant, indicating the effect of F toxicity on osteogenesis and mineralization. Also, F exposure interferes with bone metabolism through altered osteogenic differentiation, development, and mineralization in zebrafish larvae.

In vivo toxic and lethal cardiorespiratory effects of a synthetic quaternary ammonium salt derivative of haloperidol in mice.

To investigate the toxicity of N-n-butyl haloperidol iodide (F2), a quaternary ammonium salt derivative of haloperidol, in mice for potential therapeutic purposes. The acute median lethal dose (LD50) of F2 was determined using the Bliss method following intravenous administration in mice. Routine surface electrocardiograms (ECGs) and arterial blood pressures (aBPs) were recorded under general anesthesia in untreated and pharmacologically vagotomized mice injected with F2. Sublethal doses of F2 were tested for their effects on aBP, heart rate, and biochemical parameters such as lactate dehydrogenase (LDH), blood urea nitrogen (BUN), and serum lactate levels. Histopathological changes in the heart, lungs, liver, and kidneys were evaluated after F2 administration. The acute LD50 of F2 was determined to be 5.11 mg/kg. A 10 mg/kg dose of F2 caused severe hypotension, second-degree atrioventricular block, progressive prolongation of Pmurr intervals, and death due to cardiac asystole. Similar ECG and aBP changes were observed in atropine-pretreated mice, indicating that cholinergic effects do not play a major role in F2-induced toxicity. Sublethal doses of F2 (1.2 and 2.4 mg/kg) caused dose-dependent decreases in aBP and increases in heart rate. F2 induced significant, dose-dependent increases in LDH, BUN, and serum lactate levels. Histopathological analysis revealed acute lung lesions at 10 mg/kg, with no significant changes observed in the heart, liver, or kidneys. Acute intravenous injection of F2 exhibits dose-dependent cardiopulmonary toxicity, characterized by severe hypotension, arrhythmias, and biochemical changes. These findings highlight the potential risks of F2 and the need for further evaluation of its safety profile for therapeutic use.

Non-invasive quantification of bone (re) modeling dynamics in adults with osteogenesis imperfecta treated with Setrusumab using timelapse HR-pQCT.

Timelapse imaging using high-resolution peripheral quantitative computed tomography (HR-pQCT) has emerged as a noninvasive method to quantify bone (re)modelling. However, there is no consensus on how to perform the procedure. As part of the ASTEROID phase-2b multicenter trial, we used 29 same-day repeated scans from adults with osteogenesis imperfecta (OI) to identify a method that minimized measurement error. We evaluated input image type, registration method, segmentation mask, and for grayscale images various values for the voxel density difference considered formed or resorbed, minimum formation/resorption cluster size, and Gaussian smoothing sigma. We verified the accuracy of our method and then used it on longitudinal scans (baseline, 6, 12, 18 and 24month) from 78 participants to assess bone formation and resorption induced by an anabolic (setrusumab) and anti-catabolic (zoledronic acid) treatments as part of the ASTEROID trial. Regardless of image registration method, binary input images resulted in large errors ~13% and ~ 8% for first- and second-generation scanners, respectively. For the grayscale input images, errors were smaller for 3D compared to matched angle registration. For both scanner generations, a density threshold of 200mgHA/cm3 combined with Gaussian noise reduction resulted in errors <1%. We verified the method was accurate by showing that similar regions of bone formation and resorption were identified when comparing each scan from the same-day repeated scans with a scan from another timepoint. Timelapse analysis revealed a dose-dependent increase in bone formation and resorption with setrusumab treatment. Zoledronic acid altered bone changes in favor of formation, although no changes reached statistical significance. This study identifies a timelapse method that minimizes measurement error, which can be used in future studies to improve the uniformity of results. This noninvasive imaging biomarker revealed dose dependent bone (re) modeling outcomes from one year of setrusumab treatment in adults with OI.