Integrative Single Cell Atlas Revealed Intratumoral Heterogeneity Generation from an Adaptive Epigenetic Cell State in Human Bladder Urothelial Carcinoma.

Intratumor heterogeneity (ITH) of bladder cancer (BLCA) contributes to therapy resistance and immune evasion affecting clinical prognosis. The molecular and cellular mechanisms contributing to BLCA ITH generation remain elusive. It is found that a TM4SF1-positive cancer subpopulation (TPCS) can generate ITH in BLCA, evidenced by integrative single cell atlas analysis. Extensive profiling of the epigenome and transcriptome of all stages of BLCA revealed their evolutionary trajectories. Distinct ancestor cells gave rise to low-grade noninvasive and high-grade invasive BLCA. Epigenome reprograming led to transcriptional heterogeneity in BLCA. During early oncogenesis, epithelial-to-mesenchymal transition generated TPCS. TPCS has stem-cell-like properties and exhibited transcriptional plasticity, priming the development of transcriptionally heterogeneous descendent cell lineages. Moreover, TPCS prevalence in tumor is associated with advanced stage cancer and poor prognosis. The results of this study suggested that bladder cancer interacts with its environment by acquiring a stem cell-like epigenomic landscape, which might generate ITH without additional genetic diversification.

A rapid immunomagnetic beads-based sELISA method for the detection of bovine αs1-casein based on specific epitopes.

Although αs1-casein poses significant health risks to individuals with milk allergies, the availability of quantification methods for this allergen remains limited. In this study, we developed an immunomagnetic beads-based immunoassay (IMBs-ELISA) for the precise quantitative detection of bovine αs1-CN, specifically targeting epitope AA173-194. No cross-reactivity was observed with the other 7 food allergens including milk allergen. The linear detection range of the established IMBs-ELISA method was 0.125 µg/mL-2.000 µg/mL, with a limit of detection of 0.099 µg/mL. The accuracy of this method was 1.048 %, and the intra-plate and inter-plate precision achieved 4.100 % and 6.777 %, respectively. Notably, the entire IMBs-ELISA process could be completed within 75 min, representing a substantial time-saving advantage over traditional ELISA methods. These results proved the reliability and rapidity of the IMBs-ELISA method for detecting αs1-CN in real food.

Circ0087385 promotes DNA damage in benzo(a)pyrene-induced lung cancer development by upregulating CYP1A1.

Increasing environmental genotoxic chemicals have been shown to induce epigenetic alterations. However, the interaction between genetics and epigenetics in chemical carcinogenesis is still not fully understood. Here, we constructed an in vitro human lung carcinogenesis model (16HBE-T) by treating human bronchial epithelial cells with a typical significant carcinogen benzo(a)pyrene (BaP). We identified a novel circular RNA, circ0087385, which was overexpressed in 16HBE-T and human lung cancer cell lines, as well as in lung cancer tissues and serum exosomes from lung cancer patients. The upregulated circ0087385 after exposure to BaP promoted DNA damage in the early stage of chemical carcinogenesis and affected the cell cycle, proliferation, and apoptosis of the malignantly transformed cells. Overexpression of circ0087385 enhanced the expression of cytochrome P450 1A1 (CYP1A1), which is crucial for metabolically activating BaP. Interfering with circ0087385 or CYP1A1 reduced the levels of ultimate carcinogen benzo(a)pyrene diol epoxide (BPDE) and BPDE-DNA adducts. Interfering with CYP1A1 partially reversed the DNA damage induced by high expression of circ0087385, as well as decreased the level of BPDE and BPDE-DNA adducts. These findings provide novel insights into the interaction between epigenetics and genetics in chemical carcinogenesis which are crucial for understanding the epigenetic and genetic toxicity of chemicals.

Salmonella enterica serovar Typhimurium remodels mitochondrial dynamics of macrophages via the T3SS effector SipA to promote intracellular proliferation.

Mitochondrial dynamics are critical in cellular energy production, metabolism, apoptosis, and immune responses. Pathogenic bacteria have evolved sophisticated mechanisms to manipulate host cells' mitochondrial functions, facilitating their proliferation and dissemination. Salmonella enterica serovar Typhimurium (S. Tm), an intracellular foodborne pathogen, causes diarrhea and exploits host macrophages for survival and replication. However, S. Tm-associated mitochondrial dynamics during macrophage infection remain poorly understood. In this study, we showed that within macrophages, S. Tm remodeled mitochondrial fragmentation to facilitate intracellular proliferation mediated by Salmonella invasion protein A (SipA), a type III secretion system effector encoded by Salmonella pathogenicity island 1. SipA directly targeted mitochondria via its N-terminal mitochondrial targeting sequence, preventing excessive fragmentation and the associated increase in mitochondrial reactive oxygen species, loss of mitochondrial membrane potential, and release of mitochondrial DNA and cytochrome c into the cytosol. Macrophage replication assays and animal experiments showed that mitochondria and SipA interact to facilitate intracellular replication and pathogenicity of S. Tm. Furthermore, we showed that SipA delayed mitochondrial fragmentation by indirectly inhibiting the recruitment of cytosolic dynamin-related protein 1, which mediates mitochondrial fragmentation. This study revealed a novel mechanism through which S. Tm manipulates host mitochondrial dynamics, providing insights into the molecular interplay that facilitates S. Tm adaptation within host macrophages.

A multi-module structure labelled molecular network orients the chemical profiles of traditional Chinese medicine prescriptions: Xiaoyao San, as an example.

Ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) technology has emerged as a crucial tool for identifying components in traditional Chinese medicine (TCM). However, the characterization of the chemical profiles of TCM prescriptions (TCMPs) which often consist of multiple herbal medicines and contain diverse structural types, presents several challenges, such as component overlapping and time-consuming. In this study, a novel strategy known as the multi-module structure labelled molecular network (MSLMN), which integrates molecular networking, database annotation, and cluster analysis techniques, has been successfully proposed, which facilitates the identification of chemical constituents by leveraging a high-structural similarity ion list derived from the MSLMN. It has been effectively applied to analyze the chemical profile of Xiaoyao San (XYS), a classical TCMP. Through the MSLMN method, a total of 302 chemical constituents were identified, covering nine structural types in XYS. Furthermore, a validated and quantitative analytical method using UHPLC-QqQ-MS/MS technology was developed for 31 identified chemicals, encompassing all eight herbal medicines present in XYS, and the developed analytical approach was applied to investigate the content distribution across 40 different batches of commercially available XYS. In total, the proposed strategy has practical significance for improving the insight into the chemical profile of XYS and serves as a valuable approach for handling complex system data based on UHPLC-MS, particularly for TCMPs.

Weakened black carbon trans-boundary transport to the Tibetan Plateau during the COVID-19 pandemic.

The lockdowns implemented during the coronavirus disease 2019 (COVID-19) pandemic provide a unique opportunity to investigate the impact of emission sources and meteorological conditions on the trans-boundary transportation of black carbon (BC) aerosols to the Tibetan Plateau (TP). In this study, we conducted an integrative analysis, including in-situ observational data, reanalysis datasets, and numerical simulations, and found a significant reduction in the trans-boundary transport of BC to the TP during the 2020 pre-monsoon season as a result of the lockdowns and restrictive measures. Specifically, we observed a decrease of 0.0211 µgm-3 in surface BC concentration over the TP compared to the 2016 pre-monsoon period. Of this reduction, approximately 6.04 % can be attributed to the decrease in emissions during the COVID-19 pandemic, surpassing the 4.47 % decrease caused by changes in meteorological conditions. Additionally, the emission reductions have weakened the trans-boundary transport of South Asia BC to the TP by 0.0179 µgm-2s-1; indicating that the recurring spring atmospheric pollution from South Asia to the TP will be alleviated through the reduction of anthropogenic emissions. Moreover, it is important to note that BC deposition on glaciers contributes significantly to glacier melting due to its enrichment, posing a threat to the water sustainability of the TP. Therefore, urgent measures are needed to reduce emissions from adjacent regions to preserve the TP as the "Asian Water Tower."

Z3495, a LysR-Type Transcriptional Regulator Encoded in O Island 97, Regulates Virulence Gene Expression in Enterohemorrhagic Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) is an important foodborne pathogen that infects humans by colonizing the large intestine. The genome of EHEC O157:H7 contains 177 unique O islands (OIs). Certain OIs significantly contribute to the heightened virulence and pathogenicity exhibited by EHEC O157:H7. However, the function of most OI genes remains unknown. We demonstrated here that EHEC O157:H7 adherence to and colonization of the mouse large intestine are both dependent on OI-97. Z3495, which is annotated as a LysR-type transcriptional regulator and encoded in OI-97, contributes to this phenotype. Z3495 activated the locus of enterocyte effacement (LEE) gene expression, promoting bacterial adherence. Deletion of z3495 significantly decreased the transcription of ler and other LEE genes, the ability to adhere to the host cells, and colonization in the mouse large intestine. Furthermore, the ChIP-seq results confirmed that Z3495 can directly bind to the promoter region of rcsF, which is a well-known activator of Ler, and increase LEE gene expression. Finally, phylogenetic analysis revealed that Z3495 is a widespread transcriptional regulator in enterohemorrhagic and enteropathogenic Escherichia coli. As a result of this study, we have gained a deeper understanding of how bacteria control their virulence and provide another example of a laterally acquired regulator that regulates LEE gene expression in bacteria.

A review on in silico prediction of the environmental risks posed by pharmaceutical emerging contaminants.

Computer-aided (in silico) prediction has shown good potential to support the environmental risk assessment (ERA) of pharmaceutical emerging contaminants (PECs), allowing low-cost, animal-free, high-throughput screening of multiple potential risks posed by a wide variety of pharmaceuticals in the environment based on insufficient toxicity data. This review provided recent insights regarding the application of in silico approaches in prediction for environmental risks of PECs. Based on the review of 20 included articles from 8 countries published since 2018, we found that the researchers' interest and concern in this research topic were sharply aroused since 2021. Recently, in silico approaches have been widely used for the prediction of bioaccumulation and biodegradability, lethal endpoints, developmental toxicity, mutagenicity, other eco-toxicological effects such as ototoxicity and hematological toxicity, and human health hazards of exposure to PECs. Particular attention has been given to the simultaneous discernment of multiple environmental risks and health effects of PECs based on mechanistic data of pharmaceuticals using advanced bioinformatic methods such as transcriptomic analysis and network pharmacology prediction. In silico software platforms and databases used in the included studies were diversified, and there is currently no standardized and accepted in silico model for ERA of PECs. Date suggested that in silico prediction of the environmental risks posed by PECs is still in its infancy. Considerable critical challenges need to be addressed, including consideration of environmental exposure concentration for PECs, interactions among mixtures of PECs and other contaminants coexisting in environments, and development of in silico models specific to ERA of PECs.

Propensity-score matched analysis of patent foramen ovale closure in real-world study cohort with cryptogenic ischemic stroke.

INTRODUCTION: Patent foramen ovale (PFO) occurs in 25% of the general population and in 40% of cryptogenic ischemic stroke patients. Recent trials support PFO closure in selected patients with cryptogenic stroke. We examined the outcomes of transcatheter PFO closure in a real-world study cohort with cryptogenic stroke. METHODS: Consecutive ischemic stroke patients who were classified as cryptogenic on the TOAST aetiology and diagnosed with a PFO were included. All patients underwent either transcatheter PFO closure or medical therapy. A 2:1 propensity score matching by sex and Risk-of-Paradoxical-Embolism (RoPE) score was performed. Multivariable regression models adjusted for sex and RoPE score. RESULTS: Our cohort comprised 232 patients with mean age 44.3 years (SD 10.8) and median follow-up 1486.5 days. 33.2% were female. PFO closure (n=84) and medical therapy (n=148) groups were well-matched with <10% mean-difference in sex and RoPE score. Two patients in the treated group (2.4%) and seven in the control group (4.7%) had a recurrent ischemic stroke event. Multivariable Cox regression demonstrated a hazard-ratio of 0.26 (95%CI 0.03-2.13, P=0.21) for PFO closure compared to control. The incidence of atrial fibrillation (AF) detected post-PFO closure was similar between the treated and control (1.19% vs 1.35%, multivariable logistic regression odds-ratio 0.90, 95%CI 0.04-9.81, P=0.94). There were no major periprocedural complications documented. The difference in restricted mean survival-time free from stroke at two years between treated and control was 26.2 days (95%CI 5.52-46.85, P=0.013). CONCLUSIONS: In this Asian cohort, we report a low incidence of ischemic stroke recurrence and new-onset AF in patients who underwent PFO closure. When compared to the medical therapy group, there was no significant difference in the incidence of stroke recurrence and new-onset AF. Further studies involving larger real-world cohorts are warranted to identify patients who are more likely to benefit from PFO closure.

Benzo[a]pyrene-induced up-regulation of circ_0003552 via ALKBH5-mediated m6A modification promotes DNA damage in human bronchial epithelial cells.

Benzo [a]pyrene (B [a]P) is a widespread environmental chemical pollutant that has been linked to the development of various diseases. However, the specific mechanism of action remains unclear. In this study, human bronchial epithelial 16HBE and BEAS-2B cells were exposed to B [a]P at 0-32 µM to assess the DNA-damaging effects. B [a]P exposure resulted in elevated expression of γ-H2AX, a marker of DNA damage. The m6A RNA methylation assay showed that B [a]P exposure increased the extent of m6A modification and the demethylase ALKBH5 played an integral role in this process. Moreover, the results of the comet assay and Western blot analysis showed an increase in m6A modification mediated by ALKBH5 that promoted DNA damage. Furthermore, the participation of a novel circular RNA, circ_0003552, was assessed by high-throughput sequencing under the condition of high m6A modification induced by B [a]P exposure. In subsequent functional studies, an interference/overexpression system was created to confirm that circ_0003552 participated in regulation of DNA damage. Mechanistically, circ_0003552 had an m6A binding site that could regulate its generation. This study is the first to report that B [a]P upregulated circ_0003552 through m6A modification, thereby promoting DNA damage. These findings revealed that epigenetics played a key role in environmental carcinogen-induced DNA damage, and the quantitative changes it brought might provide an early biomarker for future medical studies of genetic-related diseases and a new platform for investigations of the interaction between epigenetics and genetics.

Engineered MED12 mutations drive leiomyoma-like transcriptional and metabolic programs by altering the 3D genome compartmentalization.

Nearly 70% of Uterine fibroid (UF) tumors are driven by recurrent MED12 hotspot mutations. Unfortunately, no cellular models could be generated because the mutant cells have lower fitness in 2D culture conditions. To address this, we employ CRISPR to precisely engineer MED12 Gly44 mutations in UF-relevant myometrial smooth muscle cells. The engineered mutant cells recapitulate several UF-like cellular, transcriptional, and metabolic alterations, including altered Tryptophan/kynurenine metabolism. The aberrant gene expression program in the mutant cells is, in part, driven by a substantial 3D genome compartmentalization switch. At the cellular level, the mutant cells gain enhanced proliferation rates in 3D spheres and form larger lesions in vivo with elevated production of collagen and extracellular matrix deposition. These findings indicate that the engineered cellular model faithfully models key features of UF tumors and provides a platform for the broader scientific community to characterize genomics of recurrent MED12 mutations.

The final 28 Days:Prenatal exposure to air pollution and child anthropometric outcomes.

This study examines the health consequences of prenatal exposure to air pollution by combining child health data from an original survey with the Air Pollution Index (API) from official Chinese statistics. Our results show that exposure to air pollution in late trimester (four-week windows before delivery) is negatively associated with health outcomes in children in the short and long terms. One standard deviation increase in the API in the final 28 days before delivery decreased birth weight and length by 0.388 and 0.458, respectively, in z-scores and lowered the weight-for-age and height-for-age by 0.370 and 0.441, respectively, in z-scores at 13-15 years post-exposure. Although the timing of exposure and its consequences have been the subject of debate in existing literature, our results focus on four-week windows and demonstrate that exposure during the late pregnancy period may have adverse health effects on children. We conducted analyses that accounted for potential covariates and omitted variables, and our results remain robust and statistically significant. We also found gender heterogeneous effects that girls are more vulnerable to fetal air pollution exposure than boys. Our findings uncover fetal and child health risks regarding air pollution and reinforce the importance of policies for mitigating air pollution in developing countries.

Mechanisms of autophagy and endoplasmic reticulum stress in the reversal of platinum resistance of epithelial ovarian cancer cells by naringin.

OBJECTIVE: Our previous studies showed that naringin (Nar) can effectively reverse the cisplatin resistance of ovarian cancer cells. This study aims to explore the potential mechanism by which Nar reverses cisplatin resistance in ovarian cancer. METHODS: The proliferative activity of cells was evaluated using CCK8 and cell clone formation assays. Autophagic flux in cells was evaluated via LC3B immunofluorescence and monodansylcadaverine (MDC) staining. The expression levels of autophagy, endoplasmic reticulum (ER) stress, and apoptosis-related proteins were detected via Western blotting. Autophagy and ER stress were regulated using siATG5, siLC3B, rapamycin (Rap), chloroquine (CQ), 4-phenylbutyric acid (4-PBA), and thapsigargin (TG). siATG5 and siLC3B are short interfering RNAs (siRNAs) used to knock down the expression of ATG5 and LC3B genes, respectively. RESULTS: Nar inhibited autophagy in SKOV3/DDP cells by activating the PI3K/AKT/mTOR pathway. And Nar increased the levels of ER stress-related proteins, namely, P-PERK, GRP78, and CHOP, and promoted apoptosis in SKOV3/DDP cells. Moreover, treatment with the inhibitor of ER stress alleviated apoptosis induced by Nar in SKOV3/DDP cells. In addition, compared to cisplatin or naringin alone, the combination of Nar and cisplatin significantly reduced the proliferative activity of SKOV3/DDP cells. And siATG5, siLC3B, CQ or TG pretreatment further inhibited the proliferative activity of SKOV3/DDP cells. Conversely, Rap or 4-PBA pretreatment alleviated the cell proliferation inhibition caused by Nar combined with cisplatin. CONCLUSION: Nar not only inhibited the autophagy in SKOV3/DDP cells by regulating the PI3K/AKT/mTOR signalling pathway, but also promoted apoptosis in SKOV3/DDP cells by targeting ER stress. Nar can reverse the cisplatin resistance in SKOV3/DDP cells through these two mechanisms.

Wildfire-Derived Nitrogen Aerosols Threaten the Fragile Ecosystem in Himalayas and Tibetan Plateau.

Himalayas and Tibetan Plateau (HTP) is important for global biodiversity and regional sustainable development. While numerous studies have revealed that the ecosystem in this unique and pristine region is changing, their exact causes are still poorly understood. Here, we present a year-round (23 March 2017 to 19 March 2018) ground- and satellite-based atmospheric observation at the Qomolangma monitoring station (QOMS, 4276 m a.s.l.). Based on a comprehensive chemical and stable isotope (15N) analysis of nitrogen compounds and satellite observations, we provide unequivocal evidence that wildfire emissions in South Asia can come across the Himalayas and threaten the HTP's ecosystem. Such wildfire episodes, mostly occurring in spring (March-April), not only substantially enhanced the aerosol nitrogen concentration but also altered its composition (i.e., rendering it more bioavailable). We estimated a nitrogen deposition flux at QOMS of ∼10 kg N ha-1 yr-1, which is approximately twice the lower value of the critical load range reported for the Alpine ecosystem. Such adverse impact is particularly concerning, given the anticipated increase of wildfire activities in the future under climate change.

Experimental Study on the Methane Adsorption of Massive Shale Considering the Effective Stress and the Participation of Nanopores of Varying Sizes.

To explore the shale gas occurrence mechanism in shale with an intact pore structure under actual reservoir conditions, an adsorption experiment on massive shale was performed. Considering the change in the pore volume of massive shale under effective stress, the adsorption mechanism and free gas storage space of massive shale were investigated. Based on the adsorption mechanism assumptions of micropore filling and mesopore multilayer adsorption, the adsorbed phase densities of pores of varying pore sizes were calculated and applied to the conversion of the absolute adsorption amount of massive shale. The results show the existence of isolated pores in the massive shale, resulting in a lower adsorption capacity in comparison to granular samples. When subjected to the combined effects of in situ stress and pore pressure, the pore volume of massive shale gradually decreases with the increase in effective stress. Shale gas is mainly adsorbed in micropores, but with increasing pressure, the adsorption amount of micropores approaches saturation, and the contribution of mesopores to the total adsorption amount gradually increases. The main adsorption mechanism of shale gas is based on micropore filling, and the multilayer surface adsorption of mesopores should also be considered. By combining the simplified local density model and the Ono-Kondo lattice model, the adsorption behavior of shale gas can be accurately described. To accurately estimate shale gas reserves, it is necessary to take into account the actual pore size distribution, pore volume compressibility, and connected porosity of the shale samples.

Integrated mRNA and miRNA expression analyses for Cryptocaryon irritans resistance in large yellow croaker (Larimichthys crocea).

Large yellow croaker (Larimichthys crocea) is one of the most important mariculture fish in China. However, cryptocaryonosis caused by Cryptocryon irritans infection has brought huge economic losses and threatened the healthy and sustainable development of L. crocea industry. Recently, a new C. irritans resistance strain of L. crocea (RS) has been bred using genomic selection technology in our laboratory work. However, the molecular mechanisms for C. irritans resistance of RS have not been fully understood. MicroRNAs (miRNAs) are endogenous small non-coding RNAs that are post-transcriptional regulators, and they play vital roles in immune process of bony fish. Identification of anti-C.irritans relevant miRNA signatures could, therefore, be of tremendous translational value. In the present study, integrated mRNA and miRNA expression analysis was used to explore C. irritans resistance mechanisms of the L. crocea. RS as well as a control strain (CS) of L. crocea, were artificially infected with C. irritans for 100 h, and their gill was collected at 0 h (pre-infection), 24 h (initial infection), and 72 h (peak infection) time points. The total RNA from gill tissues was extracted and used for transcriptome sequencing and small RNA sequencing. After sequencing, 23,172 known mRNAs and 289 known miRNAs were identified. The differential expression was analyzed in these mRNAs and mRNAs and the interactions of miRNA-mRNA pairs were constructed. KEGG pathway enrichment analyses showed that these putative target mRNAs of differentially expressed miRNAs (DEMs) were enriched in different immune-related pathways after C. irritans infection in RS and CS. Among them, necroptosis was the immune-related pathway that was only significantly enriched at two infection stages of RS group (RS-24 h/RS-0h and RS-72 h/RS-0h). Further investigation indicates that necroptosis may be activated by DEMs such as miR-133a-3p, miR-142a-3p and miR-135c, this promotes inflammation responses and pathogen elimination. These DEMs were selected as miRNAs that could potentially regulate the C. irritans resistance of L. crocea. Though these inferences need to be further verified, these findings will be helpful for the research of the molecular mechanism of C. irritans resistance of L. crocea and miRNA-assisted molecular breeding of aquatic animals.

Quality assessment of commercial dried ginger (Zingiber officinale Roscoe) based on targeted and non-targeted chemical profiles and anti-inflammatory activity.

Dried ginger, a well-known medicine and food homologous production, has been widely circulated in China with high health benefits and economic value. Currently, there is still a lack of quality assessment on whether dried ginger in China exhibits chemically and biologically distinct properties, which creates a barrier to its quality control in commercial circulation. In this study, the chemical characteristics of 34 batches of common dried ginger samples in China were first explored using non-targeted chemometrics based on the UPLC-Q/TOF-MS analysis, leading to the identification of 35 chemicals that contributed to clustering into two categories, with sulfonated conjugates being the key chemically distinct components. By comparing the samples before and after sulfur-containing treatment and the further synthesis of a key differentiating component of [6]-gingesulfonic acid, it was then demonstrated that sulfur-containing treatment was the primary cause of the formation of sulfonated conjugates, as opposed to regional or environmental factors. Furthermore, the anti-inflammatory efficacy of dried ginger with high presence of sulfonated conjugates was significantly decreased. Consequently, for the first time, UPLC-QqQ-MS/MS was used to develop a targeted quantification method for 10 characteristic chemicals in dried ginger, allowing researchers to quickly determine whether dried ginger has been processed with sulfur and quantitatively evaluate the quality of dried ginger. These results provided an insight into the quality of commercial dried ginger in China and a suggested method for its quality supervision as well.

Association of Shunt Size and Long-Term Clinical Outcomes in Patients with Cryptogenic Ischemic Stroke and Patent Foramen Ovale on Medical Management.

INTRODUCTION: Patent foramen ovale (PFO) is a potential source of cardiac embolism in cryptogenic ischemic stroke, but it may also be incidental. Right-to-left shunt (RLS) size may predict PFO-related stroke, but results have been controversial. In this cohort study of medically-managed PFO patients with cryptogenic stroke, we aimed to investigate the association of shunt size with recurrent stroke, mortality, newly detected atrial fibrillation (AF), and to identify predictors of recurrent stroke. METHODS: Patients with cryptogenic stroke who screened positive for a RLS using a transcranial Doppler bubble study were included. Patients who underwent PFO closure were excluded. Subjects were divided into two groups: small (Spencer Grade 1, 2, or 3; n = 135) and large (Spencer Grade 4 or 5; n = 99) shunts. The primary outcome was risk of recurrent stroke, and the secondary outcomes were all-cause mortality and newly detected AF. RESULTS: The study cohort included 234 cryptogenic stroke patients with medically-managed PFO. The mean age was 50.5 years, and 31.2% were female. The median period of follow-up was 348 (IQR 147-1096) days. The rate of recurrent ischemic stroke was higher in patients with large shunts than in those with small shunts (8.1% vs. 2.2%, p = 0.036). Multivariate analyses revealed that a large shunt was significantly associated with an increased risk of recurrent ischemic stroke [aOR 4.09 (95% CI 1.04-16.0), p = 0.043]. CONCLUSIONS: In our cohort of cryptogenic stroke patients with medically managed PFOs, those with large shunts were at a higher risk of recurrent stroke events, independently of RoPE score and left atrium diameter.

Engineered MED12 mutations drive uterine fibroid-like transcriptional and metabolic programs by altering the 3D genome compartmentalization.

Uterine fibroid (UF) tumors originate from a mutated smooth muscle cell (SMC). Nearly 70% of these tumors are driven by hotspot recurrent somatic mutations in the MED12 gene; however, there are no tractable genetic models to study the biology of UF tumors because, under culture conditions, the non-mutant fibroblasts outgrow the mutant SMC cells, resulting in the conversion of the population to WT phenotype. The lack of faithful cellular models hampered our ability to delineate the molecular pathways downstream of MED12 mutations and identify therapeutics that may selectively target the mutant cells. To overcome this challenge, we employed CRISPR knock-in with a sensitive PCR-based screening strategy to precisely engineer cells with mutant MED12 Gly44, which constitutes 50% of MED12 exon two mutations. Critically, the engineered myometrial SMC cells recapitulate several UF-like cellular, transcriptional and metabolic alterations, including enhanced proliferation rates in 3D spheres and altered Tryptophan/kynurenine metabolism. Our transcriptomic analysis supported by DNA synthesis tracking reveals that MED12 mutant cells, like UF tumors, have heightened expression of DNA repair genes but reduced DNA synthesis rates. Consequently, these cells accumulate significantly higher rates of DNA damage and are selectively more sensitive to common DNA-damaging chemotherapy, indicating mutation-specific and therapeutically relevant vulnerabilities. Our high-resolution 3D chromatin interaction analysis demonstrates that the engineered MED12 mutations drive aberrant genomic activity due to a genome-wide chromatin compartmentalization switch. These findings indicate that the engineered cellular model faithfully models key features of UF tumors and provides a novel platform for the broader scientific community to characterize genomics of recurrent MED12 mutations and discover potential therapeutic targets.

Cytochrome P450 metabolism studies of [6]-gingerol, [8]-gingerol, and [10]-gingerol by liver microsomes of humans and different species combined with expressed CYP enzymes.

Gingerols, mainly [6]-gingerol (6G), [8]-gingerol (8G), and [10]-gingerol (10G), are the functional and specific pungent phytochemicals in ginger. However, poor oral bioavailability limits their applications owing to extensive metabolism. The present study aims to characterize the cytochrome P450 (CYP) metabolic characteristics of 6G, 8G, and 10G by using pooled human liver microsomes (HLM), different animal liver microsomes, and the expressed CYP enzymes. It is shown that NADPH-dependent oxidation and hydrogenation metabolisms of gingerols are the main metabolic types in HLM. With the increase of the carbon chain, the polarity of gingerols decreases and the formation of hydrogenated metabolites is more efficient (CLint: 1.41 µL min-1 mg-1 for 6G, 7.79 µL min-1 mg-1 for 8G and 14.11 µL min-1 mg-1 for 10G), indicating that the phase I metabolism of gingerols by HLM varied with the chemical structure of the substrate. The phase I metabolism of gingerols revealed considerable species variations, and compared to HLM, novel metabolites such as (3S,5S)-gingerdiols and demethylated metabolites are generated in some animal liver microsomes. The primary enzymes involved in the oxidized and demethylated metabolism of these gingerols are CYP1A2 and CYP2C19, but their affinities for gingerols are not the same. CYP2D6 and CYP2B6 contributed significantly to the formation of (3R,5S)-[8]-gingerdiol and (3R,5S)-[10]-gingerdiol, respectively; however, the enzyme responsible for the production of (3R,5S)-[6]-gingerediol is yet to be identified. Some metabolites in microsomes cannot be detected by the 12 investigated CYP enzymes, which may be related to the combined effects of multiple enzymes in microsomes, the different affinity of mixed liver microsomes and CYP enzymes, gene polymorphisms, etc. Overall, this work provides a deeper knowledge of the influence of CYP metabolism on the gingerols, as well as the mode of action and the possibility for drug-herbal interactions.