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Studies have shown that maternal exposure to PM2.5 could potentially disrupt glucose and lipid metabolism in offspring supplied with high-fat diet, yet whether this effect is gender-dependent or not and the underlying biological mechanisms are not well understood. In our current research, female ICR mice were exposed to filtered air (FA) or concentrated ambient PM2.5 (CAP) before and during pregnancy. The offspring mice were fed with control diet (CD) or high-fat diet (HFD) for 9 weeks, and their metabolic conditions were analyzed. Our findings reveal that maternal exposure to PM2.5 induced glucose intolerance and insulin resistance in female offspring fed with HFD but not in males. Specifically, hepatic insulin resistance as indicated by significantly decreased AKT phosphorylation (p-AKT) level, changed liver structure as indicated by increased ballooning and steatosis based on H&E staining images, and impaired liver function as indicated by up-regulated ALT activity were observed in HFD-fed female offspring from CAP-exposed mothers in comparison to those from FA-exposed ones. Further analysis indicated that these impacts of prenatal PM2.5 exposure on glucose metabolism in offspring may result from disturbed gluconeogenesis and induced inflammatory response in liver. Our research underscores that prenatal PM2.5 exposure induces glucose metabolism abnormalities in offspring fed with HFD in a gender-dependent manner, and the liver potentially serves as a key player in mediating these effects of maternal PM2.5 exposure.

As an effective method to prolong the life of mature field, conformance control in water-injection well has been used wildly. Naturally, effect evaluation of conformance control has attracted great attention because it is an important guideline for the design of later enhanced oil recovery (EOR) plan. Usually, production responses such as excessive water reduction and oil production increment are widely used as the indicators. However, production responses may be unreliable due to the difficulty in determining an effective injection well which is caused by a large number of treated water-injection wells in a well group. Therefore, with the application of fuzzy comprehension evaluation (FCE), five evaluation indexes (injection pressure, injectivity index, slope of hall curve, variation coefficient and homogenization coefficient of injection profile) describe injection responses were selected to establish a new evaluation method in this paper. Based on fuzzy mathematics, FCE reflects the difference of evaluation units. Meanwhile, weights of evaluation indexes were obtained by analytic hierarchy process (AHP), which made the results more convincing. Taking Bai 239 oilfield as an example, the five injection responses indexes were used to assess treatment effect on five water-injection wells by single index evaluation and FCE. The results showed that among the five evaluation indexes mentioned above, the slope of hall curve was the most important factor affected evaluation result. In single index evaluation, opposite results may be produced easily on account of the one-sidedness of single index or human error. Furthermore, we found that effective treatment was a relative concept actually. The result of FCE was consistent with single index evaluation but FCE was more acceptable. This study suggests that FCE could be applied to another field such as water flooding, acidizing and hydraulic fracturing

Abstract HELLS/LSH (Helicase, Lymphoid Specific) is a SNF2-like chromatin remodelling protein involved in DNA methylation. Its loss-of-function in humans causes humoral immunodeficiency, called ICF4 syndrome (Immunodeficiency, Centromeric Instability, Facial anomalies). Here we show by our newly generated B-cell-specific Hells conditional knockout mouse model that HELLS plays a pivotal role in T-dependent B-cell responses. HELLS deficiency induces accelerated decay of germinal center (GC) B cells and impairs the generation of high affinity memory B cells and circulating antibodies. Mutant GC B cells undergo dramatic DNA hypomethylation and massive de-repression of evolutionary recent retrotransposons, which surprisingly does not directly affect their survival. Instead, they prematurely upregulate either memory B cell markers or the transcription factor ATF4, which is driving an mTORC1-dependent metabolic program typical of plasma cells. Treatment of wild type mice with a DNMT1-specific inhibitor phenocopies the accelerated kinetics, thus pointing towards DNA-methylation maintenance by HELLS being a crucial mechanism to fine-tune the GC transcriptional program and enable long-lasting humoral immunity.

As confusion mounts over RNA isoforms involved in phenotypic plasticity, aberrant CpG methylation-mediated disruption of alternative splicing is increasingly recognized as a driver of intratumor heterogeneity (ITH). Protease serine 3 (PRSS3), possessing four splice variants (PRSS3-SVs; PRSS3-V1–V4), is an indispensable trypsin that shows paradoxical effects on cancer development. Here, we found that PRSS3 transcripts and their isoforms were divergently expressed in lung cancer, exhibiting opposing functions and clinical outcomes, namely, oncogenic PRSS3-V1 and PRSS3-V2 versus tumor-suppressive PRSS3-V3, by targeting different downstream genes. We identified an intragenic CpG island (iCpGI) in PRSS3. Hypermethylation of iCpGI was mediated by UHRF1/DNMT1 complex interference with the binding of myeloid zinc finger 1 (MZF1) to regulate PRSS3 transcription. The garlic-derived compound diallyl trisulfide cooperated with 5-aza-2′-deoxycytidine to exert antitumor effects in lung adenocarcinoma cells through site-specific iCpGI demethylation specifically allowing MZF1 to upregulate PRSS3-V3 expression. Epigenetic silencing of PRSS3-V3 via iCpGI methylation (iCpGIm) in BALF and tumor tissues was associated with early clinical progression in patients with lung cancer but not in those with squamous cell carcinoma or inflammatory disease. Thus, UHRF1/DNMT1–MZF1 axis-modulated site-specific iCpGIm regulates divergent expression of PRSS3-SVs, conferring nongenetic functional ITH, with implications for early detection of lung cancer and targeted therapies.

LSH/HELLS is a chromatin remodeler promoting incorporation of histone variant macroH2A. Here the authors reveal a role for LSH in genome stability, in protecting nascent DNA at stalled forks mediated by macroH2A deposition and RAD51 filament formation.

ObjectivesAcute kidney injury (AKI) is a serious complication of acute hemorrhagic stroke (AHS). Early detection and early treatment are crucial for patients with AKI. We conducted a study to analyze the role of the monocyte-to-lymphocyte ratio (MLR) in predicting the development of AKI after AHS.MethodsThis retrospective observational study enrolled all subjects with AHS who attended the neurosurgical intensive care unit (NSICU) at the First Affiliated University of South China between 2018 and 2021. Patient demographics, laboratory data, treatment details, and clinical outcomes were recorded.ResultsOf the 771 enrolled patients, 180 (23.3%) patients developed AKI. Compared to patients without AKI, those with AKI had a higher MLR and the neutrophil-lymphocyte ratio (NLR) at admission (P < 0.001). The MLR and the NLR at admission were associated with an increased AKI risk, with odds ratios (ORs) of 8.27 (95% CI: 4.23, 16.17, p < 0.001) and 1.17 (95% CI: 1.12, 1.22, p < 0.001), respectively. The receiver operating characteristic curve (ROC) analysis was conducted to analyze the ability of the MLR and NLR to predict AKI, and the areas under the curve (AUCs) of the MLR and the NLR were 0.73 (95% CI: 0.69, 0.77, p < 0.001) and 0.67 (95% CI: 0.62, 0.72, p < 0.001), with optimal cutoff values of 0.5556 and 11.65, respectively. The MLR and the NLR at admission were associated with an increased in-hospital mortality risk, with ORs of 3.13 (95% CI: 1.08, 9.04) and 1.07 (95% CI: 1.00, 1.14), respectively. The AUCs of the MLR and the NLR for predicting in-hospital mortality were 0.62 (95% CI: 0.54, 0.71, p = 0.004) and 0.52 (95% CI: 0.43, 0.62, p = 0.568), respectively. The optimal cutoff value for the MLR was 0.7059, with a sensitivity of 51% and a specificity of 73.3%.ConclusionsMLR and NLR measurements in patients with AHS at admission could be valuable tools for identifying patients at high risk of early AKI. The MLR was positively associated with in-hospital mortality and the NLR showed a weak ability for the prediction of in-hospital mortality.

The human ICF 4 syndrome is caused by mutation of the chromatin remodeller LSH. Here, the authors show that LSH depletion disrupts the ability of histone variant macroH2A to insert into chromatin and silence transcription.

BackgroundHepatocellular carcinoma (HCC) is one of the most lethal human tumors with extensive intratumor heterogeneity (ITH). Serine protease 3 (PRSS3) is an indispensable member of the trypsin family and has been implicated in the pathogenesis of several malignancies, including HCC. However, the paradoxical effects of PRSS3 on carcinogenesis due to an unclear molecular basis impede the utilization of its biomarker potential. We hereby explored the contribution of PRSS3 transcripts to tumor functional heterogeneity by systematically dissecting the expression of four known splice variants of PRSS3 (PRSS3-SVs, V1~V4) and their functional relevance to HCC.MethodsThe expression and DNA methylation of PRSS3 transcripts and their associated clinical relevance in HCC were analyzed using several publicly available datasets and validated using qPCR-based assays. Functional experiments were performed in gain- and loss-of-function cell models, in which PRSS3 transcript constructs were separately transfected after deleting PRSS3 expression by CRISPR/Cas9 editing.ResultsPRSS3 was aberrantly differentially expressed toward bipolarity from very low (PRSS3Low) to very high (PRSS3High) expression across HCC cell lines and tissues. This was attributable to the disruption of PRSS3-SVs, in which PRSS3-V2 and/or PRSS3-V1 were dominant transcripts leading to PRSS3 expression, whereas PRSS3-V3 and -V4 were rarely or minimally expressed. The expression of PRSS3-V2 or -V1 was inversely associated with site-specific CpG methylation at the PRSS3 promoter region that distinguished HCC cells and tissues phenotypically between hypermethylated low-expression (mPRSS3-SVLow) and hypomethylated high-expression (umPRSS3-SVHigh) groups. PRSS3-SVs displayed distinct functions from oncogenic PRSS3-V2 to tumor-suppressive PRSS3-V1, -V3 or PRSS3-V4 in HCC cells. Clinically, aberrant expression of PRSS3-SVs was translated into divergent relevance in patients with HCC, in which significant epigenetic downregulation of PRSS3-V2 was seen in early HCC and was associated with favorable patient outcome.ConclusionsThese results provide the first evidence for the transcriptional and functional characterization of PRSS3 transcripts in HCC. Aberrant expression of divergent PRSS3-SVs disrupted by site-specific CpG methylation may integrate the effects of oncogenic PRSS3-V2 and tumor-suppressive PRSS3-V1, resulting in the molecular diversity and functional plasticity of PRSS3 in HCC. Dysregulated expression of PRSS3-V2 by site-specific CpG methylation may have potential diagnostic value for patients with early HCC.

Dynamic regulation of chromatin accessibility is a key feature of cellular differentiation during embryogenesis, but the precise factors that control access to chromatin remain largely unknown. Lsh/HELLS is critical for normal development and mutations of Lsh in human cause the ICF (Immune deficiency, Centromeric instability, Facial anomalies) syndrome, a severe immune disorder with multiple organ deficiencies. We report here that Lsh, previously known to regulate DNA methylation level, has a genome wide chromatin remodeling function. Using micrococcal nuclease (MNase)-seq analysis, we demonstrate that Lsh protects MNase accessibility at transcriptional regulatory regions characterized by DNase I hypersensitivity and certain histone 3 (H3) tail modifications associated with enhancers. Using an auxin-inducible degron system, allowing proteolytical degradation of Lsh, we show that Lsh mediated changes in nucleosome occupancy are independent of DNA methylation level and are characterized by reduced H3 occupancy. While Lsh mediated nucleosome occupancy prevents binding sites for transcription factors in wild type cells, depletion of Lsh leads to an increase in binding of ectopically expressed tissue specific transcription factors to their respective binding sites. Our data suggests that Lsh mediated chromatin remodeling can modulate nucleosome positioning at a subset of putative enhancers contributing to the preservation of cellular identity through regulation of accessibility.

Lsh is a chromatin remodeling factor that regulates DNA methylation and chromatin function in mammals. The dynamics of these chromatin changes and whether they are directly controlled by Lsh remain unclear. To understand the molecular mechanisms of Lsh chromatin controlled regulation of gene expression, we established a tethering system that recruits a Gal4-Lsh fusion protein to an engineered Oct4 locus through Gal4 binding sites in murine embryonic stem (ES) cells. We examined the molecular epigenetic events induced by Lsh binding including: histone modification, DNA methylation and chromatin accessibility to determine nucleosome occupancy before and after embryonic stem cell differentiation. Our results indicate that Lsh assists gene repression upon binding to the Oct4 promoter region. Furthermore, we detected less chromatin accessibility and reduced active histone modifications at the tethered site in undifferentiated ES, while GFP reporter gene expression and DNA methylation patterns remained unchanged at this stage. Upon differentiation, association of Lsh promotes transcriptional repression of the reporter gene accompanied by the increase of repressive histone marks and a gain of DNA methylation at distal and proximal Oct4 enhancer sites. Taken together, this approach allowed us to examine Lsh mediated epigenetic regulation as a dynamic process and revealed chromatin accessibility changes as the primary consequence of Lsh function.

Most recently, a technology termed TRIM-Away has allowed acute and rapid destruction of endogenous target proteins in cultured cells using specific antibodies and endogenous/exogenous tripartite motif 21 (TRIM21). However, the relatively large size of the full-size mAbs (150 kDa) results in correspondingly low tissue penetration and inaccessibility of some sterically hindered epitopes, which limits the target protein degradation. In addition, exogenous introduction of TRIM21 may cause side effects for treated cells. To tackle these limitations, we sought to replace full-size mAbs with the smaller format of antibodies, a nanobody (VHH, 15 kDa), and construct a new type of fusion protein named TRIMbody by fusing the nanobody and RBCC motif of TRIM21. Next, we introduced enhanced green fluorescent protein (EGFP) as a model substrate and generated αEGFP TRIMbody using a bispecific anti-EGFP (αEGFP) nanobody. Remarkably, inducible expression of αEGFP TRIMbody could specifically degrade intracellular EGFP in HEK293T cells in a time-dependent manner. By treating cells with inhibitors, we found that intracellular EGFP degradation by αEGFP TRIMbody relies on both ubiquitin–proteasome and autophagy–lysosome pathways. Taken together, these results suggested that TRIMbody-Away technology could be utilized to specifically degrade intracellular protein and could expand the potential applications of degrader technologies.

Abstract Epigenetic mechanisms are known to exert control over gene expression and determine cell fate. Genetic mutations in epigenetic regulators are responsible for several neurologic disorders. Mutations of the chromatin remodeling protein Lsh/HELLS can cause the human Immunodeficiency, Centromere instability and Facial anomalies (ICF) syndrome, which is associated with neurologic deficiencies. We report here a critical role for Lsh in murine neural development. Lsh depleted neural stem/progenitor cells (NSPCs) display reduced growth, increases in apoptosis and impaired ability of self-renewal. RNA-seq analysis demonstrates differential gene expression in Lsh−/− NSPCs and suggests multiple aberrant pathways. Concentrating on specific genomic targets, we show that ablation of Lsh alters epigenetic states at specific enhancer regions of the key cell cycle regulator Cdkn1a and the stem cell regulator Bmp4 in NSPCs and alters their expression. These results suggest that Lsh exerts epigenetic regulation at key regulators of neural stem cell fate ensuring adequate NSPCs self-renewal and maintenance during development.

Mutation of HELLS (Helicase, Lymphoid-Specific)/Lsh in human DNA causes a severe immunodeficiency syndrome, but the nature of the defect remains unknown. We assessed here the role of Lsh in hematopoiesis using conditional Lsh knockout mice with expression of Mx1 or Vav Cre-recombinase. Bone marrow transplantation studies revealed that Lsh depletion in hematopoietic stem cells severely reduced B cell numbers and impaired B cell development in a hematopoietic cell-autonomous manner. Lsh-deficient mice without bone marrow transplantation exhibited lower Ig levels in vivo compared to controls despite normal peripheral B cell numbers. Purified B lymphocytes proliferated normally but produced less immunoglobulins in response to in vitro stimulation, indicating a reduced capacity to undergo class switch recombination (CSR). Analysis of germline transcripts, examination of double-stranded breaks using biotin-labeling DNA break assay, and End-seq analysis indicated that the initiation of the recombination process was unscathed. In contrast, digestion–circularization PCR analysis and high-throughput sequencing analyses of CSR junctions and a chromosomal break repair assay indicated an impaired ability of the canonical end-joining pathway in Lsh-deficient B cells. Our data suggest a hematopoietic cell-intrinsic role of Lsh in B cell development and in CSR providing a potential target for immunodeficiency therapy. [ABSTRACT FROM AUTHOR]

DNA methylation patterns are established in early embryogenesis and are critical for cellular differentiation. To investigate the role of CG methylation in potential enhancer formation, we assessed H3K4me1 modification in murine embryonic fibroblasts (MEFs) derived from the DNA methylation mutant Lsh-/- mice. We report here de novo formation of putative enhancer elements at CG hypomethylated sites that can be dynamically altered. We found a subset of differentially enriched H3K4me1 regions clustered at neuronal lineage genes and overlapping with known cis-regulatory elements present in brain tissue. Reprogramming of Lsh-/- MEFs into induced pluripotent stem (iPS) cells leads to increased neuronal lineage gene expression of premarked genes and enhanced differentiation potential of Lsh-/- iPS cells toward the neuronal lineage pathway compared with WT iPS cells in vitro and in vivo. The state of CG hypomethylation and H3K4me1 enrichment is partially maintained in Lsh-/- iPS cells. The acquisition of H3K27ac and activity of subcloned fragments in an enhancer reporter assay indicate functional activity of several of de novo H3K4me1-marked sequences. Our results suggest a functional link of H3K4me1 enrichment at CG hypomethylated sites, enhancer formation, and cellular plasticity. [ABSTRACT FROM AUTHOR]

Background: Interleukin 1 beta (IL-1β) plays an important role in a number of chronic and acute inflammatory diseases. To understand the role of IL-1β in disease processes and develop an in vivo screening system for anti-inflammatory drugs, a transgenic mouse line was generated which incorporated the transgene firefly luciferase gene driven by a 4.5-kb fragment of the human IL-1β gene promoter. Luciferase gene expression was monitored in live mice under anesthesia using bioluminescence imaging in a number of inflammatory disease models. Results: In a LPS-induced sepsis model, dramatic increase in luciferase activity was observed in the mice. This transgene induction was time dependent and correlated with an increase of endogenous IL-1β mRNA and pro-IL-1β protein levels in the mice. In a zymosan-induced arthritis model and an oxazolone-induced skin hypersensitivity reaction model, luciferase expression was locally induced in the zymosan injected knee joint and in the ear with oxazolone application, respectively. Dexamethasone suppressed the expression of luciferase gene both in the acute sepsis model and in the acute arthritis model. Conclusion: Our data suggest that the transgenic mice model could be used to study transcriptional regulation of the IL-1β gene expression in the inflammatory process and evaluation the effect of anti-inflammatory drug in vivo. [ABSTRACT FROM AUTHOR]