Somatic mutations of MLL4/COMPASS induce cytoplasmic localization providing molecular insight into cancer prognosis and treatment.

Cancer genome sequencing consortiums have recently catalogued an abundance of somatic mutations, across a wide range of human cancers, in the chromatin-modifying enzymes that regulate gene expression. Defining the molecular mechanisms underlying the potentially oncogenic functions of these epigenetic mutations could serve as the basis for precision medicine approaches to cancer therapy. MLL4 encoded by the KMT2D gene highly mutated in a large number of human cancers, is a key histone lysine monomethyltransferase within the Complex of Proteins Associated with Set1 (COMPASS) family that regulates gene expression through enhancer function, potentially functioning as a tumor suppressor. We report that the KMT2D mutations which cause MLL4 protein truncation also alter MLL4's subcellular localization, resulting in loss-of-function in the nucleus and gain-of-function in the cytoplasm. We demonstrate that isogenic correction of KMT2D truncation mutation rescues the aberrant localization phenotype and restores multiple regulatory functions of MLL4, including COMPASS integrity/stabilization, histone H3K4 mono-methylation, enhancer activation, and therefore transcriptional regulation. Moreover, isogenic correction diminishes the sensitivity of KMT2D-mutated cancer cells to targeted metabolic inhibition. Using immunohistochemistry, we identified that cytoplasmic MLL4 is unique to the tissue of bladder cancer patients with KMT2D truncation mutations. Using a preclinical carcinogen model of bladder cancer in mouse, we demonstrate that truncated cytoplasmic MLL4 predicts response to targeted metabolic inhibition therapy for bladder cancer and could be developed as a biomarker for KMT2D-mutated cancers. We also highlight the broader potential for prognosis, patient stratification and treatment decision-making based on KMT2D mutation status in MLL4 truncation-relevant diseases, including human cancers and Kabuki Syndrome.

Carbon-based nanozymes: design, catalytic mechanisms, and environmental applications.

Carbon-based nanozymes are synthetic nanomaterials that are predominantly constituted of carbon-based materials, which mimic the catalytic properties of natural enzymes, boasting features such as tunable catalytic activity, robust regenerative capacity, and exceptional stability. Due to the impressive enzymatic performance similar to various enzymes such as peroxidase, superoxide dismutase, and oxidase, they are widely used for detecting and degrading pollutants in the environment. This paper presents an exhaustive review of the fundamental design principles, catalytic mechanisms, and prospective applications of carbon-based nanozymes in the environmental field. These studies not only serve to augment the comprehension on the intricate operational mechanism inherent in these synthetic nanostructures, but also provide essential guidelines and illuminating perspectives for advancing their development and practical applications. Future studies that are imperative to delve into the untapped potential of carbon-based nanozymes within the environmental domain was needed to be explored to fully harness their ability to deliver broader and more impactful environmental preservation and management outcomes.

Significances of viable synergistic autophagy-associated cathepsin B and cathepsin D (CTSB/CTSD) as potential biomarkers for sudden cardiac death.

BACKGROUND: The Cathepsins family, including cathepsin B and cathepsin D, potentially affects the entire processes involved in atherosclerosis. Although coronary heart disease (CHD) has been widely studied as the basis of Sudden Cardiac Death (SCD), the relationship between CHD and CTSB/D remains unclear. METHODS: We screened for differentially expressed proteins (DEPs) associated with autophagy by limma package in R. For the genes corresponding to the DEPs after screening, we used various databases to carry out functional enrichment of related DEGs to explore their possible influence on a specific aspect of the disease. Functional enrichment analysis of DEGs was performed by DAVID, Metascape and GSEA. STRING and Cytoscape were obtained the hub genes, the analysis of interaction networks through the GENMANIA and Networkanalyst. Western Blot was used to validate the protein expression level of target genes. TF and miRNA prediction were performed using Networkanalyst and visualized using Cytoscape. RESULTS: The expression levels of members of the cathepsin family were up regulated in CHD tissues compared with the control. GO and KEGG revealed that cathepsin was markedly enriched in endopeptidase activities, immune responses, lysosome pathways, et al. The correlation analysis showed that in patients with CHD, the CTSB/CTSD expression were negatively correlated with ATG4D and BNIP3, but positively with BCL2L1, CAPNS1, and TP53. In the TF-mRNA-miRNA network, has-miR-24-3p and has-miR-128-3p had higher degrees, CTSB/CTSD could be targeted by them. CONCLUSIONS: Our findings elucidated the expression and regulatory role of cathepsins in coronary heart disease induced SCD and might further explore the potential mechanisms of autophagy in CHD.

Potential Role of Glycogen Synthase Kinase-3ß in Regulation of Myocardin Activity in Human Vascular Smooth Muscle Cells.

Glycogen synthase kinase (GSK)-3ß, a serine/threonine kinase with an inhibitory role in glycogen synthesis in hepatocytes and skeletal muscle, is also expressed in cardiac and smooth muscles. Inhibition of GSK-3ß results in cardiac hypertrophy through reducing phosphorylation and increasing transcriptional activity of myocardin, a transcriptional co-activator for serum response factor. Myocardin plays critical roles in differentiation of smooth muscle cells (SMCs). This study, therefore, aimed to examine whether and how inhibition of GSK-3ß regulates myocardin activity in human vascular SMCs. Treatment of SMCs with the GSK-3ß inhibitors AR-A014418 and TWS 119 significantly reduced endogenous myocardin activity, as indicated by lower expression of myocardin target genes (and gene products), CNN1 (calponin), TAGLN1 (SM22), and ACTA2 (SM α-actin). In human SMCs overexpressing myocardin through the T-REx system, treatment with either GSK-3ß inhibitor also inhibited the expression of CNN1, TAGLN1, and ACTA2. These effects of GSK-3ß inhibitors were mimicked by transfection with GSK-3ß siRNA. Notably, both AR-A014418 and TWS 119 decreased the serine/threonine phosphorylation of myocardin. The chromatin immunoprecipitation assay showed that AR-A014418 treatment reduced myocardin occupancy of the promoter of the myocardin target gene ACTA2. Overexpression of a dominant-negative GSK-3ß mutant in myocardin-overexpressing SMCs reduced the expression of calponin, SM22, and SM α-actin. As expected, overexpression of constitutively active or wild-type GSK-3ß in SMCs without myocardin overexpression increased expression of these proteins. In summary, our results indicate that inhibition of GSK-3ß reduces myocardin transcriptional activity, suggesting a role for GSK-3ß in myocardin transcriptional activity and smooth muscle differentiation.

Upregulated CDK16 Expression in Serous Epithelial Ovarian Cancer Cells.

BACKGROUND: As CDK-16 has been shown to be upregulated in several transformed cancer lines, we hypothesized that the cyclin-dependent kinase 16 (CDK-16) may be upregulated in serous epithelial ovarian cancer (EOC) cells. Therefore, we comparatively examined the mRNA and protein expression of CDK-16 in samples resected from serous EOC patients and normal controls. MATERIAL AND METHODS: Tissue samples were collected from 70 serous EOC patients and 40 normal controls. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was conducted to assess mRNA expression. CDK-16 protein expression was assessed by semi-quantitative immunohistochemical staining. Differences in mRNA and protein expression between serous EOC cells and normal tissue cells were tested with the Kruskal-Wallis test and analysis of variance (ANOVA). RESULTS: Both CDK-16 mRNA and protein expression were significantly higher in serous EOC tumor cells as compared to normal control ovarian cells (p<0.01). Although there was no significant correlation between CDK-16 mRNA expression and serous EOC stage (p=0.0794), there was a significant correlation between CDK-16 mRNA expression and serous EOC grade (p<0.0001). Moreover, there were significant correlations between CDK-16 protein expression and serous EOC stage (p<0.0001) and grade (p<0.0001). CONCLUSIONS: CDK-16 upregulation in serous EOC cells may represent a negative feedback loop to promote ovarian cell differentiation in malignantly-transformed serous EOC cells. Further in-depth investigation on CDK-16's role in serous EOC is needed.

Change in forced vital capacity and associated subsequent outcomes in patients with newly diagnosed idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a rare and serious disease characterized by progressive lung-function loss. Limited evidence has been published on the impact of lung-function loss on subsequent patient outcomes. This study examined change in forced vital capacity (FVC) across IPF patients in the 6 months after diagnosis and its association with clinical and healthcare resource utilization (HRU) outcomes in a real-world setting in the U.S. METHODS: A retrospective chart review was conducted of patients diagnosed with IPF by U.S. pulmonologists. Patient eligibility criteria included: 1) 40 years or older with a confirmed date of first IPF diagnosis with high-resolution computed tomography and/or lung biopsy between 01/2011 and 06/2013; 2) FVC results recorded at first diagnosis (±1 month) and at 6 months (±3 months) following diagnosis. Based on relative change in FVC percent predicted (FVC%), patients were categorized as stable (decline <5%), marginal decline (decline ≥5% and <10%), or significant decline (decline ≥10%). Physician-reported clinical and HRU outcomes were assessed from ~6 months post-diagnosis until the last contact date with the physician and compared between FVC% change groups. Multivariable Cox proportional-hazards models were constructed to assess risk of mortality, suspected acute exacerbation (AEx), and hospitalization post-FVC% change. Generalized estimating equations were used to account for multiple patients contributed by individual physicians. RESULTS: The sample included 490 IPF patients contributed by 168 pulmonologists. The mean (SD) age was 61 (11) years, 68% were male, and the mean (SD) baseline FVC% was 60% (26%). 250 (51%) patients were categorized as stable, 98 (20%) as marginal decline, and 142 (29%) as significant decline. The mean (SD) observation time was 583 (287) days. In both unadjusted analysis and multivariable models, significantly worse clinical outcomes and increased HRU were observed with greater lung-function decline. CONCLUSIONS: These findings suggest that nearly half of IPF patients experienced decline in FVC% within ~6 months following IPF diagnosis. Greater FVC% decline was associated with an increased risk of further IPF progression, suspected AEx, mortality, and higher rate of HRU. Management options that slow FVC decline may help improve future health outcomes in IPF.

[Impact of glycogen synthase kinase-3ß (GSK-3ß) inhibitor on Wnt and NF-κB signal pathways in a rat model of diabetic nephropathy].

OBJECTIVE: To explore the impact of glycogen synthase kinase-3ß (GSK-3ß) on Wnt and NF-κB pathways in a rat model of diabetic nephropathy (DN). METHODS: SD rats were randomly divided into normal control group (NC), DN model group (DM) and GSK-3ß inhibitor group (DI). Blood glucose and 24-hour urine protein were monitored in three groups. Renal tissue samples were stained by HE. The expression of GSK-3ß and NF-κB proteins was studied by immunohistochemistry. GSK-3ß and NF-κB mRNAs were detected by RT-qPCR. RESULTS: Ten weeks after STZ injection, the level of blood glucose increased significantly in DM group [(23.2±5.4) mmol/L] and DI group [(25.0±4.0) mmol/L], compared with NC group, and the level of 24-hour urinary protein increased significantly in DM group [(185.2±35.6) g/24 h] and DI group [(179.6±44.7) g/24 h], compared with NC group. Two weeks after LiCl injection, the level of blood glucose and 24-hour urinary protein decreased in DI group (17.6±2.1) mmol/L, (106.9±30.0) g/24 h], compared with DM Group. Compared with NC group, pathological changes of the kidney of DM group aggravated along with increased mRNA and protein expression of GSK-3ß and NF-κB. But the pathological changes of the kidney in DI group alleviated along with declined mRNA and protein expression of GSK-3ß and NF-κB as compared with DM group (all P<0.05). CONCLUSIONS: NF-κB protein expression positively correlates with the GSK3ß expression. Wnt and NF-κB signal pathways play an important role in the development of diabetic nephropathy.

Blockade of peanut allergy with a novel Ara h 2-Fcγ fusion protein in mice.

BACKGROUND: Conventional immunotherapy for peanut allergy using crude peanut extracts is not recommended because of the unacceptably high risk of anaphylaxis. Allergen-specific immunotherapy is not currently undertaken for peanut allergy. OBJECTIVES: The objective of this study was to develop a novel peanut-human fusion protein to block peanut-induced anaphylaxis. METHODS: We genetically designed and expressed a novel plant-human fusion protein composed of the major peanut allergen Ara h 2 and human IgG Fcγ1. We tested the Ara h 2-Fcγ fusion protein (AHG2)'s function in purified human basophils. Transgenic mice expressing human FcεRIα and a murine peanut allergy model were used. RESULTS: AHG2 inhibited histamine release induced by whole peanut extract (WPE) from basophils of patients with peanut allergy, whereas the fusion protein itself did not induce mediator release. AHG2 inhibited the WPE-induced, peanut-specific, IgE-mediated passive cutaneous anaphylaxis in hFcεRIα transgenic mice. AHG2 also significantly inhibited acute anaphylactic reactivity, including the prototypical decrease in body temperature in WPE-sensitized mice challenged with crude peanut extract. Histologic evaluation of the airways showed that AHG2 decreased peanut-induced inflammation, whereas the fusion protein itself did not induce airway inflammation in peanut-sensitized mice. AHG2 did not exert an inhibitory effect in mice lacking FcγRII. CONCLUSION: AHG2 inhibited peanut-specific IgE-mediated allergic reactions in vitro and in vivo. Linking specific peanut allergen to Fcγ can provide a new approach for the allergen immunotherapy of peanut allergy.

[Expression of sonic hedgehog signaling pathw ay and its inhibition by cyclopamine in rat liver with chronic fluorosis].

OBJECTIVE: To investigate the expression of sonic hedgehog (Shh) signaling pathway in liver fluorosis and to explore related mechanism. METHODS: To establish animal model, 48 normal SD rats (aged 4-5 weeks) were randomly divided into 4 groups (12 each): control group, fluoriosis group, blocking group and blocking control group. After 6 months, the blocking group and blocking control group were injected intraperitoneally once every 2 days for 3 times with 10 mg/kg cyclopamine or dimethysulfoxide, respectively. Rats were sacrificed at the end of the experiment and the fluoride content in urine and liver function was determined. The expression of Shh and Gli1 protein and mRNA in hepatocytes was detected by immunohistochemistry and real-time fluorescence quantitative PCR, respectively. RESULTS: The fluoride contents in the urine and the incidence of dental fluorosis increased in the fluoride and blocking control groups as compared with those in the control group, but decreased in the blocking group compared with those of the fluoride and blocking control group. Compared with the control group, the titers of aspartate transaminase (AST) and alanine transaminase (ALT) significantly increased, while the activity of total protein and albumin decreased in the fluoride and blocking control groups. Compared with the fluoride and blocking control groups, the activity of the ALT slightly declined and the AST, total protein and albumin slightly increased in the blocking group. Histologically, the cells were disorganized and swollen with cytoplasmic clearing (balloon cells), compared with the control group. The expression of Shh and Gli1 significantly increased in all but the control group. Compared with the fluoride and blocking control groups, the expression of Shh and Gli1 declined in the blocking group. CONCLUSIONS: The overexpression and cyclopamine inhibition of the Shh signaling pathway are closely related to the content of fluoride in the liver. The Shh signaling pathway plays an important role in the pathogenesis of liver injury caused by fluorosis, suggesting a preventive and therapeutic target of the disease.

Spatial comparison of molecular features associated with resistance to pembrolizumab in BCG unresponsive bladder cancer.

Intravenous immune checkpoint inhibition achieves a 40% 3-month response in BCG-unresponsive non-muscle invasive bladder cancer (NMIBC) with carcinoma in situ. Yet, only half of the early responders will continue to be disease-free by 12 months, and resistance mechanisms are poorly defined. We performed spatial profiling of BCG-unresponsive tumors from patients responsive or resistant to intravenous pembrolizumab treatment, analyzing samples both before initiating and 3 months post-intravenous pembrolizumab treatment. We analyzed 119 regions of interest, which included 59 pairs of epithelial and adjacent stromal segments across five patients: two responders and three non-responders. We demonstrate that BCG unresponsive tumors with an inflamed PanCK+ tumor area and an infiltrated stromal segment respond better to intravenous pembrolizumab. Furthermore, using segment-specific gene signatures generated from a cohort of BCG unresponsive NMIBC treated with intravesical BCG+pembrolizumab, we find that non-inflamed, immune-cold tumors that do not respond to intravenous pembrolizumab exhibit a favorable outcome to the combined application of BCG and pembrolizumab. For the first time, we have identified molecular features of tumors associated with response and resistance to intravenous pembrolizumab in BCG unresponsive NMIBCs. Further research with more patients and alternative checkpoint inhibitors is essential to validate our findings. We anticipate that using a transcriptomics signature like the one described here can help identify tumors with a higher possibility of responding to intravenous pembrolizumab.

[Expression of calcineurin and nuclear factor of activated T cells 1 in testis of rats with chronic fluorosis].

OBJECTIVE: To discuss the significance of calcineurin (CaN) and nuclear factor of active T cells 1 (NFATc1) in the damage mechanism of the testis of rats with chronic fluorosis. METHODS: Eighteen clear class SD male rats, aging 6 week-old, were randomly divided into 3 groups, 6 rats in each. The rats of control group were fed with tap water (NaF < 1 mg/L) and the experimental rats were exposed to NaF (lower group: 5 mg/L, higher group: 50 mg/L) to established the chronic fluorosis model. After 8 months, we observed the occurrence of dental fluorosis among rats in different groups, and the contents of urine fluoride were detected by fluorine ion selective electrode method. The body of the rats were weighted as well as their testis. The testis tissues were stained with hematoxylin-eosin and observed under light microscope to find the morphological changes. The expression of CaN and NFATc1's protein and mRNA in testis were detected by Immunocytochemistry (IHC) and In-situ hybridization (ISH). RESULTS: The number of rats which was found dental fluorosis were separately 0, 4 and 5 in control group, low dose group and high dose group (χ(2) = 10.60, P < 0.05). The contents of urine fluoride were gradually increased in control group, low group and high group, which were (1.26 ± 0.17), (2.06 ± 0.64) and (7.69 ± 1.96)mg/L, respectively (F = 36.57, P < 0.05). The body weight were significantly different in all three groups(629.00 ± 16.00), (585.17 ± 17.27), (560.50 ± 16.07)g, F = 26.67, P < 0.05) and the testis weight were without statistical difference ((2.58 ± 0.17), (2.43 ± 0.31), (2.35 ± 0.38)g, F = 0.91, P > 0.05). Compared with the control group, the testicular structures were damaged in the experimental groups and especially significant in high dose group. The expression of CaN (59.10 ± 5.62, 77.93 ± 4.16, 101.69 ± 6.31, F = 74.18, P < 0.05) and NFATc1's (76.11 ± 4.41, 93.42 ± 3.85, 120.42 ± 9.31, F = 92.4, P < 0.05) protein in testis tissues were increased by the fluorine concentration. The mRNA expression of CaN and NFATc1 were separately (CaN: 58.76 ± 7.70, 82.01 ± 6.88, 99.47 ± 8.33, F = 42.65, P < 0.05 and NFATc1: 59.39 ± 4.74, 90.02 ± 5.37, 121.15 ± 7.69, F = 155.47, P < 0.05). There were positive correlation between the expression of CaN and NFATc1's protein and mRNA expression (r = 0.899, r = 0.908). CONCLUSION: The changes in the signaling pathway of expression of CaN may be involved in the injury mechanism of testis tissues of rats with chronic fluorosis.

[Changed expression of mito-fusion 1 and mitochondrial fragmentation in the cortical neurons of rats with chronic fluorosis].

OBJECTIVE: To observe the mitochondrial fragmentation and the expression of mito-fusion 1 gene in the cortical neurons of rats with chronic fluorosis, and to reveal their roles in mitochondria damage to neurons due to chronic fluorosis. METHODS: SD rats were divided randomly into three groups of 20 each (a half females and a half males housed individually in stainless-steel cages), and fed with the different doses of fluoride containing in drinking water (untreated control containing 0 mg/L fluoride, and low-fluoride and high supplemented with 10 and 50 mg/L fluoride, respectively). After 3 or 6 months exposure, the mitochondrial morphology of the neurons in rat brains were observed by transmission electron microscopy (TEM), then the expression of mitochondrial fusion gene, Mfn1, were detected by immunohistochemistry and western-blotting, respectively. RESULTS: Dental fluorosis was obvious in the rats exposed to excessive fluoride in their drinking water, that is, (16 rats out of 20) numbers of I° detal fluorosis in the low-fluoride group, and (11 rats out of 20) numbers of I° and (9 rats out of 20) numbers of II° detal fluorosis in the high-fluoride group were observed after 3 months exposure. Moreover, (14 rats out of 20) numbers of I° and (6 rats out of 20) numbers of II° detal fluorosis in the low-fluoride group and (6 rats out of 20) numbers of Io, (13 rats out of 20) numbers of II°, and (1 rats out of 20) numbers of III° detal fluorosis in the high-fluoride group were observed after 6 months exposure. And both of untreated controls without detal fluorosis were also observed. The urinary level of fluoride in the low-fluoride group (3.30 ± 1.18) mg/L and in the high-fluoride group (5.10 ± 0.35) were observed after 3 months exposure (F = 3.18, P < 0.05). Moreover, the urinary level of fluoride in the low-fluoride group (4.16 ± 1.39) mg/L and in the high-fluoride group (5.70 ± 1.70) mg/L were also observed after 6 months exposure (F = 3.17, P < 0.05). The normal mitochondrial morphology of neurons in rats without fluorosis was observed after 3 and 6 months, while the abnormal mitochondrial morphology of neurons with fluorosis was shown, presenting mitochondrial fragmentation with swollen cristae and even the fragmented, shortened or stacked punctuate membranes (section observation of three bullous mitochondrial-mitochondrial fission process) by TEM. As compared with controls (53.0 ± 4.54 and 1.21 ± 0.18) at the experiment period of 3 months, Mif1 protein analysis with immunocytochemical (the numbers of positive cells: 51.09 ± 6.25) and western-blotting (1.22 ± 0.26) were no significant difference for low fluoride group (t = 1.7, 1.1, P > 0.05); Mif1 protein analysis with immunocytochemical (the numbers of positive cells: 59.71 ± 5.64) and western-blotting (1.66 ± 0.20) were significantly increasing for high fluoride group (t = 2.1, 2.1, P < 0.05). As compared with controls (36.43 ± 4.04 and 1.00 ± 0.13) at the experiment period of 6 months, Mif1 protein analysis with immunocytochemical (the numbers of positive cells 20.05 ± 4.55 and 17.10 ± 3.86) and western-blotting (0.64 ± 0.08 and 0.39 ± 0.06) were significantly decreasing for the two fluoride group (t = 2.1, 2.2; 2.2, 2.2 respectively, all P value were < 0.05). CONCLUSIONS: Taking excessive amount of fluoride might result in the mitochondrial fragmentation for the changed expression of Mfn1, and the neurons damage from the chronic fluorosis might be associated with the dysfunction of mitochondrial fusion.

[Influence of chronic fluorosis on the expression of mitochondrial fission protein dynamin-related 1 in the cortical neurons of rats].

OBJECTIVE: To explore the changes of protein expression of mitochondrial fission gene dynamin-related 1(Drp 1) in the cortical neurons of rats with chronic fluorosis. METHODS: A total of 120 one-month-old SD rats (each weighing approximately 100-120 g at the beginning of the experiment) were randomly divided into three groups, and fed with the different doses of fluoride containing in drinking water (untreated control containing 0 mg/L fluoride, and low-fluoride & high-fluoride supplemented with 10 and 50 mg/L fluoride,respectively). After 3 or 6 months exposure, 20 rats from each group were killed. Then the protein expression of mitochondrial fission gene, Drp1, was detected by immunohistochemistry and western-blotting method. RESULTS: Dental fluorosis and urinary fluorosis were obviously found in the rats exposed to fluoride. At the experiment period of 3 months, the numbers of positive cells of Drp1 detected by immunohistochemistry changed. Compared with the control group (36.3 ± 5.8), the changes in low-fluoride group (34.7 ± 4.1) showed no significant difference (t = 1.5, P > 0.05),but the increase in high-fluoride group (45.0 ± 4.7) had statistical significance (t = 8.8, P < 0.05). The western-blotting method had consistent results. Compared with the control group (0.59 ± 0.03), a significant increase of the average topical density in low- fluoride (0.62 ± 0.03) and high-fluoride (0.71 ± 0.02) groups were found (t = 0.02,0.11, P < 0.05). At the experiment period of 6 months, the numbers of positive cells of Drp1 detected by immunohistochemistry significantly changed. Compared with the control group (33.2 ± 4.4), the number in low- fluoride and high-fluoride groups were separately (36.6 ± 3.8) and (39.4 ± 4.2),both increased significantly (t = 3.5,6.3, P < 0.05). Same results could be found in western-blotting method,compared with the control group (0.65 ± 0.06), the average topical density in low- fluoride (0.80 ± 0.09) and high-fluoride (0.76 ± 0.08) groups both increased significantly (t = 0.1,0.1, P < 0.05). CONCLUSIONS: Taking excessive amount of fluoride might result in the changes of expression of Drp1, and the neurons damage from the chronic fluorosis might be associated with the hyperfunction of mitochondrial fusion.

Bio- and eco-corona related to plants: Understanding the formation and biological effects of plant protein coatings on nanoparticles.

The thriving nano-enabled agriculture facilitates the interaction of nanomaterials with plants. Recently, these interactions and their biological effects are receiving increasing attention. Upon entering plants via leaves, roots, stems, and other organs, nanoparticles adsorb numerous biomolecules inside plants and form bio-corona. In addition, nanoparticles that enter plants through roots may have formed eco-corona with root exudates in the rhizosphere environment before contacting with plant exogenous proteins. The most significant biological effects of plant protein corona include changes in protein structure and function, as well as changes in nanoparticle toxicity and targeting ability. However, the mechanisms, particularly how protein corona affects plant protein function, plant development and growth, and rhizosphere environment properties, require further investigation. Our review summarizes the current understanding of the formation and biological effects of nanoparticle-plant protein corona and provides an outlook on future research.

A Spatial Comparison of Molecular Features Associated with Resistance to Pembrolizumab in BCG Unresponsive Bladder Cancer.

Background: Intravenous immune checkpoint inhibition achieves a 40% three-month response in BCG-unresponsive non-muscle invasive bladder cancer (NMIBC) with carcinoma in situ (CIS). Yet only half of early responders will continue to be disease free by 12 months, and resistance mechanisms are poorly defined. Objective: We assessed the molecular features associated with response to immunotherapy in BCG unresponsive non-muscle invasive bladder cancers treated with pembrolizumab. Design Setting and Participants: We performed digital spatial profiling (DSP) of BCG unresponsive NMIBC tumors before and after IV pembrolizumab therapy. Intervention: Pembrolizumab was administered intravenously in patients with NMIBC at the time of recurrence after BCG therapy. Biopsies were obtained before starting IV pembrolizumab and three months post-treatment. Outcomes and Statistical Analysis: Spatial gene expression profiling of the tumor niche pre- and post IV pembrolizumab. Results and Limitations: We evaluated 119 regions of interest (ROIs) from five patients, which included 60 epithelial (PanCK+) and 59 stromal segments (PanCK-). ROIs from responders had distinct expression signatures from non-responders for both the tumor and TME. Responders were more likely to have a dynamic change in expression after pembrolizumab than non-responders. A major limitation of this study was the number of patients evaluated. Conclusion: For the first time, we have identified distinct expression signatures associated with response and resistance to IV pembrolizumab in NMIBCs. Further research with more patients and alternative checkpoint inhibitors is essential to validate our findings. Patient Summary: We identify the molecular features of tumors associated with response to pembrolizumab for patients with BCG unresponsive NMIBCs.

Fluoride induces osteoblast autophagy by inhibiting the PI3K/AKT/mTOR signaling pathway in vivo and in vitro.

Fluorosis primarily manifests as bone damage in the form of dental fluorosis and skeletal fluorosis and represents a critical global public health challenge. However, few studies have examined autophagy-related signaling pathways in skeletal fluorosis. This study aimed to investigate the effect of fluoride on autophagy in osteoblasts using comprehensive methods and to explore the role of the PI3K/AKT/mTOR signaling pathway in regulating fluoride-induced autophagy in osteoblasts. Sprague-Dawley (SD) rats were exposed to different concentrations of fluoride (NaF: 5, 50, and 100 mg/L) for six months. Primary osteoblasts were treated with 0.5, 1.0, or 3.0 mM NaF. Hematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), immunohistochemistry (IHC), immunofluorescence staining, and western blotting were performed to evaluate morphological changes in bone tissues and autophagosomes and to detect the protein expression of autophagy-related markers and PI3K/AKT/mTOR signaling pathway-related molecules both in vivo and in vitro. The bone tissues of fluoride-exposed rats showed osteosclerosis, autophagosomes and autolysosomes. LC3B immunofluorescence staining revealed an increase in autophagosomes in the primary osteoblasts treated with fluoride. The LC3Ⅱ/Ⅰ ratio and levels of autophagy-related markers (Beclin 1 and Atg7) were increased, whereas P62 levels were decreased in bone tissues and primary osteoblasts in the fluoride groups. Simultaneously, p-AKT and p-mTOR levels were reduced in bone tissues and primary osteoblasts in the fluoride groups. Moreover, a PI3K inhibitor (LY294002) further downregulated p-AKT and p-mTOR protein expression but slightly increased the LC3Ⅱ/Ⅰ ratio in primary osteoblasts. These results demonstrate that fluoride induces autophagy in osteoblasts by inhibiting the PI3K/AKT/mTOR signaling pathway, which deepens our understanding of the molecular mechanisms underlying fluoride-induced bone damage and provides a theoretical basis for the prevention and treatment of skeletal fluorosis.

[Formation and characteristics of polystyrene nanoplastic-plant protein corona].

To investigate the formation of polystyrene nanoplastic-plant protein corona and its potential impact on plants, three differently modified polystyrene nanoplastics with an average particle size of 200 nm were taken to interact with the leaf proteins of Impatiens hawkeri for 2 h, 4 h, 8 h, 16 h, 24 h, and 36 h, respectively. The morphological changes were observed by scanning electron microscopy (SEM), the surface roughness was determined by atomic force microscopy (AFM), the hydrated particle size and zeta potential were determined by nanoparticle size and zeta potential analyzer, and the protein composition of the protein corona was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The proteins were classified in terms of biological processes, cellular components, and molecular functions to study the adsorption selection of nanoplastics to proteins, investigate the formation and characteristics of polystyrene nanoplastic-plant protein corona and predict the potential impact of protein corona on plants. The results showed that the morphological changes of the nanoplastics became clearer as the reaction time extends, as evidenced by the increase in size and roughness and the enhancement of stability, thus demonstrating the formation of protein corona. In addition, the transformation rate from soft to hard protein corona was basically the same for the three polystyrene nanoplastics in the formation of protein corona with leaf proteins under the same protein concentration conditions. Moreover, in the reaction with leaf proteins, the selective adsorption of the three nanoplastics to proteins with different isoelectric points and molecular weights differed, and the particle size and stability of the final formed protein corona also differed. Since a large portion of the protein fraction in protein corona is involved in photosynthesis, it is hypothesized that the formation of the protein corona may affect photosynthesis in I. hawkeri.

Chronic Fluoride Exposure Induces Ovarian Dysfunction and Potential Association with Premature Ovarian Failure in Female Rats.

Chronic fluorosis has been widely investigated for its adverse effects on skeletal and neurological health; however, its impact on reproductive health, especially in females, remains underexplored. In this study, female Sprague-Dawley rats were exposed to different fluoride concentrations (0.75, 50, and 100 mg/L) in their drinking water for six months. Dental fluorosis and increased urinary fluoride content were observed in fluoride-exposed rats, reflecting fluoride accumulation and exposure levels. Chronic fluorosis resulted in reduced ovary organ coefficient, indicating harmful effects on ovarian tissue. Additionally, the number of ovarian primordial and primary/secondary follicles decreased, while the number of atresia follicles increased. Furthermore, chronic fluorosis led to disrupted estrous cycles. Hormonal analysis revealed altered secretion of estrogen, progesterone, anti-Müllerian hormone, luteinizing hormone, follicular stimulating hormone, and inhibin B in response to fluoride exposure. Ultrastructural observation of ovarian granulosa cells showed evidence of apoptosis, which was further confirmed by flow cytometry. Caspase-3 activity was increased, and ATP levels were decreased, suggesting mitochondrial impairment and apoptosis induction. The mRNA and protein expression of BMP15 and GDF9, essential regulators of ovarian function, significantly decreased with increasing fluoride concentration. Furthermore, gene expression analysis identified a panel of premature ovarian failure-related genes that were downregulated in fluoride-exposed rat ovaries. These findings suggest that chronic fluoride exposure may contribute to ovarian dysfunction and possibly the pathogenesis of premature ovarian failure. Understanding the toxicological effects of chronic fluoride exposure on ovarian function is essential for identifying potential environmental risk factors affecting female reproductive health.

Expression-based subtypes define pathologic response to neoadjuvant immune-checkpoint inhibitors in muscle-invasive bladder cancer.

Checkpoint immunotherapy (CPI) has increased survival for some patients with advanced-stage bladder cancer (BCa). However, most patients do not respond. Here, we characterized the tumor and immune microenvironment in pre- and post-treatment tumors from the PURE01 neoadjuvant pembrolizumab immunotherapy trial, using a consolidative approach that combined transcriptional and genetic profiling with digital spatial profiling. We identify five distinctive genetic and transcriptomic programs and validate these in an independent neoadjuvant CPI trial to identify the features of response or resistance to CPI. By modeling the regulatory network, we identify the histone demethylase KDM5B as a repressor of tumor immune signaling pathways in one resistant subtype (S1, Luminal-excluded) and demonstrate that inhibition of KDM5B enhances immunogenicity in FGFR3-mutated BCa cells. Our study identifies signatures associated with response to CPI that can be used to molecularly stratify patients and suggests therapeutic alternatives for subtypes with poor response to neoadjuvant immunotherapy.

Corn baked by burning coal triggered overexpression of osteopontin in hepatocytes of rats following fluorosis.

OBJECTIVE: To observe the expression of osteopontin (OPN) in hepatocytes of rats fed with corn baked by burning coal from fluorosis areas and a deficiency of calcium/protein intake following fluorosis. METHODS: A total of 48 Wistar rats as objects were randomly assorted into four groups: dose-free fluorine group, which were mainly fed with fluorine-free corn (56% structurally), dose-free fluorine with biased dietary group, which were fed with lower contents of protein (119.41 g/kg) and calcium (0.68 g/kg), high-dose fluorine group (fluorine contents: 104.2 mg/kg), and high-dose fluorine with biased dietary group. After 180 days of cultivation, the contents of fluorine in the bones of rats were tested for the assessment of construction of fluorosis animal model. And the expression of OPN in hepatocytes of rats in different groups was detected with immunohistochemistry and reverse transcription polymerase chain reaction. RESULTS: The present study validated the result that OPN was overexpressed in hepatocytes following fluorosis after oral intake of burning coal-baked corn. OPN was expressed most significantly in high fluorine with biased dietary group, and the high-fluorine group ranked the second most; and dose-free fluorine with biased dietary group ranked the third. The dose-free fluorine group expressed the least OPN. CONCLUSION: Overexpression of OPN in hepatocytes following fluorosis after excess fluorine intake was involved in liver damage process, which was enhanced by deficiency of calcium and protein intake. The results also demonstrated that the development of fluorosis in Guizhou province was correlated with local baking staple corn as a way of excess intake of fluorine and deficiency of calcium/protein intake.