Atomic force microscopy correlates mechanical and electrical properties of HepG2 cells with curcumin concentration.

Hepatocellular carcinoma (HCC) is a highly prevalent cancer with a significant impact on human health. Curcumin, a natural compound, induces cytoskeletal changes in liver cancer cells and modifies the distribution of lipids, proteins, and polysaccharides on plasma membranes, affecting their mechanical and electrical properties. In this study, we used nanomechanical indentation techniques and Kelvin probe force microscopy (KPFM) based on atomic force microscopy (AFM) to investigate the changes in surface nanomechanical and electrical properties of nuclear and cytoplasmic regions of HepG2 cells in response to increasing curcumin concentrations. CCK-8 assays and flow cytometry results demonstrated time- and concentration-dependent inhibition of HepG2 cell proliferation by curcumin. Increasing curcumin concentration led to an initial increase and then decrease in the mechanical properties of nuclear and cytoplasmic regions of HepG2 cells, represented by the Young's modulus (E), as observed through nanoindentation. KPFM measurements indicated decreasing trends in both cell surface potential and height. Fluorescence microscopy results indicated a positive correlation between curcumin concentration and phosphatidylserine translocation from the inner to the outer membrane, which influenced the electrical properties of HepG2 cells. This study provides valuable insights into curcumin's mechanisms against cancer cells and aids nanoscale evaluation of therapeutic efficacy and drug screening.

Systematic characterization of chromodomain proteins reveals an H3K9me1/2 reader regulating aging in C. elegans.

The chromatin organization modifier domain (chromodomain) is an evolutionally conserved motif across eukaryotic species. The chromodomain mainly functions as a histone methyl-lysine reader to modulate gene expression, chromatin spatial conformation and genome stability. Mutations or aberrant expression of chromodomain proteins can result in cancer and other human diseases. Here, we systematically tag chromodomain proteins with green fluorescent protein (GFP) using CRISPR/Cas9 technology in C. elegans. By combining ChIP-seq analysis and imaging, we delineate a comprehensive expression and functional map of chromodomain proteins. We then conduct a candidate-based RNAi screening and identify factors that regulate the expression and subcellular localization of the chromodomain proteins. Specifically, we reveal an H3K9me1/2 reader, CEC-5, both by in vitro biochemistry and in vivo ChIP assays. MET-2, an H3K9me1/2 writer, is required for CEC-5 association with heterochromatin. Both MET-2 and CEC-5 are required for the normal lifespan of C. elegans. Furthermore, a forward genetic screening identifies a conserved Arginine124 of CEC-5's chromodomain, which is essential for CEC-5's association with chromatin and life span regulation. Thus, our work will serve as a reference to explore chromodomain functions and regulation in C. elegans and allow potential applications in aging-related human diseases.

TP53 R249S mutation in hepatic organoids captures the predisposing cancer risk.

BACKGROUND AND AIMS: Major genomic drivers of hepatocellular carcinoma (HCC) are nowadays well recognized, although models to establish their roles in human HCC initiation remain scarce. Here, we used human liver organoids in experimental systems to mimic the early stages of human liver carcinogenesis from the genetic lesions of TP53 loss and L3 loop R249S mutation. In addition, chromatin immunoprecipitation sequencing (ChIP-seq) of HCC cell lines shed important functional insights into the initiation of HCC consequential to the loss of tumor-suppressive function from TP53 deficiency and gain-of-function activities from mutant p53. APPROACH AND RESULTS: Human liver organoids were generated from surgical nontumor liver tissues. CRISPR knockout of TP53 in liver organoids consistently demonstrated tumor-like morphological changes, increased in stemness and unrestricted in vitro propagation. To recapitulate TP53 status in human HCC, we overexpressed mutant R249S in TP53 knockout organoids. A spontaneous increase in tumorigenic potentials and bona fide HCC histology in xenotransplantations were observed. ChIP-seq analysis of HCC cell lines underscored gain-of-function properties from L3 loop p53 mutants in chromatin remodeling and overcoming extrinsic stress. More importantly, direct transcriptional activation of PSMF1 by mutant R249S could increase organoid resistance to endoplasmic reticulum stress, which was readily abrogated by PSMF1 knockdown in rescue experiments. In a patient cohort of primary HCC tumors and genome-edited liver organoids, quantitative polymerase chain reaction corroborated ChIP-seq findings and verified preferential genes modulated by L3 mutants, especially those enriched by R249S. CONCLUSIONS: We showed differential tumorigenic effects from TP53 loss and L3 mutations, which together confer normal hepatocytes with early clonal advantages and prosurvival functions.

Role of cerebellar cortex in associative learning and memory in guinea pigs.

Time-related cognitive function refers to the capacity of the brain to store, extract, and process specific information. Previous studies demonstrated that the cerebellar cortex participates in advanced cognitive functions, but the role of the cerebellar cortex in cognitive functions is unclear. We established a behavioral model using classical eyeblink conditioning to study the role of the cerebellar cortex in associative learning and memory and the underlying mechanisms. We performed an investigation to determine whether eyeblink conditioning could be established by placing the stimulating electrode in the middle cerebellar peduncle. Behavior training was performed using a microcurrent pulse as a conditioned stimulus to stimulate the middle cerebellar peduncle and corneal blow as an unconditioned stimulus. After 10 consecutive days of training, a conditioned response was successfully achieved in the Delay, Trace-200-ms, and Trace-300-ms groups of guinea pigs, with acquisition rates of >60%, but the Trace-400-ms and control groups did not achieve a conditioned stimulus-related blink conditioned response. It could be a good model for studying the function of the cerebellum during the establishment of eyeblink conditioning.

Overexpression of nucleotide metabolic enzyme DUT in hepatocellular carcinoma potentiates a therapeutic opportunity through targeting its dUTPase activity.

Uracil misincorporation during DNA replication is a major cell toxic event, of which cancer cells overcome by activating the dUTPase enzyme. The DUT gene is the only known dUTPase in human. Despite reports on common upregulations in cancers, the role of DUT in human hepatocellular carcinoma (HCC) remains largely undetermined. In this study, we investigated the mechanism underlying DUT biology in HCC and tumor susceptibility to drug targeting dUTPase. Overexpression of DUT was found in 42% of HCC tumors and correlated with advanced stage HCC. Knockout of DUT in HCC cell lines showed suppressed proliferation through cell cycle arrest and a spontaneous induction of DNA damage. A protective effect from oxidative stress was also demonstrated in both knockout and overexpression DUT assays. Transcriptome analysis highlighted the NF-κB survival signaling as the downstream effector pathway of DUT in overriding oxidative stress-induced cell death. Interestingly, stably expressed DUT in liver progenitor organoids conferred drug resistance to TKI Sorafenib. Targeting dUTPase activity by TAS-114, could potentiate suppression of HCC growth that synergized with Sorafenib for better treatment sensitivity. In conclusion, upregulated DUT represents a nucleotide metabolic weakness and therapeutic opportunity in HCC.

Medium Chain Triglycerides Promote the Uptake of ß-Carotene in O/W Emulsions via Intestinal Transporter SR-B1 in Caco-2 Cells.

This study aimed to elucidate the impacts of carrier oil types (long chain triglycerides (LCT), medium chain triglycerides (MCT), and orange oil (indigestible oil)) on the micellization and cellular uptake of ß-carotene (BC) formulated in O/W emulsions, with an emphasis on the role of intestinal transporters. The micellization and cellular uptake of BC in the gastrointestinal tract were evaluated via an in vitro digestion model and a Caco-2 cell monolayer. And the interactions between lipids and intestinal transporters were monitored by nontargeted lipidomics, RT-PCR, and Western blot. The BC micellization rates followed a decreasing trend in emulsions: corn oil (69.47 ± 4.19%) > MCT (22.22 ± 0.89%) > orange oil (11.01 ± 2.86%), whereas the cellular uptake rate of BC was significantly higher in MCT emulsion (56.30 ± 20.13%) than in corn oil emulsion (14.01 ± 1.04%, p < 0.05). The knockdown of SR-B1 led to a 31.63% loss of BC cellular uptake from MCT micelles but had no effect on corn oil micelles. Lipidomics and transporter analysis revealed that TG (10:0/10:0/12:0) and TG (10:0/12:0/12:0) might be the fingerprint lipids that promoted the cellular absorption of BC-MCT micelles via stimulating the mRNA expression of SR-B1.

Unique molecular characteristics of NAFLD-associated liver cancer accentuate ß-catenin/TNFRSF19-mediated immune evasion.

BACKGROUND & AIMS: The hepatic manifestation of the metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), can lead to the development of hepatocellular carcinoma (HCC). Despite a strong causative link, NAFLD-HCC is often underrepresented in systematic genome explorations. METHODS: Herein, tumor-normal pairs from 100 patients diagnosed with NAFLD-HCC were subject to next-generation sequencing. Bioinformatic analyses were performed to identify key genomic, epigenomic and transcriptomic events associated with the pathogenesis of NAFLD-HCC. Establishment of primary patient-derived NAFLD-HCC culture was used as a representative human model for downstream in vitro investigations of the underlying CTNNB1 S45P driver mutation. A syngeneic immunocompetent mouse model was used to further test the involvement of CTNNB1mutand TNFRSF19 in reshaping the tumor microenvironment. RESULTS: Mutational processes operative in the livers of patients with NAFLD inferred susceptibility to tumor formation through defective DNA repair pathways. Dense promoter mutations and dysregulated transcription factors accentuated activated transcriptional regulation in NAFLD-HCC, in particular the enrichment of MAZ-MYC activities. Somatic events common in HCCs arising from NAFLD and viral hepatitis B infection underscore similar driver pathways, although an incidence shift highlights CTNNB1mut dominance in NAFLD-HCC (33%). Immune exclusion correlated evidently with CTNNB1mut. Chromatin immunoprecipitation-sequencing integrated with transcriptome and immune profiling revealed a unique transcriptional axis, wherein CTNNB1mut leads to an upregulation of TNFRSF19 which subsequently represses senescence-associated secretory phenotype-like cytokines (including IL6 and CXCL8). This phenomenon could be reverted by the Wnt-modulator ICG001. CONCLUSIONS: The unique mutational processes in the livers of patients with NAFLD and NAFLD-HCC allude to a "field effect" involving a gain-of-function role of CTNNB1 mutations in immune exclusion. LAY SUMMARY: The increasing prevalence of metabolic syndrome in adult populations means that NAFLD is poised to be the major cause of liver cancer in the 21st century. We showed a strong "field effect" in the livers of patients with NAFLD, wherein activated ß-catenin was involved in reshaping the tumor-immune microenvironment.

The SNAPc complex mediates starvation-induced trans-splicing in Caenorhabditis elegans.

Dietary restriction usually suppresses biosynthesis but activates catabolic pathways in animals. However, the short-term starvation enhances biosynthetic activities and promotes ribosomal biogenesis in adult Caenorhabditis elegans. The mechanism underlying the processes remains largely unknown. Here, we find that the short-term starvation enhances the SL1 trans-splicing of translation-related genes in adult C. elegans by transcriptome analysis. The small nuclear RNA-activating protein complex (SNAPc) promotes SL RNA production and mediates starvation-induced trans-splicing. TOFU-5, a core factor in the upstream sequence transcription complex (USTC) essential for piRNA production, is also involved in the starvation-induced trans-splicing processes. Knocking down components of the SNAPc complex and tofu-5 extends worm survival under starvation conditions. Taken together, our study highlights the importance of SL trans-splicing in the nutrition response and reveals a mechanism of the survival regulation by food deprivation via SNAPc and TOFU-5.

Venetoclax enhances T cell-mediated antileukemic activity by increasing ROS production.

Venetoclax, a Bcl-2 inhibitor, in combination with the hypomethylating agent azacytidine, achieves complete remission with or without count recovery in ∼70% of treatment-naive elderly patients unfit for conventional intensive chemotherapy. However, the mechanism of action of this drug combination is not fully understood. We discovered that venetoclax directly activated T cells to increase their cytotoxicity against acute myeloid leukemia (AML) in vitro and in vivo. Venetoclax enhanced T-cell effector function by increasing reactive oxygen species generation through inhibition of respiratory chain supercomplexes formation. In addition, azacytidine induced a viral mimicry response in AML cells by activating the STING/cGAS pathway, thereby rendering the AML cells more susceptible to T cell-mediated cytotoxicity. Similar findings were seen in patients treated with venetoclax, as this treatment increased reactive oxygen species generation and activated T cells. Collectively, this study presents a new immune-mediated mechanism of action for venetoclax and azacytidine in the treatment of AML and highlights a potential combination of venetoclax and adoptive cell therapy for patients with AML.

Promotion of growth and phytoextraction of cadmium and lead in Solanum nigrum L. mediated by plant-growth-promoting rhizobacteria.

Plant growth-promoting rhizobacteria (PGPR) are a specific category of microbes that improve plant growth and promote greater tolerance to metal stress through their interactions with plant roots. We evaluated the effects of phytoremediation combining the cadmium accumulator Solanum nigrum L. and two Cd- and Pb-resistant bacteria isolates. To understand the interaction between PGPR and their host plant, we conducted greenhouse experiments with inoculation treatments at Nanjing Agricultural University (Jiangsu Province, China), in June 2018. Two Cd- and Pb-resistant PGPR with various growth-promoting properties were isolated from heavy metal-contaminated soil. 16S rRNA analyses indicated that the two isolates were Bacillus genus, and they were named QX8 and QX13. Pot experiments demonstrated that inoculation may improve the rhizosphere soil environment and promote absorption of Fe and P by plants. Inoculation with QX8 and QX13 also enhanced the dry weight of shoots (1.36- and 1.7-fold, respectively) and roots (1.42- and 1.96-fold) of plants growing in Cd- and Pb-contaminated soil, and significantly increased total Cd (1.28-1.81 fold) and Pb (1.08-1.55 fold) content in aerial organs, compared to non-inoculated controls. We also detected increases of 23% and 22% in the acid phosphatase activity of rhizosphere soils inoculated with QX8 and QX13, respectively. However, we did not detect significant differences between inoculated and non-inoculated treatments in Cd and Pb concentrations in plants and available Cd and Pb content in rhizosphere soils. We demonstrated that PGPR-assisted phytoremediation is a promising technique for remediating heavy metal-contaminated soils, with the potential to enhance phytoremediation efficiency and improve soil quality.

Mitochondrial DNA copy number in cervical exfoliated cells and risk of cervical cancer among HPV-positive women.

BACKGROUND: Although human papillomavirus (HPV) infection has been regarded as the cause of cervical cancer in over 99% of cases, only a small fraction of HPV-infected women develop this malignancy. Emerging evidence suggests that alterations of mitochondrial DNA copy number (mtCN) may contribute to carcinogenesis. However, the relationship between mtCN and cervical cancer remains undetermined. METHODS: The current study included 591 cervical cancer cases and 373 cancer-free controls, all of whom were infected with high-risk HPV. Relative mtCN in cervical cancer exfoliated cells was measured by qRT-PCR assays, and logistic regression analysis was performed to compute odds ratios (ORs) and 95% confidence intervals (CIs). Interaction between mtCN and HPV types was assessed by using the Wald test in logistic regression models. RESULTS: HPV16, 18, 52, and 58 were the most common types in both case and control groups. Median mtCN in cases was significantly higher than that in controls (1.63 vs. 1.23, P = 0.03). After adjustment for age and HPV types, the highest quartile of mtCN was associated with increased odds of having cervical cancer (OR = 1.77, 95% CI = 1.19, 2.62; P < 0.01), as compared to the lowest quartile. A dose-response effect of mtCN on cervical cancer was also observed (Ptrend < 0.001). The interaction between mtCN and HPV types was statistically nonsignificant. CONCLUSIONS: In women who test HPV positive, the increase of mtCN in cervical exfoliated cells is associated with cervical cancer. This suggests a potential role of mtCN in cervical carcinogenesis.

Tafazzin modulates cellular phospholipid composition to regulate AML stemness.

Tafazzin is a mitochondrial enzyme necessary for the remodeling of the phospholipid cardiolipin. Seneviratne and Xu et al. demonstrated that Tafazzin-mediated phospholipid production regulates stemness in Acute Myeloid Leukemia (AML). Tafazzin influenced intracellular levels of phospholipids to control AML stemness. Thus, inhibiting mitochondrial phospholipid production could be a new therapeutic strategy for AML.

The Mitochondrial Transacylase, Tafazzin, Regulates for AML Stemness by Modulating Intracellular Levels of Phospholipids.

Tafazzin (TAZ) is a mitochondrial transacylase that remodels the mitochondrial cardiolipin into its mature form. Through a CRISPR screen, we identified TAZ as necessary for the growth and viability of acute myeloid leukemia (AML) cells. Genetic inhibition of TAZ reduced stemness and increased differentiation of AML cells both in vitro and in vivo. In contrast, knockdown of TAZ did not impair normal hematopoiesis under basal conditions. Mechanistically, inhibition of TAZ decreased levels of cardiolipin but also altered global levels of intracellular phospholipids, including phosphatidylserine, which controlled AML stemness and differentiation by modulating toll-like receptor (TLR) signaling.

[Retracted] BIRC5 is a novel target of peroxisome proliferator­activated receptor γ in brain microvascular endothelium cells during cerebral ischemia.

The authors of the above article wish to retract their paper. Subsequently to its publication, in addition to certain irreconcilable differences in opinion shared among the authors, they have realized that, in their analysis of the role of peroxisome proliferator­activated receptor Î³ in brain microvascular endothelial (bEnd.3) cells, certain of the results presented have been found to be incomplete and unrepeatable. Therefore, they wish to retract the paper in order to maintain the integrity of the scientific record. The authors regret any inconvenience that this retraction will caused to the Editor of the journal, and the readership. [the original article was published in Molecular Medicine Reports 16: 8882­8890, 2017; DOI: 10.3892/mmr.2017.7750].

BIRC5 is a novel target of peroxisome proliferator­activated receptor γ in brain microvascular endothelium cells during cerebral ischemia.

Cerebral ischemia is a leading cause of ischemic stroke, which may lead to severe disability and mortality worldwide. There are some key factors concerned in cardioprotection, such as peroxisome proliferator­activated receptor Î³ (PPARγ), a ligand binding transcription factor involved in various biological functions including atherosclerosis, vascular dysfunction and hypertension, and baculoviral IAP repeat­containing 5 (BIRC5), which may protect human brain endothelial cells from ischemia­induced apoptosis. To determine the potential roles of PPARγ in brain microvascular endothelial (bEnd.3) cells during cerebral ischemia and the relationship between PPARγ and BIRC5, a cerebral ischemia model was established with bEnd.3 cells cells by oxygen­glucose deprivation (OGD) treatment. OGD treatment reduced proliferation and enhanced apoptosis of bEnd.3 cells in a time­dependent manner. PPARγ expression levels were decreased in bEnd.3 cells following OGD treatment. Upregulation of PPARγ expression protected bEnd.3 cells from ischemia injury and also upregulated BIRC5 expression. PPARγ­specific binding sites in the BIRC5 promoter were predicted bioinformatically and verified by luciferase reporter experiments. Results from electrophoretic mobility shift/supershift and chromatin immunoprecipitation assays suggested that BIRC5 may be a novel target of PPARγ transcriptional regulation during ischemic injury. The present results indicated that PPARγ may serve a protective role on bEnd.3 cells and that BIRC5 may be a downstream target of PPARγ regulation during cerebral ischemia.

Calycosin-7-O-ß-D-glucoside regulates nitric oxide /caveolin-1/matrix metalloproteinases pathway and protects blood-brain barrier integrity in experimental cerebral ischemia-reperfusion injury.

ETHNOPHARMACOLOGY RELEVANCE: Astragali Radix (AR) has been used for thousands years to treat ischemic stroke. Calycosin and its glycoside form calycosin-7-O-ß-D-glucoside (CG) are two representative isoflavones in Astragali Radix. However, its neurological effects and related molecular mechanisms are largely unknown. The present study aims to evaluate the neuroprotective effects of CG on blood-brain barrier (BBB) integrity of ischemic brain tissue and explore the relevant signaling mechanisms. MATERIAL AND METHOD: Male adult Sprague-Daweley rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) plus 24 h or 14 days of reperfusion. CG (26.8 mg/kg) was intraperitoneally administered into the rats at 15 min before onset of ischemia. The neuroprotective effects of CG were evaluated by measuring infarct volume, histological damage and BBB permeability. Furthermore, the effects of CG on scavenging nitric oxide (NO), and modulating matrix metalloproteinases (MMPs) and caveolin-1 (cav-1) were investigated with in vitro cultured brain microvascular endothelial cells treated with NO donor or oxygen-glucose deprivation (OGD) and/or in vivo rat model of MCAO cerebral ischemia-reperfusion injury. RESULTS: CG treatment significantly reduced infarct volume, histological damage and BBB permeability in the in vivo MCAO ischemia-reperfusion rat model. CG treatment remarkably inhibited the expression and activities of MMPs, and secured the expression of cav-1 and tight junction proteins in the microvessels isolated from ischemic rat cortex. Furthermore, CG was revealed to scavenge NO, inhibit the activities of MMP-2 and MMP-9, and attenuate cell death in the in vitro cultured brain microvascular endothelial cells under OGD condition. CONCLUSION: CG could protect BBB integrity in experimental cerebral ischemia-reperfusion injury via regulating NO/cav-1/MMPs pathway.

Baicalin can scavenge peroxynitrite and ameliorate endogenous peroxynitrite-mediated neurotoxicity in cerebral ischemia-reperfusion injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Baicalin is one of the principal flavonoids isolated from the dried root of Scutellaria baicalensis Georgi that has long been used to treat ischemic stroke. However, its neuroprotective mechanisms against cerebral ischemia injury are poorly understood. AIM OF THE STUDY: To explore the neuroprotective mechanisms of baicalin against cerebral ischemia reperfusion injury. MATERIAL AND METHODS: In chemical systems, we conducted electron paramagnetic resonance (EPR) spin trapping experiments to evaluate the scavenging effects of baicalin on superoxide and nitric oxide, and mass spectrometry (MS) studies on the reaction of baicalin and peroxynitrite. In cellular experiments, we investigated the effects of baicalin against extraneous and endogenous peroxynitrite mediated neurotoxicity in SH-SY5Y cells treated with peroxynitrite donor, synthesized peroxynitrite and exposed to oxygen glucose deprivation and reoxygenation (OGD/RO) in vitro. Moreover, we studied the neuroprotective effects of baicalin by using a rat model of middle cerebral artery occlusion in vivo. FeTMPyP, a peroxynitrite decomposition catalyst, was used as positive control. Cell viability and apoptotic cell death was accessed by MTT assay and TUNEL assay respectively; 3-nitrotyrosine formation and infarction volume were detected by immunostaining experiments and TTC staining respectively. RESULTS: Baicalin revealed strong antioxidant ability by directly scavenging superoxide and reacting with peroxynitrite. Baicalin protected the neuronal cells from extraneous and endogenous peroxynitrite-induced neurotoxicity. In ischemia-reperfused brains, baicalin inhibited the formation of 3-nitrotyrosine, reduced infarct size and attenuated apoptotic cell death, whose effects were similar to FeTMPyP. CONCLUSIONS: Baicalin can directly scavenge peroxynitrite and the peroxynitrite-scavenging ability contributes to its neuroprotective mechanisms against cerebral ischemia reperfusion injury.