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Observational data suggest a link between gut microbiota and immune-related vasculitis, but causality remains unclear. A bidirectional mendelian randomization study was conducted using public genome-wide data. The inverse-variance-weighted (IVW) method identified associations and addressed heterogeneity.Families Clostridiaceae 1 and Actinomycetaceae correlated positively with granulomatosis with polyangiitis risk, while classes Lentisphaeria and Melainabacteria, and families Lachnospiraceae and Streptococcaceae showed negative associations. Behçet's disease was positively associated with the risk of family Streptococcaceae abundance. And other several gut microbiota constituents were identified as potential risk factors for immune-related vasculitis. Furthermore, combining positive association results from the IVW analysis revealed numerous shared gut microbiota constituents associated with immune-related vasculitis. MR analysis demonstrated a causal association between the gut microbiota and immune-related vasculitis, offering valuable insights for subsequent mechanistic and clinical investigations into microbiota-mediated immune-related vasculitis., (© 2024. The Author(s).)

Diabetic retinopathy (DR) is one of the leading causes of adult blindness in the United States. Although studies applying traditional statistical methods have revealed that heavy metals may be essential environmental risk factors for diabetic retinopathy, there is a lack of analyses based on machine learning (ML) methods to adequately explain the complex relationship between heavy metals and DR and the interactions between variables. Based on characteristic variables of participants with and without DR and heavy metal exposure data obtained from the NHANES database (2003-2010), a ML model was developed for effective prediction of DR. The best predictive model for DR was selected from 11 models by receiver operating characteristic curve (ROC) analysis. Further permutation feature importance (PFI) analysis, partial dependence plots (PDP) analysis, and SHapley Additive exPlanations (SHAP) analysis were used to assess the model capability and key influencing factors. A total of 1042 eligible individuals were randomly assigned to two groups for training and testing set of the prediction model. ROC analysis showed that the k-nearest neighbour (KNN) model had the highest prediction performance, achieving close to 100% accuracy in the testing set. Urinary Sb level was identified as the critical heavy metal affecting the predicted risk of DR, with a contribution weight of 1.730632 ± 1.791722, which was much higher than that of other heavy metals and baseline variables. The results of the PDP analysis and the SHAP analysis also indicated that antimony (Sb) had a more significant effect on DR. The interaction between age and Sb was more significant compared to other variables and metal pairs. We found that Sb could serve as a potential predictor of DR and that Sb may influence the development of DR by mediating cellular and systemic senescence. The study revealed that monitoring urinary Sb levels can be useful for early non-invasive screening and intervention in DR development, and also highlighted the important role of constructed ML models in explaining the effects of heavy metal exposure on DR., (© 2024. The Author(s).)

Due to global warming and the disturbance of the interannual variability of precipitation, the frequency of extreme drought events has increased. The impact of global climate change on water resources is becoming increasingly apparent, then it is particularly necessary to explore the carrying capacity of water ecological environment under extreme drought conditions, which can guarantee the ecological water security in river basins. This study takes the Guanzhong area of the Wei River Basin as an example, calculating the water environment carrying capacity of 40 areas in the Weihe Guanzhong area in different levels of years under extreme drought conditions by comprehensive evaluation model of carrying capacity and using geographic information system GIS to display the spatial distribution of water environment carrying capacity in 40 regions. According to the results of the spatial distribution of water environmental bearing capacity, four different schemes are designed to improve the bearing capacity. The first plan reduces the industrial water consumption and irrigation quota by 5%, the second plan increases the industrial water and sewage treatment rate on this basis. the third plan further improves the development and utilization rate of surface and groundwater, and the fourth plan, on the basis of the first three plans, supplies 600 million cubic meters of industrial and agricultural water to Guanzhong region. Through comparative analysis, without taking any measures, under the extreme drought conditions, the water environment carrying capacity of the 40 areas in Guanzhong is all in an unbearable state. Overall, plan 4 has the most significant improvement in the water environment-carrying capacity, especially the Dong zhuang Reservoir of the Jing River which has played a very important role in enhancing the water ecological environment carrying capacity of the downstream water of the Wei River., (© 2024. The Author(s).)

The majority of pigeon paramyxovirus type 1 (PPMV-1) strains are generally non-pathogenic to chickens; however, they can induce severe illness and high mortality rates in pigeons, leading to substantial economic repercussions. The genomes of 11 PPMV-1 isolates from deceased pigeons on meat pigeon farms during passive monitoring from 2009 to 2012 were sequenced and analyzed using polymerase chain reaction and phylogenetic analysis. The complete genome lengths of 11 isolates were approximately 15,192 nucleotides, displaying a consistent gene order of 3'-NP-P-M-F-HN-L-5'. ALL isolates exhibited the characteristic motif of 112RRQKRF117 at the fusion protein cleavage site, which is characteristic of velogenic Newcastle disease virus. Moreover, multiple mutations have been identified within the functional domains of the F and HN proteins, encompassing the fusion peptide, heptad repeat region, transmembrane domains, and neutralizing epitopes. Phylogenetic analysis based on sequences of the F gene unveiled that all isolates clustered within genotype VI in class II. Further classification identified at least two distinct sub-genotypes, with seven isolates classified as sub-genotype VI.2.1.1.2.2, whereas the others were classified as sub-genotype VI.2.1.1.2.1. This study suggests that both sub-genotypes were implicated in severe disease manifestation among meat pigeons, with sub-genotype VI.2.1.1.2.2 displaying an increasing prevalence among Shanghai's meat pigeon population since 2011. These results emphasize the value of developing pigeon-specific vaccines and molecular diagnostic tools for monitoring and proactively managing potential PPMV-1 outbreaks., (© 2024. The Author(s).)

Hepatocellular carcinoma (HCC) is a primary liver cancer with a high mortality rate that requires research and improved treatment strategies. Chemotherapy is still one of the main methods of HCC treatment, but it may lead to drug resistance and damage to normal organs. Capsaicin, a naturally occurring active ingredient in chili peppers, has demonstrated anticancer properties in a variety of malignant tumor cell lines. However, the anti-cancer mechanism of capsaicin needs to be further explored in HCC. In this study, we utilized Arvanil, a non-stimulating synthetic capsaicin analog, in place of capsaicin. We found that Arvanil induced high mitochondrial calcium flow, which contributed to a decrease in mitochondrial membrane permeability transition pore (mPTP) opening and oxidative phosphorylation levels, ultimately triggering cellular ferroptosis by live cells in real time with a high content screening (HCS) platform and confocal microscopy. It was further confirmed by vina molecular docking and point mutation experiments that Arvanil directly binds to two amino acid sites of mitochondrial calcium uptake protein 1 (MICU1), namely Ser47 and Phe128, to trigger this process, which in turn inhibits the growth of HCC cells. In addition, it was confirmed that Arvanil enhances cisplatin chemosensitivity by inducing HCC cellular ferroptosis in vivo. In conclusion, our study suggests that Arvanil induces ferroptosis in HCC cells and is a candidate drug for the treatment of HCC., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a human paracaspase protein with proteolytic activity via its caspase-like domain. The pharmacological inhibition of MALT1 by MI-2, a specific chemical inhibitor, diminishes the response of endothelial cells to inflammatory stimuli. However, it is largely unknown how MALT1 regulates the functions of vascular smooth muscle cells (SMCs). This study aims to investigate the impact of MALT1 inhibition by MI-2 on the functions of vascular SMCs, both in vitro and in vivo. MI-2 treatment led to concentration- and time-dependent cell death of cultured aortic SMCs, which was rescued by the iron chelator deferoxamine (DFO) or ferrostatin-1 (Fer-1), a specific inhibitor of ferroptosis, but not by inhibitors of apoptosis (Z-VAD-fmk), pyroptosis (Z-YVAD-fmk), or necrosis (Necrostatin-1, Nec-1). MI-2 treatment downregulated the expression of glutathione peroxidase 4 (GPX4) and ferritin heavy polypeptide 1 (FTH1), which was prevented by pre-treatment with DFO or Fer-1. MI-2 treatment also activated autophagy, which was inhibited by Atg7 deficiency or bafilomycin A1 preventing MI-2-induced ferroptosis. MI-2 treatment reduced the cleavage of cylindromatosis (CYLD), a specific substrate of MALT1. Notably, MI-2 treatment led to a rapid loss of contractility in mouse aortas, which was prevented by co-incubation with Fer-1. Moreover, local application of MI-2 significantly reduced carotid neointima lesions and atherosclerosis in C57BL/6J mice and apolipoprotein-E knockout (ApoE -/- ) mice, respectively, which were both ameliorated by co-treatment with Fer-1. In conclusion, the present study demonstrated that MALT1 inhibition induces ferroptosis of vascular SMCs, likely contributing to its amelioration of proliferative vascular diseases., (© 2023. The Author(s).)

Circadian rhythms are crucial for regulating physiological and behavioral processes. Pineal hormone melatonin is often used to measure circadian amplitude but its collection is costly and time-consuming. Wearable activity data are promising alternative, but the most commonly used measure, relative amplitude, is subject to behavioral masking. In this study, we firstly derive a feature named circadian activity rhythm energy (CARE) to better characterize circadian amplitude and validate CARE by correlating it with melatonin amplitude (Pearson's r = 0.46, P = 0.007) among 33 healthy participants. Then we investigate its association with cognitive functions in an adolescent dataset (Chinese SCHEDULE-A, n = 1703) and an adult dataset (UK Biobank, n = 92,202), and find that CARE is significantly associated with Global Executive Composite (β = 30.86, P = 0.016) in adolescents, and reasoning ability, short-term memory, and prospective memory (OR = 0.01, 3.42, and 11.47 respectively, all P < 0.001) in adults. Finally, we identify one genetic locus with 126 CARE-associated SNPs using the genome-wide association study, of which 109 variants are used as instrumental variables in the Mendelian Randomization analysis, and the results show a significant causal effect of CARE on reasoning ability, short-term memory, and prospective memory (β = -59.91, 7.94, and 16.85 respectively, all P < 0.0001). The present study suggests that CARE is an effective wearable-based metric of circadian amplitude with a strong genetic basis and clinical significance, and its adoption can facilitate future circadian studies and potential intervention strategies to improve circadian rhythms and cognitive functions., (© 2023. The Author(s).)

Animal models play crucial roles in the rapid development of vaccines/drugs for the prevention and therapy of COVID-19, but current models have some deficits when studying the pathogenesis of SARS-CoV-2 on some special tissues or organs. Here, we generated a human ACE2 and SARS-CoV-2 N F/F knockin mouse line that constitutively expresses human ACE2 and specifically expresses SARS-CoV-2 N gene induced by Cre-recombinase. By crossing with Cre transgenic lines allowing for lung-specific and constitutive expression, we generated lung-specific (Sftpc-hACE2-N F/F ) and constitutive SARS-CoV-2 N (EIIa-hACE2-N F/F ) expressing mice. Upon intranasal infection with a SARS-CoV-2 GFP/ΔN strain which can only replicate in SARS-CoV-2 N expressed cells, we demonstrated that both the Sftpc-hACE2-N F/F and EIIa-hACE2-N F/F mice support viral replication. Consistent with our design, viral replication was limited to the lung tissues in Sftpc-hACE2-N F/F mice, while the EIIa-hACE2-N F/F mice developed infections in multiple tissues. Furthermore, our model supports different SARS-CoV-2 variants infection, and it can be successfully used to evaluate the effects of therapeutic monoclonal antibodies (Ab1F11) and antiviral drugs (Molnupiravir). Finally, to test the effect of SARS-CoV-2 infection on male reproduction, we generated Sertoli cell-specific SARS-CoV-2 N expressed mice by crossing with AMH-Cre transgenic line. We found that SARS-CoV-2 GFP/ΔN strain could infect Sertoli cells, led to spermatogenic defects due to the destruction of blood-testis barrier. Overall, combining with different tissue-specific Cre transgenic lines, the human ACE2 and SARS-CoV-2 N F/F line enables us to evaluate antivirals in vivo and study the pathogenesis of SARS-CoV-2 on some special tissues or organs., (© 2023. The Author(s).)

Isoflavone reductase (IFR) is a key enzyme controlling isoflavone synthesis and widely involved in response to various stresses. In this study, the IFR genes in four Gossypium species and other 7 species were identified and analyzed in the whole genome, and the physicochemical properties, gene structures, cis-acting elements, chromosomal locations, collinearity relationships and expression patterns of IFR genes were systematically analyzed. 28, 28, 14 and 15 IFR genes were identified in Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum and Gossypium raimondii, respectively, which were divided into five clades according to the evolutionary tree and gene structure. Collinear analysis showed that segmental duplication and whole genome duplication were the main driving forces in the process of evolution, and most genes underwent pure selection. Gene structure analysis showed that IFR gene family was relatively conserved. Cis-element analysis of promoter showed that most GhIFR genes contain cis-elements related to abiotic stresses and plant hormones. Analysis of GhIFR gene expression under different stresses showed that GhIFR genes were involved in the response to drought, salt, heat and cold stresses through corresponding network mechanisms, especially GhIFR9A. Phenotypic analysis after silencing GhIFR9A gene by VIGS was shown that GhIFR9A gene was involved in the response to salt stress. This study laid a foundation for the subsequent functional study of cotton IFR genes., (© 2023. The Author(s).)

Neointima lesion and atherosclerosis are proliferative vascular diseases associated with deregulated proliferation of vascular smooth muscle cells (SMCs). CFI-400945 is a novel, highly effective anticancer drug that inhibits polo-like kinase 4 (PLK4) and targets mitosis. In this study, we aim to investigate how CFI-400945 affects the development of proliferative vascular diseases. In C57BL/6 mice, neointima formation was generated by complete carotid ligation. In apolipoprotein E knockout (ApoE -/- ) mice fed a high-fat diet, atherosclerosis was induced by partial carotid ligation. CFI-400945 was directly applied to carotid arteries via a perivascular collar. Our results showed that CFI-400945 drastically inhibited neointima formation but significantly accelerated atherosclerosis. In vitro studies showed that CFI-400945 treatment induced SMC polyploidization and arrested cells in the G2/M phase. CFI-400945 treatment upregulated p53 and p27 expression but decreased p21 and cyclin B1 expression. CFI-400945 also induced SMC apoptosis, which was inhibited by hydroxyurea, a DNA synthesis inhibitor that inhibits polyploidization. Furthermore, CFI-400945 caused supernumerary centrosomes, leading to mitotic failure, resulting in polyploidization. In conclusion, CFI-400945 prevents carotid arterial neointima formation in C57BL/6 mice but accelerates atherosclerosis in ApoE -/- mice, likely through mitotic arrest and subsequent induction of polyploidization and apoptosis., (© 2023. The Author(s).)

N 6 -methyladenosine (m 6 A) is an abundant nucleotide modification in mRNA, and its emerging roles have been gradually identified. However, the potential function of m 6 A and m 6 A-modified circular RNA (circRNA) is still unclear. Here, m 6 A-circRNA epitranscriptomic microarray analysis revealed a high-expressed m 6 A-modified circFOXK2 (hsa&#95;circ&#95;0000816, from FOXK2 gene) in oral squamous cell carcinoma (OSCC). For the biofunctions of OSCC, results revealed that circFOXK2 promoted the malignant phenotypes of OSCC cells. Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) demonstrated that a remarkable m 6 A modified site was installed on glucose transporter 1 (GLUT1) mRNA. Mechanistically, circFOXK2 promoted the GLUT1 mRNA stability through cooperating with insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) in a m 6 A-dependent manner. In summary, the present study explored the oncogenic role of m 6 A-modified circFOXK2 in OSCC through the m 6 A-dependent IGF2BP3/GLUT1 axis, indicating a potential therapeutic target for OSCC., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Identification and functional annotations of regulatory sequences play a pivotal role in heart development and function., Methods: To generate a map of human heart-specific enhancers, we performed an integrative analysis of 148 chromatin immunoprecipitation coupled to massively parallel sequencing (ChIP-seq) samples with enhancer-associated epigenetic marks from the heart, liver, brain, and kidney. Functional validation of heart-specific enhancer activity was then performed using cultured cells., Results: A 144.6-Mb candidate heart-specific enhancer compendium was generated by integrating the analysis of 148 epigenomic data sets from human and mouse hearts and control tissues. To validate in vivo enhancer activity, we tested 12 of these sequences around 45 CHD-related genes in cultured cells and found that 8 (67%) have reproducible heart-specific enhancer activity. A functional analysis demonstrated that the identified human heart-specific enhancer wf1 regulates the FBN1 gene which is involved in heart disease., Conclusions: Our study provides an integrative analysis pipeline for ChIP-seq data and identified a comprehensive catalog of human heart-specific enhancers for clinical CHD-related studies., Impact: Establishing an efficient way to analyze regulatory regions in CHD is very important. A highly qualified heart-specific enhancer compendium was generated by integrating 148 online ChIP-seq samples. Sixty-seven percent of predicted regulatory sequences have reproducible heart-specific enhancer activity in vivo. Human heart-specific enhancer wf1 regulates the CHD-related FBN1 gene., (© 2022. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Lung adenocarcinoma (LUAD) is the most frequent subtype of lung cancer globally. However, the survival rate of lung adenocarcinoma patients remains low. Immune checkpoints and long noncoding RNAs are emerging as vital tools for predicting the immunotherapeutic response and outcomes of patients with lung adenocarcinoma. It is critical to identify lncRNAs associated with immune checkpoints in lung adenocarcinoma patients. In this study, immune checkpoint-related lncRNAs (IClncRNAs) were analysed and identified by coexpression. Based on the immune checkpoint-related lncRNAs, we divided patients with lung adenocarcinoma into two clusters and constructed a risk model. Kaplan-Meier analysis, Gene Set Enrichment Analysis, and nomogram analysis of the 2 clusters and the risk model were performed. Finally, the potential immunotherapeutic prediction value of this model was discussed. The risk model consisting of 6 immune checkpoint-related lncRNAs was an independent predictor of survival. Through regrouping the patients with this model, we can distinguish between them more effectively in terms of their immunotherapeutic response, tumour microenvironment, and chemotherapy response. This risk model based on immune checkpoint-based lncRNAs may have an excellent clinical value for predicting the immunotherapeutic response and outcomes of patients with LUAD., (© 2022. The Author(s).)

Aldo-keto reductase family 1 member C3 (AKR1C3) serves as a contributor to numerous kinds of tumors, and its expression is elevated in patients with hepatocellular carcinoma (HCC). However, the biological function of AKR1C3 in HCC remains unclear. Here we investigated the role of AKR1C3 in liver carcinogenesis using in vitro and in vivo models. We determined that AKR1C3 is frequently increased in HCC tissues with poor prognosis. Genetically manipulated cells with AKR1C3 construction were examined to highlight the pro-tumoral growth of both wild-type AKR1C3 and mutant in vitro and in vivo. We observed promising treatment effects of AKR1C3 shRNA by intratumoral injection in mice. Mechanically, we demonstrated that the transcription factor heterodimer NRF2/MAFG was able to bind directly to AKR1C3 promoter to activate its transcription. Further, AKR1C3 stabilized PARP1 by decreasing its ubiquitination, which resulted in HCC cell proliferation and low sensitivity of Cisplatin. Moreover, we discovered that the tumorigenic role of AKR1C3 was non-catalytic dependent and the NRF2/MAFG-AKR1C3-PARP1 axis might be one of the important proliferation pathways in HCC. In conclusion, blockage of AKR1C3 expression provides potential therapeutic benefits against HCC., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Spermiogenesis is a complex process depending on the sophisticated coordination of a myriad of testis-enriched gene regulations. The regulatory pathways that coordinate this process are not well understood, and we demonstrate here that AXDND1, as a novel testis-enriched gene is essential for spermiogenesis and male fertility. AXDND1 is exclusively expressed in the round and elongating spermatids in humans and mice. We identified two potentially deleterious mutations of AXDND1 unique to non-obstructive azoospermia (NOA) patients through selected exonic sequencing. Importantly, Axdnd1 knockout males are sterile with reduced testis size caused by increased germ cell apoptosis and sloughing, exhibiting phenotypes consistent with oligoasthenoteratozoospermia. Axdnd1 mutated late spermatids showed head deformation, outer doublet microtubules deficiency in the axoneme, and loss of corresponding accessory structures, including outer dense fiber (ODF) and mitochondria sheath. These phenotypes were probably due to the perturbed behavior of the manchette, a dynamic structure where AXDND1 was localized. Our findings establish AXDND1 as a novel testis-enrich gene essential for spermiogenesis and male fertility probably by regulating the manchette dynamics, spermatid head shaping, sperm flagellum assembly., (© 2021. The Author(s).)

Genetic factors are one of the most important causes of non-obstructive azoospermia (NOA). ESX1 is an X-linked testis-biased expressed gene, and a potential biomarker for testicular sperm retrieval in NOA patients, yet few systematic studies have investigated its association with NOA. Here, we performed selected exonic sequencing in a large cohort of Chinese males, and four novel missense mutations (including one compound mutation), one novel synonymous mutation of ESX1 unique to NOA patients were identified. We analyzed the effects of ESX1 mutations on cyclin A degradation and cell cycle progression by immunoprecipitation assay and flow cytometry, and found that the compound mutant p.[P365R; L366V] ESX1 compromised the stabilizing effect of ESX1 on polyubiquitinated cyclin A, thereby causing the failure of M phase arrest in cells. Further studies showed that the deleterious effect of the compound mutations on ESX1 protein function was attributed to p.P365R but not p.L366V alteration. The novel ESX1 mutation p.P365R might confer high risk for NOA in Han Chinese population, probably via affecting cell cycle control.

Densely integrated active photonics is key for next generation on-chip networks for addressing both footprint and energy budget concerns. However, the weak light-matter interaction in traditional active Silicon optoelectronics mandates rather sizable device lengths. The ideal active material choice should avail high index modulation while being easily integrated into Silicon photonics platforms. Indium tin oxide (ITO) offers such functionalities and has shown promising modulation capacity recently. Interestingly, the nanometer-thin unity-strong index modulation of ITO synergistically combines the high group-index in hybrid plasmonic with nanoscale optical modes. Following this design paradigm, here, we demonstrate a spectrally broadband, GHz-fast Mach-Zehnder interferometric modulator, exhibiting a high efficiency signified by a miniscule V π L of 95 V μm, deploying a one-micrometer compact electrostatically tunable plasmonic phase-shifter, based on heterogeneously integrated ITO thin films into silicon photonics. Furthermore we show, that this device paradigm enables spectrally broadband operation across the entire telecommunication near infrared C-band. Such sub-wavelength short efficient and fast modulators monolithically integrated into Silicon platform open up new possibilities for high-density photonic circuitry, which is critical for high interconnect density of photonic neural networks or applications in GHz-fast optical phased-arrays, for example.

Therapeutic drug monitoring (TDM) is necessary for the optimal administration of anti-arrhythmic drugs in the treatment of heart arrhythmia. The present study aimed to develop and validate a direct analysis in real time tandem mass spectrometry (DART-MS/MS) method for the rapid and simultaneous determination of five anti-arrhythmic drugs (metoprolol, diltiazem, amiodarone, propafenone, and verapamil) and one metabolite (5-hydroxy(OH)-propafenone) in human serum. After the addition of isotope-labeled internal standards and protein precipitation with acetonitrile, anti-arrhythmic drugs were ionized by DART in positive mode followed by multiple reaction monitoring (MRM) detection. The use of DART-MS/MS avoided the need for chromatographic separation and allowed rapid and ultrahigh throughput analysis of anti-arrhythmic drugs in a total run time of 30 s per sample. The DART-MS/MS method yielded satisfactory linearity (R 2  ≥ 0.9906), accuracy (86.1-109.9%), and precision (≤ 14.3%) with minimal effect of biological matrixes. The method was successfully applied to analyzing 30 clinical TDM samples. The relative error (RE) of the concentrations obtained by DART-MS/MS and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was within ± 13%. This work highlights the potential usefulness of DART for the rapid quantitative analysis of anti-arrhythmic drugs in human serum and gives rapid feedback in the clinical TDM practices.

Although more than 1,000 androgen receptor (AR) mutations have been identified and these mutants are pathologically important, few theoretical studies have investigated the role of AR protein folding stability in disease and its relationship with the phenotype of the patients. Here, we extracted AR variant data from four databases: ARDB, HGMD, Cosmic, and 1,000 genome. 905 androgen insensitivity syndrome (AIS)-associated loss-of-function mutants and 168 prostate cancer-associated gain-of-function mutants in AR were found. We analyzed the effect of single-residue variation on the folding stability of AR by FoldX and guanidine hydrochloride denaturation experiment, and found that genetic disease-associated mutations tend to have a significantly greater effect on protein stability than gene polymorphisms. Moreover, AR mutants in complete androgen insensitivity syndrome (CAIS) tend to have a greater effect on protein stability than in partial androgen insensitive syndrome (PAIS). This study, by linking disease phenotypes to changes in AR stability, demonstrates the importance of protein stability in the pathogenesis of hereditary disease.

Our previous study demonstrated that the mammalian target of rapamycin complex 2 (mTORC2) signaling alleviates renal inflammation and protects against cisplatin-induced AKI. However, the underlying mechanisms for mTORC2 in regulating renal inflammation in AKI remain to be determined. In this study, we found that lipopolysaccharide (LPS) could activate mTORC2 signaling in NRK-52E cells, and blockage of mTORC2 signaling led to Yap/Taz degradation, which in turn activated NF-κB signaling and induced inflammatory cytokines secretion. Overexpression of constitutively active Taz (Taz-S89A) could attenuate the inflammation-amplified role of mTORC2 blockage. In mouse models, tubule-specific deletion of Rictor had higher blood urea nitrogen level, severe morphological injury as well as more inflammatory cells accumulation compared with those in their littermate controls. Overall, these results demonstrate that mTORC2 signaling protects against renal inflammation and dictates the outcome of AKI by modulating Yap/Taz degradation., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© The Author(s) 2020.)

In this pilot study, we explored a quantitative method to analyse characteristics of breast tumours using 3D volumetric data obtained from a three-dimensional (3D) photoacoustic/ultrasound (PA/US) functional imaging system. Imaging results from 24 Asian patients with maximum tumour diameters less than 2 cm, including 8 benign tumours, 16 T1 stage invasive breast cancers (IBCs), and 22 normal breasts, were analysed. We found that the volumetric mean oxygenation saturation (SO 2 ) in tumour regions of T1 stage IBCs was 7.7% lower than that of benign tumours (P = 0.016) and 3.9% lower than that of healthy breasts (P = 0.010). The volumetric mean SO 2 in tumour surrounding regions of T1 stage IBCs was 4.9% lower than that of benign tumours (P = 0.009). For differentiating T1 stage IBCs and benign tumours, with a cut-off SO 2 value of 78.2% inside tumours, we obtained a sensitivity of 100%, a specificity of 62.5%, and an AUC of 0.81; with a cut-off SO 2 value of 77.9% in regions surrounding tumours, we obtained a sensitivity of 100%, a specificity of 75% and an AUC of 0.84. Our preliminary results demonstrate that 3D PA/US functional imaging has the potential to provide valuable quantitative physiological information that may be useful for the detection and evaluation of breast tumours.

The objective of this study was to illustrate our specific findings for intrahepatic cholangiocarcinoma (ICC) lesions on contrast-enhanced ultrasound (CEUS). In this study, 21 patients at our hospitals with pathologically proven ICC and CEUS data were retrospectively enrolled. General clinical data of the patients, and features of lesions on conventional and contrast-enhanced ultrasound were recorded. Two experienced radiologists retrospectively reviewed all images by consensus. On gray-scale sonography, hypoechoic, isoechoic and hyperechoic lesions accounted for 85.7%, 9.5% and 4.8%, respectively, of all lesions. Hypovascular patterns were found for 95.2% of the lesions on color Doppler flow imaging. During the arterial phase of CEUS, heterogeneous hyperenhancement, homogeneous hyperenhancement, rim-like hyperenhancement, isoenhancement and hypoenhancement were observed for 61.9%, 19.0%, 9.5%, 4.8%, 4.8% of the lesions, respectively. During the portal venous and late phases, 85.7% and 95.2% of the lesions, respectively, exhibited hypoenhancement. In addition, 66.7% of the ICC lesions exhibited washed-out interiors but little decrease in enhancement at the periphery during the portal venous phase, resulting in the formation of a hyperenhanced peripheral rim. In conclusion, the rim sign in the portal venous phase of CEUS could help diagnose ICC. This trait could be related to the infiltrating growth pattern of ICC.

Kidney fibroblasts play a crucial role in dictating tubular cell fate and the outcome of acute kidney injury (AKI). The underlying mechanisms remain to be determined. Here, we found that mTOR signaling was activated in fibroblasts from mouse kidneys with ischemia/reperfusion injury (IRI). Ablation of fibroblast Rheb or Rictor promoted, while ablation of fibroblast Tsc1 protected against tubular cell death and IRI in mice. In tubular cells cultured with conditioned media (CM) from Rheb -/- or Rictor -/- fibroblasts, less hepatocyte growth factor (HGF) receptor c-met signaling activation or staurosporine-induced cell apoptosis was observed. While CM from Tsc1 -/- fibroblasts promoted tubular cell c-met signaling activation and inhibited staurosporine-induced cell apoptosis. In kidney fibroblasts, blocking mTOR signaling downregulated the expression of peroxisome proliferator-activated receptor gamma (PPARγ) and HGF. Downregulating fibroblast HGF expression or blocking tubular cell c-met signaling facilitated tubular cell apoptosis. Notably, renal PPARγ and HGF expression was less in mice with fibroblast Rheb or Rictor ablation, but more in mice with fibroblast Tsc1 ablation than their littermate controls, respectively. Together, these data suggest that mTOR signaling activation in kidney fibroblasts protects against tubular cell death and dictates the outcome of AKI through stimulating PPARγ and HGF expression.

Kidney mesenchyme (KM) and nephron progenitors (NPs) depend on WNT activity, and their culture in vitro requires extensive repertoire of recombinant proteins and chemicals. Here we established a robust, simple culture of mouse KM using a combination of 3D Matrigel and growth media supplemented with Fibroblast Growth Factor 2 (FGF2) and Src inhibitor PP2. This allows dissociated KM to spontaneously self-organize into spheres. To reassess the requirement of WNT activity in KM self-organization and NPs maintenance, cells were cultured with short pulse of high-dose GSK3β inhibitor BIO, on a constant low-dose or without BIO. Robust proliferation at 48 hours and differentiation at 1 week were observed in cultures with high BIO pulse. Importantly, dissociated KM cultured without BIO, similarly to that exposed to constant low dose of BIO, maintained NPs up to one week and spontaneously differentiated into nephron tubules at 3 weeks of culture. Our results show that KM is maintained and induced to differentiate in a simple culture system. They also imply that GSK3β/WNT-independent pathways contribute to the maintenance and induction of mouse KM. The robust and easy 3D culture enables further characterization of NPs, and may facilitate disease modeling when applied to human cells.

The class of transparent conductive oxides includes the material indium tin oxide (ITO) and has become a widely used material of modern every-day life such as in touch screens of smart phones and watches, but also used as an optically transparent low electrically-resistive contract in the photovoltaics industry. More recently ITO has shown epsilon-near-zero (ENZ) behavior in the telecommunication frequency band enabling both strong index modulation and other optically-exotic applications such as metatronics. However, the ability to precisely obtain targeted electrical and optical material properties in ITO is still challenging due to complex intrinsic effects in ITO and as such no integrated metatronic platform has been demonstrated to-date. Here we deliver an extensive and accurate description process parameter of RF-sputtering, showing a holistic control of the quality of ITO thin films in the visible and particularly near-infrared spectral region. We are able to custom-engineer the ENZ point across the telecommunication band by explicitly controlling the sputtering process conditions. Exploiting this control, we design a functional sub-wavelength-scale filter based on lumped circuit-elements, towards the realization of integrated metatronic devices and circuits.

Genome instability is a common feature of tumor cells, and the persistent presence of genome instability is a potential mechanism of tumorigenesis. The E3 ubiquitin ligase MDM2 is intimately involved in genome instability, but its mechanisms are unclear. Our data demonstrated that the transcription factor HBP1 is a target of MDM2. MDM2 facilitates HBP1 proteasomal degradation by ubiquitinating HBP1, regardless of p53 status, thus attenuating the transcriptional inhibition of HBP1 in the expression of its target genes, such as the DNA methyltransferase DNMT1 and histone methyltransferase EZH2, which results in global DNA hypermethylation and histone hypermethylation and ultimately genome instability. The repression of HBP1 by MDM2 finally promotes cell growth and tumorigenesis. Next, we thoroughly explored the regulatory mechanism of the MDM2/HBP1 axis in DNA damage repair following ionizing radiation. Our data indicated that MDM2 overexpression-mediated repression of HBP1 delays DNA damage repair and causes cell death in a p53-independent manner. This investigation elucidated the mechanism of how MDM2 promotes genome instability and enhances tumorigenesis in the absence of p53, thus providing a theoretical and experimental basis for targeting MDM2 as a cancer therapy.

Mechanisms controlling ureter lenght and the position of the kidney are poorly understood. Glial cell-line derived neurotrophic factor (GDNF) induced RET signaling is critical for ureteric bud outgrowth, but the function of endogenous GDNF in further renal differentiation and urogenital system development remains discursive. Here we analyzed mice where 3' untranslated region (UTR) of GDNF is replaced with sequence less responsive to microRNA-mediated regulation, leading to increased GDNF expression specifically in cells naturally transcribing Gdnf. We demonstrate that increased Gdnf leads to short ureters in kidneys located in an abnormally caudal position thus resembling human pelvic kidneys. High GDNF levels expand collecting ductal progenitors at the expense of ureteric trunk elongation and result in expanded tip and short trunk phenotype due to changes in cell cycle length and progenitor motility. MEK-inhibition rescues these defects suggesting that MAPK-activity mediates GDNF's effects on progenitors. Moreover, Gdnf    hyper mice are infertile likely due to effects of excess GDNF on distal ureter remodeling. Our findings suggest that dysregulation of GDNF levels, for example via alterations in 3'UTR, may account for a subset of congenital anomalies of the kidney and urinary tract (CAKUT) and/or congenital infertility cases in humans and pave way to future studies.

A novel optical injection locking amplifier with acousto-optic modulator based phase modulation and a coherent detection scheme for optical frequency transfer applications is experimentally demonstrated in this study. A commercial distributed feedback diode laser is injection-locked to the resonant frequency of the optical signal with an optical fiber path length of hundreds of kilometers. This provides approximately 59 dB gain and ensures that the input carrier frequency fractional stability can be as good as 10 -20 at 1000 s. The amplifier was tested for the transfer of a commercial narrow-linewidth laser in a 180 km fiber link to a remote site with only a single amplification step. The transferred frequency at the remote end reached 10 -20 at 20000 s, which is suitable for optical frequency distribution and remote comparison between optical atomic clocks.

As one of the major epigenetic modifications, DNA methylation is constantly regulated during embryonic development, cell lineage commitment, and pathological processes. To facilitate real-time observation of DNA methylation, we generated a transgenic zebrafish reporter of DNA methylation (zebraRDM) via knockin of an mCherry-fused methyl-CpG binding domain (MBD) probe driven by the bactin2 promoter. The probe colocalized with heterochromatin, and its intensity was positively correlated with 5 mC immunostaining at a subcellular resolution in early embryos. Biochemical assays indicated that cells with stronger fluorescence maintained a higher level of DNA methylation, and time-lapse imaging at the blastula stage showed that the level of DNA methylation was transiently strengthened during mitosis. By crossing zebraRDM with other fluorescent transgenic lines, we demonstrate that the reporter can visually distinguish different cell lineages in organs like the heart. Our zebraRDM reporter therefore serves as a convenient and powerful tool for high-resolution investigation of methylation dynamics in live animals.

The SOCS1 gene coding for suppressor of cytokine signaling 1 is frequently repressed in hepatocellular carcinoma (HCC), and hence SOCS1 is considered a tumor suppressor in the liver. However, the tumor-suppressor mechanisms of SOCS1 are not yet well understood. SOCS1 is known to inhibit pro-inflammatory cytokine production and signaling and to promote activation of the p53 tumor suppressor. However, we observed that SOCS1-deficient mice developed numerous and large liver tumor nodules following treatment with the hepatocarcinogen diethylnitrosamine (DEN) without showing increased interleukin-6 production or activation of p53. On the other hand, the livers of DEN-treated Socs1-null mice showed elevated levels of p21(CIP1/WAF1) protein (p21). Even though p21 generally functions as a tumor suppressor, paradoxically many cancers, including HCC, are known to express elevated levels of p21 that correlate with poor prognosis. We observed elevated p21 expression also in the regenerating livers of SOCS1-deficient mice and in cisplatin-treated Socs1-null hepatocytes, wherein the p21 protein showed increased stability. We show that SOCS1 interacts with p21 and promotes its ubiquitination and proteasomal degradation. Besides, the DEN-treated livers of Socs1-null mice showed increased nuclear and cytosolic p21 staining, and the latter was associated with growth factor-induced, phosphatidylinositol 3-kinase-dependent phosphorylation of p21 in SOCS1-deficient hepatocytes. Cytosolic p21 is often associated with malignancy and chemo-resistance in many cancers. Accordingly, SOCS1-deficient hepatocytes showed increased resistance to apoptosis that was reversed by shRNA-mediated p21 knockdown. In the regenerating livers of Socs1-null mice, increased p21 expression coincided with elevated cyclinD levels. Correspondingly, SOCS1-deficient hepatocytes showed increased proliferation to growth factor stimulation that was reversed by p21 knockdown. Overall, our findings indicate that the tumor-suppressor functions of SOCS1 in the liver could be mediated, at least partly, via regulation of the expression, stability and subcellular distribution of p21 and its paradoxical oncogenic functions, namely, resistance to apoptosis and increased proliferation.

This study was to explore the association between gene variants and p21 expression and investigate the TP53-independent p21 regulation in hepatitis B virus (HBV) related hepatocellular carcinoma (HCC) patients from Guangxi by genome-wide association study. 426 HBV-related HCC patients were enrolled. Results showed that, after quality control, a total of 21,643 SNPs were identified in 107 p21 positive and 298 p21 negative patients. The variants of epidermal growth factor receptor (EGFR; rs2227983 and rs6950826) and spectrin repeat containing, nuclear envelope 2 (SYNE2; rs8010699, rs4027405 and rs1890908) were associated with p21 expression. Moreover the haplotype block (rs2227983 and rs6950826, r(2) = 0.378) in EGFR and the haplotype block in SYNE2 (rs8010699 was in strong LD with rs4027405 and rs1890908 (r(2) = 0.91 and 0.70, respectively)) were identified, and the haplotype A-G of EGFR and haplotype G-A-A of SYNE2 were significantly associated with p21 expression (P < 0.01). rs4027405 and rs1890908 were significantly associated with overall survival, and patients with AG/GG genotypes of SYNE2 gene had a worse overall survival (P = 0.001, P = 0.002). Our findings indicate that variants of EGFR and SYNE2 play an important role in p21 regulation and are associated with the clinical outcome of HBV-related HCC in a TP53-indenpdent manner.

Quercetin, a flavonoid found in a wide variety of plants and presented in human diet, displays promising potential in preventing kidney fibroblast activation. However, whether quercetin can ameliorate kidney fibrosis in mice with obstructive nephropathy and the underlying mechanisms remain to be further elucidated. In this study, we found that administration of quercetin could largely ameliorate kidney interstitial fibrosis and macrophage accumulation in the kidneys with obstructive nephropathy. MTORC1, mTORC2, β-catenin as well as Smad signaling were activated in the obstructive kidneys, whereas quercetin could markedly reduce their abundance except Smad3 phosphorylation. In cultured NRK-49F cells, quercetin could inhibit α-SMA and fibronectin (FN) expression induced by TGFβ1 treatment. MTORC1, mTORC2, β-catenin and Smad signaling pathways were stimulated by TGFβ1 at a time dependent manner. Similar to those findings in the obstructive kidneys, mTORC1, mTORC2 and β-catenin, but not Smad signaling pathways were remarkably blocked by quercetin treatment. Together, these results suggest that quercetin inhibits fibroblast activation and kidney fibrosis involving a combined inhibition of mTOR and β-catenin signaling transduction, which may act as a therapeutic candidate for patients with chronic kidney diseases.

Metformin, one of the most common prescriptions for patients with type 2 diabetes, is reported to protect the kidney from gentamicin-induced nephrotoxicity. However, the role and mechanisms for metformin in preventing cisplatin-induced nephrotoxicity remains largely unknown. In this study, a single intraperitoneal injection of cisplatin was employed to induce acute kidney injury (AKI) in CD1 mice. The mice exhibited severe kidney dysfunction and histological damage at day 2 after cisplatin injection. Pretreatment of metformin could markedly attenuate cisplatin-induced acute kidney injury, tubular cell apoptosis and inflammatory cell accumulation in the kidneys. Additionally, pretreatment of metformin could enhance both AMPKα phosphorylation and autophagy induction in the kidneys after cisplatin injection. In cultured NRK-52E cells, a rat kidney tubular cell line, metformin could stimulate AMPKα phosphorylation, induce autophagy and inhibit cisplatin-induced cell apoptosis. Blockade of either AMPKα activation or autophagy induction could largely abolish the protective effect of metformin in cisplatin-induced cell death. Together, this study demonstrated that metformin may protect against cisplatin-induced tubular cell apoptosis and AKI through stimulating AMPKα activation and autophagy induction in the tubular cells.

Y-chromosomal microdeletion (YCM) serves as an important genetic factor in non-obstructive azoospermia (NOA). Multiplex polymerase chain reaction (PCR) is routinely used to detect YCMs by tracing sequence-tagged sites (STSs) in the Y chromosome. Here we introduce a novel methodology in which we sequence 1,787 (post-filtering) STSs distributed across the entire male-specific Y chromosome (MSY) in parallel to uncover known and novel YCMs. We validated this approach with 766 Chinese men with NOA and 683 ethnically matched healthy individuals and detected 481 and 98 STSs that were deleted in the NOA and control group, representing a substantial portion of novel YCMs which significantly influenced the functions of spermatogenic genes. The NOA patients tended to carry more and rarer deletions that were enriched in nearby intragenic regions. Haplogroup O2* was revealed to be a protective lineage for NOA, in which the enrichment of b1/b3 deletion in haplogroup C was also observed. In summary, our work provides a new high-resolution portrait of deletions in the Y chromosome.

Differential methylation of the homologous chromosomes, a well-known mechanism leading to genomic imprinting and X-chromosome inactivation, is widely reported at the non-imprinted regions on autosomes. To evaluate the transgenerational DNA methylation patterns in human, we analyzed the DNA methylomes of somatic and germ cells in a four-generation family. We found that allelic asymmetry of DNA methylation was pervasive at the non-imprinted loci and was likely regulated by cis-acting genetic variants. We also observed that the allelic methylation patterns for the vast majority of the cis-regulated loci were shared between the somatic and germ cells from the same individual. These results demonstrated the interaction between genetic and epigenetic variations and suggested the possibility of widespread sequence-dependent transmission of DNA methylation during spermatogenesis.

Suppressor of cytokine signaling 1 (SOCS1) is considered as a tumor suppressor protein in hepatocellular carcinoma (HCC), but the underlying mechanisms remain unclear. Previously, we have shown that SOCS1-deficient hepatocytes displayed increased responsiveness to hepatocyte growth factor (HGF) due to enhanced signaling via the MET receptor tyrosine kinase. As aberrant MET activation occurs in many tumors including HCC, here we elucidated the mechanisms of SOCS1-mediated regulation. SOCS1 attenuated HGF-induced proliferation of human and mouse HCC cell lines and their growth as tumors in NOD.scid.gamma mice. Tumors formed by SOCS1 expressing HCC cells showed significantly reduced MET expression, indicating that SOCS1 not only attenuates MET signaling but also regulates MET expression. Mechanistically, SOCS1 interacted with MET via the Src homology 2 domain and this interaction was promoted by MET tyrosine kinase activity. The SOCS1-mediated reduction in MET expression does not require the juxtamembrane Y1003 residue implicated in Cbl-mediated downmodulation. Moreover, the proteasome inhibitor MG-132, but not the inhibitors of lysosomal degradation bafilomycin and chloroquine, reversed the SOCS1-mediated reduction in MET expression, indicating that this process is distinct from Cbl-mediated downmodulation. Accordingly, SOCS1 promoted polyubiquitination of MET via K48-dependent but not K63-mediated ubiquitin chain elongation. Furthermore, siRNA-mediated downmodulation of Cbl did not abolish SOCS1-mediated reduction in MET expression in HCC cells. SOCS1-dependent ubiquitination of endogenous MET receptor occurred rapidly following HGF stimulation in HCC cells, leading to proteasomal degradation of phosphorylated MET receptor. These findings indicate that SOCS1 mediates its tumor suppressor functions, at least partly, by binding to MET and interfering with downstream signaling pathways as well as by promoting the turnover of the activated MET receptor. We propose that loss of this control mechanism due to epigenetic repression of SOCS1 could contribute to oncogenic MET signaling in HCC and other cancers, and that MET inhibitors might be useful in treating these patients.

Non-obstructive azoospermia (NOA), a severe form of male infertility, is often suspected to be linked to currently undefined genetic abnormalities. To explore the genetic basis of this condition, we successfully sequenced ~650 infertility-related genes in 757 NOA patients and 709 fertile males. We evaluated the contributions of rare variants to the etiology of NOA by identifying individual genes showing nominal associations and testing the genetic burden of a given biological process as a whole. We found a significant excess of rare, non-silent variants in genes that are key epigenetic regulators of spermatogenesis, such as BRWD1, DNMT1, DNMT3B, RNF17, UBR2, USP1 and USP26, in NOA patients (P = 5.5 × 10(-7)), corresponding to a carrier frequency of 22.5% of patients and 13.7% of controls (P = 1.4 × 10(-5)). An accumulation of low-frequency variants was also identified in additional epigenetic genes (BRDT and MTHFR). Our study suggested the potential associations of genetic defects in genes that are epigenetic regulators with spermatogenic failure in human.

The objective was to investigate the effects of valsartan on the electrophysiological characteristics of left ventricular hypertrophic myocardium in spontaneously hypertensive rats (SHR). A total of 24 10-week-old male SHR were divided into two groups: valsartan and non-valsartan groups (n=12 in each). Twelve 10-week-old Wistar-Kyoto rats were served as the control group. Kv4.2 expression was measured in left ventricular myocardium using western blots. In addition, the systolic blood pressure, left ventricular mass index (LVMI), ventricular effective refractory period and ventricular fibrillation threshold (VFT) were also measured after eight weeks. INa, ICaL, Ito and membrane capacitance were measured in left ventricular myocytes after 8 weeks by whole-cell patch clamp. Valsartan decreased LVMI compared with the non-Valsartan group (Valsartan vs. non-Valsartan: 3.2±0.03 mg g(-1) vs. 3.7±0.02 mg g(-1), P<0.01). Valsartan also enhanced the VFT compared with the non-Valsartan group (Valsartan vs. non-Valsartan: 18.6±0.3 mA vs. 15.4±0.4 mA, P<0.01). The expression of Kv4.2 was significantly lower in the non-Valsartan and Valsartan groups compared with the control group (P<0.01). The expression of Kv4.2 was significantly higher in the Valsartan group compared with the non-Valsartan group (P<0.01). Valsartan decreased the density of ICaL compared with non-Valsartan group (Valsartan vs. non-Valsartan: -5.5±0.6 pA/pF vs. -7.2±0.9 pA/pF, P<0.05). Valsartan improved the density of Ito compared with non-Valsartan group(Valsartan vs. non-Valsartan: 13.93±0.8 pA/pF vs. 11.22±1.0 pA/pF, P<0.05). Valsartan improves the electrophysiological characteristics of left ventricular hypertrophic myocardium in spontaneously hypertensive rat.

High malignancy and early metastasis are the hallmarks of hepatocellular carcinoma (HCC). Here, we report that Cezanne2 expression is downregulated in HCC cells and in HCC patients' tumorous tissues and that Cezanne2 is inversely associated with Snail1 expression in HCC patients' tumorous tissues. Chromatin immunoprecipitation assays and the reporter gene assay showed that Snail1 binds to the promoter of the Cezanne2 gene and mediates the direct consequence of Cezanne2 repression. Enhanced expression of Cezanne2 could suppress proliferation, migration and invasion in HCC cells. Further, Cezanne2 could regulate MMP (matrix metalloproteinase)2, MMP9 and ICAM1 (intercellular adhesion molecule) levels through modulation of the NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cell) signaling cascade. Co-immunoprecipitation and in vivo deubiquitination assay indicated that Cezanne2 interacts with TNF receptor-associated factor (TRAF)6 and cleaves the polyubiquitin from TRAF6 substrates. Our data reveal that Snail1-mediated suppression of Cezanne2 may have a key role in HCC malignancy.