A rat model of cirrhosis with well-differentiated hepatocellular carcinoma induced by thioacetamide.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths, and commonly associated with hepatic fibrosis or cirrhosis. This study aims to establish a rat model mimicking the progression from liver fibrosis to cirrhosis and subsequently to HCC using thioacetamide (TAA). We utilized male Lewis rats, treating them with intra-peritoneal injections of TAA. These rats received bi-weekly injections of either 200 mg/kg TAA or saline (as a control) over a period of 34 weeks. The development of cirrhosis and hepatocarcinogenesis was monitored through histopathological examinations, biochemical markers, and immunohistochemical analyses. Our results demonstrated that chronic TAA administration induced cirrhosis and well-differentiated HCC, characterized by increased fibrosis, altered liver architecture, and enhanced hepatocyte proliferation. Biochemical analyses revealed significant alterations in liver function markers, including elevated alpha-fetoprotein (AFP) levels, without affecting kidney function or causing significant weight loss or mortality in rats. This TAA-induced cirrhosis and HCC rat model successfully replicates the clinical progression of human HCC, including liver function impairment and early-stage liver cancer characteristics. It presents a valuable tool for future research on the mechanisms of antitumor drugs in tumor initiation and development.

Polymorphisms Associated With Metabolic Dysfunction-Associated Steatotic Liver Disease Influence the Progression of End-Stage Liver Disease.

BACKGROUND AND AIMS: Chronic liver injury that results in cirrhosis and end-stage liver disease (ESLD) causes more than 1 million deaths annually worldwide. Although the impact of genetic factors on the severity of metabolic dysfunction-associated steatotic liver disease (MASLD) and alcohol-related liver disease (ALD) has been previously studied, their contribution to the development of ESLD remains largely unexplored. METHODS: We genotyped 6 MASLD-associated polymorphisms in healthy (n = 123), metabolic dysfunction-associated steatohepatitis (MASH) (n = 145), MASLD-associated ESLD (n = 72), and ALD-associated ESLD (n = 57) cohorts and performed multinomial logistic regression to determine the combined contribution of genetic, demographic, and clinical factors to the progression of ESLD. RESULTS: Distinct sets of factors are associated with the progression to ESLD. The PNPLA3 rs738409:G and TM6SF2 rs58542926:T alleles, body mass index (BMI), age, and female sex were positively associated with progression from a healthy state to MASH. The PNPLA3 rs738409:G allele, age, male sex, and having type 2 diabetes mellitus were positively associated, while BMI was negatively associated with progression from MASH to MASLD-associated ESLD. The PNPLA3 rs738409:G and GCKR rs780094:T alleles, age, and male sex were positively associated, while BMI was negatively associated with progression from a healthy state to ALD-associated ESLD. The findings indicate that the PNPLA3 rs738409:G allele increases susceptibility to ESLD regardless of etiology, the TM6SF2 rs58542926:T allele increases susceptibility to MASH, and the GCKR rs780094:T allele increases susceptibility to ALD-associated ESLD. CONCLUSION: The PNPLA3, TM6SF2, and GCKR minor alleles influence the progression of MASLD-associated or ALD-associated ESLD. Genotyping for these variants in MASLD and ALD patients can enhance risk assessment, prompting early interventions to prevent ESLD.

The maize WRKY transcription factor ZmWRKY64 confers cadmium tolerance in Arabidopsis and maize (Zea mays L.).

KEY MESSAGE: ZmWRKY64 positively regulates Arabidopsis and maize Cd stress through modulating Cd uptake, translocation, and ROS scavenging genes expression. Cadmium (Cd) is a highly toxic heavy metal with severe impacts on crops growth and development. The WRKY transcription factor is a significant regulator influencing plant stress response. Nevertheless, the function of the WRKY protein in maize Cd stress response remains unclear. Here, we identified a maize WRKY gene, ZmWRKY64, the expression of which was enhanced in maize roots and leaves under Cd stress. ZmWRKY64 was localized in the nucleus and displayed transcriptional activity in yeast. Heterologous expression of ZmWRKY64 in Arabidopsis diminished Cd accumulation in plants by negatively regulating the expression of AtIRT1, AtZIP1, AtHMA2, AtNRAMP3, and AtNRAMP4, which are involved in Cd uptake and transport, resulting in Cd stress tolerance. Knockdown of ZmWRKY64 in maize led to excessive Cd accumulation in leaf cells and in the cytosol of the root cells, resulting in a Cd hypersensitive phenotype. Further analysis confirmed that ZmWRKY64 positively regulated ZmABCC4, ZmHMA3, ZmNRAMP5, ZmPIN2, ZmABCG51, ZmABCB13/32, and ZmABCB10, which may influence Cd translocation and auxin transport, thus mitigating Cd toxicity in maize. Moreover, ZmWRKY64 could directly enhance the transcription of ZmSRG7, a reported key gene regulating reactive oxygen species homeostasis under abiotic stress. Our results indicate that ZmWRKY64 is important in maize Cd stress response. This work provides new insights into the WRKY transcription factor regulatory mechanism under a Cd-polluted environment and may lead to the genetic improvement of Cd tolerance in maize.

ZmB12D, a target of transcription factor ZmWRKY70, enhances the tolerance of Arabidopsis to submergence.

Submergence stress represents a serious threat to the yield and quality of maize because it can lead to oxygen deficiency and the accumulation of toxic metabolites. However, the mechanisms by which maize resists the adverse effects of submergence stress have yet to be fully elucidated. Here, we cloned a gene from maize Balem (Barley aleurone and embryo), ZmB12D, which was expressed at significant levels in seed embryos during imbibition and in leaves under submergence stress. Subcellular localization analysis indicated that the ZmB12D protein was localized in the mitochondria. The overexpression of ZmB12D in increased the tolerance of Arabidopsis to submergence stress, probably due to a reduction in the levels of malonaldehyde (MDA), the increased activity of antioxidant enzymes (SOD, POD and CAT), enhanced electron transport by coordinating the expression of non-symbiotic hemoglobin-2 (AHb2) and Fe transport-related (AtNAS3) genes (mediating Fe and oxygen availability) and also modulated the anaerobic respiration rates through upregulated the AtPDC1, AtADH1, AtSUS4 genes under submergence. Yeast one-hybrid (Y1H) and transient transactivation assays demonstrated that ZmWRKY70 bound to the ZmB12D promoter and activated ZmB12D. Collectively, out findings indicate that ZmB12D plays an important role in the tolerance of maize to submergence stress. This research provides new insights into the genetic improvement of maize with regards to submergence tolerance.

The Genome-Wide Identification, Characterization, and Expression Analysis of the Strictosidine Synthase-like Family in Maize (Zea mays L.).

Maize is often subjected to various environmental stresses. The strictosidine synthase-like (SSL) family is thought to catalyze the key step in the monoterpene alkaloids synthesis pathway in response to environmental stresses. However, the role of ZmSSL genes in maize growth and development and its response to stresses is unknown. Herein, we undertook the systematic identification and analysis of maize SSL genes. Twenty SSL genes were identified in the maize genome. Except for chromosomes 3, 5, 6, and 10, they were unevenly distributed on the remaining 6 chromosomes. A total of 105 SSL genes from maize, sorghum, rice, Aegilops tauschii, and Arabidopsis were divided into five evolutionary groups, and ZmSSL gene structures and conserved protein motifs in the same group were similar. A collinearity analysis showed that tandem duplication plays an important role in the evolution of the SSL family in maize, and ZmSSL genes share more collinear genes in crops (maize, sorghum, rice, and Ae. tauschii) than in Arabidopsis. Cis-element analysis in the ZmSSL gene promoter region revealed that most genes contained many development and stress response elements. We evaluated the expression levels of ZmSSL genes under normal conditions and stress treatments. ZmSSL4-9 were widely expressed in different tissues and were positively or negatively regulated by heat, cold, and infection stress from Colletotrichum graminicola and Cercospora zeina. Moreover, ZmSSL4 and ZmSSL5 were localized in the chloroplast. Taken together, we provide insight into the evolutionary relationships of the ZmSSL genes, which would be useful to further identify the potential functions of ZmSSLs in maize.

Utilizing Protein-Peptide Hybrid Microarray for Time-Resolved Diagnosis and Prognosis of COVID-19.

The COVID-19 pandemic has highlighted the urgent need for accurate, rapid, and cost-effective diagnostic methods to identify and track the disease. Traditional diagnostic methods, such as PCR and serological assays, have limitations in terms of sensitivity, specificity, and timeliness. To investigate the potential of using protein-peptide hybrid microarray (PPHM) technology to track the dynamic changes of antibodies in the serum of COVID-19 patients and evaluate the prognosis of patients over time. A discovery cohort of 20 patients with COVID-19 was assembled, and PPHM technology was used to track the dynamic changes of antibodies in the serum of these patients. The results were analyzed to classify the patients into different disease severity groups, and to predict the disease progression and prognosis of the patients. PPHM technology was found to be highly effective in detecting the dynamic changes of antibodies in the serum of COVID-19 patients. Four polypeptide antibodies were found to be particularly useful for reflecting the actual status of the patient's recovery process and for accurately predicting the disease progression and prognosis of the patients. The findings of this study emphasize the multi-dimensional space of peptides to analyze the high-volume signals in the serum samples of COVID-19 patients and monitor the prognosis of patients over time. PPHM technology has the potential to be a powerful tool for tracking the dynamic changes of antibodies in the serum of COVID-19 patients and for improving the diagnosis and prognosis of the disease.

Evaluation of Human Hepatocyte Drug Metabolism Carrying High-Risk or Protection-Associated Liver Disease Genetic Variants.

Metabolic-dysfunction-associated steatotic liver disease (MASLD), which affects 30 million people in the US and is anticipated to reach over 100 million by 2030, places a significant financial strain on the healthcare system. There is presently no FDA-approved treatment for MASLD despite its public health significance and financial burden. Understanding the connection between point mutations, liver enzymes, and MASLD is important for comprehending drug toxicity in healthy or diseased individuals. Multiple genetic variations have been linked to MASLD susceptibility through genome-wide association studies (GWAS), either increasing MASLD risk or protecting against it, such as PNPLA3 rs738409, MBOAT7 rs641738, GCKR rs780094, HSD17B13 rs72613567, and MTARC1 rs2642438. As the impact of genetic variants on the levels of drug-metabolizing cytochrome P450 (CYP) enzymes in human hepatocytes has not been thoroughly investigated, this study aims to describe the analysis of metabolic functions for selected phase I and phase II liver enzymes in human hepatocytes. For this purpose, fresh isolated primary hepatocytes were obtained from healthy liver donors (n = 126), and liquid chromatography-mass spectrometry (LC-MS) was performed. For the cohorts, participants were classified into minor homozygotes and nonminor homozygotes (major homozygotes + heterozygotes) for five gene polymorphisms. For phase I liver enzymes, we found a significant difference in the activity of CYP1A2 in human hepatocytes carrying MBOAT7 (p = 0.011) and of CYP2C8 in human hepatocytes carrying PNPLA3 (p = 0.004). It was also observed that the activity of CYP2C9 was significantly lower in human hepatocytes carrying HSD17B13 (p = 0.001) minor homozygous compared to nonminor homozygous. No significant difference in activity of CYP2E1, CYP2C8, CYP2D6, CYP2E1, CYP3A4, ECOD, FMO, MAO, AO, and CES2 and in any of the phase II liver enzymes between human hepatocytes carrying genetic variants for PNPLA3 rs738409, MBOAT7 rs641738, GCKR rs780094, HSD17B13 rs72613567, and MTARC1 rs2642438 were observed. These findings offer a preliminary assessment of the influence of genetic variations on drug-metabolizing cytochrome P450 (CYP) enzymes in healthy human hepatocytes, which may be useful for future drug discovery investigations.

Human Hepatocellular response in Cholestatic Liver Diseases.

Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), the most common types of cholestatic liver disease (CLD), result in enterohepatic obstruction, bile acid accumulation, and hepatotoxicity. The mechanisms by which hepatocytes respond to and cope with CLD remain largely unexplored. This study includes the characterization of hepatocytes isolated from explanted livers of patients with PBC and PSC. We examined the expression of hepatocyte-specific genes, intracellular bile acid (BA) levels, and oxidative stress in primary-human-hepatocytes (PHHs) isolated from explanted livers of patients with PBC and PSC and compared them with control normal human hepatocytes. Our findings provide valuable initial insights into the hepatocellular response to cholestasis in CLD and help support the use of PHHs as an experimental tool for these diseases.

Unraveling roles of the intermediate settler in a microaerobic hydrolysis sludge in situ reduction process.

The roles of the intermediate settler in the sludge process reduction activated sludge process (SPRAS), and the influences of its hydraulic retention time (HRTST) on pollutant removal and sludge reduction were investigated. Prolonging HRTST from 3.0 to 4.5 and 6.0 h resulted in sludge reduction efficiencies increased from 46.8% to 61.5% and 62.7%. The sludge accumulation in the intermediate settler formed an anaerobic zone but inhibited methane production, and the alternating microaerobic and anaerobic environment in the sludge process reduction (SPR) module increased the microbial diversity and enriched the hydrolytic and fermentative bacteria. Prolonging HRTST accelerated dissolved organic matter release and elevated the degradation of refractory fraction, and improved the sludge properties of the SPRAS. Metagenomic analysis showed that the SPR module enhanced the glycolysis pathway and decoupling metabolism for sludge reduction. The results revealed that the intermediate settler plays dual roles in solid-liquid separation and sludge reduction metabolism.

Immunogenicity evaluation of viral peptides via nonspecific interactions between anti-peptide IgYs and non-cognate peptides.

Immunogenicity can be evaluated by detecting antibodies (Abs) induced by an antigen. Presently deployed assays, however, do not consider the negative impacts of Ab poly-specificity, which is well established at the monoclonal antibody level. Here, we studied antibody poly-specificity at the serum level (i.e. nonspecific Ab-probe interactions, NSIs), and ended up establishing a new platform for viral peptide immunogenicity evaluation. We first selected three peptides of high, medium and low immunogenicity, using a 'vaccine serum response rate'-based approach (i.e. the gold standard). These three peptides (Pi) in the bovine serum albumin-Pi form were used to immunize chickens, resulting in longitudinal serum samples for screening with a non-cognate peptide library. The signal intensity of Ab-peptide specific binding and 'NSI count' was used to evaluate the viral peptides' immunogenicity. Only the NSI count agreed with the gold standard. The NSI count also provides more informative data on antibody production than the aggregated signal intensity by whole-protein-based indirect enzyme-linked immunosorbent assay.

Simultaneous sludge minimization and membrane fouling mitigation in membrane bioreactors by using a microaerobic - Settling pretreatment module.

Membrane fouling is the major obstacle for membrane bioreactors operated at a long sludge retention time to reduce sludge production. In this study, a sludge process reduction (SPR) module, consisting of a microaerobic tank and a settler, was inserted before an anoxic/oxic MBR (AO-MBR) to achieve dual objectives of fouling alleviation and sludge reduction. Three SPR-MBRs were operated to investigate influences of sludge recirculation ratios from the SPR settler to the microaerobic tank on process performance. Compared to AO-MBR, the SPR-MBRs reduced sludge production by 43.1-56.4% by maintaining sludge retention times above 175 d, and decreased foulant layer resistance and pore clogging resistance. Inserting SPR reduced the accumulation of dissolved organic matters and extracellular polymeric substances, enlarged sludge flocs, and decreased sludge viscoelasticity. However, increasing RSPR stimulated outward diffusion of extracellular polymeric substances and increased sludge viscosity. SPR-MBRs achieved effective sludge reduction by enriching hydrolytic (Trichococcus and Aeromonas) and fermentative genera (Lactococcus, Paludibacter, Macellibacteroides, and Acinetobacter) in the SPR, and alleviated membrane fouling by prohibiting the growth of extracellular polymeric substance-secreting bacteria and enriching filamentous bacteria to enlarge particle size. The results revealed that the SPR-MBR maximized sludge reduction with a very long sludge retention time, and alleviated membrane fouling synchronously.

Human Induced Pluripotent Stem Cell based Hepatic-Modeling of Lipid metabolism associated TM6SF2 E167K variant.

BACKGROUND AND AIMS: TM6SF2 rs58542926 (E167K) is associated with an increase in the prevalence of Metabolic Disfunction-Associated Steatotic Liver Disease (MASLD). Despite all the investigation related to the role of this variant in lipid metabolism, conflicting results in mouse studies underscore the importance of creating a human model for understanding the TM6SF2 mechanism. Therefore, the aim of this study is to generate a reliable human in vitro model that mimic the effects of the TM6SF2 E167K mutation and can be used for future mechanism studies. APPROACH AND RESULTS: We performed gene editing on human-induced pluripotent stem cells (iPSC) derived from a healthy individual to obtain the cells carrying the TM6SF2 E167K mutation. After hepatic differentiation, a decrease in TM6SF2 protein expression was observed in the mutated-induced hepatocyte. An increase in intracellular lipid droplets and a decrease in the efflux of cholesterol and ApoB100 were also observed. Transcriptomics analysis showed up-regulation of genes related to the transport, flux, and oxidation of lipids, fatty acids, and cholesterol in TM6SF2 E167K cells. Additionally, signs of cellular stress were observed in the ER and mitochondria. CONCLUSIONS: Our findings indicate that induced hepatocytes generated from iPSC carrying the TM6SF2 E167K recapitulate the effects observed in human hepatocytes from individuals with the TM6SF2 mutation. This study characterizes an in vitro model that can be used as a platform to help in the identification of potential clinical targets and therapies and to understand the mechanism by which the TM6SF2 E167K variant leads to vulnerability to MASLD.

Sludge decay kinetics and metagenomic analysis uncover discrepant metabolic mechanisms in two different sludge in situ reduction systems.

A comparative study was conducted between an anaerobic side-stream reactor (ASSR) process and a sludge process reduction (SPR) activated sludge (SPRAS) process for uncovering crucial metabolic mechanisms governing sludge reduction. Both of two processes were efficient in removing pollutants, while the SPRAS (62.3 %) obtained much higher sludge reduction than the ASSR (27.9 %). The highest rate coefficients of sludge decay, heterotroph lysis and particles hydrolysis were 0.106, 0.219 and 0.054 d-1 in the SPR module, followed by ASSR with coefficients of 0.060, 0.135 and 0.047 d-1. The SPR module achieved an 81.9 % higher sludge decay mass with a 32.8 % smaller volume than the ASSR module. The SPR module preferentially enriched hydrolytic/fermentative and slow-growing bacteria. Metagenomic analysis revealed that SPR strengthened the key hydrolases and L-lactate dehydrogenase in the glycolysis pathways and weakened the citrate cycle, inducing metabolic uncoupling due to the reduced biosynthesis of ATP. Inserting ASSR only altered the ATP biosynthesis pathway, but maintenance metabolism was dominant for sludge reduction, with a long sludge retention time prolonging the food chain for predation.

Identification of Amplified Fragment Length Polymorphism (AFLP) Markers Tightly Associated with Drought Stress Gene in Male Sterile and Fertile Salvia miltiorrhiza Bunge.

Consistent grain yield in drought environment has attracted wide attention due to global climate change. However, the important drought-related traits/genes in crops have been rarely reported. Many near-isogenic lines (NILs) of male sterile and fertile Salvia miltiorrhiza have been obtained in our previous work through testcross and backcross in continuous field experiments conducted in 2006-2009. Both segregating sterile and fertile populations were subjected to bulked segregant analysis (BSA) and amplified fragment length polymorphism (AFLP) with 384 and 170 primer combinations, respectively. One out of 14 AFLP markers (E9/M3246) was identified in treated fertile population as tightly linked to the drought stress gene with a recombination frequency of 6.98% and at a distance of 7.02 cM. One of 15 other markers (E2/M5357) was identified in a treated sterile population that is closely associated with the drought stress gene. It had a recombination frequency of 4.65% and at a distance of 4.66 cM. Interestingly, the E9/M3246 fragment was found to be identical to another AFLP fragment E11/M4208 that was tightly linked to the male sterile gene of S. miltiorrhiza with 95% identity and e-value 4 × 10-93. Blastn analysis suggested that the drought stress gene sequence showed higher identity with nucleotides in Arabidopsis chromosome 1-5.