High Expression Research Articles

Genet identity and season drive gene expression in outplanted Acropora palmata at different reef sites

Coral reefs are experiencing decreases in coral cover due to anthropogenic influences. Coral restoration is addressing this decline by outplanting large volumes of corals onto reef systems. Understanding how outplanted corals react at a transcriptomic level to different outplant locations over time is important, as it will highlight how habitat affects the coral host and influences physiological measures. In this study, the transcriptomic dynamics of four genets of outplanted Acropora palmata were assessed over a year at three reef sites in the Florida Keys. Genet identity was more important than time of sampling or outplant site, with differing levels of baseline immune and protein production the key drivers. Once accounting for genet, enriched growth processes were identified in the winter, and increased survival and immune expression were found in the summer. The effect of the reef site was small, with hypothesized differences in autotrophic versus heterotrophic dependent on outplant depth. We hypothesize that genotype identity is an important consideration for reef restoration, as differing baseline gene expression could play a role in survivorship and growth. Additionally, outplanting during cooler winter months may be beneficial due to higher expression of growth processes, allowing establishment of outplants on the reef system.

Proteomic Analysis Unveils the Protective Mechanism of Active Modified Atmosphere Packaging Against Senescence Decay and Respiration in Postharvest Loose-Leaf Lettuce

In this study, physicochemical and proteomic analyses were performed to investigate the effect of modified atmosphere packaging (MAP) on the quality of postharvest loose-leaf lettuce. The results showed that MAP enhanced the sensory characteristics of loose-leaf lettuce and delayed the incidence of postharvest deterioration by suppressing weight loss, electrolyte leakage, and reactive oxygen species levels. MAP-inhibited storage-induced programmed cell death may be attributed to a lower expression of protein disulfide isomerase and a higher expression of oligonucleotide/oligosaccharide binding fold nucleic acid binding site protein and reducing glutamine synthase levels. Also, we explore the potential of MAP to protect against oxidative damage in loose-leaf lettuce by potentially modulating the expression levels of NAC family proteins, which may enhance signaling and the expression of cytochrome c oxidase and membrane-bound pyrophosphate in the oxidative phosphorylation pathway. In addition, MAP potentially delayed postharvest senescence and extended the shelf life of lettuce by regulating key protein metabolic pathways that may reduce respiration rates. These include the NAC family of proteins, enzymes in the oxidative phosphorylation pathway, glutamine synthetize, and other crucial metabolic routes. These findings provide a scientific basis for enhancing the postharvest preservation of leafy vegetables, such as loose-leaf lettuce, through MAP technology.

Comparative Transcriptome Analysis Reveals a Tissue-Specific Pathway Involved in Nitrogen Utilization Between Genotypes with Different Nitrogen Use Efficiencies in Tea Plants (Camellia sinensis)

Nitrogen (N) is a key nutrient which affects plant development and quality formation for tea plants. Notable genetic variation in nitrogen use efficiency (NUE) has been reported among different genotypes of Camellia sinensis. However, the molecular mechanisms underlying these differences have not been illuminated. In this study, a 15N tracing method was used to compare nitrogen use efficiency among six genotypes. The results show that there were significant differences in the NUEs among these genotypes. Among them, TC12 had the highest NUE, while LJCY had the lowest NUE. Transcriptome analysis between these two cultivars showed that differentially expressed genes (DEGs) were significantly enriched in photosynthesis—antenna proteins and zeatin biosynthesis in mature leaves and new shoots, respectively. TC12 had higher expression levels of AMT1.2, NRT2.4, and NRT3.2 in the roots, AAP6 and AAP7 in the stems and shoots, and LHC in the mature leaves than LJCY. The expression of ZOG1 and CKX, which are involved in zeatin biosynthesis, was down-regulated in the shoots of TC12 compared with LJCY. These findings will contribute to insights into the molecular mechanism of nitrogen utilization and the identified candidate genes provide a genetic resource for improving N use efficiency in tea plants.

Heterogeneous Surface CD79b Expression in Aggressive B-Cell Lymphomas Assessed by Flow Cytometry on Lymph Node Biopsies

Background: CD79b is a B-cell-specific antigen that is crucial to the B-cell receptor and is considered a key target for treatment in aggressive B-cell lymphomas. Methods: While immunohistochemical studies have shown widespread expression of CD79b in mature B-cell-derived lymphomas, flow cytometry allows for precise measurement and differentiation between surface and intracellular localization. Results: In our comparative analysis, we discovered that CD79b expression percentages and mean fluorescence intensity (MFI) were lower in a group of 127 cases of aggressive B-cell lymphomas compared to a control group of benign reactive hyperplasia. We also observed significant variability in the surface expression of CD79b among lymphoma cases, with 18% showing predominantly intracellular positivity. There was a strong correlation between the surface expression of CD79b and clonal light chains. Notably, primary mediastinal B-cell lymphomas exhibited significantly lower surface CD79b expression compared to other lymphoma subtypes (median 0.8% IQR 0–48.5 vs. 80% IQR 24–97, p = 0.0005). Furthermore, patients over 60 years old and those with a higher Revised International Prognostic Index (R-IPI) had significantly higher CD79b expression, both of which are associated with a significant benefit from adding an anti-CD79b drug conjugate to first-line chemotherapy in diffuse large B-cell lymphomas. Conclusions: In conclusion, the quantitative flow cytometric analysis of CD79b surface expression in aggressive B-cell lymphomas provides clinically relevant information, highlighting its potential usefulness in guiding therapeutic decisions.

SNA·SMNP·CBE system: A novel integrative strategy for β-hemoglobinopathies gene therapy

Here, we developed and demonstrated a novel integrative system—Silica Nanorods (SNA) substrate cell capture combined with Supramolecular Nanoparticle (SMNP) delivery mediated CBE base editing (SNA·SMNP·CBE)—achieving the synchronization of CD34+HSPCs cell capture and gene editing for β-hemoglobinopathies. First, in vitro study shows it enables efficient and precise modification of BCL11A promoter in CD34+HSPCs, yielding the highly editing efficiency of 50.4 %, thus making an alternative strategy to conventional immunomagnetic cell separation and electroporation transfection system mediated CBE editing (IMS·EP·CBE). Then, we transplanted the edited human CD34+HSPCs into severe combined immunodeficiency (SCID) mice by using intraosseous injection strategy. When compared with conventional IMS·EP·CBE methods, our results showed that significantly higher human HBG expression in the bone marrow and peripheral blood of recipient mice, and long-term engraftment, evidenced from similar gene expression profiles to naïve CD34+HSPCs at 14 weeks. Conclusively, our integrative system—SNA·SMNP·CBE·intraosseous injection—offers an appealing novel way for the unique potential of gene therapy in the clinic application for β-hemoglobinopathies patients.

Distinct immune profiles in children living with HIV based on timing and duration of suppressive antiretroviral treatment

Timely initiation of antiretroviral therapy (ART) remains a major challenge in the effort to treat children living with HIV (“CLH”) and little is known regarding the dynamics of immune normalization following ART in CLH with varying times to and durations of ART. Here, we leveraged two cohorts of virally-suppressed CLH from Nairobi, Kenya to examine differences in the peripheral immune systems between two cohorts of age-matched children (to control for immune changes with age): one group which initiated ART during early HIV infection and had been on ART for 5–6 years at evaluation (early, long-term treated; “ELT” cohort), and one group which initiated ART later and had been on ART for approximately 9 months at evaluation (delayed, short-term treated; “DST” cohort). We profiled PBMC and purified NK cells from these two cohorts by mass cytometry time-of-flight (CyTOF). Although both groups of CLH had undetectable viral RNA load at evaluation, there were marked differences in both immune composition and immune phenotype between the ELT cohort and the DST cohort. DST donors had reduced CD4 T cell percentages, decreased naive to effector memory T cell ratios, and markedly higher expression of stress-induced markers. Conversely, ELT donors had higher naive to effector memory T cell ratios, low expression of stress-induced markers, and increased expression of markers associated with an effective antiviral response and resolution of inflammation. Collectively, our results demonstrate key differences in the immune systems of virally-suppressed CLH with different ages at ART initiation and durations of treatment and provide further rationale for emphasizing early onset of ART.

Genome-wide identification and gene expression pattern reveal dominant digestive enzyme genes contributing to major enzyme activities during the molting cycle of the Chinese mitten crab, Eriocheir sinensis

Chinese mitten crab Eriocheir sinensis is an important economic species, but it largely remains unknown for the genome-wide identification of digestive enzyme genes, gene expression pattern and their activities during the molting cycle of E. sinensis. Therefore, this study was conducted to investigate the diversity of major digestive enzyme genes, their dynamic expression patterns as well as the digestive enzyme activities during the molting cycle; the binding sites and affinities of major digestive enzyme-substrate were also predicted using molecular docking analysis. The results revealed that there were 4 α-amylase genes, 14 trypsin genes, and 7 lipase genes identified in E. sinensis. Among them, Es-Amylase4, Es-Trypsin11-I7, and Es-Lipase1 exhibited the highest transcriptional expression levels respectively, with Es-Amylase4 and Es-Lipase1 peaking in stage C and Es-Trypsin11-I7 in stage D. The α-amylase and lipase activities peak in stage C, while trypsin activity was highest in stage A/B. During molting process, the transcriptional expression levels of 4 α-amylase isoforms and 3 lipase isoforms positively correlated with enzyme activity changes, and the expression of Es-lipase1-mRNA was significantly higher than the other lipase transcripts (P < 0.05). As lipase is one of most important digestive enzymes during food digestion and there are two transcription isoforms with significantly higher expression levels than the others. There were 1 and 3 hydrogen bonds for Es-lipase1- triglyceride and Es-lipase7- triglyceride complexes, respectively, while the binding affinities of Es-lipase1- triglyceride and Es-lipase7- triglyceride were − 8.0 kcal/mol and − 7.1 kcal/mol, respectively. In conclusion, crustaceans have numerous digestive enzymes with a high degree of polymorphism, but there are 3, 2, and 1 highly expressed gene in E. sinensis contributed to the major enzyme activities of α-amylase, trypsin, and lipase, respectively. For α-amylase and lipase, both gene expression and their enzyme activities peaked at stage C, which was highly related to food intake and specific molting stage.

IL-17 signaling protects against Helicobacter pylori-induced gastric cancer

ABSTRACT Helicobacter pylori infection is the predominant risk factor for the development of gastric cancer. Risk is enhanced by specific H. pylori virulence factors, diet, and the inflammatory response. Chronic activation of T helper (Th) 1 and Th17 pathways contributes to prolonged inflammation; yet, higher expression of IL-17 receptor (IL-17RA) is a favorable prognostic marker for survival after gastric cancer diagnosis. The protective impact of IL-17RA signaling is not understood. To investigate if IL-17RA signaling protects during H. pylori-induced carcinogenesis, the transgenic InsGAS tg/tg mouse, which is prone to H. pylori-induced gastric cancer, was utilized. InsGAS tg/tg mice and InsGAS tg/tg Il17ra -/- mice were infected with a cag type 4 secretion system (T4SS) positive H. pylori strain for up to 6 months. Six weeks post-infection, IL-17RA deficiency led to increased bacterial burden, increased gastritis, and development of lymphoid follicles. Increased inflammation was associated with heightened cellular proliferation and earlier loss of parietal and chief cells in InsGAS tg/tg Il17ra -/- mice. Gastric cancers developed more frequently by 3- and 6-months post-infection in H. pylori-infected InsGAS tg/tg Il17ra -/- mice compared to InsGAS tg/tg mice. Chronic inflammation was exacerbated with IL-17RA deficiency, characterized by elevated Th1/Th17 cytokines, increased B cell infiltration, and enhanced IgA production, despite reduced expression of the polymeric immunoglobulin receptor. Further, paragastric lymph nodes of InsGAS tg/tg Il17ra -/- mice were enlarged relative to controls and displayed altered gene expression profiles. Increased inflammation was accompanied by a significant increase in Cybb expression, which encodes NADPH oxidase 2, suggesting that increased oxidative damage may occur in the absence of IL-17RA. Further, there is increased phosphorylation of histone 2AX in IL-17RA deficient mice, indicating that the DNA damage response is highly activated. These data suggest that IL-17RA signaling activates a protective pathway to prevent excessive inflammation which otherwise can lead to increased oxidative stress, DNA damage, and drive gastric carcinogenesis after H. pylori infection.

Integrated analyses of multi-omic data derived from paired primary lung cancer and brain metastasis reveal the metabolic vulnerability as a novel therapeutic target

BackgroundLung cancer brain metastases (LC-BrMs) are frequently associated with dismal mortality rates in patients with lung cancer; however, standard of care therapies for LC-BrMs are still limited in their efficacy. A deep understanding of molecular mechanisms and tumor microenvironment of LC-BrMs will provide us with new insights into developing novel therapeutics for treating patients with LC-BrMs.MethodsHere, we performed integrated analyses of genomic, transcriptomic, proteomic, metabolomic, and single-cell RNA sequencing data which were derived from a total number of 154 patients with paired and unpaired primary lung cancer and LC-BrM, spanning four published and two newly generated patient cohorts on both bulk and single cell levels.ResultsWe uncovered that LC-BrMs exhibited a significantly greater intra-tumor heterogeneity. We also observed that mutations in a subset of genes were almost always shared by both primary lung cancers and LC-BrM lesions, including TTN, TP53, MUC16, LRP1B, RYR2, and EGFR. In addition, the genome-wide landscape of somatic copy number alterations was similar between primary lung cancers and LC-BrM lesions. Nevertheless, several regions of focal amplification were significantly enriched in LC-BrMs, including 5p15.33 and 20q13.33. Intriguingly, integrated analyses of transcriptomic, proteomic, and metabolomic data revealed mitochondrial-specific metabolism was activated but tumor immune microenvironment was suppressed in LC-BrMs. Subsequently, we validated our results by conducting real-time quantitative reverse transcription PCR experiments, immunohistochemistry, and multiplexed immunofluorescence staining of patients’ paired tumor specimens. Therapeutically, targeting oxidative phosphorylation with gamitrinib in patient-derived organoids of LC-BrMs induced apoptosis and inhibited cell proliferation. The combination of gamitrinib plus anti-PD-1 immunotherapy significantly improved survival of mice bearing LC-BrMs. Patients with a higher expression of mitochondrial metabolism genes but a lower expression of immune genes in their LC-BrM lesions tended to have a worse survival outcome.ConclusionsIn conclusion, our findings not only provide comprehensive and integrated perspectives of molecular underpinnings of LC-BrMs but also contribute to the development of a potential, rationale-based combinatorial therapeutic strategy with the goal of translating it into clinical trials for patients with LC-BrMs.

Exploring RPA1-ETAA1 axis via high-throughput data analysis: implications for PD-L1 nuclear translocation and tumor-immune dynamics in liver cancer

IntroductionETAA1 is recruited to DNA damage sites via its RPA -binding and ATR -activating domain (AAD) motifs, where RPA binding is crucial for ETAA1’s regulation of ATR activity. Methods &amp;amp; resultsOur findings associate Programmed Death- Ligand1 (PD-L1) with the RPA1-ETAA1 axis, suggesting that upregulated RPA1 -dependent ETAA1 may facilitate PD-L1 nuclear accumulation. We observed strong correlations between ETAA1 and RPA1 with the components involved in HDAC2-mediated deacetylation, clathrin -dependent endocytosis, and PD-L1 nucleocytoplasmic shuttling, aligning with the established regulatory pathway of PD-L1 nuclear translocation. Moreover, nuclear PD-L1 transactivates a panel of pro-inflammatory and immune response transcription factors, potentially reshaping the tumor immune microenvironment. We identified a landscape of infiltrating lymphocytes influenced by ETAA1, finding that levels of ETAA1 were negatively correlated with CD8+ T and Natural Killer T (NKT) cells, but positively correlated with CD4+ T helper 2 (Th2) cells, cancer-associated fibroblasts (CAFs), myeloid-derived suppressor cells (MDSCs), neutrophils and regulatory T cells (Tregs), suggesting a potential role in immune evasion. Further analysis shows that the RPA1-ETAA1 axis is significantly associated with multiple metastasis mediators and unfavorable liver cancer progression, with higher expression observed in advanced stages and poorly differentiated subgroups. Discussion &amp;amp; conclusionThese findings expand the role of the RPA1-ETAA1 axis beyond DNA repair, highlighting its potential as a target for cancer therapy.

MiR-200 family as new potential prognostic factor of overall survival of patients with WHO G2 and WHO G3 brain gliomas

Gliomas are the predominant cause of cancer-related deaths among the young population. Even after incorporation of IDH1/2 mutations and 1p19q codeletion there are doubts regarding adjuvant treatment in WHO G2/G3 gliomas. miRNA molecules control about 30% of all genes, also many oncogenes, tumor suppressor genes and genes responsible for the response to ionizing radiation and systemic treatment. Patients with brain gliomas exhibit miRNA disorders. We aimed to evaluate the expression of miR-200 family members in relation to selected clinico-pathological factors and their prognostic value. We enrolled 53 patients diagnosed with WHO G2/G3 brain gliomas treated between 2012–2016. RT-qPCR based expression of miR-200 family was assessed in tumor and surrounding non-cancerous tissue. An analysis of selected clinico-pathological features was carried out. A logistic regression model was prepared for the miRNA signature. The predictive potential of the signature was assessed using the ROC curve. A stepwise backward regression model was used to select variables with a significant predictive potential related to OS. It was shown that miR-200a-3p, miR-200a-5p, miR-200c-5p, miR-141-3p and miR-429 can be independent predictors of survival. Better 2- and 5-year OS was associated with higher expression of miR-200a-3p, miR141-3p and lower expression of miR-200a-5p, miR-200c-5p, miR-429. The strongest predictors of survival were miR-200a-5p, miR-200b-3p, miR-200c-5p, miR-141-3p, miR-429, tumor volume and CTV. Members of the miR-200 family exhibit prognostic value for 2- and 5-year OS. Presented predictive models of survival may be clinically useful for treatment optimization.

The Chicken HDAC4 Promoter and Its Regulation by MYC and HIF1A

Background: Histone deacetylase 4 (HDAC4) is a member of the class II histone deacetylase family, whose members play a crucial role in various biological processes. An in-depth investigation of the transcriptional characteristics of chicken HDAC4 can provide fundamental insights into its function. Methods: We examined HDAC4 expression in chicken embryonic stem cells (ESC) and spermatogonial stem cells (SSC) and cloned a 444 bp fragment from upstream of the chicken HDAC4 transcription start site. Subsequently, we constructed pEGFP-HDAC4 and a series of 5′-deletion luciferase reporter constructs, which we transfected into DF-1 cells to measure their transcriptional activity. The regulatory mechanisms of chicken HDAC4 expression were investigated by performing trichostatin A (TSA) treatment, deleting putative transcription factor binding sites, and altering transcription factor expression levels. Results: HDAC4 exhibited higher expression in SSC than in ESC. We confirmed that the upstream region from −295 bp to 0 bp is the core transcriptional region of HDAC4. TSA effectively inhibited HDAC4 transcription, and bioinformatics analysis indicated that the chicken core HDAC4 promoter sequence exhibits high homology with those of other avian species. The myelocytomatosis viral oncogene homolog (MYC) and hypoxia-inducible factor 1 α (HIF1A) transcription factors were predicted to bind to this core region. Treatment with TSA for 24 h resulted in the upregulation of MYC and HIF1A, which repressed HDAC4 transcription. Conclusions: Our results provide a basis for subsequent investigations into the regulation of HDAC4 expression and biological function.

Unsymmetrical Bisacridines’ Interactions with ABC Transporters and Their Cellular Impact on Colon LS 174T and Prostate DU 145 Cancer Cells

Multidrug resistance (MDR) is a process that constitutes a significant obstacle to effective anticancer therapy. Here, we examined whether unsymmetrical bisacridines (UAs) are substrates for ABC transporters and can influence their expression in human colon LS 174T and prostate DU 145 cancer cells. Moreover, we investigated the cytotoxicity and the cellular response induced by UAs in these cells. The ATPase activities of MDR1, MRP1, and MRP2 were measured using vesicles prepared from insect Sf9 cells expressing particular ABC transporters. The gene expression and protein levels were analyzed using qPCR and Western blotting. The cellular effects were studied by MTT (cytotoxicity), flow cytometry (cell cycle analysis and phosphatidylserine externalization), and fluorescence microscopy. We showed that UAs are substrates for MDR1. Importantly, they did not influence remarkably the expressions of the ABCB1, ABCC1, and ABCC2 genes and the levels of the MDR1 and PXR proteins in the studied cells. Furthermore, the cytotoxicity and the level of apoptosis triggered by UAs in LS 174T cells possessing higher expressions of metabolic enzymes were lower compared with DU 145 cells. These results indicate that during possible UA treatment, the occurrence of drug resistance could be limited, which could favor the use of such compounds as potential candidates for future studies.

Heterologous Expression and Polyphasic Analysis of CLA-Converting Linoleic Acid Isomerase from Bifidobacterium breve JKL2022.

The probiotic Bifidobacterium breve is known for its efficient conjugated linoleic acid (CLA) conversion, yet their CLA conversion pathway remains underexplored. This study investigated B. breve JKL2022 for its CLA conversion in actively growing cells, washed cell states, and in crude protein extracts. Moreover, the study aimed to confirm the CLA-converting enzyme in strain JKL2022 and optimize its purification through heterologous expression of fusion proteins (LAI_sGFP and MBP_LAI). JKL2022 exhibited superior CLA conversion compared to genetically similar B. breve strains (JCM7017, JCM7019, JCM1192, and JCM1273), particularly the observed CLA conversion in washed cells (60.14 ± 7.60%) and crude protein fractions (96.11 ± 6.63%). The multipass transmembrane linoleic acid isomerase (LAI) was cloned into the E. coli BL21(DE3) as free LAI or modified with superfolder-GFP or MBP tags and expressed with 0.01 mM IPTG at 37 °C, resulting in highly active protein fractions. LAI was characterized by predictive modeling, molecular docking, and phylogenetic analyses. Moreover, reverse transcription-quantitative PCR analysis revealed upregulation (20-140× higher expression) of lai in JKL2022 compared with that in the JCM strains. Nevertheless, upscaling the production and purification of LAI for downstream applications remains a challenge, primarily because of their membrane-spanning configuration.

Spatial interactions of immune cells as potential predictors to efficacy of toripalimab plus chemotherapy in locally advanced or metastatic pancreatic ductal adenocarcinoma: a phase Ib/II trial

Advanced pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis. Immunotherapy alone offers limited efficacy, but it is still unknown whether its combination with chemotherapy could offer synergistic anti-tumor effects. This phase Ib/II study evaluated the safety and efficacy of combining toripalimab with the gemcitabine plus nab-paclitaxel (GnP) regimen as first-line treatment for locally advanced or metastatic PDAC and explored predictive biomarkers (ChiCTR2000032293). The primary endpoints were safety and overall survival (OS). The secondary outcomes were objective response rate (ORR), disease control rate (DCR), and progression-free survival (PFS). Immune-related biomarkers including programmed death-ligand 1 (PD-L1) expression, genetic status, cytokine levels, and spatial features of the tumor immune microenviroment (TIME) were investigated. Neither serious treatment-related adverse events nor grade 4 immune-related adverse events were reported. Among the 72 patients, the median OS was 8.9 months, 12-month OS rate was 31.9%, with median PFS of 5.6 months, ORR of 33.3%, and DCR of 90.3%. Higher PD-L1 expression, without liver metastases were associated with higher ORR, however these factors could not effectively distinguish responders and non-responders. Importantly, dendritic cells - T helper cells - cytotoxic T lymphocytes (DC-Th-CTL) enriched immune niche and their spatial interactions were dominant predictors of response based on TIME analysis using a cyclic multiplex tissue staining assay, with an area under the curve value of 0.8. Overall, GnP plus toripalimab exhibited good safety and differentiated efficacy in selected population, and the spatial interactions of DC-Th-CTL represent promising predictors to efficacy of immunochemotherapy in locally advanced or metastatic PDAC.

Dissecting the novel molecular interactions of solute carrier family 4 member 4 (SLC4A4) for prostate cancer (PCa) progression

Prostate cancer (PCa) is the most common malignancy diagnosed in men. The purpose of this study was to report the molecular pathways of Homo sapiens solute carrier family 4 member 4 (SLC4A4) in the progression of PCa. Here, we report our findings from clinical specimens of prostatic acinar adenocarcinoma collected from patients. We found that low-grade prostate cancers have higher SLC4A4 expression. We investigated the role of SLC4A4 and the signaling mechanism underlying its role in modulating the PCa progression. Firstly, we reported the SLC4A4/GSK-3β/β-catenin signaling axis, which regulates the clonogenic potential, invasiveness, and metastasis. In this, we found reduced phosphorylation of GSK at serine 21 of α and serine 9 of the β subunit in shSLC4A4 cells of PCa, which ultimately relieved the activity of GSK-3β. This activated GSK-3β phosphorylates β-catenin at Ser33/37 with a subsequently reduced β-catenin level in PCa cells. Our functional analysis revealed that SLC4A4 knockdown retards tumor growth and lowers invasion and migration potential. Secondly, we investigated the SLC4A4/RB axis, which acts to drive cell proliferation. SLC4A4 knockdown decreases the interaction between these molecules with hypophosphorylation of RB protein and cell cycle arrest. Likewise, transcriptome sequencing using the SLC4A4 knockdown in DU145 cells regulates differentiated expressed genes and multiple metabolic pathways. Our results suggest that SLC4A4 may serve as a potential therapeutic target for prostate cancer patients in the future.

Bilirubin, a hepatoprotective agent that activates SIRT1, PGC-1α, and PPAR-α, while inhibiting NF-κB in rats with metabolic-associated fatty liver disease

Metabolic-associated fatty liver disease (MAFLD) is a chronic liver disorder characterized by fatty liver disease alongside overweight or obesity and/or type 2 diabetes mellitus (T2DM). Timely intervention is crucial for a potential cure. This study aimed to investigate the effects of bilirubin, an endogenous antioxidant, on lipid metabolism and inflammation in MAFLD. Specifically, it examined bilirubin’s impact on SIRT1, PPAR-α, and NF-κB in the livers of rats with MAFLD induced by a high-fat diet (HFD) and streptozotocin (STZ) administration. Forty eight-week adult male Sprague Dawley rats were divided into five groups (n = 8): Control, HFD-STZ, HFD-S-BR6, HFD-S-BR14, and C-BR14. In the last three groups, bilirubin administration was performed intraperitoneally for 6 and 14 weeks (10 mg/kg/day). We selected the key genes associated with MAFLD and subsequently performed GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses to explore the enriched biological processes and signaling pathways. Hence, the gene expression of SIRT1, PGC-1α, PPAR-α, and inflammatory genes (NF-κB, TNF-α, IL-6, and IL-1β) was measured using Real-time quantitative PCR. Stereological and histopathological alterations of liver structure as well as lipid profile, biochemical indices, and liver indices, were also assessed among different groups. The enrichment analysis identified that several signaling pathways and biological processes might be related to MAFLD. Bilirubin-treated rats contained higher PPAR-α, PGC-1α, and SIRT1 expression levels by approximately 5.7-, 2.1-, and 2.2-fold, respectively, compared to the HFD-receiving rats (p < 0.0001, p < 0.05, and p < 0.05). Whereas, the genes involved in the inflammatory cascades, including NF-κB, TNF-α, and IL-6, were downregulated by 0.6-fold (p < 0.05) following 14-week treatment of bilirubin, while only significantly decreased expression of NF-κB and IL-6 (approximately 0.6-fold, p < 0.05) were observed after 6-week treatment of bilirubin. Remarkably, bilirubin administration favorably reversed the effects of HFD on the liver’s volume and cell numbers and ameliorated the related structural changes. It also improved lipid profile, biochemical parameters, and liver indices of HFD-STZ rats. This study indicated that bilirubin acts as a protective/ameliorative compound against MAFLD, particularly through regulating the key genes involved in lipid metabolism and inflammation in HFD-STZ rats.

The Formation of Human Arteriovenous Malformation Organoids and Their Characteristics

The Formation of Human Arteriovenous Malformation Organoids and Their Characteristics

Differential expression of steviol glycoside pathway genes in NaCl-treated stevia genotypes with varied SG contents

The rebaudioside-A/stevioside (R/S) ratio influences the breeding and commercial values of stevia genotypes. Three stevia genotypes, “High Sugar,” “Levan,” and “ShouA3-2,” with respectively high, medium, and low steviol glycoside (SG) contents were investigated to answer the following questions: how do SG contents change when NaCl induces gene expression? Is SG-content positively correlated with NaCl tolerance? Does the genotype, with a high R/S ratio, exhibit higher expression of SG genes; compared to the genotype, with a low R/S ratio? Growth parameters, through our results, were decreased in “High Sugar” and “ShouA3-2” relative to “Levan” who showed better performance under the severe 200 mM NaCl treatment. Extendedly, “High Sugar” decreased the expression of SG-genes (cpps, ks, kh, and ugt74) under the low 50 NaCl treatments, while the “ShouA3-2” has upregulated all the genes (except ks) under the moderate (100 mM) NaCl treatments. Overall, the antioxidant sod1 gene was upregulated, similarly, the “Levan” has upregulated the cpps, while differentially expressed ks and kh across the NaCl treatments. In conclusion, high R/S ratio does not necessarily mean enhancement in responses to abiotic stresses in stevia. The R/S ratio in stevia does not solely rely on the expression of SG-pathway genes thus; other ancestral genetic elements may independently regulate them. Under salinity, the rooting characteristics improved when R/S ratio was low, while when moderate the shooting characteristics were superior. We recommend breeding for moderate R/S ratios to assure probable resilience to challenging climates (such as salinity) and potent higher agronomic traits.

Shroom3-Rock interaction and profibrotic function: Resolving mechanism of an intronic CKD risk allele.

Common intronic enhancer SNPs in Shroom3 associate with CKD in GWAS, although there is paucity of detailed mechanism. Previously, we reported a role for Shroom3 in mediating crosstalk between TGFβ1- & Wnt/Ctnnb1 pathways promoting renal fibrosis (TIF). However, beneficial roles for Shroom3 in proteinuria have also been reported suggesting pleiotropic effects. Here we focused on identifying the specific profibrotic Shroom3 motif. Given known therapeutic roles for Rho-kinase inhibitors in experimental CKD, and the established interaction between Shroom3 and Rock via its ASD2 domain, we hypothesized that Shroom3-mediated ROCK activation played a crucial role in its profibrotic function in high expressors. To test this hypothesis, we developed transgenic mice and cell lines that inducibly overexpressed wild-type- (WT-Sh3) or ASD2-domain deletion- Shroom3 (ASD2Δ-Sh3). Prior scRNAseq data showed that during TIF, Shroom3 and Rock co-expression occurred in injured tubular cells and fibroblasts, highlighting cell-types where this mechanism could be involved. Using HEK293T cells, we first confirmed absent ROCK binding and inhibited TGFβ1-signaling with ASD2Δ-Sh3-overexpression vs WT-Sh3. In mIMCD cells, ASD2Δ-Sh3 overexpression, reduced Rock activation (phospho-MYPT1), pro-fibrotic and pro-inflammatory transcripts vs WT-Sh3. Fibroblast proliferation (3T3) was also reduced with ASD2Δ-Sh3. In vivo, we studied ureteric obstruction (UUO) and Aristolochic nephropathy (AAN) as TIF models. In AAN, inducible global-, or Pan-tubular specific-, WTSh3-overexpression showed increased azotemia, and TIF vs ASD2Δ-Sh3 mice. WT-Sh3 mice consistently showed significant enrichment of Rho-GTPase, TGFβ1- and Wnt/CtnnB1- signaling in kidney transcriptome, paralleling Shroom3-coexpressed genes in tubulo-interstitial transcriptomes from human CKD. In UUO, again WT-Sh3 mice recapitulated increased fibrosis vs ASD2Δ-Sh3. Importantly, ASD2Δ-Sh3 did not develop albuminuria vs WT-Sh3, while mutating a disparate Fyn-binding Shroom3 motif induced albuminuria in mice, suggesting motif-specific roles for Shroom3 in the kidney. Hence, our data show a critical role for the Rock-binding, ASD2-domain in mediating TIF in milieu of Shroom3 excess, with relevance to human CKD.