Gene Level Research Articles

Modulatory effect of Andean blackberry polyphenols on genes related to antioxidant and inflammatory responses, the NLRP3 inflammasome, and autophagy

BACKGROUND: The Andean blackberry (Rubus glaucus Benth) is one of Ecuador’s most iconic Andean berries for which a high anthocyanin content has been described. OBJECTIVE: The aim of the present study was to determine the chemical composition and anti-inflammatory potential of the Andean blackberry from Ecuador, with an emphasis on its effects on NLRP3 inflammasome activation and autophagy processes. RESULTS: Andean blackberry extracts were rich in hydroxycinnamates (coumaric acid and derivates), in addition to quercetin and kaempferol as principal flavonols. Cyanidin and its glycosides were identified as the main anthocyanins present. Andean blackberry extracts efficiently reduced oxidative stress markers in the lipopolysaccharide-stimulated RAW 264.7 cells. The extracts also caused a moderate decrease in the expression of the pro-inflammatory and antioxidant genes NFκB1, TNF, IL-1β, IL-6, and NOS2 expression, while they significantly increased the mRNA levels of both SOD1 and NFE2L2 genes. Andean blackberry extracts significantly decreased the activation of the NLRP3 inflammasome complex, as well as p62 levels, and the LC3I/LC3II ratio increased, suggesting a direct action of Andean blackberry compounds on the inflammatory response and restoration of the autophagy process. CONCLUSIONS: These results suggest that Andean blackberries potentially have an anti-inflammatory effect through their ability to regulate genes related to the inflammatory and antioxidant response, as well as modulate the activation of the NLRP3 inflammasome complex and autophagy processes.

Identification of host protein ENO1 (alpha-enolase) interacting with Cryptosporidium parvum sporozoite surface protein, Cpgp40

BackgroundCryptosporidium parvum is an apicomplexan zoonotic parasite causing the diarrheal illness cryptosporidiosis in humans and animals. To invade the host intestinal epithelial cells, parasitic proteins expressed on the surface of sporozoites interact with host cells to facilitate the formation of parasitophorous vacuole for the parasite to reside and develop. The gp40 of C. parvum, named Cpgp40 and located on the surface of sporozoites, was proven to participate in the process of host cell invasion.MethodsWe utilized the purified Cpgp40 as a bait to obtain host cell proteins interacting with Cpgp40 through the glutathione S-transferase (GST) pull-down method. In vitro analysis, through bimolecular fluorescence complementation assay (BiFC) and coimmunoprecipitation (Co-IP), confirmed the solid interaction between Cpgp40 and ENO1. In addition, by using protein mutation and parasite infection rate analysis, it was demonstrated that ENO1 plays an important role in the C. parvum invasion of HCT-8 cells.ResultsTo illustrate the functional activity of Cpgp40 interacting with host cells, we identified the alpha-enolase protein (ENO1) from HCT-8 cells, which showed direct interaction with Cpgp40. The mRNA level of ENO1 gene was significantly decreased at 3 and 24 h after C. parvum infection. Antibodies and siRNA specific to ENO1 showed the ability to neutralize C. parvum infection in vitro, which indicated the participation of ENO1 during the parasite invasion of HCT-8 cells. In addition, we further demonstrated that ENO1 protein was involved in the regulation of cytoplasmic matrix of HCT-8 cells during C. parvum invasion. Functional study of the protein mutation illustrated that ENO1 was also required for the endogenous development of C. parvum.ConclusionsIn this study, we utilized the purified Cpgp40 as a bait to obtain host cell proteins ENO1 interacting with Cpgp40. Functional studies illustrated that the host cell protein ENO1 was involved in the regulation of tight junction and adherent junction proteins during C. parvum invasion and was required for endogenous development of C. parvum.Graphical

NEK2 affects the ferroptosis sensitivity of gastric cancer cells by regulating the expression of HMOX1 through Keap1/Nrf2.

NEK2 is a serine/threonine protein kinase that is involved in regulating the progression of various tumors. Our previous studies have found that NEK2 is highly expressed in gastric cancer and suggests that patients have a worse prognosis. However, its role and mechanism in gastric cancer are only poorly studied. In this study, we established a model of ferroptosis induced by RSL3 or Erastin in AGS cells in vitro, and konckdown NEK2, HOMX1, Nrf2 by siRNA. The assay kit was used to analyzed cell viability, MDA levels, GSH and GSSG content, and FeRhoNox™-1 fluorescent probe, BODIPY™ 581/591 C11 lipid oxidation probe, CM-H2DCFDA fluorescent probe were used to detected intracellular Fe2+, lipid peroxidation, and ROS levels, respectively. Calcein-AM/PI staining was used to detect the ratio of live and dead cells, qRT-PCR and Western blot were used to identify the mRNA and protein levels of genes in cells, immunofluorescence staining was used to analyze the localization of Nrf2 in cells, RNA-seq was used to analyze changes in mRNA expression profile, and combined with the FerrDb database, ferroptosis-related molecules were screened to elucidate the impact of NEK2 on the sensitivity of gastric cancer cells to ferroptosis. We found that inhibition of NEK2 could enhance the sensitivity of gastric cancer cells to RSL3 and Erastin-induced ferroptosis, which was reflected in the combination of inhibition of NEK2 and ferroptosis induction compared with ferroptosis induction alone: cell viability and GSH level were further decreased, while the proportion of dead cells, Fe2+ level, ROS level, lipid oxidation level, MDA level, GSSG level and GSSG/GSH ratio were further increased. Mechanism studies have found that inhibiting NEK2 could promote the expression of HMOX1, a gene related to ferroptosis, and enhance the sensitivity of gastric cancer cells to ferroptosis by increasing HMOX1. Further mechanism studies have found that inhibiting NEK2 could promote the ubiquitination and proteasome degradation of Keap1, increase the level of Nrf2 in the nucleus, and thus promote the expression of HMOX1. This study confirmed that NEK2 can regulate HMOX1 expression through Keap1/Nrf2 signal, and then affect the sensitivity of gastric cancer cells to ferroptosis, enriching the role and mechanism of NEK2 in gastric cancer.

Dapagliflozin promotes white adipose tissue browning though regulating angiogenesis in high fat induced obese mice

Browning of white adipose tissue (WAT) is become an appealing target for therapeutics in the treatment of obesity and related metabolic diseases. Dapagliflozin is widely used in the treatment of type 2 diabetes, and it is also found that the drug exhibits regulate systemic metabolism such as obesity, insulin resistance and hepatic steatosis. However, the precise role of dapagliflozin on WAT remodeling remains to be elucidated. The current study aimed to explore the role of dapagliflozin on WAT browning in high-fat diet (HFD)-induced obese mice. Male C57BL/6J mice (n = 6 per group) were used to establish obesity model by following feeding with HFD for 6 weeks. The mice were randomly treated with or without dapagliflozin for the experimental observation. The volume and fat fraction of WAT were quantified, H&E, UCP-1 staining and immunohistochemistry were conducted to investigate the white-to-brown fat conversion and angiogenesis in WAT respectively. Quantitative real-time polymerase chain reaction (qPCR) was employed to explore the mRNA expression levels of genes related to fat browning and angiogenesis in WAT. Subsequently, 3T3-L1 cells were used to explore the effect of dapagliflozin on preadipocytes differentiation in vitro. Our results demonstrated that dapagliflozin could reduce body weight gain and promote WAT browning in HFD induced obese mice via regulating lipogenesis and angiogenesis in WAT. Furthermore, dapagliflozin reduce cells differentiation, up-regulate the expression of WAT browning and angiogenesis genes in 3T3-L1 adipocytes in vitro. In conclusion, dapagliflozin can potentially promote WAT browning in HFD induced obese mice via improving lipogenesis and angiogenesis in WAT.

Cardioprotective role of royal jelly in the prevention of celecoxib-mediated cardiotoxicity in adult male albino rats

BackgroundCelecoxib, a cyclooxygenase-2 selective inhibitor non-steroidal anti-inflammatory drugs, is used for the management of short- and long-term pain as well as in other inflammatory conditions. Unfortunately, its chronic use is highly associated with serious abnormal cardiovascular events. The current study was designed to explore the effect of long-term administration of celecoxib on the cardiac tissues of male albino rats. The study also examined the alleged cardioprotective effect of royal jelly.MethodsThirty, male albino rats were randomly divided into 3 equal groups; 10 each: (1) rats served as the control group and received no drug; (2) rats received celecoxib (50 mg/kg/day, orally), for 30 consecutive days; (3) rats received celecoxib (50 mg/kg/day, orally) plus royal jelly (300 mg/kg/day, orally) for 30 consecutive days. Sera were collected to assay cardiac enzymes and oxidant/antioxidant status. Rats were euthanatized and cardiac tissues were dissected for quantitative estimation of apoptotic genes (Bax) and anti-apoptotic gene (Bcl-2).ResultsLong-term celecoxib administration caused cardiotoxicity in male albino rats as manifested by significant elevation of serum levels of creatine phosphokinase (CPK), creatine kinase-MB (CK-MB), and lactate dehydrogenase (LDH), with ameliorative effects of royal jelly against celecoxib-induced cardiotoxicity as manifested by significantly decrease in serum CPK, CK-MB, and LDH levels. It also showed a significant decrease in the oxidative stress indicator malondialdehyde (MDA) levels and the bax gene. Additionally, it demonstrated significant increases in the bcl-2 gene and superoxide dismutase (SOD) levels, which contribute to its therapeutic effects against celecoxib-induced cardiotoxicity.ConclusionLong-term celecoxib administration caused cardiotoxicity in male albino rats with protective effect of royal jelly being given together. It could be concluded that royal jelly may prove a useful adjunct in patients being prescribed celecoxib.Trial registrationNot applicable.

Identification and prognostic analysis of candidate biomarkers for lung metastasis in colorectal cancer.

Colorectal cancer (CRC) is one of the most prevalent types of malignant tumors. It's vital to explore new biomarkers and potential therapeutic targets in CRC lung metastasis through adopting integrated bioinformatics tools. Multiple cohort datasets and databases were integrated to clarify and verify potential key candidate biomarkers and signal transduction pathways in CRC lung metastasis. DAVID, STRING, UALCAN, GEPIA, TIMER, cBioPortal, THE HUMAN PROTEIN ATLAS, GSEA 4.3.2, FUNRICH 3.1.3, and R 4.2.3 were utilized in this study. The enriched biological processes and pathways modulated by the differentially expressed genes (DEGs) were determined with Gene Ontology, Kyoto Encyclopedia of Genes and Genomes. The search tool Retrieval of Interacting Genes and Cytoscape were used to construct a protein-protein interaction network among DEGs. Four hundred fifty-nine colorectal primary cancer and lung metastatic gene expression profiles were screened from 3 gene expression profiles (GSE41258, GSE68468, and GSE41568). Forty-one upregulated genes and 8 downregulated genes were identified from these 3 gene expression profiles and verified by the transcriptional levels of hub genes in other GEO datasets and The Cancer Genome Atlas database. Two pathways (immune responses and chemokine receptors bind chemokines), 13 key DEGs, 6 hub genes (MMP3, SFTPD, ABCA3, CLU, APOE, and SPP1), and 2 biomarkers (APOE, SPP1) with significantly prognostic values were screened. Forty-nine DEGs were identified as potential candidate diagnostic biomarkers for patients with CRC lung metastasis in present study. Enrichment analysis indicated that immune responses and chemokine receptors bind chemokines may play a leading role in lung metastasis of CRC, and further studies are needed to validate these findings.

Abnormal Calcium Accumulation and ROS Homeostasis-Induced Tapetal Programmed Cell Death Lead to Pollen Abortion of Petaloid-Type Cytoplasmic Male Sterility in Camellia oleifera

Cytoplasmic male sterility (CMS) plays a crucial role in the utilization of heterosis. The petaloid anther abortion in oil tea (Camellia oleifera Abel.) constitutes a CMS phenomenon, which is of great value for the hybrid breeding of oil tea. However, as the mechanism of its CMS is still poorly understood, it is necessary to study the cytology and physiological characteristics of anther abortion. In this study, a C. oleifera cultivar, Huashuo (HS), and its petalized CMS mutant (HSP) were used as materials to explore this mechanism. Compared with HS, cytological analysis demonstrated that HSP showed early-onset tapetum programmed cell death (PCD) and an organelle disorder phenotype during the tetrad stage. In HSP, anthers exhibited elevated levels of calcium deposition in anther wall tissues, tapetum layers, and microspores, and yet calcium accumulation was abnormal at the later stage. The contents of hydrogen peroxide and MDA in HSP anthers were higher, and the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were lower than those of HS, which resulted in an excessive accumulation of reactive oxygen species (ROS). Real-time quantitative PCR confirmed that the transcription levels of CoPOD and CoCAT genes encoding key antioxidant enzymes in HSP were downregulated compared with HS in early pollen development; the gene CoCPK, which encodes a calcium-dependent protein kinase associated with antioxidase, was upregulated during the critical period. Thus, we suggest that excessive ROS as a signal breaks the balance of the antioxidant system, and along with an abnormal distribution of calcium ions, leads to the early initiation of PCD in the tapetum, and ultimately leads to pollen abortion for HSP. These results lay a cytological and physiological foundation for further studies on the CMS mechanism, and provide information for breeding male-sterile lines of C. oleifera.

Cytokinin oxidase gene CKX5 is modulated in the immunity of Arabidopsis to Botrytis cinerea.

In our previous work, cytokinin (CK) signaling and biosynthesis were found to be modulated during Arabidopsis defense against infection by the necrotrophic pathogen Botrytis cinerea. Notably, the expression level of CYTOKININ OXIDASE/DEHYDROGENASE 5 (CKX5) was significantly induced in B. cinerea-infected leaves and later in distant B. cinerea-untreated leaves of the same plant. To confirm and determine how CKX5 is involved in the response to B. cinerea infection, transcript levels of CKX family genes were analyzed in B. cinerea-inoculated leaves, and only CKX5 was remarkably induced by B. cinerea infection. Furthermore, CKX5-overexpressing Arabidopsis plants were more resistant to B. cinerea than wild-type plants. Transcription factors (TFs) binding to the CKX5 promoter were then screened by yeast one-hybrid assays. Quantitative Real-Time Reverse Transcription PCR (qRT-PCR) analysis further showed that genes encoding TFs, including WRKY40, WRKY33, ERF6, AHL15, AHL17, ANAC003, TCP13 and ANAC019, were also strongly induced in infected leaves, similar to CKX5. Analysis of ERF6-overexpressing plants and ERF6-and AHL15-knockout mutants indicated that ERF6 and AHL15 are involved in plant immunity to B. cinerea. Furthermore, CKX5 upregulation by B. cinerea infection was affected when ERF6 or AHL15 levels were altered. Our work suggests that CKX5 levels are controlled by the plant defense system to defend against attack by the pathogen B. cinerea.

Construction of a prognostic model based on genome-wide methylation analysis of miRNAs for hepatocellular carcinoma.

Aim: Using the methylation level of miRNA genes to develop a prognostic model for patients with hepatocellular carcinoma (HCC). Materials & methods: least absolute shrinkage and selection operator and multivariate Cox regression analyses were performed to develop a prognostic model. One miRNA in the model was selected for verification. Results: A prognostic model was developed using eight miRNAs. The areas under the curve for predicting overall survival at 1, 3and 5years were 0.75, 0.81and 0.81. miR-223 was found to be hypomethylated in 160 HCC tissues, and its methylation level was associated with Barcelona Clinic Liver Cancerstages and the prognosis of patients with HCC. Conclusion: The prognostic model based on miRNA methylation levels has the capability to partially forecast the prognosis of patients with HCC.

Investigating the Promising Anticancer Activity of Cetuximab and Fenbendazole Combination as Dual CBS and VEGFR-2 Inhibitors and Endowed with Apoptotic Potential.

In this work, the cytotoxicity of monoclonal antibody (Cetuximab, Ce) and Fenbendazole (Fen), as well as their combination therapy were tested with the MTT assay. On the other side, Ce, Fen, and a combination between them were subjected to a colchicine-tubulin binding test, which was conducted and compared to Colchicine as a reference standard. Besides, Ce, Fen, and the combination of them were tested against the VEGFR-2 target receptor, compared to Sorafenib as the standard medication. Moreover, the qRT-PCR technique was used to investigate the levels of apoptotic genes (p53 and Bax) and anti-apoptotic gene (Bcl-2) as well. Also, the effect of Ce, Fen, and the combination of them on the level of ROS was studied. Furthermore, the cell cycle analysis and Annexin V apoptosis assay were carried out for Ce, Fen, and a combination of them. In addition, the molecular docking studies were used to describe the molecular levels of interactions for both (Fen and colchicine) or (Fen and sorafenib) within the binding pockets of the colchicine binding site (CBS) and vascular endothelial growth factor-2 receptor (VEGFR-2), respectively.

Long-chain acyl-CoA synthetase 4-mediated mitochondrial fatty acid metabolism and dendritic cell antigen presentation.

This study aims to investigate the role of Acyl-CoA synthetase 4 (ACSL4) in mediating mitochondrial fatty acid metabolism and dendritic cell (DC) antigen presentation in the immune response associated with asthma. RNA sequencing was employed to identify key genes associated with mitochondrial function and fatty acid metabolism in DCs. ELISA was employed to assess the levels of fatty acid metabolism in DCs. Mitochondrial morphology was evaluated using laser confocal microscopy, structured illumination microscopy, and transmission electron microscopy. Flow cytometry and immunofluorescence were utilized to detect changes in mitochondrial superoxide generation in DCs, followed by immunofluorescence co-localization analysis of ACSL4 and the mitochondrial marker protein COXIV. Subsequently, pathological changes and immune responses in mouse lung tissue were observed. ELISA was conducted to measure the levels of fatty acid metabolism in lung tissue DCs. qRT-PCR and western blotting were employed to respectively assess the expression levels of mitochondrial-associated genes (ATP5F1A, VDAC1, COXIV, TFAM, iNOS) and proteins (ATP5F1A, VDAC1, COXIV, TOMM20, iNOS) in lung tissue DCs. Flow cytometry was utilized to analyze changes in the expression of surface antigens presented by DCs in lung tissue, specifically the MHCII molecule and the co-stimulatory molecules CD80/86. The sequencing results reveal that ACSL4 is a crucial gene regulating mitochondrial function and fatty acid metabolism in DCs. Inhibiting ACSL4 reduces the levels of fatty acid oxidases in DCs, increases arachidonic acid levels, and decreases A-CoA synthesis. Simultaneously, ACSL4 inhibition leads to an increase in mitochondrial superoxide production (MitoSOX) in DCs, causing mitochondrial rupture, vacuolization, and sparse mitochondrial cristae. In mice, ACSL4 inhibition exacerbates pulmonary pathological changes and immune responses, reducing the fatty acid metabolism levels within lung tissue DCs and the expression of mitochondria-associated genes and proteins. This inhibition induces an increase in the expression of MHCII antigen presentation molecules and co-stimulatory molecules CD80/86 in DCs. The research findings indicate that ACSL4-mediated mitochondrial fatty acid metabolism and dendritic cell antigen presentation play a crucial regulatory role in the immune response of asthma. This discovery holds promise for enhancing our understanding of the mechanisms underlying asthma pathogenesis and potentially identifying novel targets for its prevention and treatment.

Prognostic gene landscapes and therapeutic insights in sepsis-induced coagulopathy

BackgroundSepsis is a common and critical condition encountered in clinical practice that can lead to multi-organ dysfunction. Sepsis-induced coagulopathy (SIC) significantly affects patient outcomes. However, the precise mechanisms remain unclear, making the identification of effective prognostic and therapeutic targets imperative. MethodsThe analysis of transcriptome data from the whole blood of sepsis patients, facilitated the identification of key genes implicated in coagulation. Then we developed a prognostic model and a nomogram to predict patient survival. Consensus clustering classified sepsis patients into three subgroups for comparative analysis of immune function and immune cell infiltration. Single-cell sequencing elucidated alterations in intercellular communication between platelets and immune cells in sepsis, as well as the role of the coagulation-related gene FYN. Real-time quantitative PCR determined the mRNA levels of critical coagulation genes in septic rats' blood. Finally, administration of a FYN agonist to septic rats was observed for its effects on coagulation functions and survival. ResultsThis study identified four pivotal genes—CFD, FYN, ITGAM, and VSIG4—as significant predictors of survival in patients with sepsis. Among them, CFD, FYN, and ITGAM were underexpressed, while VSIG4 was upregulated in patients with sepsis. Moreover, a nomogram that incorporates the coagulation-related genes (CoRGs) risk score with clinical features of patients accurately predicted survival probabilities. Subgroup analysis of CoRGs expression delineated three molecular sepsis subtypes, each with distinct prognoses and immune profiles. Single-cell sequencing shed light on heightened communication between platelets and monocytes, T cells, and plasmacytoid dendritic cells, alongside reduced interactions with neutrophils in sepsis. The collagen signaling pathway was found to be essential in this dynamic. FYN may affect platelet function by modulating factors such as ELF1, PTCRA, and RASGRP2. The administration of the FYN agonist can effectively improve coagulation dysfunction and survival in septic rats. ConclusionsThe research identifies CoRGs as crucial prognostic markers for sepsis, highlighting the FYN gene's central role in coagulation disorders associated with the condition and suggesting novel therapeutic intervention strategies.

The Vitis yeshanensis U-box E3 ubiquitin ligase VyPUB21 enhances resistance to powdery mildew by targeting degradation of NIM1-interacting (NIMIN) protein.

VyPUB21 plays a key role during the defense against powdery mildew in grapes. Ubiquitin-ligating enzyme (E3), a type of protein widely found in plants, plays a key role in their resistance to disease. Yet how E3 participates in the disease-resistant response of Chinese wild grapevine (Vitis yeshanensis) remains unclear. Here we isolated and identified a U-box type E3 ubiquitin ligase, VyPUB21, from V. yeshanensis. This gene's expression level rose rapidly after induction by exogenous salicylic acid (SA), jasmonic acid (JA), and ethylene (ETH) and powdery mildew. In vitro ubiquitination assay results revealed VyPUB21 could produce ubiquitination bands after co-incubation with ubiquitin, ubiquitin-activating enzyme (E1), and ubiquitin-conjugating enzyme (E2); further, mutation of the conserved amino acid site in the U-box can inhibit the ubiquitination. Transgenic VyPUB21 Arabidopsis had low susceptibility to powdery mildew, and significantly fewer conidiophores and spores on its leaves. Expression levels of disease resistance-related genes were also augmented in transgenic Arabidopsis, and its SA concentration also significantly increased. VyPUB21 interacts with VyNIMIN and targets VyNIMIN protein hydrolysis through the 26S proteasome system. Thus, the repressive effect of the NIMIN-NPR complex on the late systemic acquired resistance (SAR) gene was attenuated, resulting in enhanced resistance to powdery mildew. These results indicate that VyPUB21 encoding ubiquitin ligase U-box E3 activates the SA signaling pathway, and VyPUB21 promotes the expression of late SAR gene by degrading the important protein VyNIMIN of SA signaling pathway, thus enhancing grape resistance to powdery mildew.

Combined Plasma Olink Proteomics and Transcriptomics Identifies CXCL1 and TNFRSF12A as Potential Predictive and Diagnostic Inflammatory Markers for Acute Kidney Injury.

Acute kidney injury (AKI) poses a significant global public health challenge. Current methods for detecting AKI rely on monitoring changes in serum creatinine (Scr), blood urea nitrogen (BUN), urinary output and some commonly employed biomarkers. However, these indicators are usually neither specific nor sensitive to AKI, especially in cases of mild kidney injury. AKI is accompanied by severe inflammatory reactions, resulting in the upregulation of numerous inflammation-associated proteins in the plasma. Plasma biomarkers are a noninvasive method for detecting kidney injury, and to date, plasma inflammation-associated cytokines have not been adequately studied in AKI patients. The objective of our research was to identify novel inflammatory biomarkers for AKI. We utilized Olink proteomics to analyze the alterations in plasma inflammation-related proteins in the serum of healthy mice (n = 2) or mice treated with cisplatin (n = 6). Additionally, transcriptome datasets for the lipopolysaccharide (LPS), cisplatin, and ischemia‒reperfusion injury (IRI) groups were obtained from the National Center of Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database. We calculated the intersection of differentially expressed proteins (DEPs) and genes (DEGs) from both datasets. In the Olink proteomics analysis, the AKI group had significantly greater levels of 11 DEPs than did the control group. In addition, 56 common upregulated DEGs were obtained from the transcriptome dataset. The expression of CXCL1 and TNFRSF12A overlapped across all the datasets. The transcription and protein expression levels of CXCL1 and TNFRSF12A were detected in vivo. The gene and protein levels of CXCL1 and TNFRSF12A were significantly increased in different AKI mouse models and clinical patients, suggesting that these genes and proteins could be potential specific biomarkers for the identification of AKI.

Protective effect of crocin on peroxidation-induced oxidative stress and apoptosis in IPEC-J2 cells.

The antioxidant properties of crocin are attracting interest, yet the underlying mechanisms by which crocin mitigates oxidative stress-induced intestinal damage have not been determined. This study aimed to elucidate the effects of crocin on oxidative stress, apoptosis, and intestinal epithelial injury in intestinal epithelial cells (IPEC-J2). Using an H2O2-induced oxidative stress model in IPEC-J2 cells, crocin was added to assess its effects. Cell viability and apoptosis were evaluated using methyl thiazolyl tetrazolium assays and flow cytometry. Additionally, oxidative stress markers, such as superoxide dismutase (SOD), catalase (CAT), reactive oxygen species (ROS), and malondialdehyde (MDA), were quantified. We investigated, in which cell oxidation and apoptosis were measured at the gene and protein levels and employed transcriptome analysis to probe the mechanism of action and validate relevant pathways. The results showed that crocin ameliorates H2O2-induced oxidative stress by reducing ROS and MDA levels and by countering the reductions in CAT, total antioxidant capacity, and SOD. Crocin also attenuates the upregulation of key targets in the Nrf2 pathway. Furthermore, it effectively mitigated IPEC-J2 cell apoptosis caused by oxidative stress, as evidenced by changes in cell cycle factor expression, apoptosis rate, mitochondrial membrane potential, and apoptosis pathway activity. In addition, crocin preserves the integrity of the intestinal barrier by protecting tight junction proteins against oxidative stress. Transcriptome sequencing analysis suggested that the mitochondrial pathway may be a crucial mechanism through which crocin exerts its protective effects. In summary, crocin decreases oxidative stress molecule formation, inhibits Nrf2 pathway activity, prevents apoptosis-induced damage, enhances oxidative stress resistance in IPEC-J2 cells, and maintains redox balance in the pig intestine.

Long-Term Effects of Maternal Fat Consumption on the Brain Transcriptome of Obesogenic Diet-Fed Young Adult Mice Offspring

BackgroundSubstantial evidence has demonstrated that maternal high-fat (HF) consumption during gestation and lactation plays as a risk factor for neurodevelopmental alterations and subsequent neurological disorders. ObjectiveWe investigated the regulatory mechanisms of maternal fat consumption on brain development and function in offspring at different ages. MethodsMouse dams were fed either a control diet [low-fat (LF)] or an HF diet for 3 wk before mating and throughout pregnancy and lactation. Offspring were killed at postnatal day (PD) 21 (LF21 and HF21), and the rest were fed an HF diet for 12 wk until the killing at PD 105 (LF105 and HF105). The expression levels of genes and proteins in the brains of offspring were analyzed by microarray and immunoblotting, respectively. ResultsMaternal dietary fat content, offspring age, and their interaction affected the expression levels of 1215, 10,453, and 2105 genes, respectively. The 67 differentially expressed genes (DEGs) between the HF21 and LF21 groups were enriched in several Gene Ontology terms related to nervous system development. Among 45 DEGs of the HF105/LF105 comparison, several genes associated with neurotransmitter action are detected. In addition, we observed increased activation of the AMP-dependent protein kinase–cAMP response element binding protein signaling pathway in HF105/LF105 comparison. However, maternal fat content did not change the protein levels of amyloid-β and tau hyperphosphorylation, the markers of neuropathogenesis. ConclusionsMaternal HF feeding altered the expression of genes involved in the development and neurotransmitter system in the brains of PD 21 and HF diet-fed PD 105 offspring, respectively. Especially, the absence of overlap between DEGs at each comparison highlights the dynamic nature of alterations in gene expression in offspring of dams fed an HF diet. Further investigation on older adult offspring is necessary to elucidate the effects of maternal fat intake on the brain pathophysiology of offspring.

Mikania micrantha silver nanoparticles exhibit anticancer activities against human lung adenocarcinoma via caspase-mediated apoptotic cell death

Green-mediated synthesis of nanoparticles has earned a promising role in the area of nanotechnology due to their biomedical applications. This study describes the synthesis of silver nanoparticles (AgNPs) using Mikania micrantha leaf extract and its functional activities against cancer. The synthesis of AgNPs was confirmed using Ultraviolet-Visible (UV-Vis) spectrum that exhibited an absorption band at 459 nm. The bioactive compounds of M. micrantha leaf extract that functioned as reducing and capping agents were confirmed by a shift in the absorption bands in Fourier Transform Infra-red Spectroscopy (FT-IR). Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) studies validated the spherical shape and size of AgNPs, respectively. Energy Dispersive Spectroscopy (EDS) analysis revealed the presence of elemental silver. The crystalline nature of AgNPs was confirmed by the X-ray Diffraction Analysis (XRD). AgNPs effectively induced cytotoxicity and prevented A549 cell colony formation in a dose-dependent manner. Treatment of A549 cells with AgNPs also increased DNA damage, which was coupled with elevated lipid peroxidation and decreased antioxidant enzymes such as glutathione (GSH), glutathione-s-transferase (GST), and superoxide dismutase (SOD). Following AgNPs treatment, the mRNA expression levels of the pro-apoptotic genes as well as the activities of caspases were significantly elevated in A549 cells while the expression levels of anti-apoptotic genes were downregulated. Our study demonstrates the potential of the synthesised AgNPs for cancer therapy possibly targeting the apoptotic pathway.

Modulation of Stiffness-Dependent Macrophage Inflammatory Responses by Collagen Deposition.

Macrophages are innate immune cells that interact with complex extracellular matrix environments, which have varied stiffness, composition, and structure, and such interactions can lead to the modulation of cellular activity. Collagen is often used in the culture of immune cells, but the effects of substrate functionalization conditions are not typically considered. Here, we show that the solvent system used to attach collagen onto a hydrogel surface affects its surface distribution and organization, and this can modulate the responses of macrophages subsequently cultured on these surfaces in terms of their inflammatory activation and expression of adhesion and mechanosensitive molecules. Collagen was solubilized in either acetic acid (Col-AA) or N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid (HEPES) (Col-HEP) solutions and conjugated onto soft and stiff polyacrylamide (PA) hydrogel surfaces. Bone marrow-derived macrophages cultured under standard conditions (pH 7.4) on the Col-HEP-derived surfaces exhibited stiffness-dependent inflammatory activation; in contrast, the macrophages cultured on Col-AA-derived surfaces expressed high levels of inflammatory cytokines and genes, irrespective of the hydrogel stiffness. Among the collagen receptors that were examined, leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) was the most highly expressed, and knockdown of the Lair-1 gene enhanced the secretion of inflammatory cytokines. We found that the collagen distribution was more homogeneous on Col-AA surfaces but formed aggregates on Col-HEP surfaces. The macrophages cultured on Col-AA PA hydrogels were more evenly spread, expressed higher levels of vinculin, and exerted higher traction forces compared to those of cells on Col-HEP. These macrophages on Col-AA also had higher nuclear-to-cytoplasmic ratios of yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), key molecules that control inflammation and sense substrate stiffness. Our results highlight that seemingly slight variations in substrate deposition for immunobiology studies can alter critical immune responses, and this is important to elucidate in the broader context of immunomodulatory biomaterial design.

BPIFB1, Serving as a Downstream Effector of EBV-miR-BART4, Blocks Immune Escape of Nasopharyngeal Carcinoma via Inhibiting PD-L1 Expression.

Nasopharyngeal carcinoma (NPC) is one of the most common tumors of head and neck in the Southeast Asia. PD-L1-dependent immune escape plays a critical role involved in NPC development. BPIFB1 has previously reported to take tumor-suppressive actions on NPC cell proliferation and migration. Nonetheless, the function of BPIFB1 in immune escape remains largely elusive. Expression pattern on mRNA and protein levels of target genes in NPC patients' samples and cell lines were examined by qRT-PCR, western blot, and immunohistochemistry staining, respectively. The assessment of CD8+ T-cell apoptosis and expression was determined by flow cytometry. Molecular interactions were verified using chromatin immunoprecipitation (ChIP) and luciferase reporter assay. BPIFB1 was downregulated in NPC tumor tissues, exhibiting a negative correlation of PD-L1. Overexpression of BPIFB1 significantly inhibited the expression of PD-L1, suppressing the apoptosis and enhancing the expression of CD8+ T cells. Mechanistically, BPIFB1 was found to repress the expression of STAT1, which was identified to be an upstream activator of PD-L1. Furthermore, the EBV-encoded miR-BART4 overexpressed in NPC cells could directly target and inhibit BPIFB1. This study provided a comprehensive understanding of the molecular mechanism for the upstream and downstream pathway of BPIFB1 related with immune escape, indicating a novel approach for the treatment of NPC.

The Effect of Thymoquinone on the Protein Levels of PLA2G7, UCP2, and NEDD4L Genes Associated with Lipid Droplets Formation in Prostate Cancer

Objective: Prostate cancer (PCa) patients suffer severe side effects of standard treatment beside the resistance to castration. PCa cells shows increased lipogenesis. Thymoquinone (TQ) inhibits cell proliferation, metastasis, and invasion. However, there was no study on the effect of TQ on the levels of NEDDL4, PLA2G7, and UCP2 lipid droplets (LD) related proteins. Hence, the study aims to investigate the impact of TQ on PLA2G7, UCP2, and NEDD4L proteins on DU145 and PC3 cell lines.
 Materials and Methods: Cells were cultured and treated with TQ with a IC50 of 60 µM and 80 µM for DU145 and PC3, respectively. PLA2G7, UCP2, and NEDD4L levels were measured using the ELISA.
 Results: TQ has significantly increased the level of NEDD4L (p