Changes In Expression Profiles Research Articles

Integrated miRNA-seq and functional analyses reveal the regulatory role of sha-miR-92a_L + 2R + 4 via targeting vegfaa in rainbow trout (Oncorhynchus mykiss) responding to acute hypoxia and reoxygenation stress

BackgroundHypoxia negatively affects the behavior, growth, reproduction and survival of fish, causing serious economic losses to aquaculture. Rainbow trout (Oncorhynchus mykiss), an important economic fish worldwide, belongs to a hypoxia-sensitive fish species, however, little is known about the regulatory mechanism of microRNAs (miRNAs) under hypoxia stress.ResultsRainbow trout were subjected to hypoxia stress for 3 h (H3h_L), 12 h (H12h_L), 24 h (H24h_L) and 3 h reoxygenation (R3h_L) to systemically evaluate the changes of miRNA expression profiles in liver, and functions of sha-miR-92a_L + 2R + 4 were investigated. We found 17, 144, 57 and 55 differentially expressed (DE) miRNAs in the H3h_L vs. control (N_L), H12h_L vs. N_L, H24h_L vs. N_L and R3h_L vs. N_L comparisons, respectively. Enrichment analysis revealed that the targets of DE miRNAs were significantly enriched in HIF signaling pathway, VEGF signaling pathway, FoxO signaling pathway and glycolysis/gluconeogenesis. Through miRNA-mRNA interaction and weighted gene co-expression network analysis (WGCNA), five key DE miRNAs (sha-miR-92a_L + 2R + 4, ssa-miR-128-3p, ssa-miR-101b-3p_R + 1, ola-miR-199a-5p_R + 2 and tni-miR-199_1ss18CG) were identified, which can target at least two hypoxia-responsive genes, such as vegfaa, ho, glut1a and junb. Functional analysis found that sha-miR-92a_L + 2R + 4 directly regulated vegfaa expression by targeting its 3′-UTR, overexpression of sha-miR-92a_L + 2R + 4 significantly decreased vegfaa expression in rainbow trout liver cells, while opposite results were obtained after transfection of sha-miR-92a_L + 2R + 4 inhibitor. Furthermore, overexpressed sha-miR-92a_L + 2R + 4 promoted rainbow trout liver cell proliferation and inhibited apoptosis.ConclusionThis study deepens our understanding of the crucial roles of miRNAs under hypoxia stress in rainbow trout. These results can contribute to devise strategies for improving rainbow trout survival rate and aquaculture production during hypoxia stress and help speeding up the selective breeding of hypoxia-tolerant rainbow trout.

NADPH oxidase expression profile and PBMC immunophenotypic changes in anti-TNF-treated rheumatoid arthritis patients

The aim of this research was to prospectively evaluate the impact of NOX2 gene expression profile (including NCF1, NCF2 and NCF4 genes) in peripheral blood mononuclear cells (PBMCs) on immune signatures, clinical characteristics and responsiveness to anti-TNFα treatment in RA patients. Blood specimens were collected from 31 rheumatoid arthritis (RA) patients and 25 healthy controls, and 16 RA patients were followed at two timepoints during anti-TNF treatment. mRNA expression levels of selected genes and immunoregulatory cytokines concentrations were determined. We observed the significant upregulation of NCF4 and CD14 expression in RA group. The mRNA levels of NCF1 and CD14 positively correlated both in groups of RA patients and healthy controls. NOX2 gene expression profile was not associated with anti-TNFα responsiveness, nor with RA clinical features. TNFα inhibition has not influenced NOX2 expression either. Notably, this study indicate the novel links between expression levels of NCF1 and monocyte differentiation antigen CD14.

DENV-1 Infection of Macrophages Induces Pyroptosis and Causes Changes in MicroRNA Expression Profiles

Background: Dengue virus (DENV) is the most widespread mosquito-borne virus, which can cause dengue fever with mild symptoms, or progress to fatal dengue hemorrhagic fever and dengue shock syndrome. As the main target cells of DENV, macrophages are responsible for the innate immune response against the virus. Methods: In this study, we investigated the role of pyroptosis in the pathogenic mechanism of dengue fever by examining the level of pyroptosis in DENV-1-infected macrophages and further screened differentially expressed microRNAs by high-throughput sequencing to predict microRNAs that could affect the pyroptosis of the macrophage. Results: Macrophages infected with DENV-1 were induced with decreased cell viability, decreased release of lactate dehydrogenase and IL-1β, activation of NLRP3 inflammasome and caspase-1, cleavage of GSDMD to produce an N-terminal fragment bound to cell membrane, and finally induced macrophage pyroptosis. MicroRNA expression profiles were obtained by sequencing macrophages from all periods of DENV-1 infection and comparing with the negative control. Sixty-three microRNAs differentially expressed in both the early and later stages of infection were also identified. In particular, miR-223-3p, miR-148a-3p, miR-125a-5p, miR-146a-5p and miR-34a-5p were recognized as small molecules that may be involved in the regulation of inflammation. Conclusions: In summary, this study aimed to understand the pathogenic mechanism of DENV through relevant molecular mechanisms and provide new targets for dengue-specific therapy.

Plasma long noncoding RNA expression profiles in patients with Parkinson's disease and the role of lnc-CTSD-5:1 in a PD cell model: a ceRNA microarray-based study

To explore the key genes and long non-coding RNAs (lncRNAs) associated with Parkinson's disease (PD). Peripheral blood plasma samples were collected from 6 PD patients and 6 healthy individuals. The mRNA and lncRNA expression profiles were detected using ceRNA microarray technology, and the differentially expressed genes were analyzed using bioinformatics methods. The differentially expressed mRNAs transcribed within 10 kb upstream or downstream of the differentially expressed lncRNAs were defined as potential cis-regulatable (Cis) target genes of the lncRNAs. A PD-specific protein-protein interaction network (PPI) was constructed. Competitive endogenous RNA (ceRNA) networks were also constructed using the differential lncRNAs with mRNAs and known microRNAs. Using MPP+-treated SHSY5Y cells as a PD cell model, the expressions of the key lncRNAs and their functions were examined. We identified 316 genes and 986 lncRNAs showing significant differential expressions in PD patients (P< 0.05). The differentially expressed mRNAs and the potential cis-regulatable target genes of these lncRNAs were functionally annotated using GO and KEGG enrichment analysis, and the targeting relationship of the differentially expressed mRNAs and lncRNAs with microRNAs were predicted. Analysis of the ceRNA networks constructed based on the differentially expressed lncRNAs suggested that lncMTG2-1:1, lnc-CTSD-5:1, lnc-PCCA-3:1, lnc-VTCN1-3:1, lnc-ZNF25-7:1, and lnc-DAZ3-1:1 might be the key lncRNAs in PD. In MPP+-treated SH-SY5Y cells, the expression of lnc-CTSD-5:1 showed the most significant changes, and silencing lnc-CTSD-5:1 obviously restored the expression level of tyrosine hydroxylase. PD patients have significant changes in plasma lncRNA expression profile, and the differentially expressed genes and lncRNAs found in this study may provide new clues for exploring the pathogenesis and identifying potential biomarkers of PD.

Characterising the transcriptomic response of bovine peripheral blood mononuclear cells to a mycobacterial cell wall fraction

BackgroundInnate immune stimulants, including mycobacterium cell wall fractions (MCWF), offer an alternative control option to prevent and treat disease in livestock, by appropriately augmenting the innate immune response. However, the functional response to mycobacterium cell wall fractions in cattle is not well defined. In this study we report the transcriptomic response of bovine peripheral blood mononuclear cells to MCWF in the product Amplimune®. MethodsAmplimune-induced transcriptomic changes in bovine peripheral blood mononuclear cells were determined following an initial pilot study and a later time course experiment. These cells were cultured in vitro for 24 h. In the pilot experiment, cells were stimulated with 0, 2, 5, 12.5 or 31.25 µg/mL Amplimune. In the time course experiment, cells were stimulated with 0 or 31.25 µg/mL Amplimune. In both experiments the total RNA was extracted at 0 h, 6 h and 24 h following stimulation. Ribosomal RNA depleted samples were sequenced, and data analysed to determine differential gene expression profiles. Differential gene expression was further analysed to determine enriched biological processes and pathways and a co-expression network. Results and conclusionAmplimune induced dose- and time-dependent gene expression profile changes in bovine peripheral blood mononuclear cells, which were enriched into GO-BP regulation of signalling receptor activity, response to cytokine and inflammatory response. Enriched pathways from KEGG analysis were cytokine-cytokine receptor interaction, IL17 signalling and TNF signalling pathways. Selected genes involved in these processes and pathways included IFNG, IL17A, TNF, IL22 and IL23A. PDE1B, CSF2 and IL36G were identified as the most connected genes in a co-expression network, while the connection between SAA2 and SIGLEC5 was the most important for flow of information within the network. Genes encoding for pro-inflammatory cytokines TNF, IL1B, IL6, IL2, and IL12B, and chemokines CCL3, CCL4 and CCL20 were also upregulated at 6 and 24 h post stimulation, as was the β-defensin gene TAP. These results assist in understanding how mycobacterial cell wall fractions alter immune function and may contribute to our understanding of the immune stimulant response attributed to Amplimune.

Post-sepsis chronic muscle weakness can be prevented by pharmacological protection of mitochondria.

Sepsis, mainly caused by bacterial infections, is the leading cause of in-patient hospitalizations. After discharge, most sepsis survivors suffer from long-term medical complications, particularly chronic skeletal muscle weakness. To investigate this medical condition in detail, we previously developed a murine severe sepsis-survival model that exhibits long-term post-sepsis skeletal muscle weakness. While mitochondrial abnormalities were present in the skeletal muscle of the sepsis surviving mice, the relationship between abnormal mitochondria and muscle weakness remained unclear. Herein, we aimed to investigate whether mitochondrial abnormalities have a causal role in chronic post-sepsis muscle weakness and could thereby serve as a therapeutic target. Experimental polymicrobial abdominal sepsis was induced in 16-18months old male and female mice using cecal slurry injection with subsequent antibiotic and fluid resuscitation. To evaluate the pathological roles of mitochondrial abnormalities in post-sepsis skeletal muscle weakness, we utilized a transgenic mouse strain overexpressing the mitochondria-specific antioxidant enzyme manganese superoxide dismutase (MnSOD). Following sepsis development in C57BL/6 mice, we evaluated the effect of the mitochondria-targeting synthetic tetrapeptide SS-31 in protecting mitochondria from sepsis-induced damage and preventing skeletal muscle weakness development. In vivo and in vitro techniques were leveraged to assess muscle function at multiple timepoints throughout sepsis development and resolution. Histological and biochemical analyses including bulk mRNA sequencing were used to detect molecular changes in the muscle during and after sepsis RESULTS: Our time course study revealed that post sepsis skeletal muscle weakness develops progressively after the resolution of acute sepsis and in parallel with the accumulation of mitochondrial abnormalities and changes in the mitochondria-related gene expression profile. Transgenic mice overexpressing MnSOD were protected from mitochondrial abnormalities and muscle weakness following sepsis. Further, pharmacological protection of mitochondria utilizing SS-31 during sepsis effectively prevented the later development of muscle weakness. Our study revealed that the accumulation of mitochondrial abnormalities is the major cause of post-sepsis skeletal muscle weakness. Pharmacological protection of mitochondria during acute sepsis is a potential clinical treatment strategy to prevent post-sepsis muscle weakness.

Role of semaphorin7A in epithelial-mesenchymal transition and proliferative vitreoretinopathy

Proliferative vitreoretinopathy (PVR) is a multifactorial ocular condition characterized by the development of fibrotic membranes inside the vitreous cavity and on the detached retina, which can result in severe blindness. Semaphorin7A (Sema7a) is involved in axon growth, inflammatory responses, and immune regulation; however, its role in PVR and regulatory mechanisms in retinal pigment epithelium (RPE) cells remains unclear. This study aimed to examine Sema7a in PVR and the underlying mechanisms. Transcriptome sequencing was used to investigate the changes in mRNA expression profiles. Western blotting, immunofluorescence, and real-time polymerase chain reaction (RT-PCR) were utilized to investigate the potential mechanism of Sema7a on epithelial-mesenchymal transition (EMT) in RPE cells. Stimulating RPE cells with transforming growth factor beta-1 (TGF-β1) decreased the levels of epithelial markers but increased those of mesenchymal markers. Based on transcriptome sequencing, many molecules associated with PVR progression were regulated. PVR vitreous fluid proteomics data analysis showed that Sema7a significantly changed at different levels. Silencing Sema7a in RPE cells attenuated TGF-β1-induced EMT and their ability to induce experimental PVR; in contrast, recombinant Sema7a (rSema7a) directly triggered EMT in RPE cells. TGF-β1 induction mechanically activated the PI3k-AKT and MAPK pathways, while Sema7a knockdown by short interfering RNA lowered the phosphorylation of the PI3k-AKT/MAPK signaling pathway. Therefore, Sema7a may be a viable therapeutic target for PVR due to its crucial role in the TGF-β1-induced EMT of RPE cells.

Abstract A009: Increasing the Sensitivity to sorafenib of Resistant Hepatocellular Carcinoma Cells with Bovine Lactoferrin through Ferroptosis

Abstract Hepatocellular carcinoma (HCC) remains one of the most lethal cancers worldwide, with a high mortality rate largely due to late-stage diagnosis and the limited efficacy of current therapeutic options. Sorafenib is one of the first-line therapies for advanced HCC. However, its effectiveness is often compromised by the development of drug resistance, significantly limiting its long-term therapeutic potential. This limitation has driven extensive research into alternative therapeutic strategies that can target this resistance, thereby improving the clinical outcomes of patients. Bovine Lf (bLf), which is functionally similar to human Lf (hLf), is safe for human use and has demonstrated anticancer properties, including synergistic effects with chemotherapy. In our study, we explored the role of bovine lactoferrin (bLf) as a potential agent to increase the sensitivity of sorafenib-resistant HCC cells. We developed sorafenib-resistant HCC cell lines through repeated exposure to the drug, with IC50 calculations confirming their increased drug tolerance. These resistant cells were subsequently treated with a combination of sorafenib and bLf. This combination significantly reduced cell viability, with a sorafenib IC50 below the level of the parental sensitive cell line. RNA-seq data revealed significant changes in gene expression profiles, highlighting the differential expression of both mRNAs and long noncoding RNAs (lncRNAs) among resistant, nonresistant, and resistant bLf-treated cells. Further insights were gained through gene set enrichment analysis (GSEA), which identified ferroptosis as a critical pathway significantly enriched in resistant cells treated with bLf and sorafenib vs sorafenib treatment only. Among the differentially expressed lncRNAs, LINC02015 was the most upregulated in resistant cells, and the most downregulated in resistant cells treated with bLf and sorafenib. This lncRNA has emerged as a potentially influencing ferroptosis in other pathologies. Notably, LINC02015 was found to be correlated with HMOX1, a gene with established roles in oxidative stress and iron metabolism. Based on these findings, we conducted a more focused analysis of the ATF3/HMOX1/GPX4 axis, a signaling pathway previously implicated in the regulation of ferroptosis and cellular responses to oxidative stress. Our data suggest that this axis could play a role in modulating lactoferrin-induced ferroptosis in the context of sorafenib resistance. In conclusion, our study provides compelling evidence that bovine lactoferrin (bLf) has the potential to serve as a novel therapeutic agent for overcoming sorafenib resistance in hepatocellular carcinoma by inducing ferroptosis. By potentially targeting the LINC02015/ATF3/HMOX1/GPX4 axis, bLf not only enhances the efficacy of sorafenib but also opens new avenues for the development of combinatory treatment strategies aimed at improving outcomes for HCC patients. Citation Format: Hury V Hernandez-Galdamez, Mireya De la Garza, Karla Rubio, Ivonne Ramirez-Diaz, Josue Guzman-Linares, Theresa Azcarraga Acosta, Guillermo Barreto, stefan günther, Teresita del niño Jesus Flores-Tellez, Mario A Aguilar-Chaparro, Pablo Muriel de la torre, Magda Reyes-López, Saul Villa-Treviño, Carolina Piña-Vazquez. Increasing the Sensitivity to sorafenib of Resistant Hepatocellular Carcinoma Cells with Bovine Lactoferrin through Ferroptosis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A009.

Glyphosate contact alters the expression of genes in the head of africanized Apis mellifera bees

The increase in the world population results in a greater demand for food, intensifying the dependence on pesticides use. Glyphosate, a pesticide widely used in crops, can contaminate resources collected by bees. In this context, this study aimed to analyze the transcriptome of the head of Africanized Apis mellifera bees during the foraging phase to determine whether contact with the herbicide glyphosate, in lethal and sublethal doses, causes changes in the gene expression profile. For this, 180 marked bees were collected from their hives, aged 21 days, and distributed into three treatment groups: 1) bees in contact with the lethal dose of glyphosate (255.73 µg/bee); 2) bees in contact with the sublethal dose of glyphosate (2.5573 µg/bee); 3) control group with distilled water. Transcriptome analysis was performed on 12 bees from each treatment after one and four hours of exposure. The results show that one hour of contact with glyphosate resulted in 57 genes presenting differential expression in relation to the control (11 lethal and 53 sublethal), interfering in the signaling, communication and metabolism processes, while four hours of exposure, 38 genes (32 lethal and 11 sublethal) related, mainly, to the stimulation of the immune response. In summary, glyphosate affects the gene expression of honey bees during the foraging phase, at both doses, resulting in negative effects on behavior, metabolism and immune system, which compromises the health, activity and development of the colonies, also compromising the process of pollination.

Integration of mRNA and miRNA Analysis Sheds New Light on the Muscle Response to Heat Stress in Spotted Sea Bass (Lateolabrax maculatus).

Temperature is a crucial environmental factor for fish. Elevated temperatures trigger various physiological and molecular responses designed to maintain internal environmental homeostasis and ensure the proper functioning of the organism. In this study, we measured biochemical parameters and performed mRNA-miRNA integrated transcriptomic analysis to characterize changes in gene expression profiles in the muscle tissue of spotted sea bass (Lateolabrax maculatus) under heat stress. The measurement of biochemical parameters revealed that the activities of nine biochemical enzymes (ALP, γ-GT, AST, GLU, CK, ALT, TG, LDH and TC) were significantly affected to varying degrees by elevated temperatures. A total of 1940 overlapping differentially expressed genes (DEGs) were identified among the five comparisons in the muscle tissue after heat stress. Protein-protein interaction (PPI) analysis of DEGs indicated that heat shock protein genes (HSPs) were deeply involved in the response to heat stress. In addition, we detected 462 differential alternative splicing (DAS) events and 618 DAS genes, which are closely associated with sarcomere assembly in muscle, highlighting the role of alternative splicing in thermal response regulation. Moreover, 32 differentially expressed miRNAs (DEMs) were identified in response to heat stress, and 599 DEGs were predicted as potential target genes of those DEMs, generating 846 DEG-DEM negative regulatory pairs potentially associated with thermal response. Function enrichment analysis of the target genes suggested that lipid metabolism-related pathways and genes were regulated by miRNAs. By analyzing PPIs of target genes, we identified 28 key negative regulatory pairs, including 13 miRNAs (such as lma-miR-122, lma-miR-200b-5p and novel-miR-444) and 15 target genes (such as hspa13, dnaja1, and dnajb1a). This study elucidates the molecular mechanisms of response to high-temperature stress and offers valuable information for the selection and breeding of heat-tolerant strains of spotted sea bass.

Acupuncture-induced gene co-expression networks in postmenopausal women with osteoarthritis and osteoporosis: In-silico analysis.

Objective: Bone is a tissue that is constantly remodeled to adjust the microarchitecture and maintain the mechanical needs of bone through the balance of bone resorption and formation processes. Alterations in these processes can lead to the development of different diseases, such as osteoarthritis and osteoporosis. In recent years, it has been shown that acupuncture is an effective treatment for pain, physical dysfunctions, and the immune system, so the stimulation of acupuncture points could affect genes associated with inflammatory processes and, therefore, osteoarthritis and osteoporosis. To analyze changes in gene expression post-acupuncture in data from a group of individuals with osteoarthritis that also manifests in osteoporosis. Method: Through using microarray technology and bioinformatics analysis, potential genes associated with osteoarthritis after acupuncture treatment are identified and compared with genes implicated in osteoporosis. The genes identified in each disease were evaluated through a KEGG signaling pathway analysis, where the results allowed the generation of an in-silico model that shows interaction networks between signaling pathways and genes involved in both diseases. Results: In this interaction, 37 differentially expressed genes were identified in patients with osteoarthritis before and after acupuncture treatment, and 665 differentially expressed genes was involved in osteoporosis. In the osteoarthritis group, 15 signaling pathways involved in this disease were obtained, and for osteoporosis, 13 signaling pathways associated with immunological processes that participate in bone metabolism were obtained osteoarthritis and osteoporosis are two age-associated diseases that are characterized by alterations in the bone remodeling mechanism induced by changes in gene expression profiles. Conclusions: Treatment with acupuncture can modify various cytokines involved in diseases related to the immune system so that it can have beneficial effects on osteoarthritis and osteoporosis. In addition, bioinformatics analysis allows us to know those signaling pathways through which they could have acupuncture effects. Graphical abstract: http://links.lww.com/AHM/A137.

Analysis of Polyphyllin II alters microRNA expression profile in lung cancer A549 cells

Non-small cell lung cancer (NSCLC) is a malignant tumor that threatens the whole world, previous studies found that Polyphyllin II (PPII) can suppress NSCLC cells growth, exhibits significant antitumor activity. MicroRNAs (miRNAs) can regulate the expression of other genes and play a crucial role in the prevention and development of cancer. However, the miRNA portrait of ginsenoside PPII-treated NSCLC A549 cells has not yet been studied. In this work, miRNA-seq analysis was used to determine the changes in miRNA expression profile of NSCLC A549 cells PPII-treaded. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed on differentially expressed miRNA target genes. Then, predicted the target genes of miRNA and performed functional enrichment analysis on them. We identified 58 up-regulated and 24 down-regulated miRNAs displaying changes their expression in PPII treated A549 cells were greater than 2-fold. In addition, the expression levels of miRNAs were validated by real-time PCR. Therefore, this work has a certain promoting effect on the study of the anti-cancer mechanism of PPII in lung cells.

Impact of gonadotropin on certain testis-related genes identified through testicular transcriptome analysis in the Asian and the African catfish

Induction of spawning in catfish is well demonstrated in females while stimulation of spermiation in males seems difficult. This has led to least understanding of gene profile changes during testicular maturation. Expression of the factors after hormonal induction that control reproductive processes has become an intense research area in fish endocrinology. In view of this, de novo assemblies of testicular transcriptomes of the Asian catfish, Clarias batrachus and the African catfish, C. gariepinus, were performed to identify genes/factors involved in testicular maturation. For testicular induction, human chorionic gonadotropin (hCG) was administered in vivo to both the catfish species during active phase of reproduction. 1,68,071 and 1,26,232 assembled transcripts were obtained from C. batrachus and C. gariepinus testicular transcriptomes and were annotated using different databases. Further, in silico analysis suggested the presence of several transcripts that were involved in steroidogenesis and male reproduction. Comparison of transcriptomes of both species revealed the presence of certain unique genes related to reproduction differentially. Expression profiles after hCG induction in testis showed higher expression of certain steroidogenesis related genes such as star, cyp11b1, cyp17a and cyp21a in both the species. Further, expression levels of crucial factors related to testis, such as dmrt1/2/3, were also found to be significantly higher after gonadotropin induction. However, amh, tgfα and dmrt4/5 expression levels were significantly low. Factors related to male reproduction i.e., tekt1, tekt2, sox9, spag1, spata2 and spata7 were found to be differentially upregulated in hCG treated testicular tissues of both the species. Histology analysis indicated that the gonadotropin induction either short or long term is a better strategy to highlight expression profile changes during testicular maturation in teleost.

Epigenetic modifications in transdifferentiation of adipose tissue-derived stromal cells into spontaneously beating cardiomyocyte-like cells

Abstract Background Adipose tissue-derived stromal vascular fraction (adipose SVF) contains pluripotent mesenchymal stem cells and rarely transdifferentiate into beating cardiomyocytes. We developed a simple culture protocol under which the adult murine inguinal adipose SVF reproductively transdifferentiates into beating cardiomyocyte-like cells (beating CM) without inductions in the primary culture. We also reported that Mef2c is a crucial factor in this conversion. However, the characteristics of beating CM and the factors that regulate the differentiation of adipose SVF toward the cardiac lineage are unknown. Purpose To investigate sequential changes in global gene expression profiles of adipose SVF in primary culture and determine the mechanism of differentiation into beating CM. Methods Adipose SVF cells were isolated from adult mice's inguinal subcutaneous fat pad and cultured using the previously established beating CM induction method (Sci Rep. 2021). At 6 time points (days 0, 3, 7, 14, 21, and 28) in primary culture, total RNA was extracted, and RNA-sequencing was performed (n = 3). The data was analyzed using Subio Platform and MetaCore software. To assess the candidate gene and proteins, adipose SVF cells were transduced with the gene using a lentivirus vector and supplemented with the specific inhibitors. The cardiac differentiation was evaluated by quantitative PCR on day 28. Results The beating CM was confirmed on days 14, 21, and 28 in the primary culture. Among prefiltered 14,574 genes, the expression level of 749 genes were significantly changed (153 genes upregulated vs. 596 genes downregulated) between days 7 and 14 (2-fold, P &amp;lt; 0.05), in which beating CM appeared. The GO molecular functions analysis showed that O-GluNAC transferase and histone deacetylase (HDAC)-associated genes were expressed dominantly before day 7, and KLF4-associated genes were expressed prominently after day 14 in primary culture. The expression of cardiac troponin T (Tnnt2) and myosin light chain ventricular type (Myl2) increased on day 3 and day 14, respectively. The HDAC7, an epigenetic regulator, decreased the expression on day 14 or later. Treatment with the HDAC inhibitor (150 nmol/L) increased the expression of Tnnt2 (5-fold vs. control) on day 28 in adipose SVF. Furthermore, treatment of Mef2c-transduced adipose SVFs with HDAC inhibitors increased expression of cardiac troponin T (29-fold vs. control, P &amp;lt; 0.05). Conclusion The time course analysis demonstrated that a remarkable change occurred in the regulation of transcription on day 14 in adipose SVF primary culture. Alterations in epigenetic modifications increased beating CM in adipose SVFs. Adipose SVF could be applied to new cardiac regeneration therapies by increasing the efficiency of introduction into the beating CM.

Proangiogenic effect of thyrotropin receptor stimulating antibody in human umbilical vein endothelial cells.

This study aims to investigate the role of TRAb in the angiogenesis associated with Graves' disease (GD) and to elucidate its underlying mechanisms. Human thyroid follicular epithelial cells (Nthy-ori 3-1) and human umbilical vein endothelial cells (HUVECs) were treated with the monoclonal thyroid-stimulating antibody M22 and thyroid-stimulating hormone (TSH) at various concentrations. Cell viability, migration, and tube formation were evaluated using CCK-8, wound healing, and tube formation assays, respectively. Protein expressions of TSHR receptor (TSHR) and phosphorylated AKT (p-AKT) in M22-induced HUVECs were quantified via Western blotting. Proteomic analysis was employed to identify changes in protein expression profiles and relevant signaling pathways in GD specimens. Immunofluorescence assays were conducted to detect and localize the expressions of CD34 and PROX1 in GD specimens and normal thyroid tissues. M22 stimulated the proliferation of Nthy-ori 3-1 cells and HUVECs in a dose-dependent manner, while TSH exhibited an inverted U-shaped dose-response effect. M22 also dose-dependently promoted angiogenesis, and more effectively induced tube formation in HUVECs compared to TSH, although the difference was not statistically significant. A total of 16 proteins were significantly upregulated and 24 were downregulated in M22-induced HUVECs. Notably, PROX1, the most significantly upregulated protein, is closely associated with angiogenesis. Immunofluorescence confirmed that PROX1 was significantly more expressed in thyroid tissues from GD patients compared to normal tissues adjacent to papillary thyroid cancer (PTC), and it co-localized with CD34. TRAb enhances angiogenesis and upregulates PROX1 expression in HUVECs, suggesting a novel possible mechanism for goiter formation in GD.

Changes in gene expression profile of normal human fibroblasts on P(VDF-TrFE) scaffolds highly doped with Fe3O4-CA nanoparticles under alternating magnetic field stimulation

The design of novel hybrid magnetoactive scaffolds based on biocompatible piezopolymers and magnetic nanoparticles is of interest for medicine, mainly for tissue regeneration, because application of an external either static or alternating magnetic field to cells that settled on a magnetoactive scaffold offers an opportunity for remote control of cellular functions. This study describes fabrication of electrospun magnetoactive poly(vinylidene fluoride-co-trifluoroethylene) [P(VDF-TrFE)] scaffolds highly doped with 20 wt% of magnetite nanoparticles modified with citric acid (Fe3O4-CA). The electrospun P(VDF-TrFE)/Fe3O4-CA scaffolds have defect-free morphology with a fiber diameter of approximately 1 μm and contain both an electroactive β-phase (predominantly) and a lesser amount of an γ-phase. A high content of uniformly distributed Fe3O4-CA nanoparticles within P(VDF-TrFE) fibrous scaffolds resulted in a high saturation magnetization of 12.1 emu/g and ferrimagnetic behavior of the composite P(VDF-TrFE)/Fe3O4-CA scaffolds. They were proved to be biocompatible with normal human cells: normal human fibroblasts and human mesenchymal stem cells adhered to the scaffold and retained their viability. According to high-throughput RNA-sequencing data, the adhesion of fibroblasts to the scaffolds upregulated genes related to key stages of tissue regeneration such as coagulation (genes THBD and SERPINB2) and wound healing (IL24, PDGFB, F3, and PLAU) and affected TGFβ, BMP, and Wnt signaling pathways. Alternating-magnetic-field exposure of the magnetoactive P(VDF-TrFE)/Fe3O4-CA scaffolds with fibroblasts settled on the surface activated extracellular and intracellular cell signaling pathways.

Fasciola hepatica Excretory-Secretory Products (Fh-ES) Either Do Not Affect miRNA Expression Profile in THP-1 Macrophages or the Changes Are Undetectable by a Microarray Technique.

Fasciola hepatica is a liver fluke that resides in the bile ducts of various mammals. The parasitosis leads to economic losses in animal production estimated at USD 3.2 billion annually. It is also considered a zoonosis of great significance and a problem for public health affecting 2.4 million people worldwide. Nevertheless, besides the negative aspects of infestation, the antigens released by the fluke, F. hepatica Excretory-Secretory Products (Fh-ES) contain several immunomodulatory molecules that may be beneficial during the course of type I diabetes, multiple sclerosis, ulcerative colitis, or septic shock. This phenomenon is based on the natural abilities of adult F. hepatica to suppress proinflammatory responses. To underline the molecular basis of these mechanisms and determine the role of microRNA (miRNA) in the process, lipopolysaccharide (LPS)-activated THP-1 macrophages were stimulated with Fh-ES, followed by miRNA microarray analyses. Surprisingly, no results indicating changes in the miRNA expression profile were noted (p < 0.05). We discuss potential reasons for these results, which may be due to insufficient sensitivity to detect slight changes in miRNA expression or the possibility that these changes are not regulated by miRNA. Despite the negative data, this work may contribute to the future planning of experiments by other researchers.

Downregulation of chemokine (C‑C motif) ligand 5 induced by a novel 8‑hydroxyquinoline derivative (91b1) suppresses tumor invasiveness in esophageal carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a particularly aggressive form of cancer with high mortality. In the present study, a novel 8‑hydroxyquinoline derivative (91b1) was investigated for its anticancer activities in ESCC along with its associated mechanisms. The in vitro cytotoxic effect of 91b1 were evaluated across five ESCC cell lines using MTS assay, with cisplatin serving as a comparative standard. Changes in gene expression profile were identified by cDNA microarray and further validated by qualitative PCR and immunostaining. Additionally, protein levels of the most notably downregulated target in archival ESCC samples were also studied. 91b1 demonstrated comparable anticancer effect with cisplatin. Notably, chemokine ligand 5 (Ccl5) was identified as the most substantially downregulated gene, with its suppression at both mRNA and protein expression in ESCC cells, exhibiting a dose‑dependent manner. The recombinant human protein of CCL5 enhanced the invasion of ESCC cells using the Transwell assay. The upregulation of CCL5 protein was also detected in 50% of ESCC cell lines. CCL5 was also overexpressed in 76.9% of ESCC specimens. The overall results indicated that 91b1 could effectively induce anticancer effect on ESCC cells through downregulating CCL5 expression with suppression of tumor invasion. Overall, these findings suggested that 91b1 effectively inhibited ESCC cell proliferation and tumor invasion by downregulating CCL5 expression, highlighting its potential as a therapeutic agent for ESCC treatment.

Vδ1 T Cells Integrated in Full-Thickness Skin Equivalents: A Model for the Role of Human Skin–Resident γδT Cells

Vδ1 T cells are a subpopulation of γδT cells found in human dermis. In contrast to murine skin-resident γδT cells, much less is known regarding their role and function in skin health and disease. Here we report the successful integration of Vδ1 T cells into long-term fibroblast-derived matrix skin equivalents (SE). We isolated Vδ1 T cells from human blood, where they are rare, and established conditions for the integration and maintenance of the freshly isolated Vδ1 T cells in the SEs. Plated on top of the dermal equivalents (DEs), almost all Vδ1 T cells migrated into the dermal matrix where they exerted their influence on both the DE and the epithelium. Vδ1 T cells contributed to epidermal differentiation as indicated by histology, expression of epidermal differentiation markers and RNAseq expression profile. When complemented with the carcinoma-derived SCC13 cells instead of HaCaT, our data suggest a role for Vδ1 T cells in slowing growth of the tumor cells, as indicated by reduced stratification and changes in gene expression profiles. Together, we demonstrate the successful establishment of human Vδ1 T cell-competent skin and skin carcinoma equivalents (SE, SCE) and provide evidence for molecular and functional consequences of the Vδ1 T cells on their respective environment.

Targeting Kidney Inflammation After Brain Death and Cold Storage: Investigating the Potential of an NLRP3 Inflammasome Inhibitor (MCC950) for Preconditioning Donor Kidneys.

Brain death (BD) and cold storage (CS) are critical factors that induce inflammation in donor kidneys, compromising organ quality. We investigated whether treating kidneys from BD rats with an inflammasome Nod-like receptor family pyrin domain containing 3 (NLRP3) inhibitor (MCC950) followed by CS could reduce kidney inflammation. BD rats were assigned to MCC950-treated or nontreated (NT) groups. Kidneys were evaluated immediately before CS (T0) and after 12 h (T12) and 24 h (T24) of CS. Mean arterial pressure, serum creatinine, gene/protein expression, and histology were evaluated. At T0, MCC950 treatment did not affect mean arterial pressure but tended to reduce serum creatinine and ameliorated the histological score of acute tubular necrosis. However, MCC950 reduced NLRP3, caspase-1, interleukin (IL)-1β, IL-6, Kim-1, nuclear factor kappa B, tumor necrosis factor alpha, and caspase-3 gene expression while increasing IL-10 cytokine gene expression. After 12 h of CS, only the expression of the NLRP3 and caspase-1 genes decreased, and after 24 h of CS, no further changes in the gene expression profile were observed. The levels of the inflammasome proteins NLRP3, caspase-1, and IL-1β consistently decreased across all time points (T0, T12, and T24). These findings suggest that MCC950 treatment holds promise for mitigating the proinflammatory state observed in kidneys after BD and CS.