Relative mRNA Expression Levels Research Articles

P-522 Impact of TGFß/BMP/SMAD Pathway on Premature Ovarian Insuffiency (POI)

Abstract Study question Is TGFß/BMP/SMAD pathway altered in women with POI and can results be detected from peripheral blood samples? Summary answer The TGFß/BMP/SMAD pathway mRNA-expression-alterations are detectable in leukocytes of women with Premature Ovarian Insufficiency (POI) paving the way to use it as predictive tool. What is known already A proper ovarian function relies on a complex system of signalling molecules including several BMP/TGFß-superfamily-members expressed in the ovary at different stages of folliculogenesis acting via two types of Serine/Threonine Kinase receptors. The downstream located SMAD proteins are classified into three subclasses according to their function as activated R-SMADs1,2,3,4,9, common-partner SMAD4 or inhibitory SMADs6,7. Mutations in BMPR2 and its ligands have been shown to be related to POI. Recently neurologic studies demonstrated that FMRP(Fragile-X-Mental-Retardation-1-Protein) represses BMPR2 and its gene FMR1(Fragile X Mental-Retardation-1) is one major regulator of follicular development and the most frequent genetic cause of POI if premutated. Study design, size, duration RT-q-PCR was performed to measure the mRNA-levels of SMAD 1,3,4,5, BMPR2 and FMR1. cDNA was synthesized using RNA obtained from leucocytes of patients with POI and of the control group with a normal ovarian reserve. 163 patients diagnosed with POI and 51 women in the control group were recruited between 2008 and 2022 at Heidelberg University’s Women’s Hospital. Participants/materials, setting, methods Blood samples were drawn from every participant to extract leucocyte-RNA and synthesize non-specific cDNA. RT-q-PCR was performed with TaqMan reagents to quantify relative mRNA-expression levels of SMAD 1,3,4, 5, BMPR2 and FMR1. Statistical analysis was performed with Microsoft Excel and GraphPad Prism 10 using the 2–ΔΔCt method and Mann-Whitney-Test. Statistical significance was set to p = 0.05. Main results and the role of chance In our investigation, we observed a consistent and statistically significant decrease in the expression levels of all tested SMADs—SMAD1, SMAD3, SMAD4, and SMAD5—in leukocytes of women diagnosed with POI compared to unaffected controls. The p-values amounted to p < 0.0001 for SMAD1, p = 0.0340 for SMAD3, p < 0.0001 for SMAD4, and p = 0.0076 for SMAD5. However, we did not identify notable differences in the expression levels of FMR1 and BMPR2 between the two groups. These results suggest an alteration in the regulation of the activated downstream elements SMAD 1, 3, and 5 and their common partner SMAD4 in women with POI, emphasizing its potential role as a contributing factor to diminished ovarian function. These effects seem to be independent of BMPR2 and FMR1 and regulated by other receptors and or ligands. Limitations, reasons for caution Our data are limited to mRNA-levels. So further experiments including protein-level and their phosphorylation-status are needed to receive an overall assessment of its impact on ovarian damage. It’s worth noting that the study is concentrated on leukocytes as potential diagnostic tool and results should be cross-checked in ovarian-tissue and cells. Wider implications of the findings The identified alteration in TGFß/BMP/SMAD pathway gene expression in women with POI suggest a role in ovarian damage. Lower expression levels in the aforementioned mRNAs could serve as a potential predictive biomarker for POI risks and putatively used in consultations of women prior to fertility treatment and/or preservation. Trial registration number not applicable

Evaluation test and analysis of a microneedle and iontophoresis based medical device "CELLADEEP Patch" in skin improvement on ex vivo human-derived skin tissue models.

Microneedles are tiny needles, typically ranging from tens to hundreds of micrometers in length, used in various medical procedures and treatments. The tested medical device named "CELLADEEP Patch" a dissolvable microneedle therapy system (MTS), made of hyaluronic acid and collagen. And the iontophoresis technique is also applied in the system. The study aimed to evaluate the effectiveness of the "CELLADEEP Patch" in skin improvement. Ex vivo human-derived skin tissue models were used in this study and they were divided into three different groups, namely, the Untreated Group, the Negative Control Group, and the Test Group respectively. The Untreated Group received no treatment measures, the Negative Control Group was exposed to ultraviolet B radiation (UVB) irradiation, and the Test Group was exposed to UVB irradiation and treated with "CELLADEEP Patch". Skin moisture content, transdermal water loss, and skin elasticity were evaluated by three clinical devices. Additionally, histological staining and related mRNA expression levels were also analyzed. The results of skin moisture content, transdermal water loss, and skin elasticity evaluation consistently illustrated that the application of "CELLADEEP Patch" led to remarkable skin improvement. And the analysis of histological staining images also confirmed the effectiveness of the "CELLADEEP Patch", especially for increasing collagen density. Moreover, the upregulation of Collagen type 1 a (COL1A1) and hyaluronan synthase 3 mRNA expression and the decrease of Matrix metalloproteinase 1 (MMP-1) and Interleukin-1 beta (IL-1β) mRNA expression reflected its wrinkle improvement, moisturizing and anti-inflammation function. "CELLADEPP Patch", the MTS combined with the iontophoresis technique, exhibits its effectiveness in moisturizing, skin elasticity improvement, and anti-inflammatory function when applied to ex vivo human-derived skin tissue models in experiments. The study has contributed to the understanding of the "CELLADEPP Patch" and laid the foundation for subsequent animal experiments and clinical trials.

Saccharomyces cerevisiae and Kluyveromyces marxianus yeast co-cultures modulate the ruminal microbiome and metabolite availability to enhance rumen barrier function and growth performance in weaned lambs

In lambs, weaning imposes stress that can contribute to impaired rumen epithelial barrier functionality and immunological dysregulation. In this study, the effects of a yeast co-culture consisting of Saccharomyces cerevisiae and Kluyveromyces marxianus (NM) on rumen health in lambs was evaluated, with a focus on parameters including growth performance, ruminal fermentation, and epithelial barrier integrity, ruminal metabolic function, and the composition of the ruminal bacteria. In total, 24 lambs were grouped into four groups of six lambs including a control (C) group fed a basal diet, and N, M, and NM groups in which lambs were fed the basal diet respectively supplemented with Saccharomyces cerevisiae yeast cultures (30 g/d per head), Kluyveromyces marxianus yeast cultures (30 g/d per head), and co-cultures of both yeasts (30 g/d per head), the experiment lasted for 42 d. Subsequent analyses revealed that relative to the C group, the average daily gain (ADG) of lambs in the NM group was significantly greater (P<0.05) and exhibited significant increases in a range of mRNA relative expression including monocarboxylate transporter 1 (MCT1), (Na+)/hydrogen (H+) exchanger 1 (NHE1), (Na+)/hydrogen (H+) exchanger 3 (NHE3), proton-coupled amino acid transporter 1 (PAT1), vacuolar H+-ATPase (vH+ ATPase), Claudin-1, Occludin in the rumen epithelium (P<0.05). Compared with the C group, the pH of the rumen contents in the NM group was significantly decreased (P<0.05), and the concentrations of acetate, propionate, and butyrate were significantly increased (P<0.05). Analysis of the rumen bacteria showed that the NM group exhibited increases in the relative abundance of Prevotella, Treponema, Moryella, Fibrobacter, CF231 and Ruminococcus (P<0.05). Metabolomics analyses revealed an increase in the relative content of phthalic acid (P<0.001) and cinnamaldehyde (P=0.008) in the NM group as compared to the C group (P<0.05), together with the greater relative content of L-tyrosine, L-dopa, rosmarinic acid, and tyrosol generated by the tyrosine metabolic pathway (P<0.05). Spearman’s correlation analyses revealed relative abundance levels of Fibrobacter and Ruminococcus were positively correlated with the mRNA relative expression levels of PAT1, NHE3, and zonula occluden-1 (ZO-1), as well as with tyrosol, phthalic acid, and cinnamaldehyde levels (P<0.05). Ultimately, these results suggest that dietary supplementation with NM has a wide range of beneficial effects on weaned lambs and is superior to single bacterial fermentation. These effects include improvements in daily gain and rumen epithelial barrier integrity, as well as improvements in the composition of the rumen microbiome, and alterations in tyrosine metabolic pathways.

Targeting CCR3 with antagonist SB 328437 sensitizes 5‑fluorouracil‑resistant gastric cancer cells: Experimental evidence and computational insights.

Gastric cancer (GC) ranks fifth globally in cancer diagnoses and third for cancer-related deaths. Chemotherapy with 5-fluorouracil (5-FU), a primary treatment, faces challenges due to the development of chemoresistance. Tumor microenvironment factors, including C-C motif chemokine receptor 3 (CCR3), can contribute to chemoresistance. The present study evaluated the effect of CCR3 receptor inhibition using the antagonist SB 328437 and the molecular dynamics of this interaction on resistance to 5-FU in gastric cancer cells. The 5-FU-resistant AGS cell line (AGS R-5FU) demonstrated notable tolerance to higher concentrations of 5-FU, with a 2.6-fold increase compared with the parental AGS cell line. Furthermore, the mRNA expression levels of thymidylate synthase (TS), a molecular marker for 5-FU resistance, were significantly elevated in AGS R-5FU cells. CCR3 was shown to be expressed at significantly higher levels in these resistant cells. Combining SB 328437 with 5-FU resulted in a significant decrease in cell viability, particularly at higher concentrations of 5-FU. Furthermore, when SB 328437 was combined with 5-FU at a high concentration, the relative mRNA expression levels of CCR3 and TS decreased significantly. Computational analysis of CCR3 demonstrated dynamic conformational changes, especially in extracellular loop 2 region, which indicated potential alterations in ligand recognition. Docking simulations demonstrated that SB 328437 bound to the allosteric site of CCR3, inducing a conformational change in ECL2 and hindering ligand recognition. The present study provides comprehensive information on the molecular and structural aspects of 5-FU resistance and CCR3 modulation, highlighting the potential for therapeutic application of SB 328437 in GC treatment.

The Role of Cuproptosis Key Factor FDX1 in Gastric Cancer.

Gastric cancer is a common malignant tumor of the digestive tract, both domestically and internationally. It has high incidence and mortality rates, posing a significant threat to human health. The levels of blood copper are elevated in patients with gastric cancer. However, the exact relationship between copper overload and the malignant phenotype of gastric cancer is still unclear. This study aims to investigate the role of the Cuproptosis-related factor FDX1 in the conversion of gastric cancer to a malignant phenotype. Firstly, the relative mRNA and protein expression levels of FDX1 in gastric cancer were detected. Secondly, lentiviral transfection of gastric cancer cell lines was performed, and the effects of FDX1 functional intervention on the proliferation, invasion and migration of gastric cancer cells were assessed by CCK-8, colony formation, EdU proliferation, cell scratch and Transwell assays. Thirdly, the differential alteration of genes after overexpression of FDX1 was also analyzed by transcriptome sequencing. Finally, we assessed the tumour-forming capacity in vivo by the xenograft model. FDX1 is significantly upregulated in gastric cancer. The inhibition of FDX1 function results in the suppression of malignant phenotypic transformation in gastric cancer cells. Conversely, overexpression of FDX1 function leads to alterations in tumor-related signaling pathways and the tumor microenvironment. FDX1 plays a significant role in the malignant phenotypic transformation of gastric cancer cells. Further investigation into the regulatory mechanism of FDX1 in the malignant transformation of gastric cancer will enhance our understanding of the involvement of Cuproptosis in gastric cancer.

Network Pharmacology and Bioinformatics Analysis identify potential therapeutic effects of Berberine on Colon Cancer complicated with Radiation Enteritis

Background: Patients with colon adenocarcinoma (COAD) who undergo radiation therapy develop radiation enteritis (RE). The predictive value of RE in COAD is yet to be established. Berberine, an active compound derived from the traditional Chinese medicinal plant, Coptis chinensis, has notable anti-inflammatory properties and offers protection to the intestinal mucosa. This study aimed to evaluate the possible therapeutic effect and mechanism of berberine as a treatment for COAD complicated with RE (COAD&amp;RE). Methods: Relevant genetic features of diverse COAD&amp;RE populations were analyzed using bioinformatics and the Cox proportional hazards regression model. The therapeutic targets of berberine were predicted using network pharmacology and molecular docking. In vivo and in vitro experiments were conducted to validate the core genes identified using molecular docking. Results: RE has a certain impact on the prognosis of COAD and berberine may play an important role in the treatment of COAD&amp;RE. In addition, we identified five core therapeutic targets of berberine by network pharmacology and molecular docking: CCND1, MYC, AR, LEP, and CYP19A1. In vivo experiments showed that berberine increased short-term survival rate, body weight, and intestinal epithelial cell recovery in mice after radiation. In an in vitro study, berberine promoted the proliferation of human intestinal epithelial cells and enhanced the radiosensitivity of HT29 cells after radiation, and the relative mRNA expression levels of CCND1 and MYC closely correlated with these effects. Conclusions: This study predicted the potential therapeutic effects of berberine on COAD&amp;RE and verified the relevant mechanisms, which may provide insights and suggestions for the clinical treatment of COAD&amp;RE.

The identification of hub genes associated with pure ground glass nodules using weighted gene co-expression network analysis

BackgroundWhether there are invasive components in pure ground glass nodules(pGGNs) in the lungs is still a huge challenge to forecast. The objective of our study is to investigate and identify the potential biomarker genes for pure ground glass nodule(pGGN) based on the method of bioinformatics analysis.MethodsTo investigate differentially expressed genes (DEGs), firstly the data obtained from the gene expression omnibus (GEO) database was used.Next Weighted gene co-expression network analysis (WGCNA) investigate the co-expression network of DEGs. The black key module was chosen as the key one in correlation with pGGN. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analyses were done. Then STRING was uesd to create a protein-protein interaction (PPI) network, and the chosen module genes were analyzed by Cytoscape software.In addition the polymerase chain reaction (PCR) was used to evaluate the value of these hub genes in pGGN patients’ tumor tissues compared to controls.ResultsA total of 4475 DEGs were screened out from GSE193725, then 225 DEGs were identified in black key module, which were found to be enriched for various functions and pathways, such as extracellular exosome, vesicle, ribosome and so on. Among these DEGs, 6 overlapped hub genes with high degrees of stress method were selected. These hub genes include RPL4, RPL8, RPLP0, RPS16, RPS2 and CCT3.At last relative expression levels of CCT3 and RPL8 mRNA were both regulated in pGGN patients’ tumor tissues compared to controls.ConclusionsTo summarize, the determined DEGs, pathways, modules, and overlapped hub genes can throw light on the potential molecular mechanisms of pGGN.

Dimorphic Regulation of the MafB Gene by Sex Steroids in Hamsters, Mesocricetus auratus.

MafB is a transcription factor that regulates macrophage differentiation. Macrophages are a traditional feature of the hamster Harderian gland (HG); however, studies pertaining to MafB expression in the HG are scant. Here, the full-length cDNA of the MafB gene in hamsters was cloned and sequenced. Molecular characterization revealed that MafB encodes a protein containing 323 amino acids with a DNA-binding domain, a transactivation domain, and a leucine zipper domain. qPCR assays indicated that MafB was expressed in different tissues of both sexes. The highest relative expression levels in endocrine tissues were identified in the pancreas. Gonadectomy in male hamsters was associated with significantly higher mRNA levels in the HG; replacement with dihydrotestosterone restored mRNA expression. The HG in male hamsters contained twofold more MafB mRNA than the HG of female hamsters. Adrenals revealed similar mRNA relative expression levels during the estrous cycle. The estrous phase was associated with higher mRNA levels in the ovary. A significantly up-regulated expression and sexual dimorphism of MafB was found in the pancreas. Therefore, MafB in the HG may play an active role in the macrophage differentiation required for phagocytosis activity and intraocular repair. Additionally, sex steroids appear to strongly influence the MafB expression in the HG and pancreas. These studies highlight the probable biological importance of MafB in immunological defense and pancreatic β cell regulation.

Effects of solid-state fermentation product of yeast supplementation on liver and intestinal health, and resistance of common carp (Cyprinus carpio) against spring viraemia carp virus

This study aimed to investigate the effects of solid-state fermentation products of yeast (SFPY) on liver and intestinal health and disease resistance of common carp (Cyprinus carpio). A total of 200 common carp with an initial average weight of 2.55 ± 0.004 g were divided into 5 groups (4 replications per group and 10 fish per replication), and were fed with one of five diets, including a control diet and 4 diets supplemented with 2‰ (Y2), 3‰ (Y3), 4‰ (Y4), or 5‰ (Y5) SFPY, respectively, for 8 weeks. Results indicated that, the addition of SFPY to the diet of common carp did not affect the growth performance or survival rate of fish (P = 0.253). Interestingly, with the addition of SFPY, the triacylglycerol (TAG) content of the liver presented a linear decreasing tendency (P = 0.004), with significantly decreased in Y4 and Y5 groups (P = 0.035) compared with control. Serum lipopolysaccharide (LPS) content and diamine oxidase (DAO) activity presented a negative linear relationship with the addition of SFPY (P = 0.015, P = 0.030), while serum lipopolysaccharide binding protein (LBP) content first decreased and then increased (P < 0.001). The total antioxidant capacity (T-AOC) in the intestine of fish increased continuously with increasing SFPY supplementation (P = 0.026), reaching the highest level in Y5 group. The villus height in all experimental groups were significantly higher than that in the control group (P < 0.001). Furthermore, compared to the control, adding 3‰ SFPY to the control diet of common carp significantly increased the relative abundance of Fusobacteria (P = 0.018) and decreased that of Proteobacteria (P = 0.039) at phylum level, and increased the relative abundance of Cetobacterium (P= 0.018) and decreased that of Shewanella (P = 0.013) at genus level. Compared with the control, the relative mRNA expression level of spring viraemia of carp virus N protein (SVCV-n) in the kidney was lower than that of the control group without significance and bottomed out in Y4 group (P = 0.138). In conclusion, dietary SFPY enhanced the SVCV resistance capacity of common carp by improving liver and intestinal health and modulating the gut microbiota. Thus, SFPY is a potential feed additive to be used in aquaculture to reduce the huge economic loss of common carp due to SVCV disease. Based on liver TAG content and intestinal villus height, the optimal addition level of SFPY was 3.02‰ and 2.72‰, respectively.

The Role of Notch Signaling Pathway in Adult Patients with Epstein-Barr Virus-induced Infectious Mononucleosis

To investigate the changes of Notch signaling molecules and Th22 cells in adult patients with infectious mononucleosis (IM), and assess the regulatory function of Notch signaling inhibition to Th22 cells. Forty-two IM patients and twenty-one healthy controls were enrolled in this study. Their peripheral blood was collected, from which plasma and peripheral blood mononuclear cells (PBMCs) were isolated. Plasma interleukin (IL)-17 and IL-22 were measured by enzyme-linked immunosorbent assay. The percentages of CD3+ CD4+ IL-17+ Th17 cells and CD3+ CD4+ IL-22+ Th22 cells were investigated by flow cytometry. The mRNA relative levels corresponding to Th17 transcription factor retinoic acid related orphan receptor γt (RORγt), Th22 transcription factor aryl hydrocarbon receptor (AhR), and Notch signaling pathway molecules (including Notch receptors, Notch ligands, Notch downstream molecules) were semi-quantified by real-time PCR. CD4+ T cells were purified and stimulated with γ-secretase inhibitor (GSI). Cellular proliferation, Th17 and Th22 percentage, IL-17 and IL-22 secretion, transcription factor mRNA were measured in response to GSI stimulation. The relative expression levels of Notch1 and Notch2 mRNA in PBMCs of IM group were 13.58±3.18 and 4.73±1.16, respectively, which were significantly higher than 1.09±0.12 and 1.07±0.15 in PBMCs of control group (both P < 0.001). However, there were no significant differences in relative expression levels of Notch3 and Notch4 mRNA between IM group and control group (P >0.05). The relative expression levels of Notch ligands (including DLL1 and Jagged1 ) mRNA and Notch downstream molecules (including Hes1, Hes5, and Hey1 ) were increased in IM group compared with control group (all P < 0.001). In IM group, the Th17 and Th22 percentage were 5.03%±1.15% and 4.48%±1.29%, respectively, which were both higher than 4.36%±0.82% and 3.83%±0.55% in control group (both P < 0.05). In IM group, the IL-17 and IL-22 level were (301.1±53.82) and (101.2±16.45) pg/ml, respectively, which were both higher than (237.2±72.18) and (84.75±11.83) pg/ml in control group (both P < 0.001). In IM group, the relative expression levels of RORγt and AhR mRNA were 1.25±0.22 and 1.21±0.12, respectively, which were both higher than 0.99±0.15 and 1.04±0.11 in control group (both P < 0.001). There were no remarkable differences in CD4+ T cell proliferation, Th17 percentage, IL-17 secretion, and relative expression level of RORγt mRNA between cells with GSI stimulation and without GSI stimulation (P >0.05). GSI stimulation reduced Th22 percentage, IL-22 secretion, and relative expression level of AhR mRNA compared with non-stimulation (all P < 0.05). Notch signaling pathway regulates IL-22 secretion by CD4+ T cells via AhR in IM patients. Notch-AhR-Th22 pathway may take part in the pathogenesis of IM.

MEOX2 Participates in Hepatic Stellate Cells-Induced Liver Fibrosis by Regulating the PI3K/AKT Signaling Pathway.

Hepatic stellate cells (HSCs) serve as the crucial accelerating factor in the progression of liver fibrosis (LF). In contrast to HSCs, adult-derived human liver stem/progenitor cells (ADHLSCs) exhibit greater potency in terms of differentiation and proliferation, rendering them highly applicable in LF treatment. The objective of this study is to identify new therapeutic targets for LF by comparing differentially expressed genes (DEGs) between ADHLSCs and HSCs. We investigated DEGs between ADHLSCs and HSCs using the GSE49995 dataset obtained from the Gene Expression Omnibus (GEO) database, aiming to identify new therapeutic targets for LF. Subsequently, we activated HSCs to delve deeper into the mesenchyme homeobox 2 (MEOX2), PH domain Leucine-rich repeat protein phosphatase (PHLPP), and Phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways in LF progression, employing platelet-derived growth factor (PDGF), and conducted infection with Overexpression (OE)-MEOX2 and shRNA-MEOX2 (sh-MEOX2) lentiviruses. Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay, while cell proliferation was evaluated through 5-ethynyl-2'-deoxyuridine (EdU) staining and flow cytometry. Relative mRNA expression levels were determined via qPCR. Western blot analysis was performed to measure protein expression levels, and the regulatory role of MEOX2 was investigated using dual luciferase reporter assays. We identified 332 DEGs that were down-regulated and 201 DEGs that were up-regulated between ADHLSCs and HSCs. Notably, MEOX2 expression in ADHLSCs was significantly reduced. These DEGs primarily participated in the collagen-containing extracellular matrix and the PI3K/AKT signaling pathway. MEOX2 could inhibit cancer cell proliferation via the PI3K/AKT signaling pathway. Additionally, the JASRPAR2022 database predicted the target gene PHLPP of MEOX2. Our results indicated that OE-MEOX2 significantly inhibited HSCs' cell vitality and proliferation. Further analysis revealed that MEOX2 binds to PHLPP promoters, thereby up-regulating its transcription. This action led to the inhibition of p-AKT expression, consequently reducing HSC proliferation and slowing the progression of LF. MEOX2 up-regulates PHLPP expression and inhibits AKT phosphorylation, thereby reducing the cell activity and proliferation ability of HSCs and inhibiting the progression of LF.

Effect of TLK2 Expression Regulated by MiR-21 on Proliferation and Apoptosis of Acute Myeloid Leukemia Cells

To investigate the effect of TLK2 expression regulated by miR-21 on proliferation and apoptosis of acute myeloid leukemia cells. Seventy patients with AML admitted to our hospital from January 2019 to July 2022 were selected, while 30 patients with iron deficiency anemia were selected as the control group. Bone marrow mononuclear cells (BMMNCs) of the patients were obtained using Ficoll density gradient centrifugation. RT-qPCR was used to determine the expression levels of miR-21 and TLK2 mRNA in BMMNCs. Mimics-miR-21, mimics-NC, inhibitor-miR-21, inhibitor-NC and NC were transfected into HL-60 cells using liposome-mediated transfection technology. CCK-8 method was used to determine the activity of transfected HL-60 cells after treatment with cytarabine. The apoptosis rate of HL-60 transfected cells was determined by TUNEL method. The expression of TLK2 mRNA in HL-60 cells transfected with inhibitor-miR-21 was determined by RT-qPCR. The relative expression levels of miR-21 and TLK2 mRNA in BMMNCs of AML patients were significantly higher than those of controls (both P < 0.05). After HL-60 cells were treated with cytarabine, both the cell activity of inhibitor-miR-21 group and mimics-miR-21 group decreased significantly with the increase of cytarabine concentration (both P < 0.05). However, at each concentration point of cytarabine, the cell activity of inhibitor-miR-21 group was lower than that of control group (P < 0.05), while mimics-miR-21 group was higher than control group (P < 0.05). After HL-60 cells were treated with cytarabine, the apoptosis rate of inhibitor-miR-21 group was significantly increased (P < 0.05), while that of mimics-miR-21 group was significantly decreased (P < 0.05). After HL-60 cells were treated with inhibitor-miR-21, the relative expression of TLK2 mRNA decreased significantly (P < 0.05). miR-21 is highly expressed in AML patients, which may promote the apoptosis of AML cells by inhibiting the expression of TLK2.

The role of MASP1 in the complement system and expression characteristics in response to GCRV infection in grass carp

The grass carp (Ctenopharyngodon idella) constitutes a significant economic resource within the aquaculture sector of our nation, yet it has been chronically afflicted by the Grass Carp Reovirus (GCRV) disease. The complement system, a vital component of fish's innate immunity, plays a crucial role in combating viral infections. This research investigates the potential role of MASP1, a key molecule in the lectin pathway of the complement system, in the GCRV infection in grass carp. An analysis of the molecular characteristics of MASP1 in grass carp revealed that its identity and similarity percentages range from 35.10 to 91.00 % and 35.30–91.00 %, respectively, in comparison to other species. Phylogenetically, MASP1 in C. idella aligns closely with species such as Danio rerio, Cyprinus carpio, and Carassius carassius, exhibiting chromosomal collinearity with the zebrafish. Subsequent tissue analysis in both healthy and GCRV-infected grass carp indicated that MASP1's basal expression was predominantly in the liver. Post-GCRV infection, MASP1 expression in various tissues exhibited temporal variations: peaking in the liver on day 5, spleen on day 7, and kidney on day 14. Furthermore, employing Complement Component 3 (C3) as a benchmark for complement system activation, it was observed that MASP1 could activate and cleave C3 to C3b. MASP1 also demonstrated an inhibitory effect on GCRV replication (compared with the control group, VP2 and VP7 decreased by 6.82-fold and 4.37-fold) and enhanced the expression of antiviral genes, namely IRF3, IRF7 and IFN1 (compared with the control group, increased 2.25-fold, 45.38-fold and 22.37-fold, respectively). In vivo protein injection experiments substantiated MASP1's influence on the relative mRNA expression levels of C3 in various tissues and its protein expression in serum. This study also verified that C3 could modulate the expression of antiviral genes such as IFN1 and IRF3.

Dietary supplementation with succinic acid improves growth performance and flesh quality of adult Nile tilapia (Oreochromis niloticus) fed a high-carbohydrate diet

To evaluate the effects of dietary supplementation with succinic acid on growth performance, flesh quality, glucose, and lipid metabolism of Nile tilapia (Oreochromis niloticus) fed a high-carbohydrate diet (HCD), five iso-nitrogenous and iso-lipidic diets were prepared as follows: HCD (control group) consisting of 55% corn starch and HCD supplemented with 0.5%, 1.0%, 2.0%, and 4.0% succinic acid, respectively. Tilapia with an initial body weight of 204.90 ± 1.23 g randomly assigned to 15 tanks with 3 replicates per group and 10 fish per tank fed for 8 weeks. Increasing dietary succinic acid supplementation resulted in significant second-order polynomial relationship in the weight gain rate (WGR), specific growth rate (SGR), feed conversion ratio (FCR), protein efficiency rate (PER), viscerosomatic index, condition factor, and contents of muscular crude lipid and glycogen (P < 0.05). The hepatosomatic index, mesenteric fat index, liver glycogen content and crude lipid contents of the whole-body and liver demonstrated significantly linear and second-order polynomial relationship (P < 0.05). Quadratic curve model analysis based on WGR, SGR, PER, and FCR demonstrated that optimal supplementation with succinic acid in the HCD of Nile tilapia ranged from 1.83% to 2.43%. Fish fed with 1.0% succinic acid had higher muscular hardness, increased the contents of alkali-soluble hydroxyproline in collagen, docosahexaenoic acid (DHA) and n-3 polyunsaturated fatty acid (n-3PUFA) in muscle, and lower total fatty acid content in muscle (P < 0.05) compared with the control group. Compared to the control group, dietary supplementation with 1.0% succinic acid significantly increased the contents of total bounding amino acid (arginine, histidine, isoleucine, lysine, methionine, alanine, proline), total flavor amino acid (free aspartic acid), the catalase (CAT) activity and total antioxidant capacity, and the mRNA relative expression levels of CAT, superoxide dismutase (SOD), and nuclearfactor erythroidderived 2-like 2 (Nrf2) in muscle (P < 0.05). Furthermore, succinic acid supplementation significantly up-regulated mRNA relative expression levels of glycolysis genes (hexokinase 2 [HK2], phosphofructokinase, muscle-A [PFKMA], and phosphofructokinase, muscle-B [PFKMB]), a key glycogen synthesis gene (glycogen synthase [GYS]), and lipid catabolism genes (carnitine palmitoyltransferase-1B [CPT1B], hormone sensitive lipase [HSL], and lipoprotein lipase [LPL]), while down-regulating the mRNA relative expression level of fatty acid synthase (FASN) in muscle (P < 0.05). In conclusion, dietary supplementation with 1.83% to 2.43% succinic acid improved muscle quality by increasing muscle antioxidant capacity and hardness, changing muscle amino acid and fatty acid composition, and regulating muscle glucose and lipid metabolism.

The regulatory effect of electroacupuncture on endometrial M1-type macrophages in rats with intrauterine adhesions.

To observe the effect of electroacupuncture(EA) on endometrial fibrosis and M1-type macrophages in rats with intrauterine adhesions(IUA), so as to explore the possible mechanism of EA in the treatment of IUA. Fifteen female SD rats were randomly divided into blank group, model group and EA group, with 5 rats in each group. The IUA rat model was established by double damage method using mechanical scraping combined with lipopolysaccharide infection. Rats in the EA group were treated with acupuncture at "Guanyuan"(CV4), and EA at bilateral "Zusanli"(ST36) and "Sanyinjiao"(SP6)for 20 minutes each time, once a day, for 3 consecutive cycles of estrus. Five rats in each group were sampled during the estrous period, and the endometrial morphology, endometrial thickness and the number of blood vessels and glands were observed after HE staining. The fibrotic area of the uterus was observed after Masson staining. The positive expressions of Runt-related transcription factor(RUNX1), transforming growth factor-β1(TGF-β1), connective tissue growth factor(CTGF), α-smooth muscle actin(α-SMA), collagen type I(Col-Ⅰ), cluster of differentiation 86(CD86), interleukin-1β(IL-1β), and tumor necrosis factor-α(TNF-α) in endometrial tissue were detected by immunohistochemistry. Western blot was used to detect relative protein expressions of RUNX1, TGF-β1, α-SMA, CD86, and TNF receptor 2 (TNFR2), and real-time fluorescence quantitative PCR was used to detect mRNA expressions of RUNX1, TGF-β1, α-SMA, CD86, and TNF-α in the endometrium. During the estrous phase, the endometrial layer in the model group was damaged, with reduced folds, disordered arrangement of epithelial cells, loose fibrous connective tissue, significant narrowing and adhesions in the uterine cavity, interstitial congestion, edema, and a significant infiltration of inflammatory cells with sparse glands. While uterine tissue structure of the EA group was basically intact, resembling a normal uterus, with more newly formed glands and a small amount of inflammatory cell infiltration. In comparison with the blank group, the endometrial thickness, the number of blood vessels, and the number of glands were significantly decreased(P<0.001) in the model group, while the ratio of uterine fibrosis area, the positive expressions of RUNX1, TGF-β1, CTGF, α-SMA, Col-Ⅰ, CD86, IL-1β, and TNF-α, the protein relative expressions of RUNX1, TGF-β1, α-SMA, CD86 and TNFR2, and the mRNA relative expression levels of RUNX1, TGF-β1, α-SMA, CD86 and TNF-α in the endometrium were significantly increased (P<0.001, P<0.01). Compared to the model group, the endometrial thickness, the number of blood vessels, and the number of glands were significantly increased(P<0.01, P<0.05) in the EA group, while the ratio of uterine fibrosis area, the positive expressions of RUNX1, TGF-β1, CTGF, α-SMA, Col-Ⅰ, CD86, IL-1β and TNF-α in the endometrial tissue, the protein expressions of RUNX1, TGF-β1, α-SMA, CD86 and TNFR2, and the mRNA relative expressions of RUNX1, TGF-β1, α-SMA, CD86 and TNF-α in the endometrium were significantly decreased (P<0.001, P<0.01, P<0.05). EA can improve endometrial fibrosis in IUA rats, which may be related to its function in decreasing the level of endometrial M1-type macrophages and the secretion of related inflammatory factors.

Prediction of response to neoadjuvant chemotherapy by MammaTyper® across breast cancer subtypes: A retrospective cross-sectional study

BackgroundNeoadjuvant chemotherapy (NACT) is widely used in the treatment of triple-negative and HER2-positive breast cancer (BC), but its use in estrogen receptor (ER) and/or progesterone receptor (PR) positive/HER2-negative BC is questioned because of the low pathologic complete response (pCR) rates. This retrospective study assessed the mRNA-based MammaTyper® assay's capability of predicting pCR with NACT, and ER, PR, Ki67, and HER2 status at immunohistochemical (IHC) through transcriptomics. MethodsDiagnostic biopsies from 76 BC patients treated at the Cremona Hospital between 2012-2018 were analyzed. Relative mRNA expression levels of ERBB2, ESR1, PGR, and MKI67 were measured using the MammaTyper® kit and integrated into a pCR score. Predicting capability of pCR and standard IHC biomarkers could be assessed with ROC curves in 75 and 76 patients, respectively. ResultsOverall, 68.0% patients obtained a MammaTyper® high-score and 32.0% a MammaTyper® low-score. Among high-score patients, 62.7% achieved pCR, compared to 16.7% in the low-score group (p = 0.0003). The binary MammaTyper® score showed good prediction of pCR in the overall cohort (area under curve [AUC] = 0.756) and in HR+/HER2-negative cases (AUC = 0.774). In cases with residual disease, the continuous MammaTyper® score correlated moderately with residual tumor size and decrease in tumor size. MammaTyper® showed substantial agreement with IHC for ESR1/ER and ERBB2/HER2, and moderate agreement for PGR/PR and MKI67/Ki67. ConclusionOverall, MammaTyper® pCR score may serve as a standardized tool for predicting NACT response in HR+/HER2-negative BC, potentially guiding treatment strategies. Additionally, it could provide a more standardized and reproducible assessment of ER, PR, HER2, and Ki67 status.

De novo variation in ARID1B gene causes Coffin-Siris syndrome 1 in a Chinese family with excessive early-onset high myopia

Coffin-Siris syndrome (CSS) is a rare autosomal dominant inheritance disorder characterized by distinctive facial features, hypoplasia of the distal phalanx or nail of the fifth and additional digits, developmental or cognitive delay of varying degree, hypotonia, hirsutism/hypertrichosis, sparse scalp hair and varying kind of congenital anomalies. CSS can easily be misdiagnosed as other syndromes or disorders with a similar clinical picture because of their genetic and phenotypic heterogeneity. We describde the genotype-phenotype correlation of one patient from a healthy Chinese family with a novel genotype underlying CSS, who was first diagnosed in the ophthalmology department as early-onset high myopia (eoHM). Comprehensive ophthalmic tests as well as other systemic examinations were performed on participants to confirm the phenotype. The genotype was identified using whole exome sequencing, and further verified the results among other family members by Sanger sequencing. Real-time quantitative PCR (RT-qPCR) technology was used to detect the relative mRNA expression levels of candidate genes between proband and normal family members. The pathogenicity of the identified variant was determined by The American College of Medical Genetics and Genomics (ACMG) guidelines. STRING protein-protein interactions (PPIs) network analysis was used to detect the interaction of candidate gene-related proteins with high myopia gene-related proteins. The patient had excessive eoHM, cone-rod dystrophy, coarse face, excessive hair growth on the face, sparse scalp hair, developmental delay, intellectual disability, moderate hearing loss, dental hypoplasia, patent foramen ovale, chronic non-atrophic gastritis, bilateral renal cysts, cisterna magna, and emotional outbursts with aggression. The genetic assessment revealed that the patient carries a de novo heterozygous frameshift insertion variant in the ARID1B c.3981dup (p.Glu1328ArgfsTer5), which are strongly associated with the typical clinical features of CSS patients. The test results of RT-qPCR showed that mRNA expression of the ARID1B gene in the proband was approximately 30% lower than that of the normal control in the family, suggesting that the variant had an impact on the gene function at the level of mRNA expression. The variant was pathogenic as assessed by ACMG guidelines. Analysis of protein interactions in the STRING online database revealed that the ARID1A protein interacts with the high myopia gene-related proteins FGFR3, ASXL1, ERBB3, and SOX4, whereas the ARID1A protein antagonizes the ARID1B protein. Therefore, in this paper, we are the first to report a de novo heterozygous frameshift insertion variant in the ARID1B gene causing CSS with excessive eoHM. Our study extends the genotypic and phenotypic spectrums for ARID1B-CSS and supplies evidence of significant association of eoHM with variant in ARID1B gene. As CSS has high genetic and phenotypic heterogeneity, our findings highlight the importance of molecular genetic testing and an interdisciplinary clinical diagnostic workup to avoid misdiagnosis as some disorders with similar manifestations of CSS.

Codonopsis pilosula-derived glycopeptide dCP1 promotes the polarization of tumor-associated macrophage from M2-like to M1 phenotype

The majority of the immune cell population in the tumor microenvironment (TME) consists of tumor-associated macrophages (TAM), which are the main players in coordinating tumor-associated inflammation. TAM has a high plasticity and is divided into two main phenotypes, pro-inflammatory M1 type and anti-inflammatory M2 type, with tumor-suppressive and tumor-promoting functions, respectively. Considering the beneficial effects of M1 macrophages for anti-tumor and the high plasticity of macrophages, the conversion of M2 TAM to M1 TAM is feasible and positive for tumor treatment. This study sought to evaluate whether the glycopeptide derived from simulated digested Codonopsis pilosula extracts could regulate the polarization of M2-like TAM toward the M1 phenotype and the potential regulatory mechanisms. The results showed that after glycopeptide dCP1 treatment, the mRNA relative expression levels of some M2 phenotype marker genes in M2-like TAM in simulated TME were reduced, and the relative expression levels of M1 phenotype marker genes and inflammatory factor genes were increased. Analysis of RNA-Seq of M2-like TAM after glycopeptide dCP1 intervention showed that the gene sets such as glycolysis, which is associated with macrophage polarization in the M1 phenotype, were significantly up-regulated, whereas those of gene sets such as IL-6-JAK-STAT3 pathway, which is associated with polarization in the M2 phenotype, were significantly down-regulated. Moreover, PCA analysis and Pearson's correlation also indicated that M2-like TAM polarized toward the M1 phenotype at the transcriptional level after treatment with the glycopeptide dCP1. Lipid metabolomics was used to further explore the efficacy of the glycopeptide dCP1 in regulating the polarization of M2-like TAM to the M1 phenotype. It was found that the lipid metabolite profiles in dCP1-treated M2-like TAM showed M1 phenotype macrophage lipid metabolism profiles compared with blank M2-like TAM. Analysis of the key differential lipid metabolites revealed that the interconversion between phosphatidylcholine (PC) and diacylglycerol (DG) metabolites may be the central reaction of the glycopeptide dCP1 in regulating the conversion of M2-like TAM to the M1 phenotype. The above results suggest that the glycopeptide dCP1 has the efficacy to regulate the polarization of M2-like TAM to M1 phenotype in simulated TME.

Integrated multiple-omics reveals the regulatory mechanism underlying the effects of homologous Bacillus tequilensis (GCB-3) on growth performance of grass carp (Ctenopharyngodon idellus)

The purpose of this study was to investigate the regulatory mechanism of Bacillus tequilensis (GCB-3) in improving the growth performance of grass carp by microbiome and targeted metabolomics analysis. Fish were fed with different amounts of GCB-3 including B0 (0 CFU/g), B6 (1×106 CFU/g), B7 (1×107 CFU/g), B8 (1×108 CFU/g), and B9 (1×109 CFU/g) for 8 weeks. The results indicate that with the increase of dietary GCB-3 concentration, grass carp showed a significant linear trend in growth performance, and appetite factors showed a significant linear and quadratic trend, the growth performance and appetite-promoting factors in the B8 group reach their maximum value in the B8 group. Microbiome analysis found that Proteobacteria and Actinobacteria were the most abundant phylum in the gut microbiota of grass carp. The functional potential predicted that GCB-3 affected the synthesis and degradation of carbohydrates, amino acids, fatty acids and lipids. It found that GCB-3 also affected the tricarboxylic acid (TCA) cycle VII (acetate-producers) pathway. The results of targeted metabolomics showed a significant linear and quadratic increase in intestinal acetate with GCB-3 concentration and a maximum in the B8 group. The results of the correlation analysis showed a significant positive correlation between Actinobacteria abundance and acetic acid content, and also between TCA cycle VII (acetate-producers) pathway abundance and Actinobacteria abundance and acetic acid content. In B8 group, grass carp showed a faster regulation speed of blood glucose levels after glucose injection, and the GLP-1 content remained stable. The activities of glycolytic enzymes, lipolytic enzymes, protein metabolism enzymes, and their related mRNA expression levels in grass carp showed a linear or quadratic increasing trend. However, the expression levels of gluconeogenic enzymes and fatty acid synthesis enzyme related mRNA showed an opposite trend. In summary, the addition of 1×108 CFU/g GCB-3 in grass carp feed can improve the abundance of dominant bacterial phyla in the intestine, especially Actinobacteria, increase the concentration of intestinal acetate, provide substrate for the TCA cycle VII (acetate-producers) pathway, thereby regulating the nutrient metabolism of grass carp and ultimately promoting growth.

Transcriptomics reveals age-related changes in ion transport-related factors in yak lungs.

Yaks inhabit high-altitude, low-oxygen regions, where ion transport functions play a crucial role in maintaining intracellular and extracellular ionic balance and regulating pulmonary vascular tension. These functions affect pulmonary ventilation and blood flow rate, aiding tissue development and enhancing oxygen transfer efficiency, thus facilitating better adaptation to hypoxic environments. To investigate the regulatory mechanisms of ion transport-related factors on the growth and development of yak lungs, we employed RNA sequencing (RNA-seq)for sequencing the transcriptome in the lung tissues of neonatal (1-day-old), juvenile (1-year-old), and adult (4-year-old) yaks. We also performed differential gene expression and functional analyses. The results yielded 26 genes associated with ion transport, mainly enriched in the salivary and pancreatic secretion pathways. Finally, we used several methods including quantitative polymerase chain reaction (qRT-PCR), and Western blotting (WB), immunohistochemical (IHC) and immunofluorescence (IF) staining to determine the distribution of the expression of the ion transport genes FOXI1, KCNMA1, and SLC12A2 in yak lung tissues. qRT-PCR and WB results indicated that mRNA and protein relative expression levels of FOXI1 and SLC12A2 were significantly higher in neonatal yaks than in juvenile and adult yaks (all p < 0.05), whereas those of KCNMA1 were significantly higher in adult yaks than in neonatal and juvenile yaks (all p < 0.05). IHC and IF results demonstrated that FOXI1, KCNMA1, and SLC12A2 were distributed among the epithelial mucosal layers (including ciliated, goblet, and Clara cells) of the yaks' bronchi and their branches in the lungs across different age groups of yak. Therefore, our results suggested that FOXI1, KCNMA1, and SLC12A2 may be strongly associated with the development and aging processes in yak lungs. These results provide insights into the molecular mechanisms underlying the yak's adaptation to high-altitude environments and valuable references for further research.