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We investigated the impact of tumor burden on muscle wasting in metastatic (m) and xenograft (x) models of colorectal cancer (CRC). Male Nod SCID γ and CD2F1 mice were injected subcutaneously or intrasplenically with HCT116 or C26 tumor cells, respectively. CRC tumors resulted in significant muscle wasting regardless of tumor type or model, although muscle loss was exacerbated in mHCT116 hosts. The mHCT116 model decreased ribosomal (r)RNA content and rDNA transcription, whereas the mC26 model showed no loss of rRNA and the upregulation of rDNA transcription. The xHCT116 model reduced mTOR, RPS6, and 4E-BP1 phosphorylation, whereas the mHCT116 model had a similar effect on RPS6 and 4E-BP1 without altering mTOR phosphorylation. The C26 models caused a reduction in 4E-BP1 phosphorylation independent of mTOR. Muscle interleukin (IL)-6 mRNA was elevated in all models except xHCT116, and the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) mRNA was induced only in the mC26 model. IL-1β mRNA increased in all groups with greater expression in metastatic relative to the xenograft model regardless of tumor types. Our findings indicate that HCT116 tumor burden results in more drastic muscle wasting and anabolic deficits, whereas C26 tumor burden causes similar muscle wasting but exhibits a divergent proinflammatory phenotype. These results highlight potentially important divergence in the pathogenesis of muscle wasting among preclinical models of CRC and demonstrate that tumor burden plays a role in determining anabolic deficits and the expression of proinflammatory effectors of muscle wasting in a tumor-type-dependent manner.

Background:The harmful vascular effects of smoking are well established, but the effects of chronic use of electronic cigarettes (e-cigarettes) on endothelial function are less understood. We hypothesized that e-cigarette use causes changes in blood milieu that impair endothelial function.Methods:Endothelial function was measured in chronic e-cigarette users, chronic cigarette smokers, and nonusers. We measured effects of participants’ sera, or e-cigarette aerosol condensate, on NO and H2O2release and cell permeability in cultured endothelial cells (ECs).Results:E-cigarette users and smokers had lower flow-mediated dilation (FMD) than nonusers. Sera from e-cigarette users and smokers reduced VEGF (vascular endothelial growth factor)-induced NO secretion by ECs relative to nonuser sera, without significant reduction in endothelial NO synthase mRNA or protein levels. E-cigarette user sera caused increased endothelial release of H2O2, and more permeability than nonuser sera. E-cigarette users and smokers exhibited changes in circulating biomarkers of inflammation, thrombosis, and cell adhesion relative to nonusers, but with distinct profiles. E-cigarette user sera had higher concentrations of the receptor for advanced glycation end products (RAGE) ligands S100A8 and HMGB1 (high mobility group box 1) than smoker and nonuser sera, and receptor for advanced glycation end product inhibition reduced permeability induced by e-cigarette user sera but did not affect NO production.Conclusions:Chronic vaping and smoking both impair FMD and cause changes in the blood that inhibit endothelial NO release. Vaping, but not smoking, causes changes in the blood that increase microvascular endothelial permeability and may have a vaping-specific effect on intracellular oxidative state. Our results suggest a role for RAGE in e-cigarette-induced changes in endothelial function.

Protein kinase C-α (PKCα) plays a major role in a diverse range of cellular processes. Studies to date have defined the regulatory controls and function of PKCα entirely based upon the previously annotated ubiquitously expressed prototypical isoform. From RNA-seq-based transcriptome analysis in murine heart, we identified a previously unannotated PKCα variant produced by alternative RNA splicing. This PKCα transcript variant, which we named PKCα-novel exon (PKCα-NE), contains an extra exon between exon 16 and exon 17, and is specifically detected in adult mouse cardiac and skeletal muscle, but not other tissues; it is also detected in human hearts. This transcript variant yields a PKCα isoform with additional 16 amino acids inserted in its COOH-terminal variable region. Although the canonical PKCα enzyme is a lipid-dependent kinase, in vitro kinase assays show that PKCα-NE displays a high level of basal lipid-independent catalytic activity. Our unbiased proteomic analysis identified a specific interaction between PKCα-NE and eukaryotic elongation factor-1α (eEF1A1). Studies in cardiomyocytes link PKCα-NE expression to an increase in eEF1A1 phosphorylation and elevated protein synthesis. In summary, we have identified a previously uncharacterized muscle-specific PKCα splicing variant, PKCα-NE, with distinct biochemical properties that plays a unique role in the control of the protein synthesis machinery in cardiomyocytes.

We investigated differentially methylated and expressed genes between panic disorder (PD) and healthy controls (HCs) to determine whether DNA methylation and expression level of candidate genes can be used as biomarkers for diagnosis and early response.Illumina infiniun Methylation EPIC (850 k) Beadchip for genome-wide methylation screening and mRNA sequencing was conducted in a discovery set (30 patients with PD and 30 matched HCs). The candidate gene loci methylation and expression were verified in an independent validation sample (101 PD patients and 107 HCs).In the discovery set, there were 3613 differentially methylated cytosine phosphate guanosine sites and these differential methylation positions were located within 1938 unique genes, including 1758 hypermethylated genes, 150 hypomethylated genes, and the coexistence of hypermethylation and hypomethylation sites were found in 30 genes. There were 1111 differential transcripts in PD compared to normal controls (850 down-regulated and 261 up-regulated). Further, 212 differentially expressed genes were screened (40 up-regulated and 172 down-regulated). In the validation set, compared with HCs, there was no significant difference in DNA methylation level of Casitas B-lineage lymphoma (CBL) gene loci (cg07123846). The expression level of CBL gene in PD patients was lower (vs. HCs). After four weeks' treatment, the baseline expression level of CBL gene in the responders was higher than nonresponders.The sample size was limited. We only chose CBL as a candidate gene. Follow-up periods were short.There are differences in genome-wide DNA methylation and mRNA expression between PD patients and HCs. The changes in expression level of CBL gene may be an important molecular marker for PD diagnosis and early response.

Studies combining data from digital surveys and electronic health records (EHR) can be used to conduct comprehensive assessments on COVID-19 vaccine safety.We conducted an observational study using data from a digital survey and EHR of children aged 5-11 years vaccinated with Pfizer-BioNTech COVID-19 mRNA vaccine across Kaiser Permanente Southern California during November 4, 2021-February 28, 2022. Parents/guardians who enrolled their children were sent a 14-day survey on reactions. Survey results were combined with EHR, and medical encounters were described for children whose parents or guardians indicated seeking medical care for vaccine-related symptoms. This study describes self-reported reactions (local and systemic) and additional symptoms (chest pain, tachycardia, and pre-syncope).The study recruited 7,077 participants aged 5-11 years who received the Pfizer-BioNTech COVID-19 mRNA vaccine. Of 6,247 participants with survey responses after dose 1, 2,176 (35 %) reported at least one systemic reaction, and 1,076 (32 %) of 3,401 respondents following dose 2 reported at least one systemic reaction. Local reactions were reported less frequently following dose 2 (1,113, 33 %) than dose 1 (3,140, 50 %). The most frequently reported reactions after dose 1 were pain at the injection site (48 %), fatigue (20 %), headache (12 %), myalgia (9 %) and fever (5 %). The most frequently reported symptoms after dose 2 were also pain at the injection site (30 %), fatigue (19 %), headache (13 %), myalgia (10 %) and fever (9 %). Post-vaccination reactions occurred most frequently-one day following vaccination. Chest pain or tachycardia were reported infrequently (1 %). EHR demonstrated that parents rarely sought care for post-vaccination symptoms, and among those seeking care, the most common symptoms documented in EHR were fever and nausea, comprising0.5 % of children. No encounters were related to myocarditis.While post-vaccination reactions to the Pfizer-BioNTech COVID-19 mRNA vaccine were common in children aged 5-11 years, our data showed that in most cases they were transient and did not require medical care.

Adrenocortical carcinoma is a rare endocrine cancer with poor overall survival. Linking survival outcomes to a common target across multiple genomic datasets incorporating microRNA-long non-coding RNA dysregulation have not been well described. We hypothesized that a multi-database analysis of microRNA-long noncoding RNA-messenger RNA regulatory networks associated with survival will identify novel biomarkers.Significantly dysregulated genes or microRNA in adrenocortical carcinoma compared to normal adrenal was identified from sequencing data for 260 human adrenocortical carcinomas using GEO2R. The miRnet identified hub microRNA and genes and long noncoding RNA and microRNA associated with survival genes. The R2 generated Kaplan-Meier curves. The database miRTarBase linked genes associated with poor survival and dysregulated microRNA.Analysis of genes and microRNAs differentially regulated in50% of datasets revealed 75 genes and 12 microRNAs were upregulated, and 167 genes and 12 microRNAs were downregulated (bonf. P.05). Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed cell cycle, P53 signaling, arachidonic acid and innate immune response, and PI3/Akt are altered in adrenocortical carcinoma. A microRNA-target interaction network of differentially regulated microRNAs identified upregulated miRNA107, 103a-3p and 27a-3p, 16-5p, and downregulated 335-5p to have the highest degree of interaction with upregulated (ie, TPX2, CDK1, BIRC5, PRC1, CCNB1, GINS1) and downregulated (ie, RSPO3, NR2F1, TLR4, HOXA5, USP53, SLC16A9) hub genes as well as hub long noncoding RNAs XIST, NEAT1, KCNQ1OT1, and PAX8-AS1. Survival analysis revealed that the hub genes are associated with poor overall survival (P.05) of adrenocortical carcinoma in the Cancer Genome Atlas data.A messenger RNA-microRNA-long noncoding RNA network analysis identified the BIRC5-miR335-5p-PAX8-AS1 network as one that was associated with poor overall survival in adrenocortical carcinoma, warranting further validation as a potential therapeutic target.

Acute exacerbations due to COVID-19 vaccination in patients with interstitial lung disease (ILD) have been reported, but their incidence is unknown. We investigated the incidence of exacerbations of ILD and respiratory symptoms due to the mRNA COVID-19 vaccines. A questionnaire survey was conducted on adverse reactions to the mRNA COVID-19 vaccination in 545 patients with ILD attending our hospital and retrospectively examined whether the eligible patients actually developed acute exacerbations of ILD induced by the vaccine. Of the 545 patients, 17 (3.1%) patients were aware of the exacerbation of respiratory symptoms, and four (0.7%) patients developed an acute ILD exacerbation after vaccination. Of the four patients who experienced exacerbations, two had collagen vascular disease-associated ILD, one had nonspecific interstitial pneumonia, another had unclassifiable idiopathic pneumonia, and none had idiopathic pulmonary fibrosis. Four patients were treated using steroid pulse therapy with a steroid taper, and two of the four also received intravenous cyclophosphamide pulse therapy. Tacrolimus was started in one patient with myositis-associated interstitial lung disease. Eventually, all patients exhibited improvement with immunosuppressive treatment and were discharged. COVID-19 vaccination for patients with ILD should be noted for developing acute exacerbations of ILD with low incidence, although manageable with early diagnosis and treatment.

Human papillomavirus (HPV) infection is a causative agent of multiple human cancers, including cervical and head and neck cancers. In these HPV-positive tumors, somatic mutations are caused by aberrant activation of DNA mutators such as members of the apolipoprotein B messenger RNA–editing enzyme catalytic polypeptide-like 3 (APOBEC3) family of cytidine deaminases. APOBEC3 proteins are most notable for their restriction of various viruses, including anti-HPV activity. However, the potential role of APOBEC3 proteins in HPV-induced cancer progression has recently garnered significant attention. Ongoing research stems from the observations that elevated APOBEC3 expression is driven by HPV oncogene expression and that APOBEC3 activity is likely a significant contributor to somatic mutagenesis in HPV-positive cancers. This review focuses on recent advances in the study of APOBEC3 proteins and their roles in HPV infection and HPV-driven oncogenesis. Further, we discuss critical gaps and unanswered questions in our understanding of APOBEC3 in virus-associated cancers.

This study compares gene expression in mucosa of the terminal ileum, right colon, and left colon in patients with diarrhea- or constipation-predominant irritable bowel syndrome (IBS) and contrasts expression between these two disease entities and also between each entity and mucosa from healthy controls. The study shows there is differential expression of genes related to immune, transmitter, nociceptive, ion channel, and transporter functions, as well as reduced serine protease inhibition, in patients with IBS.

OBJECTIVE: To establish a liquid-biopsy assay to predict response to neoadjuvant therapy (NAT) in esophageal squamous cell carcinoma (ESCC) patients. SUMMARY BACKGROUND DATA: Pretreatment prediction of resistance to NAT is of great significance for the selection of treatment options in ESCC patients. In this study, we comprehensively translated tissue-based microRNA (miRNA) and messenger RNA (mRNA) expression biomarkers into a liquid biopsy assay. METHODS: We analyzed 186 clinical ESCC samples, which included 128 formalin-fixed paraffin-embedded and a matched subset of 58 serum samples, from 2 independent institutions. We performed quantitative reverse-transcription polymerase chain reaction, and developed a resistance-prediction model using the logistic regression analyses. RESULTS: We first evaluated the potential of 4-miRNAs and 3-mRNAs panel, which robustly predicted resistance to NAT (area under the curve [AUC]: 0.85). Moreover, addition of tumor size to this panel increased predictive potential to establish a combination signature (AUC: 0.92). We successfully validated this signature performance in independent cohort, and our model was more accurate when the signature was combined with clinical predictors (AUC: 0.81) to establish a NAT resistance risk (NATRR) model. Finally, we successfully translated our NATRR model into a liquid biopsy assay (AUC: 0.78), and a multivariate regression analysis revealed this model as an independent predictor for response to NAT (odds ratio: 6.10; P < 0.01). CONCLUSIONS: We successfully developed a liquid biopsy-based assay that allows robust prediction of response to NAT in ESCC patients, and our assay provides fundamentals of developing precision-medicine.

Background/Aim: Several cases of concurrent reduction of expression of polycystin 1 (PKD1) and Tuberous Sclerosis Complex 2 (TSC2) that are contiguous in chromosome 16p13 have been previously reported. This study newly addresses the concurrent reduction of expression of PKD1, TSC2 and NTHL1, which is adjacent to TSC2 and is a tumor suppressor gene. Materials and Methods: We investigated the mRNA expression levels of PKD1, TSC2, PKD2, TSC1 and NTHL1 in blood and renal cell carcinoma (RCC) tissues in a proband with autosomal dominant polycystic kidney disease (ADPKD), tuberous sclerosis complex (TSC) and multiple pathologically diverse RCCs, including clear cell, papillary and chromophobe types. Additionally, we investigated germline variants in blood using whole exome sequencing (WES) in the proband and her four siblings. Results: mRNA expression levels of PKD1, TSC2 and NTHL1 were reduced in the proband’s blood and RCCs, compared with control groups. WES identified one novel variant with amino acid changes in the PKD1 exon in the three subjects with ADPKD, including the proband. Moreover, two variants in the TSC2 intron specific to the proband were also identified. Conclusion: In this study, we report a novel pathogenic variant in the PKD1 exon which likely led to ADPKD, and two variants in the TSC2 intron, which might have led to reduction in the expression of both TSC2 and NTHL1, consequently leading to TSC and multiple pathologically diverse RCCs.

Malignant melanoma is the most aggressive form of skin cancer, and it is characterized by poor prognosis in patients with metastatic diseases. Accurate prediction of prognosis is crucial for therapeutic decisions. In this study, bioinformatics analysis was used to explore the prognostic value of growth factor receptor-bound protein 2-associated binding protein 3 (GAB3) mRNA. RNA transcriptome sequencing data and clinical data from The Cancer Genome Atlas and genotype-tissue expression (GTEx) were analyzed for differentially expressed genes in high and low GAB3 mRNA expression groups in melanoma. Performing gene enrichment analysis and constructing protein-protein interaction networks. High expression of GAB3 was significantly correlated with a lower T stage, melanoma Clark level, Breslow depth, and melanoma ulceration. And high GAB3 expression was also associated with better progression-free interval in T1 and T2 stages and N0 stage and longer overall survival in T1 and T2 stages, N0 stage, and N1 stage. GAB3 promoted high levels of infiltration of macrophages and activated natural killer cells in melanoma. High expression of GAB3 predicted a positive prognosis in early-stage melanoma that may be mediated by the anticancer immune response.

Aicardi Goutières Syndrome (AGS) is an autoinflammatory disorder resulting in sustained interferon activation through defects in nucleic acid modification and sensing pathways. Thus, mRNA-based vaccination used against SARS-CoV-2, raise disease-specific safety concerns. To assess interferon signaling, we tested mRNA SARS-CoV-2 vaccines in AGS whole blood samples. Interferon activation is measured through quantitation of interferon signaling gene (ISG) expression and is increased in AGS patients. There was no increase in ISG scores from baseline following treatment with the nucleoside modified mRNA formulation compared to an increase with unmodified. A patient-family survey reported that the vaccines were well tolerated. These findings suggest that COVID vaccination using nucleoside-modified forms of mRNA vaccines are unlikely to directly stimulate ISG expression in response to mRNA internalization in AGS tissues. With continued community spread, we recommend vaccination using nucleoside-modified mRNA vaccines in this rare disease group in individuals for whom vaccines were previously well tolerated.

We present proximity sequencing (Prox-seq) for simultaneous measurement of proteins, protein complexes and mRNAs in thousands of single cells. Prox-seq combines proximity ligation assay with single-cell sequencing to measure proteins and their complexes from all pairwise combinations of targeted proteins, providing quadratically scaled multiplexing. We validate Prox-seq and analyze a mixture of T cells and B cells to show that it accurately identifies these cell types and detects well-known protein complexes. Next, by studying human peripheral blood mononuclear cells, we discover that naïve CD8

Background: Barbaloin, found in Aloe vera, exerts broad pharmacological activities, including antioxidant, anti-inflammatory, and anti-cancer. This study aims to investigate the effects of barbaloin on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). Methods: Osteogenic induction of hBMSCs was performed in the presence or absence of barbaloin. Cell viability was determined by using the CCK-8 assay. The characteristic of hBMSCs was identified by using flow cytometry. Intracellular alkaline phosphatase (ALP) staining was performed to evaluate the ALP activity in hBMSCs. Alizarin Red S staining was performed to evaluate the matrix mineralization. The mRNA and protein levels of target genes were determined using qRT-PCR and western blotting, respectively. Results: Treatment with barbaloin (10 and 20 μg/mL) significantly increased cell viability of hBMSCs after 72 hours. In addition, treatment with barbaloin increased the mRNA expression levels of ALP and its activities. Treatment with barbaloin also increased matrix mineralization and the mRNA and protein levels of late-differentiated osteoblast marker genes BMP2, RUNX2, and SP7 in hBMSCs. The underlying mechanisms revealed that barbaloin increased the protein expressions of Wnt/β-catenin pathway-related biomarkers. Conclusion: Barbaloin promotes osteogenic differentiation of hBMSCs by the regulation of the Wnt/β-catenin signaling pathway.

BackgroundUnderstanding protection conferred by natural SARS-CoV-2 infection versus COVID-19 vaccination is important for informing vaccine mandate decisions. We compared protection conferred by natural infection versus that from the BNT162b2 (Pfizer–BioNTech) and mRNA-1273 (Moderna) vaccines in Qatar. MethodsWe conducted two matched retrospective cohort studies that emulated target trials. Data were obtained from the national federated databases for COVID-19 vaccination, SARS-CoV-2 testing, and COVID-19-related hospitalisation and death between Feb 28, 2020 (pandemic onset in Qatar) and May 12, 2022. We matched individuals with a documented primary infection and no vaccination record (natural infection cohort) with individuals who had received two doses (primary series) of the same vaccine (BNT162b2-vaccinated or mRNA-1273-vaccinated cohorts) at the start of follow-up (90 days after the primary infection). Individuals were exact matched (1:1) by sex, 10-year age group, nationality, comorbidity count, and timing of primary infection or first-dose vaccination. Incidence of SARS-CoV-2 infection and COVID-19-related hospitalisation and death in the natural infection cohorts was compared with incidence in the vaccinated cohorts, using Cox proportional hazards regression models with adjustment for matching factors. FindingsBetween Jan 5, 2021 (date of second-dose vaccine roll-out) and May 12, 2022, 104 500 individuals vaccinated with BNT162b2 and 61 955 individuals vaccinated with mRNA-1273 were matched to unvaccinated individuals with a documented primary infection. During follow-up, 7123 SARS-CoV-2 infections were recorded in the BNT162b2-vaccinated cohort and 3583 reinfections were recorded in the matched natural infection cohort. 4282 SARS-CoV-2 infections were recorded in the mRNA-1273-vaccinated cohort and 2301 reinfections were recorded in the matched natural infection cohort. The overall adjusted hazard ratio (HR) for SARS-CoV-2 infection was 0·47 (95% CI 0·45–0·48) after previous natural infection versus BNT162b2 vaccination, and 0·51 (0·49–0·54) after previous natural infection versus mRNA-1273 vaccination. The overall adjusted HR for severe (acute care hospitalisations), critical (intensive care unit hospitalisations), or fatal COVID-19 cases was 0·24 (0·08–0·72) after previous natural infection versus BNT162b2 vaccination, and 0·24 (0·05–1·19) after previous natural infection versus mRNA-1273 vaccination. Severe, critical, or fatal COVID-19 was rare in both the natural infection and vaccinated cohorts. InterpretationPrevious natural infection was associated with lower incidence of SARS-CoV-2 infection, regardless of the variant, than mRNA primary-series vaccination. Vaccination remains the safest and most optimal tool for protecting against infection and COVID-19-related hospitalisation and death, irrespective of previous infection status. FundingThe Biomedical Research Program and the Biostatistics, Epidemiology, and Biomathematics Research Core, Weill Cornell Medicine-Qatar; Qatar Ministry of Public Health; Hamad Medical Corporation; Sidra Medicine; Qatar Genome Programme; and Qatar University Biomedical Research Center. The Biomedical Research Program and the Biostatistics, Epidemiology, and Biomathematics Research Core, Weill Cornell Medicine-Qatar; Qatar Ministry of Public Health; Hamad Medical Corporation; Sidra Medicine; Qatar Genome Programme; and Qatar University Biomedical Research Center.

Background: Colon cancer is a gastrointestinal malignancy with high incidence and poor prognosis. Objective: Saikosaponin B4 (SSB4) is a monomeric component of the Traditional Chinese medicine (TCM), Bupleurum. The current study investigates the therapeutic effect and mechanisms of SSB4 in colon cancer. Methods: The proliferation of two colon cancer cell lines, SW480 and SW620, were assessed using CCK8 and expression of regulatory molecules, including Bax, Caspase3, Caspase9, Cleaved Caspase3, Cleaved Caspase9 and Bcl2 by flow cytometry and Western blotting. Results: Survival rates, assessed by CCK8, of SW480 and SW620 cells decreased significantly when the SSB4 concentration was in the range 12.5–50 μg/ml. Flow cytometry measurements indicated apoptosis rates of 55.07% ± 1.63% for SW480 cells and 33.07% ± 1.28% for SW620 cells treated with 25 μg/ml SSB4. Western blotting revealed upregulation of the proapoptotic proteins, Bax, Caspase3, Caspase9, Cleaved Caspase3 and Cleaved Caspase9, and downregulation of the anti-apoptotic protein, Bcl2, in the presence of SSB4. Network pharmacology and molecular docking predicted that the PI3K/Akt/mTOR pathway might be the main regulatory target for the antitumor effect of SSB4. Further Western blotting experiments showed that SSB4 downregulated (p < 0.01) expression of PI3K, Akt, mTOR and the phosphorylated proteins, P-PI3K, P-Akt and P-MTOR. Expression of PI3K, Akt and mTOR mRNA was found to be downregulated by SSB4 (P < 0.01) as the result of RT-PCR measurements. Conclusion: SSB4 is a potent anti-colon cancer agent. Its effects are likely to be mediated by suppression of the PI3K/AKT/mTOR pathway.

Preeclampsia is a life-threatening disorder of pregnancy unique to humans. Poor placentation in the first trimester of pregnancy is widely accepted to be an underlying cause of preeclampsia. Galectin-7 is abnormally elevated in chorionic villous samples and serum from women that subsequently develop pre-term preeclampsia. Administration of exogenous galectin-7 to pregnant mice causes preeclampsia-like features (hypertension, proteinuria), associated with dysregulation of the renin-angiotensin system (RAS). In this study investigated the mechanism by which galectin-7 induces alterations to tissue RAS homeostasis and ROS production. We hypothesized that galectin-7 induces alterations in the production of either placental RAS or NADPH oxidases (or both) to drive the dysregulated RAS and ROS production seen in preeclampsia.Mated female mice (n = 5-6/group) received single (embryonic day [E]12/13) or multiple (E8-12) subcutaneous injections of 400 μg/kg/day galectin-7 or vehicle control and killed on E13 or E18. Human first trimester placental villous and decidual tissue (n = 11) was cultured under 8 % oxygen with 1 µg/mL galectin-7 or vehicle control for 16 h.Galectin-7 administration to pregnant mice impaired placental labyrinth formation, suppressed circulating aldosterone and altered placental RAS (Agt, Renin) and NADPH oxidase (Cyba, Cybb and Icam1) mRNA expression. In vitro, galectin-7 regulated human placental villous RAS (AGT) and NADPH oxidase (CYBA, ICAM1 and VCAM1) mRNA expression.Overall, galectin-7 likely drives hypertension in preeclampsia via its direct regulation of multiple pathways associated with preeclampsia in the placenta. Galectin-7 may therefore be a therapeutic target to improve placental function and prevent preeclampsia.

Colorectal cancer (CRC) is a leading cause of cancer-related death. There is an urgent need for new methods of early CRC detection and monitoring to improve patient outcomes. Extracellular vesicles (EVs) are secreted, lipid-bilayer bound, nanoparticles that carry biological cargo throughout the body and in turn exhibit cancer-related biomarker potential. RNA binding proteins (RBPs) are posttranscriptional regulators of gene expression that may provide a link between host cell gene expression and EV phenotypes. Insulin-like growth factor 2 RNA binding protein 1 (IGF2BP1/IMP1) is an RBP that is highly expressed in CRC with higher levels of expression correlating with poor prognosis. IMP1 binds and potently regulates tumor-associated transcripts that may impact CRC EV phenotypes. Our objective was to test whether IMP1 expression levels impact EV secretion and/or cargo. We used RNA sequencing, in vitro CRC cell lines, ex vivo colonoid models, and xenograft mice to test the hypothesis that IMP1 influences EV secretion and/or cargo in human CRC. Our data demonstrate that IMP1 modulates the RNA expression of transcripts associated with extracellular vesicle pathway regulation, but it has no effect on EV secretion levels in vitro or in vivo. Rather, IMP1 appears to affect EV regulation by directly entering EVs in a transformation-dependent manner. These findings suggest that IMP1 has the ability to shape EV cargo in human CRC, which could serve as a diagnostic/prognostic circulating tumor biomarker.

Autosomal recessive nonsyndromic auditory neuropathy is attributed to a genetic etiology. We identified a compound heterozygous missense variant, c.G736A (p.G246S) and c.C2954T (p.T985 M) in TNN of affected patients in a pedigree via candidate gene screening and exome sequencing. To determine the genetic etiology of deafness in the pedigree with a heterozygous missense variant in the gene TNN encoding tenascin-W associated with autosomal recessive nonsyndromic auditory neuropathy, the cochlear expression of tenascin-W was evaluated at mRNA and protein levels in mice, and Tnn knock out mice were generated and utilized to study the function of Tnn in the auditory system. Immunofluorescence stainings showed that tenascin-W was mainly expressed in the somatic cytoplasm of spiral ganglion neurons of mice. Homozygous Tnn knockout was lethal in mice, whereas Tnn heterozygous mice showed decreases in spiral ganglion neuron density and progressive hearing loss. We demonstrate that tenascin-W is expressed in the murine cochleae and is essential for the development of spiral ganglion neurons. An abnormal expression of tenascin-W can influence the development and function of SGNs and affect the function of the auditory system.

Visual disturbances that can heal after a complete resection of orbital meningiomas are only about 2.9%. Grading and expression of the progesterone receptor (PR) in orbital meningiomas, according to World Health Organization (WHO) is a useful predictive value of recurrence in the treatment management of orbital meningiomas. This study aims to determine the relationship of PR expression on the grading of orbital meningiomas as tumour prognostic factors.This cross-sectional observational analysis observed 44 orbital meningioma in Cicendo Eye Hospital Bandung and Hasan Sadikin Hospital between 2017-2020. We performed of mRNA PR with RT-qPCR technique and calculation with the 2∆∆Ct formula. Statistical analysis used the Kruskal-Wallis Test, followed by the Mann-Whitney post hoc test with p0.005.Relative expression of mRNA PR in meningioma orbita grade I to grade III decreased significantly the expression of relative mRNA PR at grade I, II, III of 21.69±44.35, 20.39±26.30 and 1.25±0.85, with Kruskal-Wallis test, p =0.007. Mann Whitney's test results showed relative mRNA PR expression between grades I and II not different (p = 0.055), relative expression mRNA PR between grades I and III differed significantly (p = 0.024), and relative expression mRNA PR between grades I and III was not different (p = 0.638).mRNA PR expression is viable for prognostic value, predicting recurrence and implementing more effective management of subsequent therapy, it must be combined with other markers to determine the nature of the orbital meningioma.

Numerous cases of glomerulonephritis manifesting shortly after SARS-CoV-2 vaccination have been reported, but causality remains unproven. Here, we studied the association between mRNA-based SARS-CoV-2 vaccination and new-onset glomerulonephritis using a nationwide retrospective cohort and a case-cohort design. Data from all Swiss pathology institutes processing native kidney biopsies served to calculate incidence of IgA nephropathy, pauci-immune necrotizing glomerulonephritis, minimal change disease, and membranous nephropathy in the adult Swiss population. The observed incidence during the vaccination campaign (January to August 2021) was not different from the expected incidence calculated using a Bayesian model based on the years 2015 to 2019 (incidence rate ratio 0.86, 95% credible interval 0.73-1.02) and did not cross the upper boundary of the 95% credible interval for any month. Among 111 patients 18 years and older with newly diagnosed glomerulonephritis between January and August 2021, 38.7% had received at least one vaccine dose before biopsy, compared to 39.5% of the general Swiss population matched for age and calendar-time. The estimated risk ratio for the development of new-onset biopsy-proven glomerulonephritis was not significant at 0.97 (95% confidence interval 0.66-1.42) in vaccinated vs. unvaccinated individuals. Patients with glomerulonephritis manifesting within four weeks after vaccination did not differ clinically from those manifesting temporally unrelated to vaccination. Thus, vaccination against SARS-CoV-2 was not associated with new-onset glomerulonephritis in these two complementary studies with most temporal associations between SARS-CoV-2 vaccination and glomerulonephritis likely coincidental.

Cell-cell fusion studies provide an experimental platform for evaluating disease progression and investigating cell infection. However, to realize sensitive and quantitative detection on cell-cell fusion is still a challenge. Herein, we report a facile molecular beacon (MB)-based method for precise detection on cell-cell fusion. By transfection of the spike protein (S protein) and enhanced green fluorescent protein (EGFP) in HEK 293 cells, the virus-mimicking fusogenic effector cells 293-S-EGFP cells were constructed to interact with target cells. Before mixing the effector cells with the target cells, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in 293-S-EGFP cells was silenced, and the MB for GAPDH mRNA detection was delivered into the GAPDH silenced 293-S-EGFP cells. Once cell-cell fusion occurred, MB migrated from the GAPDH silenced effector cells to the target cells and hybridized with GAPDH mRNA in the target cells to induce fluorescence emission. The cell-cell fusion can be easily visualized and quantitated by fluorescence microscopy and flow cytometry. The fluorescence intensity is strongly dependent on the number of fused target cells. This MB-based method can easily identify the differences in the cell fusions for various target cells with different angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) expression levels, resulting in dramatically different fluorescence intensities in fused target cells. Our study provides a convenient and efficient quantitative detection approach to study cell-cell fusion.

There have been limited studies focused on validation of swine microRNAs (miRNA) with mRNA targets. The objective of this study was to validate a defined set of targets using artificial miRNA mimics transfected into cell lines to confirm specific targets of endogenous miRNAs after administration of Escherichia coli lipopolysaccharide (LPS). Sixteen hours after mimic transfection of 3D4/21 cell lines, the cells were stimulated with 1 μg/ml LPS or phosphate-buffered saline (PBS). The cells were harvested and collected at 0, 1, 3, and 8 h post administration. The selected genes DAD1, IL8, and ESR, which are involved in known pathways of inflammation. and are predicted or validated human targets of either miR-146a, let-7a, or miR-22-3p. These were then evaluated by quantitative real-time-PCR (qRT-PCR) to verify microRNA-mRNA interaction in swine. Using the ROX reference dye, mRNA changes in expression were assessed using the comparative CT Method (ΔΔCT method) for normalization against the PBS control group. DAD1 and ESR1 were negatively regulated by miR-22-3p and miR-146a-5p, respectively in 3D4/21 cells after LPS stimulation. However, miR-146a-5p may play an indirect positive regulatory role of both DAD1 and IL8 mRNA expression. Furthermore, we found an inverse relationship between LPS stimulation compared with the let-7a-5p overexpression with DAD1. Our inflammation study provides new evidence on the roles and predicted targets of miR-146a, let-7a, and miR-22-3p in swine.

Breast cancer is one of the most frequent malignancies in females. The molecular mechanism of how breast cancer development and recurrence still need to be explored. Peroxisome gamma coactivator-1β (PGC-1β) was engaged in cancer energy metabolism and tumor genesis. However, the mechanisms of PGC-1β in breast cancer have not been fully understood. In this study, PCG-1β overexpressed and knockdown vectors were transferred into MCF-7 cells. With the association-quantitative connection analysis, the different expressions of mRNAs and proteins were examined. Additionally, the terms on differentially expressed mRNAs and proteins were enriched by GO and KEGG. Based on the results, 1872 differentially expressed genes were identified in the up-regulated of PGC-1β group, and 1318 genes were found in the down-regulated of PGC-1β cells. With the label-free technique, 221 differentially expressed proteins were screened in PGC-1β up-regulated group, and 459 proteins were identified in PGC-1β down-regulated group. Correlation analysis showed that 49 significantly expressed mRNA-protein pairs in OV vs CT groups and 25 paired in SI vs CT groups. Combined analysis of transcriptome and proteome demonstrated that PGC-1β plays a important role in cancer energy metabolism and boosting the pace of chemical processes in the proliferation of breast cancer cells. Additional investigation about PGC-1β and energy metabolism in cancer cells may shed fresh light on the growth and treatment of breast cancer cells.

The safety profiles of COVID-19 vaccines are incompletely evaluated in Japan.To examine the risk of serious adverse effects after COVID-19 mRNA vaccination (BNT162b2 and mRNA-1273) in cohort studies and self-controlled case series (SCCS).Using an administrative claims database linked with the COVID-19 vaccination registry in a city in Japan between September 2020 and September 2021, we identified health insurance enrolees aged ≥ 18 years. We evaluated the risk of acute myocardial infarction, appendicitis, Bell's palsy, convulsions/seizures, disseminated intravascular coagulation, immune thrombocytopenia, pulmonary embolism, haemorrhagic or ischemic stroke, venous thromboembolism, and all-cause mortality, 21 days following any COVID-19 mRNA vaccination, compared with non-vaccination periods. For the cohort studies, we estimated incidence rate ratios (IRRs) by Poisson regression and rate differences (IRDs) by weighted least-squares regression, adjusting for sex, age, and Charlson comorbidity index. We applied a modified SCCS design to appropriately treat outcome-dependent exposures. For the modified SCCS, we estimated within-subject IRRs by weighted conditional Poisson regression. Subgroup analyses stratified by sex and age were also conducted.We identified 184,491 enrolees [male: 87,218; mean (standard deviation) age: 64.2 (19.5) years] with 136,667 first and 127,322 s dose vaccinations. The risks of any outcomes did not increase in any analyses, except for the fact that the modified SCCS indicated an increased risk of pulmonary embolism after the first dose in women (within-subject IRR [95%CI]: 3.97 [1.18-13.32]).The findings suggested that the COVID-19 mRNA vaccine was generally safe, whilst a signal of pulmonary embolism following the first dose of the COVID-19 mRNA vaccine was observed.

Satellite cells (SCs) and ribosomes are key determinants of the skeletal muscle adaptive response. Both are thought to increase acutely after resistance exercise and chronically with resistance training. However, the acute SC and ribosome exercise response with prior aerobic conditioning is unknown. Fourteen young men and women underwent 6 wk of single-legged aerobic conditioning followed by an acute bout of 300 eccentric contractions on each leg. Muscle biopsies were taken from the vastus lateralis of the aerobically conditioned (AC) and the control (CTL) legs before (Pre), 24 (24 h), and 48 (48 h) h post-contractions. Pre-eccentric contractions, 45S pre-rRNA and 5.8S internal transcribed spacer (ITS) expression were lower in the AC leg compared with the CTL leg. SC content (PAX7+ cells/100 fibers) in type I and mixed fibers showed a main effect of condition, where values were greater in the AC leg compared with the CTL. A main effect of condition for Pax7 and MyoD1 mRNA expression was observed where expression was greater in the AC leg compared with the CTL. AC had greater RNA concentration and mRNA expression of Ubf and Tif-1a compared with CTL. Only the AC leg increased (Pre-24h) 45S pre-rRNA, 5.8S ITS, and 28S ITS following eccentric contractions. We discovered that aerobic conditioning increased type-I SC abundance and the acute increase in ribosome content following eccentric contractions.

Breast cancer is a complex heterogeneous disease with unique molecular subtypes, which limits the development of optimized treatment strategies for each subtype. Cancer gene therapy and potential therapeutics for advanced/refractory cancers can be promising for breast cancer. Combining tumor-tropic lipid nanoparticles (LNPs) and inducible caspase-9 (iC9) mRNA, we aimed to develop a novel treatment strategy for refractory breast cancer. LNP's anti-tumor effects were tested in vitro in three breast cancer cell lines: MDA-MB231, SKBR3, and MCF-7. Tumor cells were treated with LNPs encapsulated with eGFP or iC9 mRNA and chemical inducers of dimerization (CID). Apoptosis-related genes were evaluated by reverse transcriptase quantitative PCR. LNPs could efficiently deliver encapsulated GFP mRNA to all three cancer cell lines (80% GFP expression. in target cells). Furthermore, LNPs encapsulated with iC9 mRNA (iC9-LNPs) and CID showed cytotoxic activity against all cancer cell lines in vitro. Interestingly, susceptibility to iC9 gene therapy was heterogeneous among cancer cell lines. iC9-LNPs with CID-induced potent cytotoxic effects against SKBR3 and MDA-MB231 cells, but only a mild cytotoxic effect on MCF7 cells. Quantification of apoptosis-related genes suggested that a high BAX/Bcl-2 ratio might be associated with iC9-LNP + CID susceptibility. Thus, cancer gene therapy using iC9-LNPs and CID could be a promising alternative for the treatment of breast cancers, especially for aggressive breast cancers.

In this study, we aim to investigate the effect of metformin on cholesterol synthesis and efflux-related genes in chondrocytes during osteoarthritis (OA) and explore the underlying mechanisms. Primary chondrocytes were harvested from Wistar rat cartilage and divided into control and treatment groups. Chondrocytes in the treatment group were treated with interleukin-1β (IL-1β) mimicking the inflammatory environment of osteoarthritis. Subsequently, RT-qPCR, Western blotting, immunofluorescence staining, and Cell Counting Kit-8 (CCK-8) were conducted. Significant reductions in phosphorylated AMP-activated protein kinase (p-AMPK) and silent information regulator 1 (SIRT1) protein expression were observed in both human OA chondrocytes and cultured primary murine chondrocytes treated with IL-1β, while AMP-activated protein kinase (AMPK) was not inhibited. Moreover, in the presence of IL-1β, metformin significantly increased the expression of p-AMPK and SIRT1 at the protein and mRNA level. Meanwhile, metformin could reverse IL-1β-induced cartilage extracellular matrix degradation in chondrocytes from the rat model of OA (treated by IL-β) by activating the AMPK/SIRT1 pathway. Moreover, metformin activated AMPK and SIRT1, mediated by the activation of SREBP-2 and HMGCR in OA chondrocytes. Inhibiting AMPK/SIRT1 activity by its specific inhibitor could suppress IL-1β-induced expression of LXRα, ABCA1 and ApoA1 and cholesterol efflux. Thus, metformin inhibits cholesterol synthesis and promotes cholesterol efflux by activating the AMPK/SIRT1 pathway in OA chondrocytes. This study improves our understanding of the effect of metformin on cholesterol accumulation in OA chondrocytes.

Objective Transglutaminase (TGM)2 and peroxisome proliferator-activated receptor (PPAR)γ are thought to participate in the pathogenesis of nasal polyp formation in cystic fibrosis (CF). We herein investigated expressions of cystic fibrosis transmembrane conductance regulator (CFTR), TGM2, PPARγ and isopeptide bonds, a reaction product of TGM, in non-CF nasal polyps. Methods Nasal polyps and inferior turbinates were collected from chronic rhinosinusitis patients without CF during transnasal endoscopic sinonasal surgery. Expressions of CFTR, TGM2, isopeptide bonds and PPARγ were examined by fluorescence immunohistochemistry and quantitative RT-PCR. Expression of CFTR was also analyzed by Western blot. Results Immunohistochemical fluorescence of the nasal polyp was significantly lower for CFTR and PPARγ, and significantly higher for TGM2 and isopeptide bonds than that of the turbinate mucosa. Lower expression of CFTR in the nasal polyp than in the turbinate mucosa was also observed in Western blot. Expression of PPARG mRNA was significantly lower in the nasal polyp than in the turbinate mucosa, whereas expressions of CFTR mRNA or TGM2 mRNA did not differ between the two tissues. Immunohistochemical fluorescence for CFTR showed significant negative correlation with that for TGM2 and isopeptide bonds, and significant positive correlation with that for PPARγ. The fluorescence for TGM2 was positively correlated with that for isopeptide bonds and negatively correlated with that for PPARγ. The fluorescence for isopeptide bonds tended to be negatively correlated with that for PPARγ. Conclusions These results suggest a possible role of the CFTR-TGM2-PPARγ cascade in the pathogenesis of nasal polyp formation in non-CF patients as in CF patients.

Large intestine dampness-heat syndrome (LIDHS) is frequently-occurring in the inflammatory intestinal disease of animals and human. Yujin powder (YJP) is a classical prescription for treating LIDHS. To explore the pathological mechanism of intestinal mucosal barrier injury of LIDHS and the protection of YJP, the LIDHS rat model was established through imitating the inducing conditions of LIDHS and treated with YJP. The integrity of ileal and colonic mucosa was detected through histopathological examination. The serum DAO, D-LA and ET levels were detected by ELISA. The mRNA and protein expression levels of Occludin, ZO-1 and MUC2 in ileum and colon were detected using RT-PCR and immunohistochemistry methods, respectively. The results showed that the ileal and colonic epithelium of LIDHS rats were destroyed; the serum DAO, D-LA and ET levels were significantly increased; the mRNA and protein expression levels of Occludin, ZO-1 and MUC2 in ileum and colon were all abnormally expressed. After treatment with YJP, the mucosal integrity was restored; the levels of serum DAO, D-LA and ET, mRNA and protein levels of Occludin and ZO-1 in ileum and colon and MUC2 in ileum were back-regulated; however, MUC2 level in colon was further increased. The results demonstrated that the intestinal mucosal barrier was damaged in LIDHS rats and Occludin, ZO-1 and MUC2 were abnormally expressed, and YJP could repair the intestinal mucosal barrier through up-regulating the expression of Occludin and ZO-1 in ileum and colon as well as MUC2 in colon and down-regulating MUC2 in ileum.

Ribosomes composed of genome-encoded heterogeneous rRNAs are implicated in the rapid adaptation of bacterial cells to environmental changes. A previous study showed that ribosomes bearing the most heterogeneous rRNAs expressed from the rrnI operon (I-ribosomes) are implicated in the preferential translation of a subset of mRNAs, including hspA and tpiA, in Vibrio vulnificus CMCP6. In this study, we show that HspA nascent peptides were predominantly bound to I-ribosomes. Specifically, I-ribosomes were enriched more than two-fold in ribosomes that were pulled down by immunoprecipitation of HspA peptides compared with the proportion of I-ribosomes in crude ribosomes and ribosomes pulled down by immunoprecipitation of RNA polymerase subunit β peptides in the wild-type (WT) and rrnI-completed strains. Other methods that utilized the incorporation of an affinity tag in 23S rRNA or chimeric rRNA tethering 16S and 23S rRNAs, which generated specialized functional ribosomes in Escherichia coli, did not result in functional I-ribosomes in V. vulnificus CMCP6. This study provides direct evidence of the preferential translation of hspA mRNA by I-ribosomes.

Oncoproteins of the MYC family drive the development of numerous human tumours

Long noncoding RNAs (lncRNAs) are revealed to be involved in the tumorigenesis and progression of human malignancies mediated by microRNA (miRNA) via the competing endogenous RNA (ceRNA) mechanism, a newly proposed “RNA language.” However, the lncRNA-associated competing triplet (lncACT) network among ceRNA transcripts in clear cell renal cell carcinoma (ccRCC) is currently lacking. We carried out differential expression analysis to identify aberrantly expressed lncRNAs, miRNAs, and mRNAs by analyzing the RNA-seq data of 420 ccRCC tissues and 71 noncancerous kidney tissues obtained from The Cancer Genome Atlas (TCGA). Then, a ccRCC-specific ceRNA network was built using computational algorithms, including miRcode, TargetScan, miRanda, and miRTarBase. In total, 1491 dysregulated lncRNAs were found between normal renal tissues and ccRCC (fold change > 4 and false discovery rate < 0.01 ). A ceRNA network that comprised of 46 DElncRNAs, 11 DEmiRNAs, and 55 DEmRNAs was established by integrating the lncRNA/miRNA and miRNA/mRNA interactions into lncACTs. Several lncRNAs were identified to be significantly associated with clinical features of ccRCC patients. Notably, four key lncRNAs (TCL6, HOTTIP, HULC, and PCGEM1) were tightly correlated with both patients’ clinical characteristics and overall survival (log-rank P < 0.05 ), indicating their potential important roles in ccRCC. HOTTIP may be a potential prognostic and therapeutic molecular marker for ccRCC patients. Collectively, our results provide a comprehensive view of the lncRNA-associated ceRNA regulatory network for a better understanding of the mechanisms and prognosis biomarkers for ccRCC.

The analysis of cytochrome P450 transcripts was carried out by the nanopore sequencing in liver tissue samples of three donors and HepG2 line cells. It has been demonstrated that direct mRNA sequencing with a MinION nanopore sequencer (Oxford Nanopore Technologies) allows one to obtained quantitative profiles for transcripts (and their splice variants) of cytochrome P450 superfamily genes encoding isoforms involved in metabolism of the large (~80%) part of drugs. The splice variant profiles substantially differ for donors. The cytochrome P450 gene expression at the transcript level is significantly weaker in cells of the HepG2 line compared with that in the normal liver tissue. This limits the capability of the direct mRNA nanopore sequencing for studying alternative splicing of cytochrome P450 transcripts in HepG2 cells. Both quantitative and qualitative profiles of the cytochrome P450 gene expression at the transcript level are notably differ in human liver tissue and HepG2 cells.Metodom nanoporovogo sekvenirovaniia proveden analiz transkriptov tsitokhromov R450 v obraztsakh tkani pecheni trekh donorov i kletkakh linii HepG2 (gepatoblastoma cheloveka). Pokazano, chto priamoe sekvenirovanie mRNK s ispol'zovaniem nanoporovogo sekvenatora MinION (Oxford Nanopore Technologies) pozvoliaet poluchat' kolichestvennye profili transkriptov (i ikh splaĭs-variantov) dlia genov supersemeĭstva tsitokhroma R450, kodiruiushchikh izoformy, pri uchastii kotorykh metaboliziruetsia bol'shaia chast' (~80%) lekarstvennykh veshchestv. Profili splaĭs-variantov sushchestvenno razlichaiutsia dlia raznykh donorov. Ékspressiia genov tsitokhromov R450 na urovne transkriptov v kletkakh linii HepG2 znachitel'no slabee v sravnenii s ikh ékspressieĭ v normal'noĭ tkani pecheni, chto zatrudniaet issledovanie al'ternativnogo splaĭsinga transkriptov tsitokhromov R450 v kletkakh HepG2 metodom priamogo nanoporovogo sekvenirovaniia mRNK. Profili ékspressii genov supersemeĭstva tsitokhroma R450 na urovne trankriptov v tkani pecheni cheloveka i kletkakh HepG2 znachitel'no razlichaiutsia kak kolichestvenno, tak i kachestvenno.

Transthyretin-mediated amyloidosis (ATTR) is a rare, under-recognized, progressively debilitating, fatal disease caused by the aggregation and extracellular deposition of amyloid transthyretin (TTR) fibrils in multiple organs and tissues throughout the body. TTR is predominantly synthesized by the liver and normally circulates as a homotetramer, while misfolded monomers aggregate to form amyloid fibrils. One strategy to treat ATTR amyloidosis is to reduce the amount of TTR produced by the liver using drugs that directly target the TTR mRNA or gene. This narrative review focuses on how TTR gene silencing tools act to reduce TTR production, describing strategies for improved targeted delivery of these agents to hepatocytes where TTR is preferentially expressed. Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), termed RNA silencers, cause selective degradation of TTR mRNA, while a TTR gene editing tool reduces TTR expression by introducing nonsense mutations into the TTR gene. Two strategies to facilitate tissue-specific delivery of these nucleic acid-based drugs employ endogenous receptors expressed by hepatocytes. Lipid nanoparticles (LNPs) that recruit apolipoprotein E support low-density lipoprotein receptor-mediated uptake of unconjugated siRNA and are now used for CRISPR gene editing tools. Additionally, conjugating N-acetylgalactosamine (GalNAc) moieties to ASOs or siRNAs facilitates receptor-mediated uptake by the asialoglycoprotein receptor. In summary, ATTR is a progressive disease with various clinical manifestations due to TTR aggregation, deposition, and amyloid formation. Receptor-targeted ligands (eg, GalNAc) and nanoparticle encapsulation (eg, LNPs) are technologies to deliver ASOs, siRNAs, and gene editing tools to hepatocytes, the primary location of TTR synthesis.

The tsRNAs (tRNA-derived small RNAs) are new types of small noncoding RNAs derived from tRNAs. Gliomas are well-known malignant brain tumors. The study focused on tsRNA characterizations within gliomas. Datasets processing, bioinformatics analyses, and visualizations were performed with the packages of Python and R. Cell proliferations were demonstrated via CCK8 assays and colony formation assays, and in vivo xenograft experiments. Dual-luciferase reporter assay was performed to confirm the binding of tsRNA with its targets. Via using bioinformatics approaches, the hundreds of tsRNAs with available expression abundance were identified in gliomas dataset, most of them derived from D-loop or T-loop fragments of tRNAs. Among tsRNAs derived from tRNA-Cys-GCA, tRFdb-3003a and tRFdb-3003b (tRFdb-3003a/b) were remarkably down-regulated in gliomas. The survival outcome of gliomas patients with low tRFdb-3003a/b expressions was notably worse than that of high-expression patients. In glioma cells, tRFdb-3003a could suppress cells proliferation and colony formation ability. In vivo, tRFdb-3003a suppressed the tumor growth of xenograft gliomas. Enrichment analyses displayed the tRFdb-3003a-related mRNAs were enriched in the specific GO terms, spliceosome and autophagy pathways, and three GSEA molecular signatures. Mechanically, 3’-UTR regions of VAV2 mRNA were predicted to contain the binding positions of tRFdb-3003a/b, tRFdb-3003a and tRFdb-3003b was effective to reduce the relative luciferase activity of cells with VAV2 wild-type reporter. Overexpression of tRFdb-3003a/b could down-regulated the expression levels of VAV2 protein and mRNA in glioma cells. The tRNA-Cys-GCA derived tRFdb-3003a and tRFdb-3003b might act as key player in tumor progressions of gliomas; tRFdb-3003a/b might directly bind to VAV2 and regulate VAV2 expressions in gliomas.

Background: Hypoxia is recognized as a key feature of cancer growth and is involved in various cellular processes, including proliferation, angiogenesis, and immune surveillance. Besides hypoxia-inducible factor 1-alpha (HIF-1α), which is the main mediator of hypoxia effects and can also be activated under normoxic conditions, little is known about its counterpart, HIF-2. This study focused on investigating the clinical and molecular landscape of HIF-2-altered urothelial carcinoma (UC). Methods: Publicly available next-generation sequencing (NGS) data from muscle-invasive UC cell lines and patient tumor samples from the MSK/TCGA 2020 cohort (n = 476) were interrogated for the level of expression (mRNA, protein) and presence of mutations, copy number variations, structural variants in the EPAS1 gene encoding HIF-2, and findings among various clinical (stage, grade, progression-free and overall survival) and molecular (tumor mutational burden, enriched gene expression) parameters were compared between altered and unaltered tumors. Results: 19% (7/37) of UC cell lines and 7% (27/380) of patients with muscle-invasive UC display high EPAS1 mRNA and protein expression or/and EPAS1 alterations. EPAS1-altered tumors are associated with higher stage, grade, and lymph node metastasis as well as with shorter PFS (14 vs. 51 months, q = 0.01) and OS (15 vs. 55 months, q = 0.01). EPAS1 mRNA expression is directly correlated with that of its target-genes, including VEGF, FLT1, KDR, DLL4, CDH5, ANGPT1 (q < 0.001). While there is a slightly higher tumor mutational burden in EPAS1-altered tumors (9.9 vs. 4.9 mut/Mb), they are enriched in and associated with genes promoting immune evasion, including ARID5B, SPINT1, AAK1, CLIC3, SORT1, SASH1, and FGFR3, respectively (q < 0.001). Conclusions: HIF-2-altered UC has an aggressive clinical and a distinct genomic and immunogenomic profile enriched in angiogenesis- and immune evasion-promoting genes.

Low-grade stage I endometrioid endometrial carcinomas should have an excellent prognosis, but a small subset of these cancers can relapse. The search for putative immunohistochemical prognostic markers for relapse in low-risk/low-grade endometrioid endometrial cancers remains open. Among the candidate molecules that may implicate the roles of immunohistochemical risk markers, we focused our attention on human epididymis protein 4 (HE4) after a review of the literature. Few authors have devoted themselves to this topic, and none have found a correlation between the tissue expression of HE4 and the molecular classification of endometrial cancer. Five different variants of HE4 mRNA and multiple protein isoforms of HE4 were identified many years ago, but current HE4 assays only measure the total HE4 expression and do not distinguish the different proteins encoded by different mRNA variants. It is important to have an approach to distinguish specific variants in the future.

Asymmetric small interfering RNAs (asiRNAs) that mediate RNA interference have been investigated for therapeutic use in various tissues, including skin tissue. Androgenetic alopecia (AGA) is caused by a combination of genetic factors, resulting in sensitivity to dihydrotestosterone (DHT), which binds to the androgen receptor (AR) to mediate a series of biomolecular changes leading to hair loss. This study aimed to evaluate the therapeutic potential of a cell-penetrating, AR-targeting asiRNA (cp-asiAR) for AGA treatment, which was designed to silence the

No current RNA-targeted interference tools have been reported to simultaneously up and down-regulate different gene expressions. Here we characterized an RNA-targeted genetic regulatory strategy composed of a flap endonuclease 1 (FEN1) and specific mis-hairpin DNA probes (mis-hpDNA), to realize the orthogonal genetic regulation. By targeting mRNA, the strategy hindered the translation and silenced genes in human cells with efficiencies of ~60%. By targeting miRNA, the strategy prevented the combination of miRNA to its specific mRNA and increased this mRNA expression by about 3-folds. In combination, we simultaneously performed CXCR4 gene knock-down (~50%) and EGFR gene activation (1.5-folds) in human cells. Although the functional property can be further improved, this RNA-targeted orthogonal genetic regulating strategy is complementary to classical tools.

As the most prevalent primary CNS tumor, glioma is characterized by high mortality and morbidity. This research aims to investigate glioma-associated microRNAs (miRNAs) and their target mRNAs, as well as to explore their biological functions in gliomas. The Gene Expression Omnibus (GEO) database was applied to acquire the GSE112264 miRNA microarray dataset and the GSE15824 mRNA dataset. We selected samples from the GSE112264 dataset and the GSE15824 to identify differently expressed miRNAs (DE-miRNAs) as well as differentially expressed mRNAs (DEGs), respectively. Next, the intersections of mRNA and target mRNAs of miRNA were selected, and we constructed miRNA-mRNA regulation networks. These DEGs were selected for Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses by conducting the package clusterProfiler. After conducting Cytoscape software, a protein-protein interaction (PPI) network was created. Next, survival analysis of the miR-423-3p was confirmed by conducting TCGA database. Subsequently, Quantitative real-time PCR (qRT-PCR) was conducted to verify miR-423-3p's expression. Finally, miR-423-3p's biological functions of in effecting the cell proliferative, migratory, and invasive capabilities of glioma were investigated by performing Cell Counting Kit-8 (CCK-8) and Transwell assays. Our analysis elucidated a novel miRNA-mRNA regulatory network related to glioma carcinogenesis, which may be considered as future therapeutic biomarkers for glioma.

To investigate the regulatory mechanism of pterygium formation, we detected differentially expressed messenger RNAs (DE-mRNAs) and differentially expressed circular RNAs (DE-circRNAs) in pterygium-associated conjunctival epithelium (PCE) and normal conjunctival epithelium (NCE). Genome-wide mRNA and circRNA expression profiles of PCE and NCE were determined using high-throughput sequencing. Bioinformatics analyses, including Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, gene set enrichment analysis (GSEA), and protein–protein interaction (PPI) analysis, were conducted. The microRNAs (miRNAs) interacting with the hub DE-mRNAs and DE-circRNAs were predicted and verified using real-time quantitative PCR (RT-qPCR). The data showed that there were 536 DE-mRNAs (280 upregulated and 256 downregulated mRNAs) and 78 DE-circRNAs (20 upregulated and 58 downregulated circRNAs) in PCE. KEGG enrichment analysis indicated that the DE-mRNAs were mainly involved in the following biological processes: IL-17 signalling pathway, viral protein interaction with cytokine and cytokine receptor, cytokine–cytokine receptor interaction, ECM-receptor interaction, and focal adhesion. The GSEA results revealed that the epithelial mesenchymal transition (EMT) process was significantly enriched in upregulated mRNAs. The pterygium-associated circRNA–miRNA–mRNA network was established based on the top 10 DE-circRNAs, 4 validated miRNAs (upregulated miR-376a-5p and miR-208a-5p,downregulated miR-203a-3p and miR-200b-3p), and 31 DE-mRNAs. We found that miR-200b-3p, as a regulator of FN1, SDC2, and MEX3D, could be regulated by 5 upregulated circRNAs. In addition, we screened out EMT-related DE-mRNAs, including 6 upregulated DE-mRNAs and 6 downregulated DE-mRNAs. The EMT-related circRNA–miRNA–mRNA network was established with the top 10 circRNAs, 8 validated miRNAs (upregulated miR-17-5p, miR-181a-5p, and miR-106a-5p, downregulated miR-124-3p, miR-9-5p, miR-130b-5p, miR-1-3p, and miR-26b-5P), and 12 EMT-related DE-mRNAs. We found that hsa_circ_0002406 might upregulate FN1 and ADAM12 by sponging miR-26b-5p and miR-1-3p, respectively, thus promoting EMT in pterygium. Briefly, the study provides a novel viewpoint on the molecular pathological mechanisms in pterygium formation. CircRNA–miRNA–mRNA regulatory networks participate in the pathogenesis of pterygium and might become promising targets for pterygium prevention and treatment.

Bladder cancer is a highly prevalent disease worldwide. We aimed to investigate the effect of miRNA/mRNA signaling on bladder urothelial carcinoma (BUC).MiRNA-139-3p wasselected from The Cancer Genome Atlas database, and its downstream target gene was predicted. The correlation between miRNA-139-3p and intersected mRNAs was analyzed. The mRNA expression levels of miRNA-139-3p and KIF18B in BUC were assayed via quantitative real-time polymerase chain reaction. Effects of miRNA-139-3p on cell proliferation, invasion, migration and cell cycle were detected via Cell Counting Kit-8, colony formation, transwell, wound healing and flow cytometry assays, respectively. Binding relationship between miRNA-139-3p and KIF18B was verified by dual-luciferase reporter gene detection. The protein expression levels of KIF18B, β-catenin and Cyclin D1 were detected by Western blot. Rescue assays were performed for verifying the interaction among miRNA-139-3p, KIF18B and Wnt/β-catenin signaling pathway, which revealed effects of miRNA-139-3p/KIF18B on BUC cells.MiRNA-139-3p was remarkably underexpressed, and KIF18B was dramatically overexpressed in BUC cells, respectively. It was also demonstrated that overexpressing miRNA-139-3p could prominently inhibit proliferation, invasion and migration of BUC, and block BUC cells at G0-G1 phase. Afterwards, we found that miRNA-139-3p could bind to KIF18B mRNA 3'UTR, and miRNA-139-3p had a negative regulatory effect with KIF18B. Subsequent experimental results presented that overexpressing KIF18B could reverse inhibitory effect of overexpressing miRNA-139-3p on BUC. Finally, this study also ascertained that miRNA-139-3p/KIF18B could repress oncogenic effects of BUC via modulating Wnt/β-catenin signaling pathway.MiRNA-139-3p/KIF18B/Wnt/β-catenin could significantly inhibit the malignant progression of BUC, and its targeting mechanism might provide an effective therapeutic target for BUC patients.

Exosomes serve as a promising therapeutic nanoplatform. However, the exosomes produced by donor cells are a heterogeneous group, with only a small portion having high therapeutic efficacy. Specific isolation of the subpopulation with high efficacy is important for lowering the dose and minimizing toxicity. In this study, we loaded target mRNA and displayed specific Flag in engineered exosomes simultaneously. Briefly, the donor cells were transfected with plasmid expressing a fusion protein Flag-TCS-PTGFRN-CTSL-MCP, namely, exosome sorter. During biogenesis, the RNA-binding motif MCP can specifically bind with MS2-containing RNA and sort the target RNA into the lumen of exosomes. Anti-Flag magnetic beads can capture and thus purify the engineered exosomes

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is characterized by congenital absence of the uterus, cervix, and the upper part of the vagina in females. Whole-gene deletion and loss-of-function variants in TBX6 have been identified in association with MRKHS. We aimed to expand the spectrum of TBX6 variants in MRKHS and explore the biological effect of the variant alleles.Rare variants in TBX6 were called from a combined multiethnic cohort of 622 probands with MRKHS who underwent exome sequencing or genome sequencing. Multiple in vitro functional experiments were performed, including messenger RNA analysis, western blotting, transcriptional activity assay, and immunofluorescence staining.We identified 16 rare variants in TBX6 from the combined cohort, including 1 protein-truncating variant reported in our previous study and 15 variants with unknown effects. By comparing the prevalence of TBX6 variants in the Chinese MRKHS cohort vs 1038 female controls, we observed a significant mutational burden of TBX6 in affected individuals (P = .0004, odds ratio = 5.25), suggesting a causal role of TBX6 variants in MRKHS. Of the 15 variants with uncertain effects, 7 were shown to induce a loss-of-function effect through various mechanisms. The c.423GA (p.Leu141=) and c.839+5GA variants impaired the normal splicing of TBX6 messenger RNA, c.422TC (p.Leu141Pro) and c.745GA (p.Val249Met) led to decreased protein expression, c.10CT (p.Pro4Ser) and c.400GA (p.Glu134Lys) resulted in perturbed transcriptional activity, and c.356GA (p.Arg119His) caused protein mislocalization. We observed incomplete penetrance and variable expressivity in families carrying deleterious variants, which indicates a more complex genetic mechanism than classical Mendelian inheritance.Our study expands the mutational spectrum of TBX6 in MRKHS and delineates the molecular pathogenesis of TBX6 variants, supporting the association between deleterious variants in TBX6 and MRKHS.

Atherosclerosis (AS) is the pathological basis of many cardiovascular and cerebrovascular diseases, in which macrophage-derived foam cells are the critical step and a typical pathological feature of early atherosclerosis. We previously confirmed that low-molecular-weight fucoidan (LMWF) had a good anti-AS effect, but the mechanism is still unclear. Here with aim to investigate the inhibitory effect of LMWF on foam cells and its molecular mechanism. Oil red O staining showed that LMWF effectively alleviated lipid accumulation and the formation of foam cells. Flow cytometry detection showed that LMWF promoted foam cells apoptosis. In addition, immunofluorescence showed that LMWF inhibited macrophage scavenger receptor A1 (SR-A1)-mediated lipid uptake and promoted ATP-binding cassette transporter A1 (ABCA1)-mediated cholesterol outflow. Western blot showed that LMWF downregulated SR-A1 protein expression and upregulated ABCA1 protein expression by inhibiting p38 mitogen activated protein kinase (p38MAPK) phosphorylation. Moreover, the mRNA transcriptions of Stat1, Elk-1, and Myc were downregulated when treated with LMWF. It concluded that, LMWF achieved bidirectional regulation of SR-A1 and ABCA1, then prevented the formation of foam cells, finally ameliorated the development of AS.