RNA Therapeutics for the Cardiovascular System.

RNA therapeutics hold significant promise in the treatment of cardiovascular diseases. RNAs are biologically diverse and functionally specific and can be used for gain- or loss-of-function purposes. The effectiveness of mRNA-based vaccines in the recent COVID-19 pandemic has undoubtedly proven the benefits of an RNA-based approach. RNA-based therapies are becoming more common as a treatment modality for cardiovascular disease. This is most evident in hypertension where several small interfering RNA-based drugs have proven to be effective in managing high blood pressure in several clinical trials. As befits a rapidly burgeoning field, there is significant interest in other classes of RNA. Revascularization of the infarcted heart through an mRNA drug is under clinical investigation. mRNA technology may provide the platform for the expression of paracrine factors for myocardial protection and regeneration. Emergent technologies on the basis of microRNAs and gene editing are tackling complex diseases in a novel fashion. RNA-based gene editing offers hope of permanent cures for monogenic cardiovascular diseases, and long-term control of complex diseases such as essential hypertension, as well. Likewise, microRNAs are proving effective in regenerating cardiac muscle. The aim of this review is to provide an overview of the current landscape of RNA-based therapies for the treatment of cardiovascular disease. The review describes the large number of RNA molecules that exist with a discussion of the clinical development of each RNA type. In addition, the review also presents a number of avenues for future development.

New-onset Seropositive Rheumatoid Arthritis Post-mRNA COVID-19 Vaccine: A Case Report.

Growing evidence in the medical literature has linked the COVID-19 vaccine as a potential trigger for the development or exacerbation of various autoimmune rheumatic diseases. To the best of our knowledge, we report one of the first cases of seropositive rheumatoid arthritis diagnosed after the messenger RNA COVID-19 vaccine.

Simultaneous unilateral central retinal vein occlusion and branch retinal artery occlusion after Coronavirus Disease 2019 (COVID-19) mRNA vaccine / Oclusão unilateral simultânea da veia retiniana central e artéria retiniana ramificada após vacina mRNA COVID-19

ABSTRACT A 51-year-old non-obese woman presented with a one-week history of progressive blurry vision within the inferior visual field of her left eye. Her only relevant past medical history was long-standing hypothyroidism and recent vaccination against Coronavirus Disease 2019 (COVID-19) with an mRNA vaccine 12 days before the onset of symptoms. At examination, the anterior segment was unremarkable, but the retinal fundus revealed a central retinal vein occlusion associated with a branch retinal artery occlusion of the superior temporal branch in her left eye. Ancillary tests to rule out thrombophilia, hyperviscosity, hypercoagulability, or inflammation were negative. Ultrasound tests were also negative for a cardiac or carotid origin of the branch retinal artery occlusion. At two-month follow-up, no new retinal vascular occlusive events were observed. Although the best-corrected visual acuity at presentation was 8/10 in the left eye, the final best-corrected visual acuity remained 3/10.

RESUMO Uma mulher de 51 anos, não obesa, apresentou história de uma semana de visão embaçada progressiva no campo visual inferior do olho esquerdo. Seu único histórico médico anterior relevante era hipotireoidismo de longa data e uma recente vacinação contra a Doença de Coronavírus 2019 (COVID-19), com vacina de mRNA, 12 dias antes do início dos sintomas. O exame mostrou segmento anterior normal, mas o fundo da retina revelou uma oclusão da veia central da retina associada a uma oclusão de ramo arterial da retina do ramo temporal superior no olho esquerdo. Testes auxiliares para descartar trombofilia, hiperviscosidade, hipercoagulabilidade ou inflamação apresentaram resultados negativos. Testes de ultrassom também foram negativos quanto a uma origem cardíaca ou da carótida da oclusão do ramo da artéria da retina. Após dois meses de acompanhamento, nenhum novo evento vascular oclusivo retiniano foi observado. Embora, a acuidade visual melhor corrigida na apresentação tenha sido de 8/10 no olho esquerdo, a acuidade visual final melhor corrigida permaneceu em 3/10.

Differentially Expressed mRNA in Streptozotocin-Induced Diabetic Bladder Using RNA Sequencing Analysis.

PURPOSE: To detect elements governing the pathogenesis of diabetic cystopathy (DC), mRNA sequencing was carried out for bladder tissues from normal rats and those with induced diabetes mellitus (DM). This research therefore offers possible underlying molecular pathways for the advancement of DC in relation to differential mRNA expression, together with visceral functional and architectural alterations noted in individuals with this condition. METHODS: An intraperitoneal injection of streptozotocin (STZ) was utilized to provoke DM in male Sprague-Dawley rats. Dysregulation and significant variations between normal rats and those with induced DM were then identified by a fold change of ≥ 1.5 with a false discovery rate P < 0.05. Hierarchical clustering/heat map and Gene Ontology/DAVID reference sources were generated. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and protein-protein interaction analysis were then performed. RESULTS: The diabetic rodent group exhibited a greater residual urine volume (4.0 ± 0.4 mL) than their control counterparts (0.7 ± 0.2 mL, P < 0.01) at 12 weeks after diagnosis of diabetes. Expression analysis revealed 16 upregulated and 4 downregulated genes in STZDM bladder samples. A notable increase in expression was seen in PTHLH, TNFAIP6, PRC1, MAPK10, LOC686120, CASQ2, ACTG2, PDLIM3, FCHSD1, DBN1, NKD2, PDLIM7, ATF4, RBPMS2, ITGB1 and HSPB8. A notable decrease in expression was seen in SREBLF1, PBGFR1, PBLD1 and CELF1. Major genetic themes associated with mRNA upregulation and downregulation ware identified via Gene Ontology analysis and KEGG pathways. Protein to protein interaction analysis detected PDLIM3, PDLIM7, ITGB1, ACTG2 as core high frequency nodes within the network. CONCLUSION: Changes in mRNA expression together with biological process and pathways that contribute to the etiologies underlying visceral impairment of the bladder in DM are evident. Our strategy is promising for recognizing mRNAs exclusive to the bladder in DM that might offer useful targets for diagnosis and treatment.

Bioinformatic and experimental methods to identify and validate bacterial RNA-human RNA interactions.

Ample evidence supports the importance of the microbiota on human health and disease. Recent studies suggest that extracellular vesicles are an important means of bacterial-host communication, in part via the transport of small RNAs (sRNAs). Bacterial sRNAs have been shown to co-precipitate with human and mouse RNA-induced silencing complex, hinting that some may regulate gene expression as eukaryotic microRNAs do. Bioinformatic tools, including those that can incorporate an sRNA's secondary structure, can be used to predict interactions between bacterial sRNAs and human messenger RNAs (mRNAs). Validation of these potential interactions using reproducible experimental methods is essential to move the field forward. This review will cover the evidence of interspecies communication via sRNAs, bioinformatic tools currently available to identify potential bacterial sRNA-host (specifically, human) mRNA interactions, and experimental methods to identify and validate those interactions.

Progress of expression and function of messenger RNA CCT6A in malignancies / 肿瘤研究与临床

Messenger RNA(mRNA) CCT6A can encode chaperone proteins and plays an important role in malignant tumors such as colorectal cancer, lung cancer and breast cancer. CCT6A is highly expressed in malignant tumors, which can be used as a biomarker to assess patients' prognosis, and promote malignant biological behaviors such as tumor proliferation and metastasis by regulating transforming growth factor β signals, cell cycles, and other pathways. CCT6A can also modulate immune cell infiltration in the tumor microenvironment and may be a potential target for tumor immunotherapy. The paper reviews the expression and function of CCT6A in malignancies.

[Industrial property and basic research in the genesis of the mRNA vaccine against COVID-19]. / Propriété industrielle et recherche fondamentale dans la genèse du vaccin ARNm contre la COVID-19.

The extreme speed of the design and provisional approval of the covid-19 mRNA vaccines, as well as the speed of their mass production one year after the start of the pandemic, surprised and contradicted all the experts and big pharmaceutical companies who expected years of development. Their superior efficacy to other vaccines should make mRNA vaccines indispensable, but their diffusion remains low outside developed countries. Several reasons are given, ranging from logistical difficulties to mistrust of populations or the impossibility of producing them locally without lifting patents. Considering that, in these debates, these patents may be challenged because they do not meet the necessary criteria of inventiveness, it appeared necessary to review the history, over more than three decades, of the academic research and entrepreneurial spirit that led to the advent of mRNA platforms, to examine their mutual merits and the role of industrial property for biotechnology companies.

A New Possible Cut-Off of Cytokeratin 19 mRNA Copy Number by OSNA in the Sentinel Node of Breast Cancer Patients to Avoid Unnecessary Axillary Dissection: A 10-Year Experience in a Tertiary Breast Unit.

(1) Background: The main discriminant in breast cancer prognosis is axillary lymph node status. In a select cohort of patients, axillary lymph node dissection (ALND) may be safely spared. This study aimed to determine a new possible cut-off of cytokeratin (CK) 19 mRNA copy number in the SLN to predict cases at high risk of positive ALND. (2) Methods: Clinical records of 1339 patients were retrospectively reviewed and were separated into two groups according to the axillary status (negative: ALNs- and positive ALNs+). Receiver operative characteristic (ROC) curves were used to identify a new optimal cut-off of CK19 mRNA copy number in SLN; (3) Results: Large tumor size and high grade were found mostly in ALNs+. Results from the ROC analyses, with an AUC of 82.1%, identified a new cut-off (9150 CK19 mRNA copies) showing 94% sensitivity, 67.3% specificity, 61.2% positive, and 95.3% negative predictive values; (4) OSNA remains the most-important intra-operative tool to identify patients who can benefit from ALND but with the traditional cut-off, many patients undergo needless ALND. The results of the present study suggest a new cut-off helpful to personalize surgical treatment and avoid unnecessary invasive procedures.

Role of TLR9 methylation on its active transcription in apical inflammation.

AIM: To explore the methylation pattern, its role in transcriptional regulation and potential modifiers of methylation of the TLR9 gene in chronic periapical inflammation. METHODOLOGY: In this cross-sectional study, apical lesions of endodontic origin (ALEO, n = 61) and healthy periodontal ligaments (HPL, n = 15) were included. Products from bisulfited and PCR-amplified DNA were analysed for their methylation profiles in the promoter region and at each CpG island. Additionally, TLR9 mRNA levels were quantified by qPCR and bivariate and multiple modelling were performed to better understand the influence of methylation on gene transcription. RESULTS: TLR9 mRNA levels were upregulated in ALEO compared to HPL (p < .001). TLR9 promoter CpG sites and CpG +2086 in the intragenic island 1 were demethylated in ALEO compared to HPL (p < .05). Multivariate analysis, adjusted by smoking and gender, revealed that demethylation of TLR9 promoter sites enhanced transcriptional activity, specifically demethylated CpGs at positions -736 and -683, (p = .02), which are close to CRE binding. Although ALEO reduced the global methylation of the gene promoter and intragenic-island 2 (p < .05) by -42.5 and -9.5 percentage points, respectively, age reduced the global methylation of intragenic-island 3 within the exon 2. CONCLUSIONS: Demethylations of TLR9 promoter CpG sites, along with the intragenic DNA methylation status, were involved in higher transcription in ALEO. Hence, chronic periapical inflammation and ageing modify the methylation status both in the gene promoter and in intragenic CpG islands.

CpG Single-Site Methylation Regulates TLR2 Expression in Proinflammatory PBMCs From Apical Periodontitis Individuals.

Introduction: Apical periodontitis (AP) is a common oral disease caused by the inflammatory destruction of the periapical tissues due to the infection of the root canal system of the tooth. It also contributes to systemic bacterial translocation, where peripheric mononuclear blood cells (PBMCs) can act as carriers. Toll-like receptor (TLR) 2 mediates the response to infection and activates inflammatory responses. DNA methylation can be induced by bacteria and contributes to the modulation of this response. Despite the evidence that supports the participation of PBMCs in immune-inflammatory disorders, the inflammatory profile and epigenetic regulatory mechanisms of PBMCs in AP individuals are unknown. Aim: To determine TLR2 gene methylation and inflammatory profiles of PBMCs in AP. Methods: Cross-sectional exploratory study. Otherwise, healthy individuals with AP (n=27) and controls (n=30) were included. PMBCs were isolated by a Ficoll gradient, cultured for 24 hours, and both RNA and DNA were extracted. DNA was bisulfite-treated, and specific sites at the promoter region of the TLR2 gene were amplified by qPCR using validated primers. To verify its amplification, agarose gels were performed. Then, the PCR product was sequenced. mRNA expression of TLR2 was determined by qPCR. The soluble levels of 105 inflammatory mediators were first explored with Proteome Profiler Human Cytokine Array Kit. Consequently, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, IL-6Rα, IL-1ß, and IL-12p70 levels were measured by Multiplex assay. Results: PBMCs from individuals with AP demonstrated a proinflammatory profile showing higher soluble levels of TNF-α, IL-6, and IL-1ß compared to controls (p<0.05). Higher TLR2 expression and higher global methylation pattern of the promoter region of the gene were found in AP compared to controls (p<0.05). The CpGs single-sites at positions -166 and -146 were completely methylated, while the site -102 was totally unmethylated, independently of the presence of AP. DNA methylation of CpG single-sites in positions -77 and +24 was positively associated with TLR2 expression. Conclusions: PBMCs from AP subjects show a hyperinflammatory phenotype and TLR2 upregulation in association with single CpG-sites' methylation from the TLR2 gene promoter, thereby contributing to a sustained systemic inflammatory load in individuals with periapical endodontic diseases.

Identification of stemness and differentially expressed genes in human cementum-derived cells.

PURPOSE: Periodontal treatment aims at complete regeneration of the periodontium, and developing strategies for periodontal regeneration requires a deep understanding of the tissues composing the periodontium. In the present study, the stemness characteristics and gene expression profiles of cementum-derived cells (CDCs) were investigated and compared with previously established human stem cells. Candidate marker proteins for CDCs were also explored. METHODS: Periodontal ligament stem cells (PDLSCs), pulp stem cells (PULPSCs), and CDCs were isolated and cultured from extracted human mandibular third molars. Human bone marrow stem cells (BMSCs) were used as a positive control. To identify the stemness of CDCs, cell differentiation (osteogenic, adipogenic, and chondrogenic) and surface antigens were evaluated through flow cytometry. The expression of cementum protein 1 (CEMP1) and cementum attachment protein (CAP) was investigated to explore marker proteins for CDCs through reverse-transcription polymerase chain reaction. To compare the gene expression profiles of the 4 cell types, mRNA and miRNA microarray analysis of 10 samples of BMSCs (n=1), PDLSCs (n=3), PULPSCs (n=3), and CDCs (n=3) were performed. RESULTS: The expression of mesenchymal stem cell markers with a concomitant absence of hematopoietic markers was observed in PDLSCs, PULPSCs, CDCs and BMSCs. All 4 cell populations also showed differentiation into osteogenic, adipogenic, and chondrogenic lineages. CEMP1 was strongly expressed in CDCs, while it was weakly detected in the other 3 cell populations. Meanwhile, CAP was not found in any of the 4 cell populations. The mRNA and miRNA microarray analysis showed that 14 mRNA genes and 4 miRNA genes were differentially expressed in CDCs vs. PDLSCs and PULPSCs. CONCLUSIONS: Within the limitations of the study, CDCs seem to have stemness and preferentially express CEMP1. Moreover, there were several up- or down-regulated genes in CDCs vs. PDLSCs, PULPSCs, and BMSCs and these genes could be candidate marker proteins of CDCs.

Blood mRNA Expression Profiles of Autophagy, Apoptosis, and Hypoxia Markers on Blood Cardioplegia and Custodiol Cardioplegia Groups

Abstract Introduction: Blood cardioplegia (BC) and Custodiol cardioplegia (CC) have been used for a long time in open heart surgery and are highly effective solutions. The most controversial issue among these two is whether there is any difference between them regarding myocardial damage after ischemia surgery. In this study, autophagy, apoptosis, and hypoxia markers were investigated and that way we evaluated the differences between BC and CC patients. Methods: A total of 30 patients were included in this study, using two different cardioplegic solutions. Three different whole blood samples of the patients were taken from a central vein (preoperatively, immediately postoperatively, and one day after surgery). Total ribonucleic acid was extracted from these samples. Quantitative real-time polymerase chain reaction was performed, and changes in gene expression were determined by the 2-∆∆Ct method of relative quantification. Results: In the CC group, Beclin gene expression level was found to be higher and this difference was statistically significant (P=0.0024). Similarly, cysteine-aspartic acid protease (caspase) 9 and hypoxia-inducible factor 1α messenger ribonucleic acid (mRNA) gene expression level increased and were significantly different in the CC group. In the BC group, Beclin and microtubule-associated protein light chain 3 expressions were higher in the samples taken one day after surgery. Caspases 3 and 8 gene expressions were significantly different in the BC group. Conclusion: As a result of the analysis performed between the two cardioplegia groups, it has been shown that CC harms the myocardium more than BC at the level of mRNA expression of related markers.

Current state of diagnostic, screening and surveillance testing methods for COVID-19 from an analytical chemistry point of view.

Since December 2019, we have been in the battlefield with a new threat to the humanity known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this review, we describe the four main methods used for diagnosis, screening and/or surveillance of SARS-CoV-2: Real-time reverse transcription polymerase chain reaction (RT-PCR); chest computed tomography (CT); and different complementary alternatives developed in order to obtain rapid results, antigen and antibody detection. All of them compare the highlighting advantages and disadvantages from an analytical point of view. The gold standard method in terms of sensitivity and specificity is the RT-PCR. The different modifications propose to make it more rapid and applicable at point of care (POC) are also presented and discussed. CT images are limited to central hospitals. However, being combined with RT-PCR is the most robust and accurate way to confirm COVID-19 infection. Antibody tests, although unable to provide reliable results on the status of the infection, are suitable for carrying out maximum screening of the population in order to know the immune capacity. More recently, antigen tests, less sensitive than RT-PCR, have been authorized to determine in a quicker way whether the patient is infected at the time of analysis and without the need of specific instruments.

Blood mRNA Expression Profiles of Autophagy, Apoptosis, and Hypoxia Markers on Blood Cardioplegia and Custodiol Cardioplegia Groups.

INTRODUCTION: Blood cardioplegia (BC) and Custodiol cardioplegia (CC) have been used for a long time in open heart surgery and are highly effective solutions. The most controversial issue among these two is whether there is any difference between them regarding myocardial damage after ischemia surgery. In this study, autophagy, apoptosis, and hypoxia markers were investigated and that way we evaluated the differences between BC and CC patients. METHODS: A total of 30 patients were included in this study, using two different cardioplegic solutions. Three different whole blood samples of the patients were taken from a central vein (preoperatively, immediately postoperatively, and one day after surgery). Total ribonucleic acid was extracted from these samples. Quantitative real-time polymerase chain reaction was performed, and changes in gene expression were determined by the 2-∆∆Ct method of relative quantification. RESULTS: In the CC group, Beclin gene expression level was found to be higher and this difference was statistically significant (P=0.0024). Similarly, cysteine-aspartic acid protease (caspase) 9 and hypoxia-inducible factor 1α messenger ribonucleic acid (mRNA) gene expression level increased and were significantly different in the CC group. In the BC group, Beclin and microtubule-associated protein light chain 3 expressions were higher in the samples taken one day after surgery. Caspases 3 and 8 gene expressions were significantly different in the BC group. CONCLUSION: As a result of the analysis performed between the two cardioplegia groups, it has been shown that CC harms the myocardium more than BC at the level of mRNA expression of related markers.

Automated immunohistochemical assessment ability to evaluate estrogen and progesterone receptor status compared with quantitative reverse transcription-polymerase chain reaction in breast carcinoma patients.

BACKGROUND: This study aimed to investigate the capability of an automated immunohistochemical (IHC) evaluation of hormonal receptor status in breast cancer patients compared to a well-validated quantitative reverse transcription-polymerase chain reaction (RT-qPCR) method. METHODS: This study included 93 invasive breast carcinoma cases that had both standard IHC assay and Oncotype Dx assay results. The same paraffin blocks on which Oncotype Dx assay had been performed were selected. Estrogen receptor (ER) and progesterone receptor (PR) receptor status were evaluated through IHC stains using SP1 monoclonal antibody for ER, and 1E2 monoclonal antibody for PR. All ER and PR immunostained slides were scanned, and invasive tumor areas were marked. Using the QuantCenter image analyzer provided by 3DHISTECH, IHC staining of hormone receptors was measured and converted to histochemical scores (H scores). Pearson correlation coefficients were calculated between Oncotype Dx hormone receptor scores and H scores, and between Oncotype Dx scores and Allred scores. RESULTS: H scores measured by an automated imaging system showed high concordance with RT-qPCR scores. ER concordance was 98.9% (92/93), and PR concordance was 91.4% (85/93). The correlation magnitude between automated H scores and RT-qPCR scores was high and comparable to those of Allred scores (for ER, 0.51 vs. 0.37 [p=.121], for PR, 0.70 vs. 0.72 [p=.39]). CONCLUSIONS: Automated H scores showed a high concordance with quantitative mRNA expression levels measured by RT-qPCR.

Differentially expressed mRNA involved in the resistance of liver cancer to anlotinib / 临床肝胆病杂志

ObjectiveTo screen out the mRNAs involved in the resistance of hepatoma cells to anlotinib using ceRNA microarray. MethodsHigh-dose shock combined with low-dose induction was used to culture hepatoma cells resistant to anlotinib, and CCK8 assay was used to verify the difference in the proliferation of drug-resistant hepatoma cells treated by anlotinib. The ceRNA microarray was used to screen out the differentially expressed genes between drug-resistant hepatoma cells and normal hepatoma cells, and real-time PCR was used to verify the differentially expressed genes detected by some microarrays. the independent samples t-test was used for comparison of continuous data between two groups, and the Kaplan-Meier method was used to analyze the overall survival of hepatoma cells samples, and the log-rank test was used to compare survival rates. Fisher’s exact test was used for chip screening. ResultsThere was a significant difference in gene expression between drug-resistant hepatoma cells and normal hepatoma cells, and 10 genes with the greatest difference were screened out for analysis by reducing the range. There were 4 genes associated with drug resistance and tumor growth, i.e., BIRC2, BIRC7, ABCC2, and MAPK8. There were significant reductions in the expression levels of BIRC2, ABCC2, and MAPK8 (P=0001 4, 0001 2, and 0.011 8), and there was a significant increase in the expression of BIRC7 (P＜0.001). The results of real-time PCR were consistent with those of microarray (t=10.74,32.65,18.34, and 2.80; P=0.000 4, 0.000 1, 0.000 1, and 0.044 8). The high expression of BIRC7 and the low expression of MAPK8 were associated with the significant reduction in survival time (P=0.022 0 and 0.005 6). ConclusionBIRC2, BIRC7, ABCC2, and MAPK8 are differentially expressed between anlotinib-resistant hepatoma cells and normal hepatoma cells and may be involved in the resistance of hepatoma cells to anlotinib.

Antiviral effects of Bovine antimicrobial peptide against TGEV in vivo and in vitro.

BACKGROUND: In suckling piglets, transmissible gastroenteritis virus (TGEV) causes lethal diarrhea accompanied by high infection and mortality rates, leading to considerable economic losses. This study explored methods of preventing or inhibiting their production. Bovine antimicrobial peptide-13 (APB-13) has antibacterial, antiviral, and immune functions. OBJECTIVES: This study analyzed the efficacy of APB-13 against TGEV through in vivo and in vitro experiments. METHODS: The effects of APB-13 toxicity and virus inhibition rate on swine testicular (ST) cells were detected using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT). The impact of APB-13 on virus replication was examined through the 50% tissue culture infective dose (TCID50). The mRNA and protein levels were investigated by real-time quantitative polymerase chain reaction and western blot (WB). Tissue sections were used to detect intestinal morphological development. RESULTS: The safe and effective concentration range of APB-13 on ST cells ranged from 0 to 62.5 µg/mL, and the highest viral inhibitory rate of APB-13 was 74.1%. The log10TCID50 of 62.5 µg/mL APB-13 was 3.63 lower than that of the virus control. The mRNA and protein expression at 62.5 µg/mL APB-13 was significantly lower than that of the virus control at 24 hpi. Piglets in the APB-13 group showed significantly lower viral shedding than that in the virus control group, and the pathological tissue sections of the jejunum morphology revealed significant differences between the groups. CONCLUSIONS: APB-13 exhibited good antiviral effects on TGEV in vivo and in vitro.