Extraction and identification of exosomes from three different sources of human ovarian granulosa cells and analysis of their differential miRNA expression profiles.

OBJECTIVE: As important functional cells in the ovary, ovarian granulosa cells are involved in the regulation of oocyte growth and development and play an important role in the study of female fertility preservation. Based on the importance of granulosa cell functionalism, in this study, we analyzed the exosome secretion capacity of human ovarian granulosa cells (SVOG/KGN-cell line, PGC-primary cells) and the differences in their miRNA expression. METHODS: Cells were identified by hematoxylin-eosin staining (HE) and FSHR immunofluorescence staining; CCK8 and colony-forming assay were performed to compare cell proliferation capacity; exosomes were extracted and identified by ultra-high speed centrifugation, transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot analysis (WB), and the expression profile of each cellular exosomal miRNA was analyzed by miRNA high-throughput sequencing. RESULTS: The proliferative abilities of the three granulosa cells differed, but all had the ability to secrete exosomes. In the exosomes of SVOG, KGN, and PGC cells, 218, 327, and 471 miRNAs were detected, respectively. When compared to the exosomal miRNAs of PGC cells, 111 miRNAs were significantly different in SVOG, and 70 miRNAs were washed two significantly different in KGN cells. These differential miRNA functions were mainly enriched in the cell cycle, cell division/differentiation, multicellular biogenesis, and protein binding. CONCLUSION: Human ovarian granulosa cells of different origins are capable of secreting exosomes, but there are still some differences in their exosomes and exosomal miRNAs, and experimental subjects should be selected rationally according to the actual situation.

Heat inactivation does not alter host plasma cell-free DNA characteristics in infectious disease research.

BACKGROUND: Cell-free DNA (cfDNA) is a promising analyte for non-invasive liquid biopsy, carrying abundant signatures for disease diagnosis and monitoring. In infectious disease researches, blood plasma samples are routinely heat-inactivated before proceeding with downstream analyses. However, the effects of heat inactivation on cfDNA fragmentomic analysis remain largely unclear, potentially introducing biases or altering the characteristics of cfDNA. METHODS: We performed a comprehensive investigation of cfDNA concentrations and fragmentomics in 21 plasma samples from 7 healthy individuals, by comparing the sample group without the heat inactivation to those exposed to once or twice heat-inactivation at 56 °C for 30 min and following freeze-thaw. RESULTS: Plasma samples with once and twice heat inactivation displayed no significant deviations in primary characteristics, including cfDNA concentrations, size profiles, end motif features, and genome-wide distributions, compared to samples without heat treatment. CONCLUSIONS: Heat-inactivated cfDNA can be utilized for liquid biopsy in infectious disease researches, without substantial impact on cfDNA concentrations and fragmentomic properties. This study provides essential insights into the effects of heat inactivation on cfDNA properties and will contribute to the development of reliable non-invasive biomarkers for infectious disease.

The fragmentomic property of plasma cell-free DNA enables the non-invasive detection of diabetic nephropathy in patients with diabetes mellitus.

Background: Diabetic nephropathy (DN) is one of the most prevalent complications of diabetes mellitus (DM). However, there is still a lack of effective methods for non-invasive diagnosis of DN in clinical practice. We aimed to explore biomarkers from plasma cell-free DNA as a surrogate of renal biopsy for the differentiation of DN patients from patients with DM. Materials and methods: The plasma cell-free DNA (cfDNA) was sequenced from 53 healthy individuals, 53 patients with DM but without DN, and 71 patients with both DM and DN. Multidimensional features of plasma DNA were analyzed to dissect the cfDNA profile in the DM and DN patients and identify DN-specific cfDNA features. Finally, a classification model was constructed by integrating all informative cfDNA features to demonstrate the clinical utility in DN detection. Results: In comparison with the DM patients, the DN individuals exhibited significantly increased cfDNA concentration in plasma. The cfDNA from the DN patients showed a distinct fragmentation pattern with an altered size profile and preferred motifs that start with "CC" in the cfDNA ending sites, which were associated with deoxyribonuclease 1 like 3 (DNASE1L3) expression in the kidney. Moreover, patients with DM or DN were found to carry more alterations in whole-genome cfDNA coverage when compared with healthy individuals. We integrated DN-specific cfDNA features (cfDNA concentration, size, and motif) into a classification model, which achieved an area under the receiver operating characteristic curve (AUC) of 0.928 for the differentiation of DN patients from DM patients. Conclusion: Our findings showed plasma cfDNA as a reliable non-invasive biomarker for differentiating DN patients from DM patients. The utility of cfDNA in clinical practice in large prospective cohorts is warranted.

Could the pulp be preserved in tooth with root resorption caused by embedded tooth?

Background/purpose: At present, there are no recognized guidelines or consensus for the treatment strategy of the asymptomatic tooth with external root resorption caused by an embedded tooth (et-ERR). Most clinicians would like prophylactic or concomitant root canal therapy (RCT) along with the extraction of the embedded tooth. The purpose of this study was to report the prognosis of external root resorption (ERR) and investigate the possibility to preserve the vital pulp of ERR tooth. Materials and methods: The patients who had asymptomatic et-ERR teeth were included. After extraction of the embedded tooth, the clinical process, prognosis, and adverse events were observed, including symptoms, clinical, and radiographic examination throughout the follow-up period. Results: A total of four cases with special features were reported. Over a follow-up period of up to 12 months, on clinical examination, 3 ERR teeth preserved pulp vitality without additional intervention except for tooth extraction and have kept normal function free from any symptoms. Radiographic examination showed bone regeneration and recovery of periodontal tissue. While one case failed to keep the vital pulp and ended in intentional replantation. Conclusion: As to et-ERR, if the embedded tooth can be promptly extracted with a minimally invasive technique and effective infection control, the pulp vitality of the et-ERR tooth is likely to be preserved. In this situation, the preferred management of asymptomatic et-ERR tooth is just followed up without prophylactic RCT.

[Mechanism of Liuwei Dihuang Pills in treatment of mice with diminished ovarian reserve based on proteomics].

This study aims to investigate the efficacy and possible mechanism of Liuwei Dihuang Pills in the treatment of diminished ovarian reserve(DOR) by using proteomic techniques. Firstly, cyclophosphamide(60 mg·kg~(-1)) combined with busulfan(6 mg·kg~(-1)) was injected intraperitoneally to establish the mouse model of DOR. After drug injection, the mice were continuously observed and the success of modeling was evaluated by the disturbance of the estrous cycle. After successful modeling, the mice were administrated with the suspension of Liuwei Dihuang Pills by gavage for 28 days. At the end of the gavage, four female mice were selected and caged together with males at a ratio of 2∶1 for the determination of the pregnancy rate. Blood and ovary samples were collected from the remaining mice on the next day after the end of gavage. Hematoxylin-eosin(HE) staining and transmission electron microscopy(TEM) were then employed to observe the morphological and ultrastructural changes in the ovaries. The serum levels of hormones and oxidation indicators were measured by enzyme-linked immunosorbent assay. Quantitative proteomics techniques were used to compare the ovarian protein expression before and after modeling and before and after the intervention with Liuwei Dihuang Pills. The results showed that Liuwei Dihuang Pills regulated the estrous cycle of DOR mice, elevated the serum levels of hormones and anti-oxidation indicators, promoted follicle development, protected the mitochondrial morphology of ovarian granulosa cells, and increased the litter size and survival of DOR mice. Furthermore, Liuwei Dihuang Pills negatively regulated the expression of 12 differentially expressed proteins associated with DOR, which were mainly involved in lipid catabolism, inflammatory response, immune regulation, and coenzyme biosynthesis. These differentially expressed proteins were significantly enriched in sphingolipid metabolism, arachidonic acid metabolism, ribosomes, ferroptosis, and cGMP-PKG signaling pathway. In summary, the occurrence of DOR and the treatment of DOR with Liuwei Dihuang Pills are associated with multiple biological pathways, mainly including oxidative stress response, inflammatory response, and immune regulation. "Mitochondria-oxidative stress-apoptosis" is the key to the treatment of DOR by Liuwei Dihuang Pills. YY1 and CYP4F3 may be the key upstream targets that trigger mitochondrial dysfunction and ROS accumulation, and the metabolism of arachidonic acid is the main signaling pathway of drug action.

Plasma cell-free DNA promise monitoring and tissue injury assessment of COVID-19.

Coronavirus 2019 (COVID-19) is a complex disease that affects billions of people worldwide. Currently, effective etiological treatment of COVID-19 is still lacking; COVID-19 also causes damages to various organs that affects therapeutics and mortality of the patients. Surveillance of the treatment responses and organ injury assessment of COVID-19 patients are of high clinical value. In this study, we investigated the characteristic fragmentation patterns and explored the potential in tissue injury assessment of plasma cell-free DNA in COVID-19 patients. Through recruitment of 37 COVID-19 patients, 32 controls and analysis of 208 blood samples upon diagnosis and during treatment, we report gross abnormalities in cfDNA of COVID-19 patients, including elevated GC content, altered molecule size and end motif patterns. More importantly, such cfDNA fragmentation characteristics reflect patient-specific physiological changes during treatment. Further analysis on cfDNA tissue-of-origin tracing reveals frequent tissue injuries in COVID-19 patients, which is supported by clinical diagnoses. Hence, our work demonstrates and extends the translational merit of cfDNA fragmentation pattern as valuable analyte for effective treatment monitoring, as well as tissue injury assessment in COVID-19.

Prognosis of Second Molars with External Root Resorption Caused by Adjacent Embedded Third Molars.

INTRODUCTION: This prospective study was designed to determine the prognosis of second molars with external root resorption (ERR-M2s) caused by embedded third molars (EM3s) following EM3 removal and explore related predictors. METHODS: This study consecutively enrolled 58 participants who had asymptomatic second molars with apical external root resorption caused by EM3s. EM3s were extracted, and follow-up examinations were conducted at 1 week, 4 weeks, and 6 months. The primary outcome was prognosis of ERR-M2s after third molar extraction without any further intervention, as assessed via clinical and radiographic examinations. Potential predictors (sex, age, number of roots, EM3s position, type and degree of external root resorption) of prognosis were analyzed via univariable and multivariable analyses. RESULTS: A total of 63 ERR-M2s from 58 patients (16 males and 42 females; 19-57 years of age) were evaluated. At the last follow-up, 56 teeth (89%) remained asymptomatic and normal response to heat and cold tests. According to univariable analyses, ERR-M2s were more likely to remain normal response to heat and cold tests in younger patients. In the multivariable analysis, older age (odds ratio, 1.118; 95% confidence interval, 1.026-1.219; P < .05) and the type of all roots affected (odds ratio, 0.073; 95% confidence interval, 0.007-0.754; P < .05) were significantly associated with poor prognosis. CONCLUSIONS: Asymptomatic ERR-M2s have a high probability of remaining normal pulp without further intervention after EM3 extraction, especially in younger patients. No intervention except follow-up and observation is necessary.

Rare Variants in Inborn Errors of Immunity Genes Associated With Covid-19 Severity.

Host genetic factors have been shown to play an important role in SARS-CoV-2 infection and the course of Covid-19 disease. The genetic contributions of common variants influencing Covid-19 susceptibility and severity have been extensively studied in diverse populations. However, the studies of rare genetic defects arising from inborn errors of immunity (IEI) are relatively few, especially in the Chinese population. To fill this gap, we used a deeply sequenced dataset of nearly 500 patients, all of Chinese descent, to investigate putative functional rare variants. Specifically, we annotated rare variants in our call set and selected likely deleterious missense (LDM) and high-confidence predicted loss-of-function (HC-pLoF) variants. Further, we analyzed LDM and HC-pLoF variants between non-severe and severe Covid-19 patients by (a) performing gene- and pathway-level association analyses, (b) testing the number of mutations in previously reported genes mapped from LDM and HC-pLoF variants, and (c) uncovering candidate genes via protein-protein interaction (PPI) network analysis of Covid-19-related genes and genes defined from LDM and HC-pLoF variants. From our analyses, we found that (a) pathways Tuberculosis (hsa:05152), Primary Immunodeficiency (hsa:05340), and Influenza A (hsa:05164) showed significant enrichment in severe patients compared to the non-severe ones, (b) HC-pLoF mutations were enriched in Covid-19-related genes in severe patients, and (c) several candidate genes, such as IL12RB1, TBK1, TLR3, and IFNGR2, are uncovered by PPI network analysis and worth further investigation. These regions generally play an essential role in regulating antiviral innate immunity responses to foreign pathogens and in responding to many inflammatory diseases. We believe that our identified candidate genes/pathways can be potentially used as Covid-19 diagnostic markers and help distinguish patients at higher risk.

Plasma cell-free RNA characteristics in COVID-19 patients.

The pathogenesis of COVID-19 is still elusive, which impedes disease progression prediction, differential diagnosis, and targeted therapy. Plasma cell-free RNAs (cfRNAs) carry unique information from human tissue and thus could point to resourceful solutions for pathogenesis and host-pathogen interactions. Here, we performed a comparative analysis of cfRNA profiles between COVID-19 patients and healthy donors using serial plasma. Analyses of the cfRNA landscape, potential gene regulatory mechanisms, dynamic changes in tRNA pools upon infection, and microbial communities were performed. A total of 380 cfRNA molecules were up-regulated in all COVID-19 patients, of which seven could serve as potential biomarkers (AUC > 0.85) with great sensitivity and specificity. Antiviral (NFKB1A, IFITM3, and IFI27) and neutrophil activation (S100A8, CD68, and CD63)-related genes exhibited decreased expression levels during treatment in COVID-19 patients, which is in accordance with the dynamically enhanced inflammatory response in COVID-19 patients. Noncoding RNAs, including some microRNAs (let 7 family) and long noncoding RNAs (GJA9-MYCBP) targeting interleukin (IL6/IL6R), were differentially expressed between COVID-19 patients and healthy donors, which accounts for the potential core mechanism of cytokine storm syndromes; the tRNA pools change significantly between the COVID-19 and healthy group, leading to the accumulation of SARS-CoV-2 biased codons, which facilitate SARS-CoV-2 replication. Finally, several pneumonia-related microorganisms were detected in the plasma of COVID-19 patients, raising the possibility of simultaneously monitoring immune response regulation and microbial communities using cfRNA analysis. This study fills the knowledge gap in the plasma cfRNA landscape of COVID-19 patients and offers insight into the potential mechanisms of cfRNAs to explain COVID-19 pathogenesis.

Effect of periodontal treatments on blood pressure.

BACKGROUND: An association has been hypothesized between periodontitis and hypertension. Periodontal therapy is believed to reduce systemic inflammatory mediators and increase endothelial function, thus having the potential to prevent and treat hypertension. OBJECTIVES: To assess the effect and safety of different periodontal treatment modalities on blood pressure (BP) in people with chronic periodontitis. SEARCH METHODS: The Cochrane Hypertension Information Specialist searched for randomized controlled trials (RCTs) up to November 2020 in the Cochrane Hypertension Specialised Register, CENTRAL, MEDLINE, Embase, seven other databases, and two clinical trials registries. We contacted the authors of relevant papers regarding further published and unpublished work. SELECTION CRITERIA: RCTs and quasi-RCTs aiming to detect the effect of periodontal treatment on BP were eligible. Participants should have been diagnosed with chronic periodontitis and hypertension (or no hypertension if the study explored the preventive effect of periodontal treatment). Participants in the intervention group should have undergone subgingival scaling and root planing (SRP) and any other type of periodontal treatments, compared with either no periodontal treatment or alternative periodontal treatment in the control group. DATA COLLECTION AND ANALYSIS: We used standard methodological procedures expected by Cochrane for study identification, data extraction, and risk of bias assessment. We used a formal pilot-tested data extraction form for data extraction, and the Cochrane risk of bias tool for risk of bias assessment. We planned the meta-analysis, test for heterogeneity, sensitivity analysis, and subgroup analysis. We assessed the certainty of evidence using GRADE. The primary outcome was change in systolic BP (SBP) and diastolic BP (DBP). MAIN RESULTS: We included eight RCTs. Five had low risk of bias, one had unclear risk of bias, and two had high risk of bias. Four trials compared periodontal treatment with no treatment. We found no evidence of a difference in the short-term change of SBP and DBP for people diagnosed with periodontitis and other cardiovascular diseases except hypertension (very low-certainty evidence). We found no evidence of a difference in long-term changes in SBP (mean difference [MD] -2.25 mmHg, 95% confidence interval [CI] -9.41 to 4.92; P = 0.54; studies = 2, participants = 108; low-certainty evidence) and DBP (MD -2.55 mmHg, 95% CI -6.90 to 1.80; P = 0.25; studies = 2, participants = 103; low-certainty evidence). Concerning people diagnosed with periodontitis, in the short term, two studies of low certainty reported no changes in SBP (MD -0.14 mmHg, 95% CI -4.05 to 3.77; P = 0.94; participants = 294) and DBP (MD -0.15 mmHg, 95% CI -2.47 to 2.17; P = 0.90; participants = 294), and we found no evidence of a difference in SBP and DBP over a long period based on low certainty of evidence. Three studies compared intensive periodontal treatment with supra-gingival scaling. We found no evidence of a difference in changes in SBP and DBP for any length of time in people diagnosed with periodontitis (very low-certainty evidence). In people diagnosed with periodontitis and hypertension, we found one study reporting a significant reduction in the short term in SBP (MD -11.20 mmHg, 95% CI -15.40 to -7.00; P < 0.001; participants = 101; moderate-certainty evidence) and DBP (MD -8.40 mmHg, 95% CI -12.19 to -4.61; P < 0.0001; participants = 101; moderate-certainty evidence). AUTHORS' CONCLUSIONS: We found no evidence of a difference of an impact of periodontal treatments on BP in most comparisons assessed in this review, and given the low certainty of evidence and the lack of relevant studies we could not draw conclusions about the effect of periodontal treatment on BP in people with chronic periodontitis. We found only one study suggesting that periodontal treatment may reduce SBP and DBP over a short period in people with hypertension and chronic periodontitis, but the certainty of evidence was moderate.

A Chinese host genetic study discovered IFNs and causality of laboratory traits on COVID-19 severity.

The COVID-19 pandemic has caused over 220 million infections and 4.5 million deaths worldwide. Current risk factor cannot fully explain the diversity in disease severity. Here, we present a comprehensive analysis of a broad range of patients' laboratory and clinical assessments to investigate the genetic contributions to COVID-19 severity. By performing GWAS analysis, we discovered several concrete associations for laboratory traits and used Mendelian randomization (MR) analysis to further investigate the causality of traits on disease severity. Two causal traits, WBC counts and cholesterol levels, were identified based on MR study, and their functional genes are located at genes MHC complex and ApoE, respectively. Our gene-based analysis and GSEA revealed four interferon pathways, including type I interferon receptor binding and SARS coronavirus and innate immunity. We hope that our work will contribute to studying the genetic mechanisms of disease and serve as a useful reference for COVID-19 diagnosis and treatment.

Transcriptional Start Site Coverage Analysis in Plasma Cell-Free DNA Reveals Disease Severity and Tissue Specificity of COVID-19 Patients.

Symptoms of coronavirus disease 2019 (COVID-19) range from asymptomatic to severe pneumonia and death. A deep understanding of the variation of biological characteristics in severe COVID-19 patients is crucial for the detection of individuals at high risk of critical condition for the clinical management of the disease. Herein, by profiling the gene expression spectrum deduced from DNA coverage in regions surrounding transcriptional start site in plasma cell-free DNA (cfDNA) of COVID-19 patients, we deciphered the altered biological processes in the severe cases and demonstrated the feasibility of cfDNA in measuring the COVID-19 progression. The up- and downregulated genes in the plasma of severe patient were found to be closely related to the biological processes and functions affected by COVID-19 progression. More importantly, with the analysis of transcriptome data of blood cells and lung cells from control group and cases with severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection, we revealed that the upregulated genes were predominantly involved in the viral and antiviral activity in blood cells, reflecting the intense viral replication and the active reaction of immune system in the severe patients. Pathway analysis of downregulated genes in plasma DNA and lung cells also demonstrated the diminished adenosine triphosphate synthesis function in lung cells, which was evidenced to correlate with the severe COVID-19 symptoms, such as a cytokine storm and acute respiratory distress. Overall, this study revealed tissue involvement, provided insights into the mechanism of COVID-19 progression, and highlighted the utility of cfDNA as a noninvasive biomarker for disease severity inspections.

Anxiety and oral lichen planus.

OBJECTIVES: To investigate the association between oral lichen planu(OLP) and anxiety. SUBJECTS AND METHODS: This study included 174 OLP patients and 174 healthy controls. We assessed anxiety by Self-rating Anxiety Scale (SAS) and recorded OLP lesion type and severity. t test and analysis of variance were applied for continuous variants and chi-square test was performed for categorical variants. Multiple linear regression and logistic regression analysis were used for multi-variable analysis. RESULTS: he SAS score of OLP patients was higher than that of healthy individuals. There was no significant difference in SAS score between the OLP subgroups, obtained according to age, type, and severity, respectively. Multiple linear regression analysis showed gender was the only factor that affected the SAS score of OLP patients. Compared with weakly anxiety-related groups, the SAS score and female ratio of highly anxiety-related group were obviously higher. Logistic regression analysis demonstrated that males were less exposed to highly anxiety-related types than females. CONCLUSIONS: OLP patients tend to be more anxious compared with healthy individuals, and female patients are more anxious than male patients. There might be two types of OLP patients: weakly anxiety-related or highly anxiety-related. These results highlight the significance of psychological counseling in OLP disease management.

[Research progress in the relationship between Veillonella and oral diseases].

Veillonella species, known as the early colonizer of oral biofilm, are prevalent in oral microbiota. Seven Veillonella species have been isolated from oral cavity. Their distribution varies not only with different people but also with different sites in the oral cavity. Oral Veillonella are associated with oral diseases. They contribute to the adhesion of Streptococcus mutans and consume the lactate generated by streptococci. Veillonella species play an important role in the occurrence and development of periodontal diseases by providing adhesion sites for Porphyromonas gingivalis and boosting immune responses. The production of lipopolysaccharide and H2S is related to other oral diseases, such as pulpitis, periapical periodontitis, and halitosis. Several studies have been conducted on the relationship between Veillonella and oral diseases and the interaction between Veillonella and other pathological microorganisms, but limited knowledge is available at the molecular level. This article reviews the research progress in the relationship between Veillonella and oral infectious diseases, such as dental caries and periodontal diseases.

[Association of CACNA1C gene genetic polymorphism with the susceptibility as well as prognosis for chronic spontaneous urticaria].

OBJECTIVE: To investigate the relationship between single nucleotide polymorphisms (SNPs) of CACNA1C (SNPs rs58619945, rs7316246 and rs216008) and susceptibility of chronic spontaneous urticaria (CSU) as well as the curative effect of non-sedating antihistamine drugs.
 Methods: Peripheral blood were extracted from 191 CSU patients to collect DNA. Urticaria Activity Score 7 (UAS7) and Dermatology Life Quality Index (DLQI) changes were collected from these patients with different non-sedating antihistamine drugs. PubMed retrieval system was used to select the 3 SNPs (rs58619945, rs7316246 and rs216008) of CACNA1C. Susceptibility of CSU and curative effect of non-sedating antihistamine drugs (desloratadine, mizolastine, fisofenadine) in 189 CSU patients and 105 controls with different SNPs were compared with Chi-squared test. Data of 105 southern Chinese controls were extracted from the 1 000 genome database.
 Results: Frequency of rs58619945 G allele in the CSU patients was significantly higher than that in the controls [OR(95%CI)=0.660(0.470-0.925), P=0.016]. However, there was no significant differences in rs7316246 and rs216008 between the CSU patients and the controls. Meanwhile there was no significant difference in general curative effect of the 3 drugs in the 3 SNPs (rs58619945: OR=0.843, P=0.454; rs7316246: OR=2.103, P=0.102; rs216008: OR=0.237, P=0.363). There was significant difference in different alleles of rs216008 in the patients administered by desloratadine [OR(95%CI)=0.480(0.247-0.933), P=0.029]. No difference was shown in the 3 SNPs in patients administered by mizolastine.
 Conclusion: The rs58619945 A/G might be related to susceptibility of CSU, and the rs216008 mutation might affect drug response of desloratadine.