METTL3 and N6-Methyladenosine Promote Homologous Recombination-Mediated Repair of DSBs by Modulating DNA-RNA Hybrid Accumulation.

Double-strand breaks (DSBs) are the most deleterious DNA lesions, which, if left unrepaired, may lead to genome instability or cell death. Here, we report that, in response to DSBs, the RNA methyltransferase METTL3 is activated by ATM-mediated phosphorylation at S43. Phosphorylated METTL3 is then localized to DNA damage sites, where it methylates the N6 position of adenosine (m6A) in DNA damage-associated RNAs, which recruits the m6A reader protein YTHDC1 for protection. In this way, the METTL3-m6A-YTHDC1 axis modulates accumulation of DNA-RNA hybrids at DSBs sites, which then recruit RAD51 and BRCA1 for homologous recombination (HR)-mediated repair. METTL3-deficient cells display defective HR, accumulation of unrepaired DSBs, and genome instability. Accordingly, depletion of METTL3 significantly enhances the sensitivity of cancer cells and murine xenografts to DNA damage-based therapy. These findings uncover the function of METTL3 and YTHDC1 in HR-mediated DSB repair, which may have implications for cancer therapy.

UBQLN1 deficiency mediates telomere shortening and IPF through interacting with RPA1.

Premature telomere shortening is a known factor correlated to idiopathic pulmonary fibrosis (IPF) occurrence, which is a chronic, progressive, age-related disease with high mortality. The etiology of IPF is still unknown. Here, we found that UBQLN1 plays a key role in telomere length maintenance and is potentially relevant to IPF. UBQLN1 involves in DNA replication by interacting with RPA1 and shuttling it off from the replication fork. The deficiency of UBQLN1 retains RPA1 at replication fork, hinders replication and thus causes cell cycle arrest and genome instability. Especially at telomere regions of the genome, where more endogenous replication stress exists because of G rich sequences, UBQLN1 depletion leads to rapid telomere shortening in HeLa cells. It revealed that UBQLN1 depletion also shortens telomere length at mouse lung and accelerates mouse lung fibrosis. In addition, the UBQLN1 expression level in IPF patients is downregulated and correlated to poor prognosis. Altogether, these results uncover a new role of UBQLN1 in ensuring DNA replication and maintaining telomere stability, which may shed light on IPF pathogenesis and prevention.

Using nomogram scores to predict the early regression of coronary artery aneurysms of Kawasaki disease.

BACKGROUND: Coronary artery aneurysms have been considered the most serious complication of Kawasaki disease. However, some coronary artery aneurysms do regress. Therefore, the ability to predict the expected time of coronary artery aneurysm regression is critical. Herein, we have created a nomogram prediction system to determine the early regression (<1 month) among patients with small to medium coronary artery aneurysms. METHODS: Seventy-six Kawasaki disease patients identified with coronary artery aneurysms during the acute or subacute phase were included. All the patients who met inclusion criteria demonstrated regression of coronary artery aneurysms within the first-year post Kawasaki disease diagnosis. The clinical and laboratory parameters were compared between the groups of coronary artery aneurysms regression duration within and beyond 1 month. Multivariate logistic regression analysis was used to identify the independent parameters for early regression based on the results from the univariable analysis. Then nomogram prediction systems were established with associated receiver operating characteristic curves. RESULTS: Among the 76 included patients, 40 cases recovered within 1 month. Haemoglobin, globulin, activated partial thromboplastin time, the number of lesions, location of the aneurysm, and coronary artery aneurysm size were identified as independent factors for early regression of coronary artery aneurysms in Kawasaki disease patients. The predictive nomogram models revealed a high efficacy in predicting early regression of coronary artery aneurysms. CONCLUSION: The size of coronary artery aneurysms, the number of lesions, and the location of aneurysms presented better predictive value for predicting coronary artery aneurysms regression. The nomogram system created from the identified risk factors successfully predicted early coronary artery aneurysm regression.

Application of deep machine learning for the radiographic diagnosis of periodontitis.

OBJECTIVE: Successful application of deep machine learning could reduce time-consuming and labor-intensive clinical work of calculating the amount of radiographic bone loss (RBL) in diagnosing and treatment planning for periodontitis. This study aimed to test the accuracy of RBL classification by machine learning. MATERIALS AND METHODS: A total of 236 patients with standardized full mouth radiographs were included. Each tooth from the periapical films was evaluated by three calibrated periodontists for categorization of RBL and radiographic defect morphology. Each image was pre-processed and augmented to ensure proper data balancing without data pollution, then a novel multitasking InceptionV3 model was applied. RESULTS: The model demonstrated an average accuracy of 0.87 ± 0.01 in the categorization of mild (< 15%) or severe (≥ 15%) bone loss with fivefold cross-validation. Sensitivity, specificity, positive predictive, and negative predictive values of the model were 0.86 ± 0.03, 0.88 ± 0.03, 0.88 ± 0.03, and 0.86 ± 0.02, respectively. CONCLUSIONS: Application of deep machine learning for the detection of alveolar bone loss yielded promising results in this study. Additional data would be beneficial to enhance model construction and enable better machine learning performance for clinical implementation. CLINICAL RELEVANCE: Higher accuracy of radiographic bone loss classification by machine learning can be achieved with more clinical data and proper model construction for valuable clinical application.

α-Lactalbumin mRNA-LNP Evokes an Anti-Tumor Effect Combined with Surgery in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) has been considered a huge clinical unmet need due to its aggressive progression and highly frequent metastasis. mRNA therapeutics supply a potential and versatile immunotherapy of oncology treatment. Here, we developed α-lactalbumin mRNA-lipid nanoparticles (α-LNP) as a potential therapeutical strategy for TNBC. The α-LNP induced the specific IgG antibodies and activated IFN γ-secreting-T cells in vivo. Additionally, the safety of α-LNP also had been demonstrated in vivo. When vaccinated prior to tumor implantation, α-LNP showed a preventive effect against 4T1 tumor growth and extended the survival of the tumor model by activating the memory immune responses. Furthermore, α-LNP administration in combination with surgical removal of neoplasm effectively inhibited the progression and metastasis in the TNBC model. Taken together, our results indicate that the α-LNP vaccine is a promising novel treatment for both therapeutics and prophylactics in TNBC.

Novel mRNA adjuvant ImmunER enhances prostate cancer tumor-associated antigen mRNA therapy via augmenting T cell activity.

Prostate cancer (PCa) is characterized as a "cold tumor" with limited immune responses, rendering the tumor resistant to immune checkpoint inhibitors (ICI). Therapeutic messenger RNA (mRNA) vaccines have emerged as a promising strategy to overcome this challenge by enhancing immune reactivity and significantly boosting anti-tumor efficacy. In our study, we synthesized Tetra, an mRNA vaccine mixed with multiple tumor-associated antigens, and ImmunER, an immune-enhancing adjuvant, aiming to induce potent anti-tumor immunity. ImmunER exhibited the capacity to promote dendritic cells (DCs) maturation, enhance DCs migration, and improve antigen presentation at both cellular and animal levels. Moreover, Tetra, in combination with ImmunER, induced a transformation of bone marrow-derived dendritic cells (BMDCs) to cDC1-CCL22 and up-regulated the JAK-STAT1 pathway, promoting the release of IL-12, TNF-α, and other cytokines. This cascade led to enhanced proliferation and activation of T cells, resulting in effective killing of tumor cells. In vivo experiments further revealed that Tetra + ImmunER increased CD8+T cell infiltration and activation in RM-1-PSMA tumor tissues. In summary, our findings underscore the promising potential of the integrated Tetra and ImmunER mRNA-LNP therapy for robust anti-tumor immunity in PCa.

Efficacy of Xpert in tuberculosis diagnosis based on various specimens: a systematic review and meta-analysis.

Objective: The GeneXpert MTB/RIF assay (Xpert) is a diagnostic tool that has been shown to significantly improve the accuracy of tuberculosis (TB) detection in clinical settings, with advanced sensitivity and specificity. Early detection of TB can be challenging, but Xpert has improved the efficacy of the diagnostic process. Nevertheless, the accuracy of Xpert varies according to different diagnostic specimens and TB infection sites. Therefore, the selection of adequate specimens is critical when using Xpert to identify suspected TB. As such, we have conducted a meta-analysis to evaluate the effectiveness of Xpert for diagnosis of different TB types using several specimens. Methods: We conducted a comprehensive search of several electronic databases, including PubMed, Embase, the Cochrane Central Register of Controlled Trials, and the World Health Organization clinical trials registry center, covering studies published from Jan 2008 to July 2022. Data were extracted using an adapted version of the Checklist for Critical Appraisal and Data Extraction for Systematic Reviews of Prediction Modeling Studies. Where appropriate, meta-analysis was performed using random-effects models. The risk of bias and level of evidence was assessed using the Quality in Prognosis Studies tool and a modified version of the Grading of Recommendations Assessment, Development, and Evaluation. RStudio was utilized to analyze the results, employing the meta4diag, robvis, and metafor packages. Results: After excluding duplicates, a total of 2163 studies were identified, and ultimately, 144 studies from 107 articles were included in the meta-analysis based on predetermined inclusion and exclusion criteria. Sensitivity, specificity and diagnostic accuracy were estimated for various specimens and TB types. In the case of pulmonary TB, Xpert using sputum (0.95 95%CI 0.91-0.98) and gastric juice (0.94 95%CI 0.84-0.99) demonstrated similarly high sensitivity, surpassing other specimen types. Additionally, Xpert exhibited high specificity for detecting TB across all specimen types. For bone and joint TB, Xpert, based on both biopsy and joint fluid specimens, demonstrated high accuracy in TB detection. Furthermore, Xpert effectively detected unclassified extrapulmonary TB and tuberculosis lymphadenitis. However, the Xpert accuracy was not satisfactory to distinguish TB meningitis, tuberculous pleuritis and unclassified TB. Conclusions: Xpert has exhibited satisfactory diagnostic accuracy for most TB infections, but the efficacy of detection may vary depending on the specimens analyzed. Therefore, selecting appropriate specimens for Xpert analysis is essential, as using inadequate specimens can reduce the ability to distinguish TB. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=370111, identifier CRD42022370111.

Mpox virus mRNA-lipid nanoparticle vaccine candidates evoke antibody responses and drive protection against the Vaccinia virus challenge in mice.

In response to the human Mpox (hMPX) epidemic that began in 2022, there is an urgent need for a monkeypox vaccine. Here, we have developed a series of mRNA-lipid nanoparticle (mRNA-LNP)-based vaccine candidates that encode a collection of four highly conserved Mpox virus (MPXV) surface proteins involved in virus attachment, entry, and transmission, namely A29L, A35R, B6R, and M1R, which are homologs to Vaccinia virus (VACV) A27, A33, B5, and L1, respectively. Despite possible differences in immunogenicity among the four antigenic mRNA-LNPs, administering these antigenic mRNA-LNPs individually (5 µg each) or an average mixture of these mRNA-LNPs at a low dose (0.5 µg each) twice elicited MPXV-specific IgG antibodies and potent VACV-specific neutralizing antibodies. Furthermore, two doses of 5 µg of A27, B5, and L1 mRNA-LNPs or a 2 µg average mixture of the four antigenic mRNA-LNPs protected mice against weight loss and death after the VACV challenge. Overall, our data suggest that these antigenic mRNA-LNP vaccine candidates are both safe and efficacious against MPXV, as well as diseases caused by other orthopoxviruses.

Exome sequencing contributes to identify comorbidities in a rare case of infant ARDS induced by the CD40LG mutation.

BACKGROUND: Acute respiratory distress syndrome (ARDS) causes significant mortality in young children with certain diseases. Early diagnosis and treatment can reduce infant mortality. Here, we report a rare case of exome sequencing in the early diagnosis of immunodeficiency in an infant. CASE PRESENTATION: A four-month-old full-term male infant presented with severe shortness of breath, hypoxemia, and unexplained parenchymal lung lesions. A series of examinations were performed to search for potential culprit viruses but negative results were obtained with the only exception being the rhinovirus that tested positive. The child's family history revealed he had a brother who died of severe infection at the age of two years. We performed an exome sequencing analysis and a mutation analysis of CD40LG to obtain genetic data on the patient. Besides, we used flow cytometry to measure the CD40LG expression levels of activated T cells. A retrospective review of all the CD40LG mutant-induced X-linked hyper IgM syndromes (XHIGM) had been conducted to assess the differences between clinical and genetic molecular features. Finally, a regular intravenous immunoglobulin (IVIG) regimen led to steady breathing, the correction of hypoxemia, and a progressive improvement of lung CT scans. During follow-up, the patient received an IVIG regimen and his CT images improved. Moreover, his parents took advantage of pre-implantation genetic testing with in vitro fertilization to have a healthy twin offspring who did not carry such a mutation according to the early exome sequencing for the proband. Compared with other CD40LG mutant cases in our center, this proband displayed a normal plasma immunoglobulin level and he should be the youngest infant to have a molecular diagnosis of XHIGM. CONCLUSION: Usually, XHIGM would not be suspected with a normal plasma immunoglobulin concentration. However, as we could not identify a potential comorbidity or risk factor, exome sequencing helps target this patient's real facts. Thus, this case report calls for exome sequencing to be performed in the case of unexplained infections when immunodeficiency is suspected after general immunological tests, especially for cases with a contributive family history among infants as the maternal transfused immunoglobulin might mask immune deficiency.

Controversial molecular functions of CBS versus non-CBS domain variants of PRKAG2 in arrhythmia and cardiomyopathy: A case report and literature review.

BACKGROUND: PRKAG2 cardiac syndrome is a rare autosomal dominant genetic disorder caused by a PRKAG2 gene variant. There are several major adverse cardiac presentations, including hypertrophic cardiomyopathy (HCM) and life-threatening arrhythmia. Two cases with pathogenic variants in the PRKAG2 gene are reported here who presents different cardiac phenotypes. METHODS: Exome sequencing and variant analysis of PRKAG2 were performed to obtain genetic data, and clinical characteristics were determined. RESULTS: The first proband was a 9-month-old female infant (Case 1), and was identified with severe DCM and resistant heart failure. The second proband was a 10-year-old female infant (Case 2), and presented with HCM and ventricular preexcitation. Exome sequencing identified a de novo c.425C > T (p.T142I) heterozygous variant in the PRKAG2 gene for Case 1, and a c.869A > T (p.K290I) for Case 2. The mutated sites in the protein were labeled and identified as p.K290 in the CBS domain and p.T142 in the non-CBS domain. Differences in the molecular functions of CBS and non-CBS domains have not been resolved, and variants might lead to the different cardiomyopathy phenotypes. Single-cell RNA analysis demonstrated similar expression levels of PRKAG2 in cardiomyocytes and conductive tissues. These results suggest that the arrhythmia induced by the PRKAG2 variant was the primary change, and not secondary to cardiomyopathy. CONCLUSION: In summary, this is the first case report to describe a DCM phenotype with early onset in patients possessing a PRKAG2 c.425C > T (p.T142I) pathogenic variant. Our results aid in understanding the molecular function of non-CBS variants in terms of the disordered sequence of transcripts. Moreover, we used scRNA-seq to show that electrically conductive cells express a higher level of PRKAG2 than do cardiomyocytes. Therefore, variants in PRKAG2 are expected to also alter the biological function of the conduction system.

SLC10A2 deficiency-induced congenital chronic bile acid diarrhea and stunting.

BACKGROUND: Diarrhea is a common occurrence in children below the age of 5 years. In chronic cases, it induces malnutrition that severely stunts growth. Bile acid diarrhea (BAD), caused by malabsorption of bile acid (BA), is a rare form of chronic diarrhea seldom observed in pediatric patients. Here, we present a clinical report on a novel case of chronic BAD, with severe stunting in an infant, induced by a homozygous mutation of SLC10A2. METHODS: We performed DNA extraction, whole-exome sequencing analysis, and mutation analysis of SLC10A2 to obtain genetic data on the patient. We subsequently analyzed the patient's clinical and genetic data. RESULTS: The patient's clinical manifestations were chronic diarrhea with increased BAs in the feces and extreme stunting, which was diagnosed as BAD. A homozygous mutation of SLC10A2 at the c.313T>C (rs201206937) site was detected. CONCLUSION: Our report reveals the youngest case illustrating the characteristics of BAD induced by genetic variant at 313T>C, and the second case entailing a clear association between a SLC10A2 genetic mutation and the onset of BAD. Our findings expand the mutant spectrum of the SLC10A2 gene and contribute to the refinement of the genotype-phenotype mapping of severe stunting induced by pediatric BAD. Moreover, they highlight the value of molecular genetic screening for diagnosing BAD in young patients.

Protective efficient comparisons among all kinds of respirators and masks for health-care workers against respiratory viruses: A PRISMA-compliant network meta-analysis.

BACKGROUND: There is no definite conclusion about comparison of better effectiveness between N95 respirators and medical masks in preventing health-care workers (HCWs) from respiratory infectious diseases, so that conflicting results and recommendations regarding the protective effects may cause difficulties for selection and compliance of respiratory personal protective equipment use for HCWs, especially facing with pandemics of corona virus disease 2019. METHODS: We systematically searched MEDLINE, Embase, PubMed, China National Knowledge Infrastructure, Wanfang, medRxiv, and Google Scholar from initiation to November 10, 2020 for randomized controlled trials, case-control studies, cohort studies, and cross-sectional studies that reported protective effects of masks or respirators for HCWs against respiratory infectious diseases. We gathered data and pooled differences in protective effects according to different types of masks, pathogens, occupations, concurrent measures, and clinical settings. The study protocol is registered with PROSPERO (registration number: 42020173279). RESULTS: We identified 4165 articles, reviewed the full text of 66 articles selected by abstracts. Six randomized clinical trials and 26 observational studies were included finally. By 2 separate conventional meta-analyses of randomized clinical trials of common respiratory viruses and observational studies of pandemic H1N1, pooled effects show no significant difference between N95 respirators and medical masks against common respiratory viruses for laboratory-confirmed respiratory virus infection (risk ratio 0.99, 95% confidence interval [CI] 0.86-1.13, I2 = 0.0%), clinical respiratory illness (risk ratio 0.89, 95% CI 0.45-1.09, I2 = 83.7%, P = .002), influenza-like illness (risk ratio 0.75, 95% CI 0.54-1.05, I2 = 0.0%), and pandemic H1N1 for laboratory-confirmed respiratory virus infection (odds ratio 0.92, 95% CI 0.49-1.70, I2 = 0.0%, P = .967). But by network meta-analysis, N95 respirators has a significantly stronger protection for HCWs from betacoronaviruses of severe acute respiratory syndrome, middle east respiratory syndrome, and corona virus disease 2019 (odds ratio 0.43, 95% CI 0.20-0.94). CONCLUSIONS: Our results provide moderate and very-low quality evidence of no significant difference between N95 respirators and medical masks for common respiratory viruses and pandemic H1N1, respectively. And we found low quality evidence that N95 respirators had a stronger protective effectiveness for HCWs against betacoronaviruses causative diseases compared to medical masks. The evidence of comparison between N95 respirators and medical masks for corona virus disease 2019 is open to question and needs further study.

Association Between the Circulating Level of 25-Hydroxyvitamin D and Clinical Results After Cardiac Surgery: A Meta-Analysis and Systematic Review.

Background: Vitamin D (VitD) is an important pleiotropic hormone for organ systems. Studies have focused on the level of VitD, especially that of 25-hydroxyvitamin D (25-(OH)-VitD), in patients after cardiac surgery and the relationship between VitD deficiency and adverse outcomes, but the results have been inconsistent. We carried out a meta-analysis to evaluate differences in the 25-(OH)-VitD level before and after cardiac surgery, and evaluated the predictive value of 25-(OH)-VitD level in the clinical outcomes of patients undergoing cardiac surgery. Methods: Studies related to VitD level and cardiac surgery were searched from PubMed, EMBASE, Web of Science, and Cochrane Central Register of Controlled Trials databases from inception to October 2020. We applied the Newcastle-Ottawa Scale to assess the risk of a bias in individual studies. We examined the heterogeneity and publication bias and performed subgroup analyses and sensitivity analyses. Results: Fifteen studies were included in our analysis. The 25-(OH)-VitD level was significantly lower immediately after surgery [stand mean difference (SMD), 0.69; 95%CI (0.1, 1.28), P = 0.023] and 24-h after surgery [0.84; (0.47, 1.21), 0.000] compared with that before surgery. A higher prevalence of 25-(OH)-VitD deficiency was recorded 24 h after surgery [RR, 0.59; 95%CI (0.47, 0.73), P = 0.00]. Pooled results demonstrated a significant relationship between the preoperative 25-(OH)-VitD level and vasoactive-inotropic score (VIS) [SMD, -3.71; 95%CI (-6.32, -1.10); P = 0.005], and patients with 25-(OH)-VitD deficiency revealed a comparatively poor prognosis and severe condition after cardiac surgery [-0.80; (-1.41, -0.19), 0.01]. However, 25-(OH)-VitD deficiency was not associated with the duration of stay in the intensive care unit. Conclusions: Cardiac surgery would leads to deficiency of 25-(OH)-VitD. And the preoperative and postoperative levels of 25-(OH)-VitD are associated with adverse events, which is eligible to work as an indicator to demonstrate clinical outcomes.