Complexities of mammalian transcriptome revealed by targeted RNA enrichment techniques.

Studies using highly sensitive targeted RNA enrichment methods have shown that a large portion of the human transcriptome remains to be discovered and that most of the genome is transcribed in a complex, interleaved fashion characterized by a complex web of transcripts emanating from protein coding and noncoding loci. These results resonate with those from single-cell transcriptome profiling endeavors that reveal the existence of multiple novel, cell type-specific transcripts and clearly demonstrate that our understanding of the complexities of the human transcriptome is far from being complete. Here, we review the current status of the targeted RNA enrichment techniques, their application to the discovery of novel cell type-specific transcripts, and their impact on our understanding of the human genome and transcriptome.

Co(II) Metal-Organic Complex: Fluorescence Performances and Loaded With Drug Vitamin B2 Hydrogels Against Recurrent Oral Ulcers.

A new coordination polymer (CP) based on Co(II), namely, {[Co3(L)2(4,4'-bipy)(DMA)2]·H2O}n (1) has been synthesized after reacting Co(NO3)2·6H2O with H3L ligand in the existence of N-donor ligand 4,4'-bipyridine (4,4'-bipy), via utilizing a flexible tricarboxylic acid ligand 5-((formic acid-3-sulfur)methyl)isophthalic acid (H3L) with -S-CH2- joint. Additionally, the excellent blue fluorescence properties of CP 1 were confirmed through fluorescence spectroscopy compared to the original ligand. Using natural polysaccharide hyaluronic acid (HA) and carboxymethyl chitosan (CMCS) as raw materials, HA/CMCS hydrogel was prepared by chemical synthesis method. Taking vitamin B2 as the drug model, we designed and synthesized gels loaded with vitamin B2 metal framework and evaluated their efficacy in the treatment of recurrent oral ulcer.

Evidence for widespread existence of functional novel and non-canonical human transcripts.

BACKGROUND: Fraction of functional sequence in the human genome remains a key unresolved question in Biology and the subject of vigorous debate. While a plethora of studies have connected a significant fraction of human DNA to various biochemical processes, the classical definition of function requires evidence of effects on cellular or organismal fitness that such studies do not provide. Although multiple high-throughput reverse genetics screens have been developed to address this issue, they are limited to annotated genomic elements and suffer from non-specific effects, arguing for a strong need to develop additional functional genomics approaches. RESULTS: In this work, we established a high-throughput lentivirus-based insertional mutagenesis strategy as a forward genetics screen tool in aneuploid cells. Application of this approach to human cell lines in multiple phenotypic screens suggested the presence of many yet uncharacterized functional elements in the human genome, represented at least in part by novel exons of known and novel genes. The novel transcripts containing these exons can be massively, up to thousands-fold, induced by specific stresses, and at least some can represent bi-cistronic protein-coding mRNAs. CONCLUSIONS: Altogether, these results argue that many unannotated and non-canonical human transcripts, including those that appear as aberrant splice products, have biological relevance under specific biological conditions.

Lessons from discovery of true ADAR RNA editing sites in a human cell line.

BACKGROUND: Conversion or editing of adenosine (A) into inosine (I) catalyzed by specialized cellular enzymes represents one of the most common post-transcriptional RNA modifications with emerging connection to disease. A-to-I conversions can happen at specific sites and lead to increase in proteome diversity and changes in RNA stability, splicing, and regulation. Such sites can be detected as adenine-to-guanine sequence changes by next-generation RNA sequencing which resulted in millions reported sites from multiple genome-wide surveys. Nonetheless, the lack of extensive independent validation in such endeavors, which is critical considering the relatively high error rate of next-generation sequencing, leads to lingering questions about the validity of the current compendiums of the editing sites and conclusions based on them. RESULTS: Strikingly, we found that the current analytical methods suffer from very high false positive rates and that a significant fraction of sites in the public databases cannot be validated. In this work, we present potential solutions to these problems and provide a comprehensive and extensively validated list of A-to-I editing sites in a human cancer cell line. Our findings demonstrate that most of true A-to-I editing sites in a human cancer cell line are located in the non-coding transcripts, the so-called RNA 'dark matter'. On the other hand, many ADAR editing events occurring in exons of human protein-coding mRNAs, including those that can recode the transcriptome, represent false positives and need to be interpreted with caution. Nonetheless, yet undiscovered authentic ADAR sites that increase the diversity of human proteome exist and warrant further identification. CONCLUSIONS: Accurate identification of human ADAR sites remains a challenging problem, particularly for the sites in exons of protein-coding mRNAs. As a result, genome-wide surveys of ADAR editome must still be accompanied by extensive Sanger validation efforts. However, given the vast number of unknown human ADAR sites, there is a need for further developments of the analytical techniques, potentially those that are based on deep learning solutions, in order to provide a quick and reliable identification of the editome in any sample.

Understanding the impact of chronic diseases on COVID-19 vaccine hesitancy using propensity score matching: Internet-based cross-sectional study.

AIMS AND OBJECTIVES: To investigate whether chronic diseases are associated with higher COVID-19 vaccine hesitancy and explore factors that influence COVID-19 vaccine hesitancy in patients with chronic diseases. BACKGROUND: Vaccine hesitancy has been acknowledged as one of the greatest hazards to public health. However, little information is available about COVID-19 vaccine hesitancy among patients with chronic diseases who may be more susceptible to COVID-19 infection, severe disease or death. METHODS: From 6 to 9 August 2021, we performed an internet-based cross-sectional survey with 22,954 participants (14.78% participants with chronic diseases). Propensity score matching with 1:1 nearest neighbourhood was used to reduce confounding factors between patients with chronic diseases and the general population. Using a multivariable logistic regression model, the factors impacting COVID-19 vaccine hesitancy were identified among patients with chronic diseases. RESULTS: Both before and after propensity score matching, patients with chronic diseases had higher COVID-19 vaccine hesitancy than the general population. In addition, self-reported poor health, multiple chronic diseases, lower sociodemographic backgrounds and lower trust in nurses and doctors were associated with COVID-19 vaccine hesitancy among patients with chronic diseases. CONCLUSIONS: Patients with chronic diseases were more hesitant about the COVID-19 vaccine. Nurses should focus on patients with chronic diseases with poor health conditions, low socioeconomic backgrounds and low trust in the healthcare system. RELEVANCE TO CLINICAL PRACTICE: Clinical nurses are recommended to not only pay more attention to the health status and sociodemographic characteristics of patients with chronic diseases but also build trust between nurses and patients by improving service levels and professional capabilities in clinical practice. PATIENT OR PUBLIC CONTRIBUTION: Patients or the public were not involved in setting the research question, the outcome measures, or the design or implementation of the study. However, all participants were invited to complete the digital informed consent and questionnaires.

Hovlinc is a recently evolved class of ribozyme found in human lncRNA.

Although naturally occurring catalytic RNA molecules-ribozymes-have attracted a great deal of research interest, very few have been identified in humans. Here, we developed a genome-wide approach to discovering self-cleaving ribozymes and identified a naturally occurring ribozyme in humans. The secondary structure and biochemical properties of this ribozyme indicate that it belongs to an unidentified class of small, self-cleaving ribozymes. The sequence of the ribozyme exhibits a clear evolutionary path, from its appearance between ~130 and ~65 million years ago (Ma), to acquiring self-cleavage activity very recently, ~13-10 Ma, in the common ancestors of humans, chimpanzees and gorillas. The ribozyme appears to be functional in vivo and is embedded within a long noncoding RNA belonging to a class of very long intergenic noncoding RNAs. The presence of a catalytic RNA enzyme in lncRNA creates the possibility that these transcripts could function by carrying catalytic RNA domains.

Dynamic Patterns of Mammalian Mitochondrial DNA Replication Uncovered Using SSiNGLe-5'ES.

The proper replication of mitochondrial DNA is key to the maintenance of this crucial organelle. Multiple studies aimed at understanding the mechanisms of replication of the mitochondrial genome have been conducted in the past several decades; however, while highly informative, they were conducted using relatively low-sensitivity techniques. Here, we established a high-throughput approach based on next-generation sequencing to identify replication start sites with nucleotide-level resolution and applied it to the genome of mitochondria from different human and mouse cell types. We found complex and highly reproducible patterns of mitochondrial initiation sites, both previously annotated and newly discovered in this work, that showed differences among different cell types and species. These results suggest that the patterns of the replication initiation sites are dynamic and might reflect, in some yet unknown ways, the complexities of mitochondrial and cellular physiology. Overall, this work suggests that much remains unknown about the details of mitochondrial DNA replication in different biological states, and the method established here opens up a new avenue in the study of the replication of mitochondrial and potentially other genomes.

Genome-Wide Profiling of Endogenous Single-Stranded DNA Using the SSiNGLe-P1 Method.

Endogenous single-stranded DNA (essDNA) can form in a mammalian genome as the result of a variety of molecular processes and can both play important roles inside the cell as well as have detrimental consequences to genome integrity, much of which remains to be fully understood. Here, we established the SSiNGLe-P1 approach based on limited digestion by P1 endonuclease for high-throughput genome-wide identification of essDNA regions. We applied this method to profile essDNA in both human mitochondrial and nuclear genomes. In the mitochondrial genome, the profiles of essDNA provide new evidence to support the strand-displacement model of mitochondrial DNA replication. In the nuclear genome, essDNA regions were found to be enriched in certain types of functional genomic elements, particularly, the origins of DNA replication, R-loops, and to a lesser degree, in promoters. Furthermore, interestingly, many of the essDNA regions identified by SSiNGLe-P1 have not been annotated and thus could represent yet unknown functional elements.

Very long intergenic non-coding (vlinc) RNAs directly regulate multiple genes in cis and trans.

BACKGROUND: The majority of the human genome is transcribed in the form of long non-coding (lnc) RNAs. While these transcripts have attracted considerable interest, their molecular mechanisms of function and biological significance remain controversial. One of the main reasons behind this lies in the significant challenges posed by lncRNAs requiring the development of novel methods and concepts to unravel their functionality. Existing methods often lack cross-validation and independent confirmation by different methodologies and therefore leave significant ambiguity as to the authenticity of the outcomes. Nonetheless, despite all the caveats, it appears that lncRNAs may function, at least in part, by regulating other genes via chromatin interactions. Therefore, the function of a lncRNA could be inferred from the function of genes it regulates. In this work, we present a genome-wide functional annotation strategy for lncRNAs based on identification of their regulatory networks via the integration of three distinct types of approaches: co-expression analysis, mapping of lncRNA-chromatin interactions, and assaying molecular effects of lncRNA knockdowns obtained using an inducible and highly specific CRISPR/Cas13 system. RESULTS: We applied the strategy to annotate 407 very long intergenic non-coding (vlinc) RNAs belonging to a novel widespread subclass of lncRNAs. We show that vlincRNAs indeed appear to regulate multiple genes encoding proteins predominantly involved in RNA- and development-related functions, cell cycle, and cellular adhesion via a mechanism involving proximity between vlincRNAs and their targets in the nucleus. A typical vlincRNAs can be both a positive and negative regulator and regulate multiple genes both in trans and cis. Finally, we show vlincRNAs and their regulatory networks potentially represent novel components of DNA damage response and are functionally important for the ability of cancer cells to survive genotoxic stress. CONCLUSIONS: This study provides strong evidence for the regulatory role of the vlincRNA class of lncRNAs and a potentially important role played by these transcripts in the hidden layer of RNA-based regulation in complex biological systems.

Death by histone deacetylase inhibitor quisinostat in tongue squamous cell carcinoma via apoptosis, pyroptosis, and ferroptosis.

Tongue cancer is one of the most common oral malignancies. Quisinostat is a histone deacetylase inhibitor with antitumor activity. The aim of this study was to evaluate the effects of quisinostat on the viability of tongue squamous cell carcinoma (TSCC) cells (CAL-27, TCA-8113) in vitro and in vivo. Cell viability, cell morphological observation, scratch wound-healing assay, transwell migration assay, transmission electron microscope, flow cytometry and cellular reactive oxygen species were assessed in vitro. The results showed that quisinostat can significantly inhibit the viability, growth and migration of TSCC cells. And quisinostat could significantly induce TSCC cells apoptosis, pyroptosis, and ferroptosis. Quisinostat significantly inhibited tumor tissue growth in animal experiments. Up-regulation of the expression of Bax, cleaved-caspase3, caspase-1, p53, phospho-p53 and down-regulated of the expression of caspase-3, Bcl-2, GPX4 in cell lines and tumor tissues of nude mice were observed by Western blotting analysis. Up-regulation of the expression of caspase-1, Bax, cleaved-caspase3, p53 and down-regulated of the expression of ki67, caspase-3, Bcl-2, GPX4 in tumor tissues of nude mice were observed by immunohistochemistry. TUNEL analysis showed that quisinostat could increase the apoptosis rate in the tumor tissues of nude mice. Up-regulation of the expression of p53 and down-regulated expression of GPX4 in cell lines were observed by immunofluorescent staining, and the expression locations of p53 and GPX4 proteins in TSCC cells were observed. Based on these findings, quisinostat may be a potential drug for the treatment of tongue squamous cell carcinoma.

Asymmetric synthesis of 9-alkyl tetrahydroxanthenones via tandem asymmetric Michael/cyclization promoted by chiral phosphoric acid.

A tandem asymmetric Michael-addition/cyclization of cyclic 1,3-dicarbonyl compounds to ß,γ-unsaturated α-ketoesters catalyzed by chiral phosphoric acid is presented. This protocol provides a facile approach for the construction of enantioenriched 9-alkyl tetrahydroxanthenones, an ubiquitous framework found in a number of natural products and pharmaceutical molecules, in high yields with good to high enantioselectivities.

Characterization of high- and low-molecular-weight glutenin subunits from Chinese Xinjiang wheat landraces and historical varieties.

Landraces and historical varieties are necessary germplasms for genetic improvement of modern cereals. Allelic variations at the Glu-1 and Glu-3 loci in 300 common wheat landraces and 43 historical varieties from Xinjiang, China, were evaluated by Sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and allele-specific molecular markers. Among the materials investigated, three, nine, and seven alleles were identified from the Glu-A1, Glu-B1, and Glu-D1 loci, respectively, and a total of 26 high-molecular-weight glutenin subunit (HMW-GS) combinations were found, of which 18 combinations were identified in landraces and historical varieties. Allelic frequency of HMW-GS combinations null, 7 + 8, 2 + 12 was found to be the highest in both the landraces (63.3%) and historical varieties (39.5%). Besides, some distinctive HMW-GS alleles, such as the novel Glu-B1 allele 6.1* + 8.1* and Glu-D1 alleles 2.6 + 12, 2.1 + 10.1, and 5** + 10 were observed in Xinjiang wheat landraces. Among the Glu-A3 and Glu-B3 loci of landraces and historical varieties, a total of eight and nine alleles were found, respectively. At each locus, two novel alleles were identified. A total of 33 low-molecular-weight glutenin subunit (LMW-GS) combinations of Glu-A3 and Glu-B3 were identified, with 31 and 14 combinations occurring in landraces and historical varieties, respectively, but only 10 combinations shared by both of them. As Glu-D1, Glu-A3, and Glu-B3 have highest contribution to the end-use quality and processing properties as compared to Glu-A1, Glu-B1, and Glu-D3 locus, the novel or distinctive HMW-GS and LMW-GS alleles in these loci could potentially be utilized for the improvement in the quality of modern wheat.

Reliable multiplex sequencing with rare index mis-assignment on DNB-based NGS platform.

BACKGROUND: Massively-parallel-sequencing, coupled with sample multiplexing, has made genetic tests broadly affordable. However, intractable index mis-assignments (commonly exceeds 1%) were repeatedly reported on some widely used sequencing platforms. RESULTS: Here, we investigated this quality issue on BGI sequencers using three library preparation methods: whole genome sequencing (WGS) with PCR, PCR-free WGS, and two-step targeted PCR. BGI's sequencers utilize a unique DNA nanoball (DNB) technology which uses rolling circle replication for DNA-nanoball preparation; this linear amplification is PCR free and can avoid error accumulation. We demonstrated that single index mis-assignment from free indexed oligos occurs at a rate of one in 36 million reads, suggesting virtually no index hopping during DNB creation and arraying. Furthermore, the DNB-based NGS libraries have achieved an unprecedentedly low sample-to-sample mis-assignment rate of 0.0001 to 0.0004% under recommended procedures. CONCLUSIONS: Single indexing with DNB technology provides a simple but effective method for sensitive genetic assays with large sample numbers.

Thermomechanical response of copper films irradiated by femtosecond-pulsed lasers with dynamic optical properties.

By taking into account the dynamic thermophysical and optical properties, the ultrafast thermoelastic response of thin copper film irradiated by femtosecond lasers has been researched. The temperature and stress fields of copper film irradiated by femtosecond lasers are analyzed in this work. The simulation results reveal that the degree of thermomechanical response is much underestimated, especially with higher laser fluence and smaller pulse duration. It is necessary to employ dynamic properties in ultrafast thermoelastic simulation for accuracy.

Coresistance to Benzalkonium Chloride Disinfectant and Heavy Metal Ions in Listeria monocytogenes and Listeria innocua Swine Isolates from China.

The development of coresistance to disinfectants and heavy metals contributes to the fitness of Listeria spp. in foods or food processing environments, where life-threatening Listeria monocytogenes coexist and coevolve with other Listeria spp. Despite extensive research on L. monocytogenes, coresistance to disinfectants and heavy metals is less documented for other Listeria spp. In this study, we screened 30 L. monocytogenes and 27 Listeria innocua isolates recovered from 273 swine samples for resistance to quaternary ammonium compound benzalkonium chloride (BC) and to heavy metals cadmium (Cd) and arsenic (As). Moreover, we evaluated the potential mechanisms of resistance by detecting the efflux pump activity in BC resistance and the presence of resistance determinants. The average minimum inhibitory concentrations of BC in L. innocua (10.7 ± 2.0) were significantly higher than that in L. monocytogenes (6.9 ± 3.7) (p < 0.05). Resistance to BC and heavy metals was correlated, where all BC-resistant L. innocua and As-resistant L. monocytogenes isolates were coresistant to BC and Cd. Twenty percent and 66.7% of BC resistance in L. monocytogenes and L. innocua were related to reserpine-associated efflux pumps, whereas all cases of BC resistance were related to carbonyl cyanide 3-chlorophenylhydrazone-associated efflux pumps. The cadA1 and cadA2 genes were present in Cd-resistant isolates but not in Cd-sensitive isolates, and cadA3 was undetectable in all isolates examined. cadA4 conferring lower level of Cd resistance was copresent with arsA1 and arsA2 in the Cd-resistant and As-susceptible L. monocytogenes isolate LM3. Our findings suggest that swine serves as a reservoir for developing resistance to disinfectant and heavy metals in L. monocytogenes and L. innocua, which share common resistance mechanisms such as efflux pumps and resistance genes. This work provides new insight into the coresistance events of other Listeria as a potential contributor of the resistance in L. monocytogenes.

Analysis of novel high-molecular-weight prolamins from Leymus multicaulis (Kar. et Kir.) Tzvelev and L. chinensis (Trin. ex Bunge) Tzvelev.

Nine novel high-molecular-weight prolamins (HMW-prolamins) were isolated from Leymus multicaulis and L. chinensis. Based on the structure of the repetitive domains, all nine genes were classified as D-hordeins but not high-molecular-weight glutenin subunits (HMW-GSs) that have been previously isolated in Leymus spp. Four genes, Lmul 1.2, 2.4, 2.7, and Lchi 2.5 were verified by bacterial expression, whereas the other five sequences (1.3 types) were classified as pseudogenes. The four Leymus D-hordein proteins had longer N-termini than those of Hordeum spp. [116/118 vs. 110 amino acid (AA) residues], whereas three (Lmul 1.2, 2.4, and 2.7) contained shorter N-termini than those of the Ps. juncea (116 vs. 118 AA residues). Furthermore, Lmul 1.2 was identified as the smallest D-hordein, and Lmul 1.2 and 2.7 had an additional cysteines. Phylogenetic analysis supported that the nine D-hordeins of Leymus formed two independent clades, with all the 1.3 types clustered with Ps. juncea Ns 1.3, whereas the others were clustered together with the D-hordeins from Hordeum and Ps. juncea and the HMW-GSs from Leymus. Within the clade of four D-hordein genes and HMW-GSs, the HMW-GSs of Leymus formed a separated branch that served as an intermediate between the D-hordeins of Ps. juncea and Leymus. These novel D-hordeins may be potentially utilized in the improvement of food processing properties particularly those relating to extra cysteine residues. The findings of the present study also provide basic information for understanding the HMW-prolamins among Triticeae species, as well as expand the sources of D-hordeins from Hordeum to Leymus.

YUCCA9-Mediated Auxin Biosynthesis and Polar Auxin Transport Synergistically Regulate Regeneration of Root Systems Following Root Cutting.

Recovery of the root system following physical damage is an essential issue for plant survival. An injured root system is able to regenerate by increases in lateral root (LR) number and acceleration of root growth. The horticultural technique of root pruning (root cutting) is an application of this response and is a common garden technique for controlling plant growth. Although root pruning is widely used, the molecular mechanisms underlying the subsequent changes in the root system are poorly understood. In this study, root pruning was employed as a model system to study the molecular mechanisms of root system regeneration. Notably, LR defects in wild-type plants treated with inhibitors of polar auxin transport (PAT) or in the auxin signaling mutant auxin/indole-3-acetic acid19/massugu2 were recovered by root pruning. Induction of IAA19 following root pruning indicates an enhancement of auxin signaling by root pruning. Endogenous levels of IAA increased after root pruning, and YUCCA9 was identified as the primary gene responsible. PAT-related genes were induced after root pruning, and the YUCCA inhibitor yucasin suppressed root regeneration in PAT-related mutants. Therefore, we demonstrate the crucial role of YUCCA9, along with other redundant YUCCA family genes, in the enhancement of auxin biosynthesis following root pruning. This further enhances auxin transport and activates downstream auxin signaling genes, and thus increases LR number.

A comprehensive evaluation of regional water safety systems based on a similarity cloud model.

Regional water safety systems are affected by social, economic, ecological, hydrological and other factors, and their effects are complicated and variable. Studying water safety systems is crucial to promoting the coordinated development of regional water safety systems and anthropogenic processes. Thus, a similarity cloud model is developed to simulate the evolution mechanisms of fuzzy and complex regional systems of water security and overcome the uncertainty that is associated with the indices that are used in water safety index systems. This cloud generator is used to reciprocally transform a qualitative cloud image with a quantitative cloud characteristic value, and the stochastic weight assignment method is used to determine the weight of the evaluation indices. The results of case studies show that Jiansanjiang's water safety systems were in a safe state in 2002-2011, but the water safety systems in the arid area of Yinchuan City were in a dangerous state in 2006-2007 because of climate factors and a lack of effective water and soil resource protection. The experimental results are consistent with the research subjects' actual situations, and the proposed model provides a tool for decision makers to better understand the security issues that are associated with regional water safety systems.

Functionally conservative substitutions at cardiac troponin I S43/45.

A phospho-null Ala substitution at protein kinase C (PKC)-targeted cardiac troponin I (cTnI) S43/45 reduces myocyte and cardiac contractile function. The goal of the current study was to test whether cTnIS43/45N is an alternative, functionally conservative substitution in cardiac myocytes. Partial and more extensive endogenous cTnI replacement was similar at 2 and 4 days after gene transfer, respectively, for epitope-tagged cTnI and cTnIS43/45N. This replacement did not significantly change thin filament stoichiometry. In functional studies, there were no significant changes in the amplitude and/or rates of contractile shortening and re-lengthening after this partial (2 days) and extensive (4 days) replacement with cTnIS43/45N. The cTnIS43/45N substitution also was not associated with adaptive changes in the myocyte Ca(2+) transient or in phosphorylation of the protein kinase A and C-targeted cTnIS23/24 site. These results provide evidence that cTnIS43/45N is a functionally conservative substitution, and may be appropriate for use as a phospho-null in rodent models designed for studies on PKC modulation of cardiac performance.

Developing an AI-assisted digital auscultation tool for automatic assessment of the severity of mitral regurgitation: protocol for a cross-sectional, non-interventional study.

INTRODUCTION: Mitral regurgitation (MR) is the most common valvular heart disorder, with a morbidity rate of 2.5%. While echocardiography is commonly used in assessing MR, it has many limitations, especially for large-scale MR screening. Cardiac auscultation with electronic stethoscope and artificial intelligence (AI) can be a fast and economical modality for assessing MR severity. Our objectives are (1) to establish a deep neural network (DNN)-based cardiac auscultation method for assessing the severity of MR; and (2) to quantitatively measure the performance of the developed AI-based MR assessment method by virtual clinical trial. METHODS AND ANALYSIS: In a cross-sectional design, phonocardiogram will be recorded at the mitral valve auscultation area of outpatients. The enrolled patients will be checked by echocardiography to confirm the diagnosis of MR or no MR. Echocardiographic parameters will be used as gold standard to assess the severity of MR, classified into four levels: none, mild, moderate and severe. The study consists of two stages. First, an MR-related cardiac sound database will be created on which a DNN-based MR severity classifier will be trained. The automatic MR severity classifier will be integrated with the Smartho-D2 electronic stethoscope. Second, the performance of the developed smart device will be assessed in an independent clinical validation data set. Sensitivity, specificity, precision, accuracy and F1 score of the developed smart MR assessment device will be evaluated. Agreement on the performance of the smart device between cardiologist users and patient users will be inspected. The interpretability of the developed model will also be studied with statistical comparisons of occlusion map-guided variables among the four severity groups. ETHICS AND DISSEMINATION: The study protocol was approved by the Medical Ethics Committee of Huzhou Central Hospital, China (registration number: 202302009-01). Informed consent is required from all participants. Dissemination will be through conference presentations and peer-reviewed journals. TRIAL REGISTRATION NUMBER: ChiCTR2300069496.