A Programmable Microfluidic Paper-Based Analytical Device for Simultaneous Colorimetric and Photothermal Visual Sensing of Multiple Enzyme Activities.

New strategies for the simultaneous and portable detection of multiple enzyme activities are highly desirable for clinical diagnosis and home care. However, the methods developed thus far generally suffer from high costs, cumbersome procedures, and heavy reliance on large-scale instruments. To satisfy the actual requirements of rapid, accurate, and on-site detection of multiple enzyme activities, we report herein a smartphone-assisted programmable microfluidic paper-based analytical device (µPAD) that utilizes colorimetric and photothermal signals for simultaneous, accurate, and visual quantitative detection of alkaline phosphatase (ALP) and butyrylcholinesterase (BChE). Specifically, the operation of this µPAD sensing platform is based on two sequential steps. Cobalt-doped mesoporous cerium oxide (Co-m-CeO2) with remarkable peroxidase-like activities under neutral conditions first catalytically decomposes H2O2 for effectively converting colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxidized TMB (oxTMB). The subsequent addition of ALP or BChE to their respective substrates produces a reducing substance that can somewhat inhibit the oxTMB transformation for compromised colorimetric and photothermal signals of oxTMB. Notably, these two-step bioenzyme-nanozyme cascade reactions strongly support the straightforward and excellent processability of this platform, which exhibit lower detection limits for ALP and BChE with a detection limit for BChE an order of magnitude lower than those of the other reported paper-based detection methods. The practicability and efficiency of this platform are further demonstrated through the analysis of clinical serum samples. This innovative platform exhibits great potential as a facile yet robust approach for simultaneous, accurate, and on-site visual detection of multiple enzyme activities in authentic samples.

Characterization of a Lactobacillus gasseri strain as a probiotic for female vaginitis.

Vaginitis, a prevalent gynecological condition in women, is mainly caused by an imbalance in the vaginal micro-ecology. The two most common types of vaginitis are vaginal bacteriosis and vulvovaginal candidiasis, triggered by the virulent Gardnerella vaginalis and Candida albicans, respectively. In this study, a strain capable of inhibiting G. vaginalis and C. albicans was screened from vaginal secretions and identified as Lactobacillus gasseri based on 16S rRNA sequences. The strain, named L. gasseri VHProbi E09, could inhibit the growth of G. vaginalis and C. albicans under co-culture conditions by 99.07% ± 0.26% and 99.95% ± 0.01%, respectively. In addition, it could significantly inhibit the adhesion of these pathogens to vaginal epithelial cells. The strain further showed the ability to inhibit the enteropathogenic bacteria Escherichia coli and Salmonella enteritidis, to tolerate artificial gastric and intestinal fluids and to adhere to intestinal Caco-2 cells. These results suggest that L. gasseri VHProbi E09 holds promise for clinical trials and animal studies whether administered orally or directly into the vagina. Whole-genome analysis also revealed a genome consisting of 1752 genes for L. gasseri VHProbi E09, with subsequent analyses identifying seven genes related to adhesion and three genes related to bacteriocins. These adhesion- and bacteriocin-related genes provide a theoretical basis for understanding the mechanism of bacterial inhibition of the strain. The research conducted in this study suggests that L. gasseri VHProbi E09 may be considered as a potential probiotic, and further research can delve deeper into its efficacy as an agent which can restore a healthy vaginal ecosystem.

Single-cell transcriptome sequencing reveals aberrantly activated inter-tumor cell signaling pathways in the development of clear cell renal cell carcinoma.

BACKGROUND: Aberrant intracellular or intercellular signaling pathways are important mechanisms that contribute to the development and progression of cancer. However, the intercellular communication associated with the development of ccRCC is currently unknown. The purpose of this study was to examine the aberrant tumor cell-to-cell communication signals during the development of ccRCC. METHODS: We conducted an analysis on the scRNA-seq data of 6 ccRCC and 6 normal kidney tissues. This analysis included sub clustering, CNV analysis, single-cell trajectory analysis, cell-cell communication analysis, and transcription factor analysis. Moreover, we performed validation tests on clinical samples using multiplex immunofluorescence. RESULTS: This study identified eleven aberrantly activated intercellular signaling pathways in tumor clusters from ccRCC samples. Among these, two of the majors signaling molecules, MIF and SPP1, were mainly secreted by a subpopulation of cancer stem cells. This subpopulation demonstrated high expression levels of the cancer stem cell markers POU5F1 and CD44 (POU5F1hiCD44hiE.T), with the transcription factor POU5F1 regulating the expression of SPP1. Further research demonstrated that SPP1 binds to integrin receptors on the surface of target cells and promotes ccRCC development and progression by activating potential signaling mechanisms such as ILK and JAK/STAT. CONCLUSION: Aberrantly activated tumor intercellular signaling pathways promote the development and progression of ccRCC. The cancer stem cell subpopulation (POU5F1hiCD44hiE.T) promotes malignant transformation and the development of a malignant phenotype by releasing aberrant signaling molecules and interacting with other tumor cells.

Aspartyl-tRNA synthetase 2 orchestrates iron-sulfur metabolism in hematopoietic stem cells via fine-tuning alternative RNA splicing.

Aspartyl-tRNA synthetase 2 (Dars2) is involved in the regulation of mitochondrial protein synthesis and tissue-specific mitochondrial unfolded protein response (UPRmt). The role of Dars2 in the self-renewal and differentiation of hematopoietic stem cells (HSCs) is unknown. Here, we show that knockout (KO) of Dars2 significantly impairs the maintenance of hematopoietic stem and progenitor cells (HSPCs) without involving its tRNA synthetase activity. Dars2 KO results in significantly reduced expression of Srsf2/3/6 and impairs multiple events of mRNA alternative splicing (AS). Dars2 directly localizes to Srsf3-labeled spliceosomes in HSPCs and regulates the stability of Srsf3. Dars2-deficient HSPCs exhibit aberrant AS of mTOR and Slc22a17. Dars2 KO greatly suppresses the levels of labile ferrous iron and iron-sulfur cluster-containing proteins, which dampens mitochondrial metabolic activity and DNA damage repair pathways in HSPCs. Our study reveals that Dars2 plays a crucial role in the iron-sulfur metabolism and maintenance of HSPCs by modulating RNA splicing.

Retrospective analysis of urinary tract stone composition in a Chinese ethnic minority colony based on Fourier transform infrared spectroscopy.

To analyze the relationship between the composition of urinary stones and various influencing factors in the Enshi region. We used FT-IR to examine the composition of 1092 stone samples. Combined with the relevant clinical materials, the data were analyzed using both one-dimensional statistical methods and multivariate statistical methods. The study included 1092 stone samples, classified as follows: 457 (41.8%) with a single component, 453 (41.5%) with two components, 149 (13.6%) with three components, and 33 (3.0%) with four components. Stones were categorized into five types: Calcium Oxalate (CaOx) (76.4%), carbapatite (CaP) (9.3%), Struvite (ST) (8.3%), Uric Acid (UA) (4.9%), and Others (1.0%). Age, gender, urinary tract infection (UTI), family history of urinary stones (FH), hyperuricemia (HUA) and stone location were significantly associated with stone type. Logistic regression revealed that females and UTI were relative risk factors for predicting CaP and ST, while FH and HUA were relative risk factors for predicting UA. Our study indicates that the overall composition of urinary tract stones in the Enshi region is consistent with that of the entire China. Additionally, the predisposing factors for stone formation vary in terms of gender, age, FH, UTI, hyperuricemia HUA, and stone location.

Characterization of the anti-pathogenic, genomic and phenotypic properties of a Lacticaseibacillus rhamnosus VHProbi M14 isolate.

A strain of lactic acid bacteria from cheese was isolated, that showed strong growth inhibitory effects on Streptococcus mutans. The API 50CH system and 16S rDNA sequencing verified that this was a novel strain, and was named Lacticaseibacillus rhamnosus VHProbi M14. The strain inhibited the growth of S. mutans and Fusobacterium nucleatum under mixed culture conditions, coaggregated with S. mutans and F. nucleatum, and reduced the adhesion of S. mutans and F. nucleatum on cultured human primary gingival epithelial (HPGE) cells. The pH, peroxidase and protease sensitivity testing found antibacterial substances of protein- and peptide-like structures in addition to organic acids. The antimicrobial substances were sensitive to hydrolysis with trypsin, papain and pineapple protease and were inactived at temperatures above 100°C. Ammonium sulphate-precipitated proteins from the M14 strain retained the ability to inhibit the growth of S. mutans and F. nucleatum. The M14 strain contained 23 bacteriocin-related genes encoding for metabolites, belonging to class II bacteriocins. The M14 strain also showed inhibitory effects on 8 other pathogenic strains (A. actinomycetemcomitans, C. albicans, E. coli, G. vaginalis, P. acnes, P. gingivalis, S. aureus, S. enteritids), and thus has a broad spectrum of bacterial inhibition. This new isolate has been identified as having potential to be used as a probiotic bacterium in clinical applications.

Pd(II)-based coordination polymer nanosheets for ratiometric colorimetric and photothermal dual-mode assay of serum alkaline phosphatase.

Fabrication of a multi-signal readout assay with high sensitivity and selectivity is highly desirable for clinical and biochemical analysis, but remains a challenge due to laborious procedures, large-scale instruments, and inadequate accuracy. Herein, a straightforward, rapid, and portable detection platform based on palladium(II) methylene blue (MB) coordination polymer nanosheets (PdMBCP NSs) was unveiled for the ratiometric dual-mode detection of alkaline phosphatase (ALP) with temperature and colorimetric signal readout properties. The sensing mechanism is the ALP-catalyzed generation of ascorbic acid for competitive binding and etching PdMBCP NSs to release free MB in a quantitive means for detection. Specifically, ALP addition led to the decrease of temperature signal readout from the decomposed PdMBCP NSs under 808 nm laser excitation, and simultaneous increase of the temperature from the generated MB with a 660 nm laser, together with the corresponding absorbance changes at both wavelengths. Notably, this ratiometric nanosensor exhibited a detection limit of 0.013 U/L (colorimetric) and 0.095 U/L (photothermal) within 10 min, respectively. The reliability and satisfactory sensing performance of the developed method were further confirmed by clinic serum samples. Therefore, this study provides a new insight for the development of dual-signal sensing platforms for convenient, universal, and accurate detection of ALP.

Complete Genome Sequence of Lactobacillus bulgaricus VHProbi R03, a Potential Fermentation Strain Isolated from Fermented Milk.

The Lactobacillus bulgaricus strain VHProbi R03 is a novel starter culture that was isolated from naturally fermented milk. Whole-genome sequencing-based analysis is an ideal approach to elucidate the probiotic mechanism of action of this strain. Its genome contains a circular chromosome with 1,873,403 bp, and no plasmids exist in the genome.

Complete Genome Sequence of Streptococcus thermophilus VHProbi R08, Isolated from Fermented Sour Porridge.

Streptococcus thermophilus VHProbi R08 is a bacterial strain isolated from fermented sour porridge in northern China. Here, we report the complete genome sequence of VHProbi R08, which comprises 1,848,461 bp, 1,906 protein-coding genes, 57 tRNA genes, and 15 rRNA genes.

Advanced Functions Embedded in the Second Version of Database, Global Evaluation of SARS-CoV-2/hCoV-19 Sequences 2.

The Global Evaluation of SARS-CoV-2/hCoV-19 Sequences 2 (GESS v2 https://shiny.ph.iu.edu/GESS_v2/) is an updated version of GESS, which has offered a handy query platform to analyze single-nucleotide variants (SNVs) on millions of high coverages and high-quality severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) complete genomes provided by the Global Initiative on Sharing Avian Influenza Data (GISAID). Including the tools in the first version, the GESS v2 is embedded with new functions, which allow users to search SNVs, given the viral nucleotide or amino acid sequence. The GESS v2 helps users to identify SNVs or SARS-CoV-2 lineages enriched in countries of user's interest and show the migration path of a selected lineage on a world map during specific time periods chosen by the users. In addition, the GESS v2 can recognize the dynamic variations of newly emerging SNVs in each month to help users monitor SNVs, which will potentially become dominant soon. More importantly, multiple sets of analyzed results about SNVs can be downloaded directly from the GESS v2 by which users can conduct their own independent research. With these significant updates, the GESS v2 will continue to serve as a public open platform for researchers to explore SARS-CoV-2 evolutionary patterns from the perspectives of the prevalence and impact of SNVs.

Rora Regulates Neutrophil Migration and Activation in Zebrafish.

Neutrophil migration and activation are essential for defense against pathogens. However, this process may also lead to collateral tissue injury. We used microRNA overexpression as a platform and discovered protein-coding genes that regulate neutrophil migration. Here we show that miR-99 decreased the chemotaxis of zebrafish neutrophils and human neutrophil-like cells. In zebrafish neutrophils, miR-99 directly targets the transcriptional factor RAR-related orphan receptor alpha (roraa). Inhibiting RORα, but not the closely related RORγ, reduced chemotaxis of zebrafish and primary human neutrophils without causing cell death, and increased susceptibility of zebrafish to bacterial infection. Expressing a dominant-negative form of Rorα or disrupting the roraa locus specifically in zebrafish neutrophils reduced cell migration. At the transcriptional level, RORα regulates transmembrane signaling receptor activity and protein phosphorylation pathways. Our results, therefore, reveal previously unknown functions of miR-99 and RORα in regulating neutrophil migration and anti-microbial defense.

Forecasting greenhouse gas emissions with the new information priority generalized accumulative grey model.

Air pollution and other environmental problems caused by excessive emissions of greenhouse gases have become a comprehensive problem requiring joint global treatment. To consider the characteristics of different regions and different countries in terms of greenhouse gas emissions for accurate prediction, a new information priority generalized accumulative grey model (NIPGAGM(1,1,k)) is proposed. The new model maintains the structure of the traditional grey model and the basic result characteristics of its features. This research further deduces the calculation formulas of the model's time response sequence and parameter estimation. Furthermore, an optimization model is established to search the parameters using a detailed optimization algorithm. The optimization value of the new model is determined by the intelligent optimization algorithm. Then, the new model is applied to the greenhouse gas emission prediction of the Shanghai Cooperation Organization (SCO) member states. The numerical results are compared with those of existing models. Finally, according to the forecast results of greenhouse gas emissions in these regions, reasonable suggestions for clean energy production are proposed.

Ternary supramolecular nanocomplexes for superior anticancer efficacy of natural medicines.

The discovery of effective anticancer drug delivery systems and elucidation of the mechanism are enormous challenges. Using two drug administration-approved biomaterials, we constructed a natural medicine (NM)-loaded ternary supramolecular nanocomplex (TSN) suitable for large-scale production. The TSN has a better effect against cancer cells/stem cells than NM with differentially upregulated (27 versus 59) and downregulated (165 versus 66) proteins, respectively. Treatment with the TSN induced apoptosis and G2/M arrest, inhibited cell proliferation, metastasis and invasion, reduced colony/sphere formation, and decreased the frequency of side population cells and CD133+CD44+ABCG2+ cells. These results were revealed by multiple analyses (proteomic analysis, transwell migration and colony/sphere formation assays, biomarker profiling, etc.). We first reported the proteomic analysis of small lung cancer cells responding to a drug or its nanovesicles. We first conducted a proteomic evaluation of tumor cells responding to a drug supramolecular nanosystem. The supramolecular conformation of the TSN and the interactions of the TSN with albumin were verified by molecular docking experiments. The dominant binding forces in the TSN complexation process were electrostatic interactions, van der Waalsinteractions and bond stretching. The TSN binds to albumin more readily than NM does. The TSN has good in situ absorptive and in vitro/vivo kinetic properties. The relative bioavailability of the TSN to EA was 458.39%. The NM-loaded TSN is a supramolecular vesicle that can be produced at an industrial scale for efficient cancer therapy.

Liquid chromatography-tandem mass spectrometry analysis for identification and quantification of antimicrobial compounds in distillery wastewater.

A high-resolution mass spectrometry (HR-MS) method was developed to analyze and identify small molecule compounds in distillery wastewater. According to identification confidence levels, 4 levels of compounds were identified. The five antimicrobial compounds (lactic acid, succinic acid, acetophenone, cinnamic acid, and phenyllactic acid), which shown in high concentrations, were at the highest level of confidence (level 1, confirmed structure). Thus, a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to simultaneously quantify these antimicrobial compounds. The analysis was performed in the selective reaction monitoring (SRM) mode via the electrospray ionization (ESI) source operating in the negative ionization mode. Linear calibration curves were obtained over the concentration range of 50-1000.0 ng/mL for succinic acid, acetophenone, cinnamic acid, phenyllactic acid, and 375-7500 ng/mL for lactic acid. Precision and recovery of the analytes were all satisfactory (relative standard deviation < 10%). The validated method was successfully applied to quantitative analysis of the five antimicrobial compounds in distillery wastewater.•Analyze and identify 4 levels of small molecule compounds in distillery wastewater.•Simple method for quantification of five antimicrobial compounds.•Column temperature affected the lactic and succinic acid chromatographs significantly.

Updated SARS-CoV-2 single nucleotide variants and mortality association.

By analyzing newly collected SARS-CoV-2 genomes and comparing them with our previous study about SARS-CoV-2 single nucleotide variants (SNVs) before June 2020, we found that the SNV clustering had changed remarkably since June 2020. Apart from that the group of SNVs became dominant, which is represented by two nonsynonymous mutations A23403G (S:D614G) and C14408T (ORF1ab:P4715L), a few emerging groups of SNVs were recognized with sharply increased monthly incidence ratios of up to 70% in November 2020. Further investigation revealed sets of SNVs specific to patients' ages and/or gender, or strongly associated with mortality. Our logistic regression model explored features contributing to mortality status, including three critical SNVs, G25088T(S:V1176F), T27484C (ORF7a:L31L), and T25A (upstream of ORF1ab), ages above 40 years old, and the male gender. The protein structure analysis indicated that the emerging subgroups of nonsynonymous SNVs and the mortality-related ones were located on the protein surface area. The clashes in protein structure introduced by these mutations might in turn affect the viral pathogenesis through the alteration of protein conformation, leading to a difference in transmission and virulence. Particularly, we explored the fact that nonsynonymous SNVs tended to occur in intrinsic disordered regions of Spike and ORF1ab to significantly increase hydrophobicity, suggesting a potential role in the change of protein folding related to immune evasion.

Antiviral Drug Delivery System for Enhanced Bioactivity, Better Metabolism and Pharmacokinetic Characteristics.

Antiviral drugs (AvDs) are the primary resource in the global battle against viruses, including the recent fight against corona virus disease 2019 (COVID-19). Most AvDs require multiple medications, and their use frequently leads to drug resistance, since they have poor oral bioavailability and low efficacy due to their low solubility/low permeability. Characterizing the in vivo metabolism and pharmacokinetic characteristics of AvDs may help to solve the problems associated with AvDs and enhance their efficacy. In this review of AvDs, we systematically investigated their structure-based metabolic reactions and related enzymes, their cellular pharmacology, and the effects of metabolism on AvD pharmacodynamics and pharmacokinetics. We further assessed how delivery systems achieve better metabolism and pharmacology of AvDs. This review suggests that suitable nanosystems may help to achieve better pharmacological activity and pharmacokinetic behavior of AvDs by altering drug metabolism through the utilization of advanced nanotechnology and appropriate administration routes. Notably, such AvDs as ribavirin, remdesivir, favipiravir, chloroquine, lopinavir and ritonavir have been confirmed to bind to the severe acute respiratory syndrome-like coronavirus (SARS-CoV-2) receptor and thus may represent anti-COVID-19 treatments. Elucidating the metabolic and pharmacokinetic characteristics of AvDs may help pharmacologists to identify new formulations with high bioavailability and efficacy and help physicians to better treat virus-related diseases, including COVID-19.

Smart Stimuli-Responsive and Mitochondria Targeting Delivery in Cancer Therapy.

Dysfunction in the mitochondria (Mc) contributes to tumor progression. It is a major challenge to deliver therapeutic agents specifically to the Mc for precise treatment. Smart drug delivery systems are based on stimuli-responsiveness and active targeting. Here, we give a whole list of documented pathways to achieve smart stimuli-responsive (St-) and Mc-targeted DDSs (St-Mc-DDSs) by combining St and Mc targeting strategies. We present the formulations, targeting characteristics of St-Mc-DDSs and clarify their anti-cancer mechanisms as well as improvement in efficacy and safety. St-Mc-DDSs usually not only have Mc-targeting groups, molecules (lipophilic cations, peptides, and aptamers) or materials but also sense the surrounding environment and correspondingly respond to internal biostimulators such as pH, redox changes, enzyme and glucose, and/or externally applied triggers such as light, magnet, temperature and ultrasound. St-Mc-DDSs exquisitely control the action site, increase therapeutic efficacy and decrease side effects of the drug. We summarize the clinical research progress and propose suggestions for follow-up research. St-Mc-DDSs may be an innovative and sensitive precision medicine for cancer treatment.

The utilization status of neoadjuvant chemotherapy in muscle-invasive bladder cancer: a systematic review and meta-analysis.

INTRODUCTION: To give a comprehensive depiction of the utilization status of neoadjuvant chemotherapy (NAC) in muscle invasive bladder cancer (MIBC) worldwide. EVIDENCE ACQUISITION: Potential relevant research papers of Pubmed, Embase, Web of Science, and the Cochrane Library were reviewed to identify eligible studies. Primary outcomes of this meta-analysis were utilization rate of NAC and its utility distribution in different genders, races, ages, countries and temporal trends. The utilization rates of NAC were calculated as 'Proportion (s)' with 95% confidence intervals (CIs) and pooled estimates were calculated by using a random-effect model. EVIDENCE SYNTHESIS: A total of thirteen studies and 35,738 patients were included. The total proportion of NAC applied in MIBC populations prior to radical cystectomy (RC) was 17.2% (95% CI: 12.5-21.9%, I2=99.7%). The comparative analyses showed there were no significant differences existing in different genders or races on NAC utilization rates. In terms of age distribution, <60 age group conferred higher utilization rate of NAC than the older (OR=1.919, 95% CI: 1.671-2.202, P=0.0001). As for regional distribution, our meta-analysis showed that Japan (Proportion: 44.0%, 95% CI: 6.5-81.5%, I2=99.6%) and Sweden (37.9%, 95% CI: 34.9-40.8%) were the top two leading countries which contributed to the most frequent application of NAC. In respect of pathologic responses after NAC, complete, partial and down-staged pathologic responses were achieved in 16.6% (95% CI: 7.4-25.9%, I2=89.7%), 14.6% (95% CI: 0.8-28.5%, I2=89.7%) and 45.0% (95% CI: 17.8-72.2%, I2=98.8%) patients, respectively. CONCLUSIONS: The present study shows the low utilization rate of NAC in MIBC patients. Standardization of the treatment modality of MIBC and promotion of guidelines might be necessary to expedite the adoption of NAC in near future.

GESS: a database of global evaluation of SARS-CoV-2/hCoV-19 sequences.

The COVID-19 outbreak has become a global emergency since December 2019. Analysis of SARS-CoV-2 sequences can uncover single nucleotide variants (SNVs) and corresponding evolution patterns. The Global Evaluation of SARS-CoV-2/hCoV-19 Sequences (GESS, https://wan-bioinfo.shinyapps.io/GESS/) is a resource to provide comprehensive analysis results based on tens of thousands of high-coverage and high-quality SARS-CoV-2 complete genomes. The database allows user to browse, search and download SNVs at any individual or multiple SARS-CoV-2 genomic positions, or within a chosen genomic region or protein, or in certain country/area of interest. GESS reveals geographical distributions of SNVs around the world and across the states of USA, while exhibiting time-dependent patterns for SNV occurrences which reflect development of SARS-CoV-2 genomes. For each month, the top 100 SNVs that were firstly identified world-widely can be retrieved. GESS also explores SNVs occurring simultaneously with specific SNVs of user's interests. Furthermore, the database can be of great help to calibrate mutation rates and identify conserved genome regions. Taken together, GESS is a powerful resource and tool to monitor SARS-CoV-2 migration and evolution according to featured genomic variations. It provides potential directive information for prevalence prediction, related public health policy making, and vaccine designs.

Genetic Spectrum and Distinct Evolution Patterns of SARS-CoV-2.

Four signature groups of frequently occurred single-nucleotide variants (SNVs) were identified in over twenty-eight thousand high-quality and high-coverage SARS-CoV-2 complete genome sequences, representing different viral strains. Some SNVs predominated but were mutually exclusively presented in patients from different countries and areas. These major SNV signatures exhibited distinguishable evolution patterns over time. A few hundred patients were detected with multiple viral strain-representing mutations simultaneously, which may stand for possible co-infection or potential homogenous recombination of SARS-CoV-2 in environment or within the viral host. Interestingly nucleotide substitutions among SARS-CoV-2 genomes tended to switch between bat RaTG13 coronavirus sequence and Wuhan-Hu-1 genome, indicating the higher genetic instability or tolerance of mutations on those sites or suggesting that major viral strains might exist between Wuhan-Hu-1 and RaTG13 coronavirus.