Shine: A novel strategy to extract specific, sensitive and well-conserved biomarkers from massive microbial genomic datasets.

BACKGROUND: Concentrations of the pathogenic microorganisms' DNA in biological samples are typically low. Therefore, DNA diagnostics of common infections are costly, rarely accurate, and challenging. Limited by failing to cover updated epidemic testing samples, computational services are difficult to implement in clinical applications without complex customized settings. Furthermore, the combined biomarkers used to maintain high conservation may not be cost effective and could cause several experimental errors in many clinical settings. Given the limitations of recent developed technology, 16S rRNA is too conserved to distinguish closely related species, and mosaic plasmids are not effective as well because of their uneven distribution across prokaryotic taxa. RESULTS: Here, we provide a computational strategy, Shine, that allows extraction of specific, sensitive and well-conserved biomarkers from massive microbial genomic datasets. Distinguished with simple concatenations with blast-based filtering, our method involves a de novo genome alignment-based pipeline to explore the original and specific repetitive biomarkers in the defined population. It can cover all members to detect newly discovered multicopy conserved species-specific or even subspecies-specific target probes and primer sets. The method has been successfully applied to a number of clinical projects and has the overwhelming advantages of automated detection of all pathogenic microorganisms without the limitations of genome annotation and incompletely assembled motifs. Using on our pipeline, users may select different configuration parameters depending on the purpose of the project for routine clinical detection practices on the website https://bioinfo.liferiver.com.cn with easy registration. CONCLUSIONS: The proposed strategy is suitable for identifying shared phylogenetic markers while featuring low rates of false positive or false negative. This technology is suitable for the automatic design of minimal and efficient PCR primers and other types of detection probes.

Development of a panel of seven duplex real-time PCR assays for detecting 13 streptococcal superantigens.

BACKGROUND: Streptococcal superantigens (SAgs) are the major virulence factors of infection in humans for group A Streptococcus (GAS) bacteria. A panel consisting of seven duplex real-time PCR assays was developed to simultaneously detect 13 streptococcal SAgs and one internal control which may be important in the control of GAS-mediated diseases. METHODS: Primer and probe sequences were selected based on the highly conserved region from an alignment of nucleotide sequences of the 13 streptococcal SAgs. The reaction conditions of the duplex real-time PCR were optimized and the specificity of the duplex assays was evaluated using SAg positive strains. The limit of detection of the duplex assays was determined by using 10-fold serial dilutions of the DNA of 13 streptococcal SAgs and compared to a conventional polymerase chain reaction (PCR) method for evaluating the duplex assays sensitivity. RESULTS: Using the duplex assays, we were able to differentiate between 13 SAgs from Streptococcus strains and other non-Streptococcus bacteria without cross-reaction. On the other hand, the limit of detection of the duplex assays was at least one or two log dilutions lower than that of the conventional PCR. CONCLUSIONS: The panel was highly specific (100%) and the limit of detection of these duplex groups was at least ten times lower than that obtained by using a conventional PCR method.

An Engineered IgG-VHH Bispecific Antibody against SARS-CoV-2 and Its Variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies are shown to be effective therapeutics for providing coronavirus disease 2019 (COVID-19) protection. However, recurrent variants arise and facilitate significant escape from current antibody therapeutics. Bispecific antibodies (bsAbs) represent a unique platform to increase antibody breadth and to reduce neutralization escape. Herein, a novel immunoglobulin G-variable domains of heavy-chain-only antibody (IgG-VHH) format bsAb derived from a potent human antibody R15-F7 and a humanized nanobody P14-F8-35 are rationally engineered. The resulting bsAb SYZJ001 efficiently neutralizes wild-type SARS-CoV-2 as well as the alpha, beta, gamma, and delta variants, with superior efficacy to its parental antibodies. Cryo-electron microscopy structural analysis reveals that R15-F7 and P14-F8-35 bind to nonoverlapping epitopes within the RBD and sterically hindered ACE2 receptor binding. Most importantly, SYZJ001 shows potent prophylactic and therapeutic efficacy against SARS-CoV-2 in three established mouse models. Collectively, the current results demonstrate that the novel bsAb format is feasible and effective, suggesting great potential as an inspiring antiviral strategy.

Rational development of a human antibody cocktail that deploys multiple functions to confer Pan-SARS-CoVs protection.

Structural principles underlying the composition and synergistic mechanisms of protective monoclonal antibody cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic antibody cocktail against SARS-CoV-2. On the basis of our previously identified humanized cross-neutralizing antibody H014, we systematically analyzed a fully human naive antibody library and rationally identified a potent neutralizing antibody partner, P17, which confers effective protection in animal model. Cryo-EM studies dissected the nature of the P17 epitope, which is SARS-CoV-2 specific and distinctly different from that of H014. High-resolution structure of the SARS-CoV-2 spike in complex with H014 and P17, together with functional investigations revealed that in a two-antibody cocktail, synergistic neutralization was achieved by S1 shielding and conformational locking, thereby blocking receptor attachment and viral membrane fusion, conferring high potency as well as robustness against viral mutation escape. Furthermore, cluster analysis identified a hypothetical 3rd antibody partner for further reinforcing the cocktail as pan-SARS-CoVs therapeutics.

[Analysis on the effect of secondary mutations on Leber's hereditary optic neuropathy].

OBJECTIVE: To investigate the effect of secondary mutations on Leber's hereditary optic neuropathy (LHON). METHODS: Three primary and 24 secondary mutations were identified in 4 Chinese families which included male offspring. RESULTS: All of the four pedigrees carried classic LHON mutations at nucleotide (nt) 11778, and did not carry any point of 24 secondary mutations. Nevertheless many polymorphic points were found in the nearby fragments of these pedigrees, such as 5178, 5108, 3705, 3721, 13734, etc. CONCLUSION: Male offspring sequences should be analyzed in pedigrees with LHON to avoid the influence of familial inheritance characteristic which mitochondrial DNA polymorphism carried. Existence of the "repair genes" may affect the development of LHON.

[Detection of mtDNA*LHON G11778A mutation by real-time polymerase chain reaction using TaqMan-MGB probe technology].

OBJECTIVE: To develop a simple, rapid and reliable real-time PCR assay based on TaqMan technology using a new MGB probe for detecting mtDNA(*)LHON G11778A mutation and heteroplasmy directly. METHODS: Twenty patients with suspicion of Leber hereditary optic neuropathy (LHON) and their maternal relatives had undergone molecular genetic evaluation. Seventeen normal individuals were used as the controls. A real-time PCR involved two MGB probes (wild-type and mutation-type) in a single tube on the iCycler IQ real-time detection system was used to detect the mtDNA(*)LHON G11778A mutation. The results were then compared with the DNA sequence analysis of the PCR products. A linear standard curve was obtained by pUCm LHON-G and pUCm LHON-A clone. RESULTS: In the controls (wild type), the reaction of VIC-labeled MGB probe was positive and the channel of FAM reaction was negative, the DNA sequence was 100% matched to previously published data. In 20 LHON patients and their maternal relatives, 12 cases showed mutations in DNA sequence analysis, all of them were LHON mtDNA mutation. While 5 other cases showed the combination of LHON mtDNA mutation and wide type gene phenotype, the rate of Ct value in wild type versus gene mutation was over 25%. DNA sequence analysis showed 8 of LHON mtDNA belonged to wild types and 3 cases were heteroplasmy, and the rate of Ct value in gene mutation versus wild type was lower than 25%. CONCLUSION: This real-time PCR assay is a simple, rapid and reliable method for the detection of genotyping mtDNA mutations as well as for quantifying heteroplasmy.

Inhibition of hepatitis B virus surface antigen expression by small hairpin RNA in vitro.

AIM: To explore the anti-hepatitis B virus effect of RNA interference (RNAi) using small hairpin RNA (shRNA) expression vector. METHODS: Hepatitis B virus surface antigen green fluorescent protein (HBs-GFP) fusion vector and shRNA expression vectors were constructed and cotransfected transiently into HepG2 cells. mRNAs extracted from HepG2 cells were detected by real-time PCR. Fluorescence of HBs-GFP protein was detected by fluorescence-activated cell sorting (FACS). The effective shRNA expression vector was transfected into HepG2.2.15 cells. HBsAg and HBeAg in HepG2.2.15 cells were analyzed by radioimmunoassay (RIA) method. RESULTS: FACS revealed that shRNA targeting at HBsAg reduced the GFP signal by 56% compared to the control. Real-time PCR showed that HBs-GFP mRNA extracted from HepG2 cells cotransfected with pAVU6+27 and HBs-GFP expression plasmids decreased by 90% compared to the empty vector control. The expressions of HBsAg and HBeAg were also inhibited by 43% and 64%, respectively. CONCLUSION: RNAi using shRNA expression vector can inhibit the expression of HBsAg, providing a fresh approach to screening the efficient small interfering RNAs (siRNAs).

Relationship between dengue viral load and clinical characteristics / 预防医学

Objective@#To determine the relationship between dengue virus load and clinical characteristics, so as to provide basis for dengue fever prevention and treatment.@*Methods @#The dengue viral load and typing of 120 patients in Gongshu District of Hangzhou from June to November 2017 were detected by real-time fluorescent quantitative RT-PCR;the clinical indicators of these dengue patients were collected and their correlation with the viral load was analyzed.@*Results@#The DNA detection of dengue virus in 120 patients showed that they were all typeⅡ. The median dengue virus load was 3.91×104 copies/mL. All the patients had fever, the average peak temperature was（38.96 ± 0.69）℃. There were 102（85.00%）cases with asthenia;116（96.67%）cases with white blood cell count（WBC）less than 4× 109/L;119（99.17%）cases with platelet count（PLT）less than 100×109/L;114（95.00%）cases with glutamic oxaloacetate transaminase（GOT）more than 40 U/L;81（67.50%）cases with glutamic pyruvate transaminase（GPT）more than 52 U/L;58（48.33%）cases with creatine kinase（CK）more than 210 U/L. There was no significant correlation of dengue virus load with length of hospitalization, peak temperature,duration of fever, WBC,PLT, GOT, GPT and CK（P>0.05）. There were 75（62.50%）severe patients, and their median viral load was 9.29×104copies/mL, which was higher than 5.33×103copies/mL in non-severe patients（P<0.05）. @*Conclusion @#The dengue virus load is not related with length of hospitalization,peak temperature,WBC,PLT,GOT,GPT and CK,but with the severity of the disease.

[Preparation of cationic liposomes and its role in enhancing cellular uptake of antisense oligonucleotides].

AIM: To prepare the liposomes which protect antisense oligodeoxynucleotides (ASON) against nuclease degradation and delivery ASON into cytoplasmic efficiently. METHODS: A cationic derivative of cholesterol, 3 beta-[N-(N',N'-dimethylaminoethan)-carbamoyl] cholesterol (DC-Chol) was synthesized and used to prepare cationic liposome. The characteristics of liposomes/ASON complexes including size, drug loaded efficiency and structure were investigated. Cellular uptake of fluorescence labled ASON (FAM-ASON) under different condition was determined by flow cytometric analysis. Denatured polyacryamide gel electrophoresis (DPGE) was used to analyze the role of liposomes in protecting ASON. RESULTS: The mean values of preliposomes and liposomes/ASON complexes size were 185.7 and 228.2 nm, respectively. Cationic liposomes showed a high adsorption capacity for ASON. When the +/- charge ratio exceeded 2:1, more than 90% of the ASON was loaded into liposomes. Agarose gel electrophoresis showed three different existence of ASON in liposomes formulation: free, absorbed and encapsulated types. Concerning cellular uptake, DC-Chol liposomes indicated high efficient effect of increasing cellular uptake of ASON. Compared with free ASON, the total fluorescence intensity in cytoplasma was significantly enhanced. The level of increasing was largely depended on +/- charge ratio. The cellular uptake of FAM-ASON decreased in the presence of serum. The cellular total fluorescence intensity in 10% and 30% fetal bovine serum of cultured medium were only 22.3% and 15.5% as that of serum-free media, respectively. DPGE confirmed that free ASON was rapidly degraded by DNase I while ASON encapsulated into liposomes was efficiently protected. CONCLUSION: The cationic DC-Chol liposomes are shown to be promising carriers to deliver ASON into cytoplasma.

[Expression of MAGE, GAGE, and BAGE genes in human hepatocellular carcinoma].

OBJECTIVE: To observe the expression of MAGE, GAGE and BAGE genes in human hepatocellular carcinoma (HCC) cell lines. METHODS: The expression levels of MAGE-1, MAGE-3, GAGE1-8, GAGE1-2 and BAGE mRNAs in HCC cell lines SMMC-7721, QQY-7701, BEL-7402 were studied by reverse transcription polymerase chain reaction and were compared with those in biopsied liver tissues. RESULTS: MAGE-1 and BAGE mRNAs were expressed in SMMC-7721 cells. MAGE-3 and BAGE mRNAs were expressed in QQY-7701 cells. MAGE-1 and GAGE1-2 mRNAs were expressed in BEL-7402 cells. None of these genes was expressed in biopsied liver tissues. CONCLUSIONS: MAGE-1, MAGE-3, GAGE1-8, GAGE1-2 and BAGE are expressed in hepatocellular carcinoma cell lines. These tumor-specific antigens can be used as molecular markers for early diagnosis and possible targets for immunotherapy of human HCC.