Reversed-Phase-HPLC Assay Method for Simultaneous Estimation of Sorbitol, Sodium Lactate, and Sodium Chlorides in Pharmaceutical Formulations and Drug Solution for Infusion

A rapid, straightforward, sensitive, efficient, and cost-effective reverse-phase high-performance liquid chromatographic method was employed for the simultaneous determination of Sorbitol, Sodium Lactate, and Chlorides in a drug solution for infusion. Sorbitol, Sodium lactate, and Chloride are all officially recognized in the USP monograph. Assay methods are provided through various techniques, with titrations being ineffective for trace-level quantification. Alternatively, IC, AAS, and ICP-MS, though highly accurate, are costly and often unavailable to most testing facilities. When considering methods, it’s important to prioritize both quality control requirements and user-friendly techniques. A simple HPLC simultaneous method was developed for the quantification of Chlorides, Sorbitol, and Sodium Lactate with a shorter run time. The separation utilized a Shimpack SCR-102(H) ion exclusion analytical column (7.9 mm × 300 mm, 7 μm), with a flow rate of 0.6 mL per min. The column compartment temperature was maintained at 40°C, and the injection volume was set at 10 μL, with detection at 200 nm. All measurements were conducted in a 0.1% solution of phosphoric acid. The analytical curves demonstrated linearity (r > 0.9999) in the concentration range of 0.79 to 3.8 mg per mL for Sodium Lactate (SL), 0.16 to 0.79 mg per mL for Sodium Chloride (SC), and 1.5 to 7.2 mg per mL for Sorbitol. Validation of the developed method followed the guidelines of the International Conference on Harmonization (ICH Q2B) and USP. The method exhibited precision, robustness, accuracy, and selectivity. In accelerated stability testing over 6 months, no significant variations were observed in organoleptic analysis and pH. Consequently, the developed method is deemed suitable for routine quality control analyses, enabling the simultaneous determination of Sodium Lactate, Sodium Chloride, and Sorbitol in pharmaceutical formulations and infusions.

Interferon-gamma release assays as a tool for differential diagnosis of gastrointestinal tuberculosis

In this editorial,we comment on an article published in a recent issue of the World Journal of Clinical Cases.There is a pressing need for reliable tools for diagnosing tuberculosis(TB)of the gastrointestinal tract.Despite advancements in the diagnosis and treatment,TB remains a global health challenge.Ali et al demon-strated that TB may mimic gastrointestinal conditions,such as gastric outlet obstruction,causing a delay in the diagnosis.Furthermore,the latter complication is frequently observed during infections,including Helicobacter pylori,and rarely is related to TB,as in the presented case.In line with this,we think that laboratory tests based on interferon-gamma release assays can be a helpful tool for diagnosing latent TB paced in the gastrointestinal tract.Innovative strategies and approaches for diagnosing latent/active extra pulmonary TB are crucial for establishing the diagnosis early and enhancing treatment strategies to mitigate the global burden of TB.

Evaluation of Interferon-Gamma Release Assay Testing and Tuberculin Skin Test for Early Diagnosis of Tuberculosis in Children and Adolescents

Background: This study aimed to evaluate the diagnostic value of interferon-γ release assay (IGRA), a sensitive microbiological diagnostic method, in children and adolescents with suspected tuberculosis in a country with a high burden of tuberculosis. Method: This study included 581 children and adolescents aged 4 - 19 years who were suspected of having tuberculosis, were latently infected with Mycobacterium tuberculosis, and had received at least one dose of BCG vaccine between April 17, 2019, and February 24, 2021. The study evaluated the TST results of 106 patients who had a positive Quantiferon test and were suspected of having tuberculosis. Results: The study included 581 patients aged between 4 and 19 years. Of these, 106 patients tested positive for the Quantiferon test, while 19 were indeterminate and 456 were negative. The Quantiferon test positivity rate was 18.24%. Among the 106 QFT-Plus-positive cases, 23 patients also tested positive for TST. The difference in distribution was found to be statistically significant. Conclusion: The QFT-Plus test is considered an alternative to TST and other microbiological diagnostic methods for early tuberculosis diagnosis, particularly in children and adolescents.

Colloidal Gold Immunochromatographic Assay for Rapid On-Site Detection of Tetracycline in Seawater

Recently increasing concerns from the scientists and public have been paid for seawater pollution due to tetracycline(TC)overuse in maricultural area.However,there are few methods or instruments that can be used for specific and rapid detection of this antibiotic in seawater.In this study,the colloidal gold immunochromatographic assay(CG-ICA)was used to achieve this goal.A commercialized monoclonal antibody against TC(anti-TC mAb)was selected because of its higher sensitivity(half-maximal inhibitory concentration of 2.38μgL^(-1)).The prepared CG particles(average diameter of 20 nm)were used to label anti-TC mAb at pH 8.0.The conjugate pad was formed by spraying the CG-labeled anti-TC mAb on a glass fibre membrane followed by proper dryness.The test pad was made by immobilizing artificial antigen and anti-mouse mAb in the test line and the control line,respectively,in a nitrocellulose membrane.The test strip,assembled with sample pad,conjugate pad,test pad and absorbent pad,could be used to detect TC during seawater sample flowing through these components in turn.The results could be observed by the naked eye in 10min.The visible limit of detection(vLOD)was 20μgL^(-1) for TC in seawater.The CG-ICA test results were in good agreement with those of liquid chromatography-tandem mass spectrometry(LC-MS/MS).The assay also showed that,oxytetracycline(OTC)and chlortetracycline(CTC),as the structural analogues of TC,did not interfere with TC determination.Furthermore,the TC concentration given by test strip could not be affected by the fluctuation of temperature(10℃–30℃),pH(7–9)and salinity(0–40)of seawater.Therefore,CG-ICA is a suitable tool for rapid,on-site,and semi-quantitative detection of TC in seawater.

A novel pathogenic splicing mutation of RPGR in a Chinese family with X-linked retinitis pigmentosa verified by minigene splicing assay

AIM:To report a novel splicing mutation in the RPGR gene(encoding retinitis pigmentosa GTPase regulator)in a three-generation Chinese family with X-linked retinitis pigmentosa(XLRP).METHODS:Comprehensive ophthalmic examinations including best corrected visual acuity,fundus photography,vision field,and pattern-visual evoked potential were performed to identify the disease phenotype of a six-yearold boy from the family(proband).Genomic DNA was extracted from peripheral blood of five available members of the pedigree.Whole-exome sequencing(WES),Sanger sequencing,and pSPL3-based exon trapping were used to investigate the aberrant splicing of RPGR.Human Splice Finder v3.1 and NNSPLICE v0.9 were used for in silico prediction of splice site variants.RESULTS:The proband was diagnosed as having retinitis pigmentosa(RP).He had severe symptoms with early onset.A novel splicing mutation,c.619+1G>C in RPGR was identified in the proband by WES and in four family members by Sanger sequencing.Minigene splicing assays verified that c.619+1G>C in RPGR would result in the formation of a damaging alternative transcript in which the last 91 bp of exon 6 were skipped,leading to the subsequent deletion of 623 correct amino acids(c.529_619del p.Val177Glnfs*16).CONCLUSION:We identify a novel splice donor site mutation causing aberrant splicing of RPGR.Our findings add to the catalog of pathological mutations of RPGR and further emphasize the functional importance of RPGR in RP pathogenesis and its complex clinical phenotypes.

The Contribution of the Xpert® MTB/RIF Assay to the Surveillance of Drug-Resistant Tuberculosis in the Central African Republic

Introduction: The Central African Republic is one of the 30 high Tuberculosis burden countries in the world, with an incidence of 540 cases per 100,000 population and a mortality of 91 deaths per 100,000 population. Since 2020, following WHO recommendations, the National Reference Laboratory for Tuberculosis has been using the Xpert® MTB/RIF assay as a first-line diagnostic test for the early detection of Drug Resistance Tuberculosis. The goal of this study was to evaluate the contribution of the Xpert® MTB/RIF assay to the surveillance of rifampicin resistance in new and previously treated tuberculosis cases. Materials and Methods: The data relative to the Xpert® MTB/RIF assay carried out on various categories of tuberculosis patients registered at the National Reference Laboratory for Tuberculosis in 2020 were analyzed retrospectively. The categories of tuberculosis patients were new cases, failed treatment cases, relapse cases, lost-to-follow-up cases and multidrug-resistant tuberculosis contact cases. Results: A total of 1404 tuberculosis patients were registered at the NRL-TB in 2020;the mean age was 39.2 years (2 - 90 years) and the male-to-female sex ratio was 1.16:1. Overall, 32.7% (454/1404) proved infected with tuberculosis, of which 22.5% (102/454) cases showed resistance to rifampicin. The primary resistance rate was 9.1% (27/298) and the secondary resistance rate was 46.6% (75/161). Treatment failures and relapsed cases were significantly associated with rifampicin resistance (p 0.005). Conclusion: Large-scale use of Xpert® MTB/RIF, especially in the provinces of the Central African Republic, will help the Ministry of Health to better control Drug Resistance Tuberculosis in the country.

Comparative assessment of nitrogen fixation rate by ^(15)N_(2) tracer assays in the South China Sea

Nitrogen fixation is one of the most important sources of new nitrogen in the ocean and thus profoundly affects the nitrogen and carbon biogeochemical processes.The distribution,controlling factors,and flux of N2 fixation in the global ocean remain uncertain,partly because of the lack of methodological uniformity.The^(15)N_(2)tracer assay(the original bubble method→the^(15)N_(2)-enriched seawater method→the modified bubble method)is the mainstream method for field measurements of N2 fixation rates(NFRs),among which the original bubble method is the most frequently used.However,accumulating evidence has suggested an underestimation of NFRs when using this method.To improve the availability of previous data,we compared NFRs measured by three^(15)N_(2)tracer assays in the South China Sea.Our results indicate that the relationship between NFRs measured by the original bubble method and the^(15)N_(2)-enriched seawater method varies obviously with area and season,which may be influenced by incubation time,diazotrophic composition,and environmental factors.In comparison,the relationship between NFRs measured by the original bubble method and the modified bubble method is more stable,indicating that the N2 fixation rates based on the original bubble methods may be underestimated by approximately 50%.Based on this result,we revised the flux of N2 fixation in the South China Sea to 40 mmol/(m2·a).Our results improve the availability and comparability of literature NFR data in the South China Sea.The comparison of the^(15)N_(2)tracer assay for NFRs measurements on a larger scale is urgently necessary over the global ocean for a more robust understanding of the role of N2 fixation in the marine nitrogen cycle.

Elispot assay检测牛奶中庆大霉素

为了建立快速检测牛奶中庆大霉素的Elispot assay,采用制备GM免疫抗原获得抗GM多克隆抗体。将检测抗原点阵在PVDF膜上,通过检测抗原和样品中GM竞争结合抗GM多克隆抗体,酶标结合物催化底物显色,根据颜色的有无及深浅判读结果,从而建立了检测牛奶中GM的Elispot assay。该方法的检测阈值为10 ng/mL,检测时间为40 min,可对样品实现半定量检测。该方法制备的试纸条于4℃密封保存90 d仍可用于检测;与多种结构类似物未见交叉反应;其结果与酶联免疫方法一致。

子宫实验和E-SCREEN实验在检测雌激素活性中的相关性

目的 以 1 7β 雌二醇 (1 7β E2 )和四种植物提取物为受试物 ,研究子宫实验和E SCREEN实验在检测雌激素活性中的相关性。方法 断乳雌性小鼠 (1 0～ 1 2g)按体重分为 6组 ,分别给予小茴香、山豆根、补骨脂和川牛膝提取物 (1 0g kgBW ,ig)、1 7β E2 (0 5mg kgBW ,sc)和蒸馏水 (ig) ,持续 9天后处死动物剥离子宫称重。分别在人类乳腺癌MCF 7细胞培养液中加入四种植物提取物 (终浓度 1 0mg L)和 1 7β E2 (终浓度0 3μmol L) ,计算各组细胞在 1 2 0h内的平均群体倍增时间 ,分析小鼠子宫重量与细胞群体倍增时间的相关性。结果 4种植物提取物均可使小鼠子宫重量增加 (P <0 0 5或P <0 0 1 ) ,细胞群体倍增时间缩短。子宫重量与群体倍增时间呈显著负相关 (r=- 0 96 7,P <0 0 1 )。结论 子宫实验和E

Single Cell Gel Electrophoresis Assay of Porcine Leydig Cell DNA Damage Induced by Zearalenone

[Objective] This study aimed to investigate the effect of zearalenone (ZEN) on DNA damage of porcine leydig cells. [Method] Porcine leydig cells cultured in vitro were collected to determine the median lethal dose (LD50) of ZEN with tetrazolium-based colorimetric assay (MTT assay). Comet assay was carried out to detect the DNA damage of porcine leydig cells exposed to at 0 (negative group), 1, 5, 10, 20, 40 μmol/L of ZEN. [Result] The percentage of cell tail was 16.67%, 34.00%, 40.67%, 52.00% and 64.67% under 0, 1, 5, 10 and 20 μmol/L of ZEN, respectively; the differences between the percentages of cell tail in various experimental groups had extremely significant statistical significance compared with the negative group (P<0.01), showing a significant dose-effect relationship; Tail length in various groups was 57.60±4.78, 57.75±6.25, 78.97±5.83, 100.50±6.94 and 146.83±12.31 μm, respectively; Tail DNA % in various groups was 21.29±2.25%, 22.24±2.43%, 31.21±6.27%, 37.45±4.33% and 60.68±9.83%, respectively; Tail length and Tail DNA % in experimental groups with ZEN concentration above 5 μmol/L showed significant differences (P<0.05) compared with the negative group, which showed an upward trend with the increase of ZEN concentration. [Conclusion] ZEN has genotoxic effect on porcine leydig cells, which can cause DNA damage, with a significant dose-effect relationship.

TM9SF1 promotes bladder cancer cell growth and infiltration

BACKGROUND Bladder cancer(BC)is the most common urological tumor.It has a high recur-rence rate,displays tutor heterogeneity,and resists chemotherapy.Furthermore,the long-term survival rate of BC patients has remained unchanged for decades,which seriously affects the quality of patient survival.To improve the survival rate and prognosis of BC patients,it is necessary to explore the molecular mechanisms of BC development and progression and identify targets for treatment and intervention.Transmembrane 9 superfamily member 1(TM9SF1),also known as MP70 and HMP70,is a member of a family of nine transmembrane superfamily proteins,which was first identified in 1997.TM9SF1 can be expressed in BC,but its biological function and mechanism in BC are not clear.AIM To investigate the biological function and mechanism of TM9SF1 in BC.Overexpression of TM9SF1 increased the in vitro proliferation,migration,and invasion of BC cells by promoting the entry of BC cells into the G2/M phase.Silencing of TM9SF1 inhibited in vitro proliferation,migration,and invasion of BC cells and blocked BC cells in the G1 phase.CONCLUSION TM9SF1 may be an oncogene in BC.

Machine learning and bioinformatics to identify biomarkers in response to Burkholderia pseudomallei infection in mice

Objective:In the realm of Class I pathogens,Burkholderia pseudomallei(BP)stands out for its propensity to induce severe pathogenicity.Investigating the intricate interactions between BP and host cells is imperative for comprehending the dynamics of BP infection and discerning biomarkers indicative of the host cell response process.Methods:mRNA extraction from BP-infected mouse macrophages constituted the initial step of our study.Employing gene expression arrays,the extracted RNA underwent conversion into digital signals.The percentile shift method facilitated data processing,with the identification of genes manifesting significant differences accomplished through the application of the t-test.Subsequently,a comprehensive analysis involving Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway was conducted on the differentially expressed genes(DEGs).Leveraging the ESTIMATE algorithm,gene signatures were utilized to compute risk scores for gene expression data.Support vector machine analysis and gene enrichment scores were instrumental in establishing correlations between biomarkers and macrophages,followed by an evaluation of the predictive power of the identified biomarkers.Results:The functional and pathway associations of the DEGs predominantly centered around G protein-coupled receptors.A noteworthy positive correlation emerged between the blue module,consisting of 416 genes,and the StromaScore.FZD4,identified through support vector machine analysis among intersecting genes,indicated a robust interaction with macrophages,suggesting its potential as a robust biomarker.FZD4 exhibited commendable predictive efficacy,with BP infection inducing its expression in both macrophages and mouse lung tissue.Western blotting in macrophages confirmed a significant upregulation of FZD4 expression from 0.5 to 24 h post-infection.In mouse lung tissue,FZD4 manifested higher expression in the cytoplasm of pulmonary epithelial cells in BP-infected lungs than in the control group.Conclusion:Thesefindings underscore the upregulation of FZD4 expression by BP in both macrophages and lung tissue,pointing to its prospective role as a biomarker in the pathogenesis of BP infection.

Real-world utility of serological tests in patients with suspected scrub typhus in the Republic of Korea:A single-center,retrospective,observational study

Objective:Serological tests are widely used for scrub typhus diagnosis;however,their limitations are evident.This study aims to assess their practical value in clinical settings.Methods:We analyzed the data of adult patients with suspected scrub typhus who visited a tertiary care hospital in the Republic of Korea from September to December from 2019 to 2021.The included patients had an acute fever and at least one of the following ten secondary findings:myalgia,skin rash,eschar,headache,thrombocytopenia,increased liver enzyme levels,lymphadenopathy,hepatomegaly,splenomegaly,and pleural effusion.The diagnoses were grouped as scrub typhus or other diseases by two infectious disease physicians.Results:Among 136 patients who met the eligibility criteria,109 had scrub typhus and 27 had different diseases.Single and paired total antibodies using immunofluorescence assay(IFA),and total antibodies using immunochromatography-based rapid diagnostic testing(ICT)were measured in 98%,22%,and 75%of all patients,respectively.Confirmation using paired samples for scrub typhus was established at a median of 11[interquartile range(IQR)10-16]days following the first visit.Among the 82 admitted patients,the median admission time was 9(IQR 7-13)days.According to IFA,58(55%)patients with scrub typhus had total immunoglobulin titers≥1:320,while 23(85%)patients with other disease had titers<1:320.Positive ICT results were observed in 64(74%)patients with scrub typhus and 10(67%)patients with other diseases showed negative ICT results.Conclusions:Serological testing for scrub typhus is currently insufficient for decision-making in clinical practice.

Membrane tension evolution and mechanical regulation of melittin-induced membrane poration

Membrane tension plays a crucial role in various fundamental cellular processes,with one notable example being the T cell-mediated elimination of tumor cells through perforin-induced membrane perforation by amplifying cellular force.However,the mechanisms governing the regulation of biomolecular activities at the cell interface by membrane tension remain elusive.In this study,we investigated the correlation between membrane tension and poration activity of melittin,a prototypical pore-forming peptide,using dynamic giant unilamellar vesicle leakage assays combined with flickering tension analysis,molecular dynamics simulations,and live cell assays.The results demonstrate that an increase in membrane tension enhances the activity of melittin,particularly near its critical pore-forming concentration.Moreover,peptide actions such as binding,insertion,and aggregation in the membrane further influence the evolution of membrane tension.Live cell experiments reveal that artificially enhancing membrane tension effectively enhances melittin’s ability to induce pore formation and disrupt membranes,resulting in up to a ten-fold increase in A549 cell mortality when exposed to a concentration of 2.0-μg·mL^(-1)melittin.Our findings elucidate the relationship between membrane tension and the mechanism of action as well as pore-forming efficiency of melittin,while providing a practical mechanical approach for regulating functional activity of molecules at the cell-membrane interface.

Antioxidant and lipoxygenase inhibitory properties of a novel flavonoid from Pistacia chinensis Bunge and its molecular docking analysis

Background:Pistacia chinensis Bunge has been traditionally used to manage various conditions,including asthma,pain,inflammation,hepatoprotection,and diabetes.The study was conducted to investigate the antioxidant and anti-lipoxygenase(LOX)properties of the isolated compound 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one from Pistacia chinensis.Methods:LOX assay and antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl(DPPH)assay were performed.Molecular docking studies were conducted using a molecular operating environment.Results:The LOX assay revealed significant inhibitory effects at 0.2µM concentration,with an IC50 value of 37.80µM.The antioxidant effect demonstrated dose-dependency across 5 to 100µg/mL concentrations,reaching 93.09%at 100µg/mL,comparable to ascorbic acid’s 95.43%effect.Molecular docking studies highlighted strong interactions with the lipoxygenase enzyme,presenting an excellent docking score of-10.98 kcal/mol.Conclusion:These findings provide valuable insights into Pistacia chinensis’chemical components and biological effects,reinforcing its traditional medicinal applications.

Passive neutron multiplicity device for^(240)Pu measurement based on FPGA

A passive neutron multiplicity measurement device,FH-NCM/S1,based on field-programmable gate arrays(FPGAs),is developed specifically for measuring the mass of plutonium-240(^(240)Pu)in mixed oxide fuel.FH-NCM/S1 adopts an inte-grated approach,combining the shift register analysis mode with the pulse-position timestamp mode using an FPGA.The optimal effective length of the^(3)He neutron detector was determined to be 30 cm,and the thickness of the graphite reflector was ascertained to be 15 cm through MCNP simulations.After fabricating the device,calibration measurements were per-formed using a^(252)Cf neutron source;a detection efficiency of 43.07%and detector die-away time of 55.79μs were observed.Nine samples of plutonium oxide were measured under identical conditions using the FH-NCM/S1 in shift register analysis mode and a plutonium waste multiplicity counter.The obtained double rates underwent corrections for detection efficiency(ε)and double gate fraction(f_(d)),resulting in corrected double rates(D_(c)),which were used to validate the accuracy of the shift register analysis mode.Furthermore,the device exhibited fluctuations in the measurement results,and within a single 20 s measurement,these fluctuations remained below 10%.After 30 cycles,the relative error in the mass of^(240)Pu was less than 5%.Finally,correlation calculations confirmed the robust consistency of both measurement modes.This study holds specific significance for the subsequent design and development of neutron multiplicity devices.

Electrochemical and colorimetric dual-signal detection of Staphylococcus aureus enterotoxin B based on AuPt bimetallic nanoparticles loaded Fe-N-C single atom nanocomposite

Sensitive detection of Staphylococcus aureus enterotoxin B(SEB)is of importance for preventing food poisoning from threatening human health.In this work,an electrochemical and colorimetric dual-signal detection assay of SEB was developed.The probe(Ab2/AuPt@Fe-N-C)was bound to SEB captured by Ab1,where the Ab2/AuPt@Fe-N-C triggered methylene blue degradation and resulted in the decrease of electrochemical signal.Furthermore,the probe catalyzed the oxidation of 3,3’,5,5’-tetramethyl biphenyl to generate a colorimetric absorbance at 652 nm.Once the target was captured and formed a sandwich-like complex,the color changed from colorless to blue.SEB detection by colorimetric and electrochemical methods showed a linear relationship in the concentration ranges of 0.0002-10.0000 and 0.0005-10.0000 ng/mL,with limits of detection of 0.0667 and 0.1670 pg/mL,respectively.The dual-signal biosensor was successfully used to detect SEB in milk and water samples,which has great potential in toxin detection in food and the environment.

One stone two birds:electrochemical and colorimetric dual-mode biosensor based on copper peroxide/covalent organic framework nanocomposite for ultrasensentive norovirus detection

Norovirus(NoV)is regarded as one of the most common causes of foodborne diarrhea in the world.It is urgent to identify the pathogenic microorganism of the diarrhea in short time.In this work,we developed an electrochemical and colorimetric dual-mode detection for NoV based on the excellent dual catalytic properties of copper peroxide/COF-NH_(2)nanocomposite(CuO_(2)@COF-NH_(2)).For the colorimetric detection,NoV can be directly detected by the naked eye based on CuO_(2)@COF-NH_(2)as a laccase-like nonazyme using“peptide-NoV-antibody”recognition mode.The colorimetric assay displayed a wide and quality linear detection range from 1 copy/mL to 5000 copies/mL of NoV with a low limit of detection(LOD)of 0.125 copy/mL.For the electrochemical detection of NoV,CuO_(2)@COF-NH_(2)showed an oxidation peak of copper ion from Cu^(+)to Cu^(2+)using“peptide-NoV-antibody”recognition mode.The electrochemical assay showed a linear detection range was 1-5000 copies/mL with a LOD of 0.152 copy/mL.It's worthy to note that this assay does not need other electrical signal molecule,which provide the stable and sensitive electrochemial detection for NoV.The electrochemical and colorimetric dual-mode detection was used to detect NoV in foods and faceal samples,which has the potential for improving food safety and diagnosing of NoV-infected diarrhea.

Establishment of a Multiplex Detection Method for Common Bacteria in Blood Based on Human Mannan-Binding Lectin Protein-Conjugated Magnetic Bead Enrichment Combined with Recombinase-Aided PCR Technology

Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus(SA),Pseudomonas aeruginosa(PA),and Acinetobacter baumannii(AB)in the bloodstream based on recombinant human mannanbinding lectin protein(M1 protein)-conjugated magnetic bead(M1 bead)enrichment of pathogens combined with RAP.Methods Recombinant plasmids were used to evaluate the assay sensitivity.Common blood influenza bacteria were used for the specific detection.Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR(M-RAP)and quantitative PCR(qPCR)assays.Kappa analysis was used to evaluate the consistency between the two assays.Results The M-RAP method had sensitivity rates of 1,10,and 1 copies/μL for the detection of SA,PA,and AB plasmids,respectively,without cross-reaction to other bacterial species.The M-RAP assay obtained results for<10 CFU/mL pathogens in the blood within 4 h,with higher sensitivity than qPCR.M-RAP and qPCR for SA,PA,and AB yielded Kappa values of 0.839,0.815,and 0.856,respectively(P<0.05).Conclusion An M-RAP assay for SA,PA,and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.

Polypyrimidine Tract-Binding Protein Enhances Zika Virus Translation by Binding to the 5'UTR of Internal Ribosomal Entry Site

Objectives To identify the 5'untranslated region of Zika virus(ZIKV 5'UTR)RNA-binding proteins and to investigate the impact of the binding protein on the activity of internal ribosomal entry site(IRES)located in ZIKV 5'UTR and virus production.Methods Interacting proteins in U251 cells were captured using tRSA-tagged ZIKV 5'UTR RNA and tRSA-ZIKV 5'UTR RNA-binding proteins were visualized by SDS-PAGE silver staining,Subsequently,liquid chromatographytandem mass spectrometry(LC-MS/MS),bioinformatics analysis,and Western blot were used to identify the candidate proteins binding to ZIKV 5'UTR.Dicistronic expression assay and plaque forming assay were performed to analyze the effect of the binding protein on ZIKV IRES activity and ZIKV production,respecitvely.Results tRSA RNA pull-down assay,LC-MS/MS,and Western blot analysis showed that polypyrimidine tractbinding protein(PTB)bound to the ZIKV 5'UTR.Furthermore,dual luciferase reporter assay revealed that overexpression of PTB significantly enhanced the IRES activity of ZIKV(t=10.220,P<0.001),while PTB knockdown had the opposite effect(t=4.897,P<0.01).Additionally,virus plaque forming assay demonstrated that up-regulation of PTB expression significantly enhanced viral titer(t=6.400,P<0.01),whereas reducing PTB expression level weakened virus infectivity(t=5.055,P<0.01).Conclusion PTB positively interacts with the ZIKV 5'UTR and enhances IRES activity and virus production.