Fast infectious diseases diagnostics based on microfuidic biochip system

Molecular diagnostics is one of the most important tools currently in use for clinical pathogen detection due to its high sensitivity,specificity,and low consume of sample and reagent is keyword to low cost molecular diagnostics.In this paper,a sensitive DNA isothermal amplifi-cation method for fast clinical infectious diseases diagnostics at aM concentrations of DNA was developed using a polycarbonate(PC)microfuidic chip.A portable confocal optical fuo-rescence detector was specifically developed for the microfuidic chip that was capable of highly sensitive real-time detection of amplified products for sequence-specific molecular identification near the optical diffraction limit with low background.The molecular diagnostics of Listeria monocytogenes with nucleic acid extracted from stool samples was performed at a minimum DNA template concentration of 3.65 aM,and a detection limit of less than five copies of genomic DNA.Contrast to the general polymerase chain reaction(PCR)at eppendorf(EP)tube,the detection time in our developed method was reduced from 1.5h to 45 min for multi-target parallel detection,the consume of sample and reagent was dropped from 25μL to 1.45μL.This novel microfuidic chip system and method can be used to develop a micro total analysis system as a clinically relevant pathogen molecular diagnostics method via the amplification of targets,with potential applications in biotechnology,medicine,and clinical molecular diagnostics.

Mechanism of mitochondrial protection by the Buyin Qianzheng formula in a Parkin overexpression cell model

Objective:To identify the molecular mechanisms of the effects of the Buyin Qianzheng formula(BYQZF)on the mitochondrial dynamics in a Parkin overexpression Parkinson's disease(PD)cell model.Methods:First,a stable Parkin overexpression cell model was constructed using plasmid transfection.Then,we examined the protective effect of BYQZF on the mitochondrial dysfunction of the Parkin overexpression PD cell model induced by neurotoxin 1-methyl-4-phenylpyridinium ion(MPPþ).The mRNA expression level of Parkin was evaluated using real-time quantitative PCR.The cell survival rate was detected using the Cell Counting Kit-8 assay.We evaluated the cellular adenosine triphosphate(ATP)levels using luciferase assays.A laser scanning confocal microscope was used to observe the mitochondrial morphology,activity,and mitochondrial membrane potential(DJm).Western blot was conducted to evaluate the levels of the fusion proteins mitofusin1,mitofusin2,optic atrophy 1,dynaminrelated protein 1,and mitochondrial fission protein 1.Results:Parkin overexpression attenuated MPPþ-induced mitochondrial damage,increased mitochondrial activity and DJm.BYQZF increased the survival of MPPþ-induced cells that overexpressed Parkin and upregulated the mitochondrial form factor and activity.It also inhibited a decrease in the DJm and ATP levels.These findings suggested that BYQZF protected against MPPþ-induced mitochondrial dysfunction and enhanced the protective effect of Parkin overexpression.Furthermore,the formula upregulated the expression of the fusion proteins mitofusin1,mitofusin2,and optic atrophy 1(closely related to mitochondrial quality remodeling),and reduced the expression of the fission protein dynamicrelated protein 1,as well as mitochondrial fission protein 1.Conclusion:The mechanism by which BYQZF increased the mitochondrial protective effect of Parkin gene overexpression in MPPþ-induced cells may be related to improving mitochondrial function and regulating the balance of mitochondrial division and fusion proteins.

3D spiral channels combined with flexible micro-sieve for high-throughput rare tumor cell enrichment and assay from clinical pleural effusion samples

The sieving and enrichment of rare tumor cells from large-volume pleural effusion(PE)samples is a promising technique for cell-based lung tumor diagnosis and drug tests,which features high throughput and recovery,purification,as well as viability rates of rare target cells as the prerequisites for high sensitivity,specificity,and accuracy of tumor cell analysis.In this paper,we propose a three-dimensional(3 D)sieving method for rare tumor cell enrichment,which effectively eliminates the"dead zones"in traditional two-dimensional(2 D)cell filters with a dimension-raising strategy to satisfy the requirements mentioned above.The prototype device was combined with a funnel-shaped holder,a flexible micropore membrane in the middle,and a3 D spiral fluid channel covered on the membrane as a three-layer ice-creaming cone composite structure.Driven by gravity alone,the device performed as follows:(1)20-fold throughput compared with the 2 D commercial planee hich was up to 20 mL/min for a threefold dilution of whole blood sample;(2)high recovery rates of 84.5%±21%,86%±25%,83%±14%for 100,1000,and 10000 cells/mL,respectively,in 30 mL phosphate buffer saline(PBS)sample,and a 100%positive detection rate in the case of≤5 A549 cells in 1 mL PBS;(3)a typical purification rate of 85.5%±9.1%;and(4)a viability rate of>93%.In the demonstration application,this device effectively enriched rare target cells from large volumes(>25 mL)of clinical pleural effusions.The following results indicated that tumor cells were easy-to-discover in the enriched PE samples,and the proliferation capability of purified cells was(>4.6 times)significantly stronger than that of unprocessed cells in the subsequent 6-day culture.The above evaluation indicates that the proposed easily reproducible method for the effective execution of rare cell enrichments and assays is expected to become a practical technique for clinical cell-based tumor diagnosis.

Electroacupuncture improves neurovascular unit reconstruction by promoting collateral circulation and angiogenesis

Acupuncture at Shuigou(GV26) shows good clinical efficacy for treating stroke, but its mechanism remains poorly understood. In this study, a cerebral infarction model of ischemia/reperfusion injury received electroacupuncture at GV26(15 Hz and 1 m A, continuous wave [biphasic pulses], for 5 minutes). Electroacupuncture effectively promoted regional cerebral blood flow on the infarct and non-infarct sides, increased infarct lesions, lectin, and number of blood vessels, upregulated von Willebrand factor and cell proliferation marker Ki67 expression, and diminished neurological severity score. These findings confirm that electroacupuncture at GV26 promotes establishment of collateral circulation and angiogenesis, and improves neurological function.

一款用于快速检测包括SARS-CoV-2在内的19种常见呼吸道病毒的高通量、多指标等温扩增平台

快速、准确地诊断以及及时对感染严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的患者进行隔离被认为是抗击新冠病毒肺炎(COVID-19)大流行的最有效的措施。本文介绍了一款高通量、多指标的等温扩增检测平台(RTisochip^TM-W),该平台使用离心式微流控芯片,在90min内即可完成一次运行,从16个样本中检测出包括SARS-CoV-2在内的19种常见呼吸道病毒。通过RTisochip^TM-W系统检测的所有病毒的检出限均等于或小于50拷贝·μL^-1,与常规逆转录聚合酶链反应(RT-PCR)灵敏度相当。该系统具有重复性好、鲁棒性强和特异性高的优点。最后,使用RTisochip^TM-W系统对201例临床前样本、14例COVID-19阳性临床样本、25例临床确诊样本以及614例来自呼吸道感染或疑似感染的患者的临床样本进行了测试。测试结果与临床诊断结果吻合良好,也反映出呼吸道传染病的流行特征。在对SARS-CoV-2的诊断上,RTisochip^TM-W系统与参考试剂盒的一致性达到98.15%。基于大量的测试结果,我们相信RTisochip^TM-W系统是一款抗击COV ID-19大流行的强有力工具。

DETECTION AND APPLICATION OF MICROFLUIDIC ISOTHERMAL AMPLIFICATION ON CHIP

Loop-mediated isothermal amplification(LAMP)is a novel nucleic acid amplification method.Compared with the widely utilized polymerase chain reaction(PCR),LAMP has higher speed and efficiency as well as lower requirement for system temperature control because the whole amplification process is isothermal and no efforts are needed to switch between different temperatures.In this paper,we designed and fabricated different kinds of polycarbonate(PC)microfluid chips,explored appropriate reaction condition for LAMP in microenvironment(1 nL→10μL),and developed a microfluidic isothermal amplification detection system.The DNA optimal amplification temperature is obtained;the starting time of exponential amplification of DNA is put forward farther.The optimal condition of DNA amplification in microenvironment,with a little reaction materials and early starting exponential amplification time of DNA are very important for clinic DNA detection and the application of Lab-on-a-Chip.

Original askiatic imaging used in Chinese medicine eye-feature diagnosis of visceral diseases

Eye-feature diagnosis is a time-homored met hod for studying many diseases in tradit ional Chinese medicine.There is a dlose relationship between eye feature and viscera,and eye feature is a reflect ion of viscer al health status.Commercially used ophthalmology diagnosis instr uments have disadvantages and cannot satisfy the requirements of eye feature diagnosis.In this paper,we proposed a novel askiatic imaging method that removes the interference of an ilumination source's reflection shadow and is free from image splicing.We developed a novel imaging system to implement this method,and some eye feature characteristics to analyze visceral diseases were obtained.

LABEL-FREE DETECTION OF PROTEIN MICROARRAY WITH HIGH THROUGHPUT SURFACE PLASMON RESONANCE IMAGING(SPRI)

A surface plasmon resonance imaging(SPRI)system was developed for the discrimination of proteins on a gold surface.As a label-free and high-throughput technique,SPRI enables simultaneously monitoring of the biomolecular interactions at low concentrations.We used SPRI as a label-free and parallel method to detect different proteins based on protein microarray.Bovine Serum Albumin(BSA),Casein and Immunoglobulin G(IgG)were immobilized onto the Au surface of a gold-coated glass chip as spots forming a 6×6 matrix.These proteins can be discriminated directly by changing the incident angle of light.Excellent reproducibility for label-free detection of protein molecules was achieved.This SPRI platform represents a simple and robust method for performing high-sensitivity detection of protein microarray.

Evidence of compliance with and effectiveness of guidelines for noninvasive prenatal testing in China:a retrospective study of 189,809 cases

In China,the medical guidelines recommend performing noninvasive prenatal testing(NIPT)with caution for pregnant women aged 35 years or older.However,the Mother and Child Health Care Law suggests that all primiparous women whose age is older than 35 years undergo prenatal diagnosis.These two inconsistent suggestions/recommendations have made obstetricians confused about whether to offer NIPT to these older pregnant women.To face this issue and find out the solution we performed a retrospective study of 189,809 NIPT samples collected from 28 provincial-leveled administrative units in China.Of 1,564 women with high-risk pregnancies who underwent NIPT,459(29.3%)did not participate in follow-up.The compound sensitivity and specificity of NIPT for trisomies 21,18 and 13 detection was 99.1%(95%CI,98.0%–99.6%)and 99.9%(95%CI,98.8%–99.9%),respectively.In secundiparous women,NIPT showed high sensitivity and specificity similar to that in primiparous women.The observed risk for trisomies 21 and 18 significantly increased when the maternal age was 39 and older.After the publication of the current NIPT policy,the follow-up rate at our center was 97.9%;however,a large number of women are not in maternal and infant care networks nationwide,and that makes the follow-up rate outside our center relatively low.Our study shows that to balance the prevention of major aneuploidies and the limited resources for prenatal diagnosis,the cut-off age of 35 for invasive prenatal diagnosis might be unnecessary.Although the NIPT guidelines are well written,how to practice it effectively,especially in less industrialized areas,is worth discussing.

Evaluation of the immunomodulatory effects of anti-COVID-19 TCM formulae by multiple virus-related pathways

Dear Editor,The coronavirus infectious disease 2019(COVID-19)outbreak is seriously endangering human health.Most patients with severe COVID-19 are characterized by sustained cytokine production and hyper-inflammation,which is known as cytokine storm syndrome.1-3 Elaborating the anti-inflammatory response is crucial to these patients,and interieukin-6(IL6)inhibitors and steroids have been recommended in clinical practice.1 However,after the cytokine storm phase,the host immune response to sepsis may develop into a protracted immunosuppressive phase.

CCGD-ESCC： A Comprehensive Database for Genetic Variants Associated with Esophageal Squamous Cell Carcinoma in Chinese Population

Esophageal squamous-cell carcinoma(ESCC) is one of the most lethal malignancies in the world and occurs at particularly higher frequency in China. While several genome-wide association studies(GWAS) of germline variants and whole-genome or whole-exome sequencing studies of somatic mutations in ESCC have been published, there is no comprehensive database publically available for this cancer. Here, we developed the Chinese Cancer Genomic Database-Esophageal Squamous Cell Carcinoma(CCGD-ESCC) database, which contains the associations of 69,593 single nucleotide polymorphisms(SNPs) with ESCC risk in 2022 cases and 2039 controls, survival time of 1006 ESCC patients(survival GWAS) and gene expression(expression quantitative trait loci,eQTL) in 94 ESCC patients. Moreover, this database also provides the associations between8833 somatic mutations and survival time in 675 ESCC patients. Our user-friendly database is a resource useful for biologists and oncologists not only in identifying the associations of genetic variants or somatic mutations with the development and progression of ESCC but also in studying the underlying mechanisms for tumorigenesis of the cancer. CCGD-ESCC is freely accessible at http://db.cbi.pku.edu.cn/ccgd/ESCCdb.

Dendrocalamus latiflorus and its component rutin exhibit glucose-lowering activities by inhibiting hepatic glucose production via AKT activation

The potential medicinal value of Ma bamboo(Dendrocalamus latiflorus), one of the most popular and economically important bamboo species in China, has been underestimated. In the present study, we found that D. latiflorus leaf extract(DLE) reduced fasting blood glucose levels, body weight,and low-density lipoprotein cholesterol with low liver toxicity in db/db mice. In addition, gene expression profiling was performed and pathway enrichment analysis showed that DLE affected metabolic pathways.Importantly, DLE activated the AKT signaling pathway and reduced glucose production by downregulating glucose-6-phosphatase(G6 PC) and phosphoenolpyruvate carboxykinase 1(PCK1) expression. Moreover, network pharmacology analysis identified rutin as an active component in DLE through targeting insulin growth factor 1 receptor(IGF1 R), an upstream signaling transducer of AKT. Due to its hypoglycemic effects and low toxicity, DLE may be considered an adjuvant treatment option for type 2 diabetes patients.

COVID-19背景下生物芯片进入稳步发展与快速转化的新阶段

Severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)has placed a heavy burden on the global healthcare system,especially in developing and underdeveloped countries where professionals and specialized laboratories are scarce.And due to the increased demand for large-scale detection and the shortage of animal models for vaccines and antiviral drugs under the coronavirus disease 2019(COVID-19)pandemic.

A genome wide association study between copy number variation (CNV) and human height in Chinese population

Copy number variation (CNV) is a type of genetic variation which may have important roles in phenotypic variability and disease susceptibility. To hunt for genetic variants underlying human height variation, we performed a genome wide CNV association study for human height in 618 Chinese unrelated subjects using Affymetrix 500K array set. After adjusting for age and sex, we found that four CNVs at 6p21.3, 8p23.3-23.2, 9p23 and 16p12.1 were associated with human height (with borderline significant p value: 0.013, 0.011, 0.024, 0.049; respectively). However, after multiple tests correction, none of them was associated with human height. We observed that the gain of copy number (more than 2 copies) at 8p23.3-23.2 was associated with lower height (normal copy number vs. gain of copy number: 161.2 cm vs. 153.7 cm, p = 0.011), which accounted for 0.9% of height variation. Loss of copy number (less than 2 copies) at 6p21.3 was associated with 0.8% lower height (loss of copy number vs. normal copy number: 154.5 cm vs. 161.1 cm, p = 0.013). Since no important genes influencing height located in CNVs at loci of 8p23.3-23.2 and 6p21.3, the two CNVs may cause the structural rear- rangements of neighbored important candidate genes, thus regulates the variation of height. Our results expand our knowledge of the genetic factors underlying height variation and the biological regulation of human height.

促肿瘤长链非编码RNA LINC00973通过增强LDHA酶活促进肿瘤细胞有氧糖酵解

有氧糖酵解在肿瘤细胞内被特异激活的调控机制尚不清楚.本研究通过大规模的功能筛选,发现了一个能显著促肿瘤细胞增殖的长链非编码RNA LINC00973.敲低该RNA的表达,能够抑制多种肿瘤细胞的体外增殖及体内(动物)生长.LINC00973能够结合到糖酵解关键酶LDHA(乳酸脱氢酶A)上并增强其酶活,进而加速肿瘤细胞的有氧糖酵解,最终促进肿瘤细胞增殖.LINC00973在多种肿瘤的样本中显著高表达,且高表达LINC00973的病人预后较差.本研究提示在肿瘤细胞中高表达的LINC00973促进了肿瘤细胞中的有氧糖酵解的特异激活,揭示了肿瘤细胞中特异存在的糖酵解调控方式,为靶向糖酵解开发广谱抗肿瘤药物提供了新靶标.

Multiplexed detection of respiratory pathogens with a portable analyzer in a “raw-sample-in and answer-out” manner

Coronavirus disease 2019(COVID-19)has emerged,rapidly spread and caused significant morbidity and mortality worldwide.There is an urgent public health need for rapid,sensitive,specific,and on-site diagnostic tests for severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection.In this study,a fully integrated and portable analyzer was developed to detect SARS-CoV-2 from swab samples based on solid-phase nucleic acid extraction and reverse transcription loop-mediated isothermal amplification(RT-LAMP).The swab can be directly inserted into a cassette for multiplexed detection of respiratory pathogens without pre-preparation.The overall detection process,including swab rinsing,magnetic bead-based nucleic acid extraction,and 8-plex real-time RT-LAMP,can be automatically performed in the cassette within 80 min.The functionality of the cassette was validated by detecting the presence of a SARS-CoV2 pseudovirus and three other respiratory pathogens,i.e.,Klebsiella pneumoniae,Pseudomonas aeruginosa,and Stenotrophomonas maltophilia.The limit of detection(LoD)for the SARS-CoV-2 pseudovirus was 2.5 copies/μL with both primer sets(N gene and ORF1ab gene),and the three bacterial species were successfully detected with an LoD of 2.5 colony-forming units(CFU)/μL in 800μL of swab rinse.Thus,the analyzer developed in this study has the potential to rapidly detect SARS-CoV-2 and other respiratory pathogens on site in a“raw-sample-in and answer-out”manner.

Miniaturized Cell Lysis Device Using Spherically Focused Ultrasound

A prototype of a miniaturized cell lysis device developed using a concave spherical transducer is capable of lysing bacteria without added chemical denaturants, enzymes or microparticles and is capable of efficiently lysing yeast without any mechanical or enzymatic pretreatment. The device is designed for miniature bio analysis systems where cell lysing is needed to obtain intracellular materials for further analysis such as DNA identification. The device lysis efficiency was evaluated using viable cell counts and microscopy. Additionally, the device efficiency was compared with that of traditional chemical cell lysis methods using standard molecular biological techniques such as agarose gels and ultraviolet (UV) spectroscopy. The results indicate that efficient bacteria and cell disruption can be achieved through a low voltage driven and spherically focused high frequency ultrasonic device.

Detection of the apoptosis of Jurkat cell using an electrorotation chip

The apoptosis of cells is one of the fields that attract increasing attention in biology today.Usually,the cells are treated with chemicals when detecting apoptosis.It is highly desired to detect apoptosis in a real-time basis.Apoptosis of Jurkat cells was studied using a real-time electrorotation chip.This chip allows the detection of the cell membrane capacitance changes during the course of apoptosis and therefore facilitates the analysis of apoptosis in a real-time basis without involving any chemical treatment.

A transcriptome-based analysis reveals functional differences among Dendrobium officinale Kimura&Migo species from different growing regions and with different quality levels

Dendrobium officinale Kimura&Migo(D.officinale)is a common,high medicinal value food and herbal medicine used in the treatment of yin-deficient diseases.Adulterants obtained with similar inexpensive species of Dendrobium plants are available in the market.However,Dendrobium species with different quality levels or from different regions as well as analogous species have a similar appearance,which makes the discrimination of Dendrobium species difficult.Reliable objective methods for authenticating D.officinale are lacking.Here,samples of Dendrobium officinale Kimura&Migo(D.officinale)from two regions with three quality levels and sample of the analogous species Dendrobium compactum Rolfe ex W.Hackett(D.compactum)were subjected to genome-wide biological response fingerprinting(BioReF)in RAW264.7 cells by RNA sequencing.The biological pathways affected by these samples could distinguish D.officinale from D.compactum.The high-quality D.officinale samples from Yunnan exhibited the strongest inhibition of lipopolysaccharide(LPS)induced inflammatory genes and pathways,followed by samples from Myanmar.The medium-and low-quality samples from Yunnan exhibited weaker effects,and D.compactum exerted mild effects.The RNA sequencing results were strengthened by the results from analyses of the expression of the inflammation-related genes Il6,Ccl5,and Cox2 and the production of nitric oxide.Thus,transcriptomic profiling can be used to evaluate the quality of D.officinale and distinguish its origin.