A developmental validation of the Quick TargSeq 1.0 integrated system for automated DNA genotyping in forensic science for reference samples.

Analysis of short tandem repeats (STRs) is a global standard method for human identification. Insertion/Deletion polymorphisms (DIPs) can be used for biogeographical ancestry inference. Current DNA typing involves a trained forensic worker operating several specialized instruments in a controlled laboratory environment, which takes 6-8 h. We developed the Quick TargSeq 1.0 integrated system (hereinafter abbreviated to Quick TargSeq) for automated generation of STR and DIP profiles from buccal swab samples and blood stains. The system fully integrates the processes of DNA extraction, polymerase chain reaction (PCR) amplification, and electrophoresis separation using microfluidic biochip technology. Internal validation studies were performed using RTyper 21 or DIP 38 chip cartridges with single-source reference samples according to the Scientific Working Group for DNA Analysis Methods guidelines. These results indicated that the Quick TargSeq system can process reference samples and generate STR or DIP profiles in approximately 2 h, and the profiles were concordant with those determined using traditional STR or DIP analysis methods. Thus, reproducible and concordant DNA profiles were obtained from reference samples. Throughout the study, no lane-to-lane or run-to-run contamination was observed. The Quick TargSeq system produced full profiles from buccal swabs with at least eight swipes, dried blood spot cards with two 2-mm disks, or 10 ng of purified DNA. Potential PCR inhibitors (i.e., coffee, smoking tobacco, and chewing tobacco) did not appear to affect the amplification reactions of the instrument. The overall success rate and concordance rate of 153 samples were 94.12% and 93.44%, respectively, which is comparable to other commercially available rapid DNA instruments. A blind test initiated by a DNA expert group showed that the system can correctly produce DNA profiles with 97.29% genotype concordance with standard bench-processing methods, and the profiles can be uploaded into the national DNA database. These results demonstrated that the Quick TargSeq system can rapidly generate reliable DNA profiles in an automated manner and has the potential for use in the field and forensic laboratories.

17ß-Estradiol Mediates Staphylococcus aureus Adhesion in Vaginal Epithelial Cells via Estrogen Receptor α-Associated Signaling Pathway.

Staphylococcus aureus, a major opportunistic pathogen in aerobic vaginitis (AV), can potentially invade the host and occasionally cause infections. Estrogen is associated with an altered immune response of vaginal epithelial cells and prevention of certain vaginal infectious diseases. However, the molecular mechanisms involving estrogen and S. aureus adhesion to vaginal epithelial cells remain unclear. Thus, here, VK2/E6E7 vaginal epithelial cells were infected with S. aureus, and the role of the estrogen receptor α-associated signaling pathway (ERα/FAK/Src/iNOS axis) in S. aureus adhesion was evaluated. The estrogen-associated phosphorylation status of ERα, FAK, and Src and the protein level of iNOS were assessed by western blotting. We used a specific ERα inhibitor to validate the involvement of the ERα-associated signaling pathway. The results showed that with exposure to 1 nM estrogen for 24 h, transient ERα-associated pathway activation was observed, and the protein expression upregulation was accompanied by a dose-dependent increase in 17-ß-estradiol (E2) content and increased S. aureus adherence to vaginal epithelial cells. Estrogen-induced activation of the ERα/FAK/Src/iNOS axis was notably inhibited by the specific ERα inhibitor (ICI 182780). Simultaneously, a significant decrease in the number of adherent S. aureus was observed. However, this inhibitory effect diminished after inhibitor treatment for 24 h. Our findings suggested that the ERα-associated signaling pathway might be involved in S. aureus adherence to vaginal epithelial cells, which appeared to be linked to enhanced cell adhesion leading to AV.

Observation of the clinical efficacy of isotope phosphorus-32 dressing combined with diprospan and mucopolysaccharide polysulphate cream in the treatment of keloids.

OBJECTIVE: The present study aims to investigate the effectiveness, recurrence, and adverse reaction rates of isotope phosphorus-32 dressings combined with diprospan and mucopolysaccharide polysulphate cream in the treatment of keloids. METHODS: A total of 80 patients with keloids admitted to the Dermatology Clinic of the Fourth Affiliated Hospital of Harbin Medical University between June 2019 and June 2021 were included in the present study and randomly divided into three groups: Control Group 1 (n = 27), Control Group 2 (n = 25), and the treatment group (n = 28). Patients in Control Group 1 were treated with diprospan combined with mucopolysaccharide polysulphate cream, patients in Control Group 2 were treated with an isotopic phosphorus-32 dressing combined with mucopolysaccharide polysulphate cream, and patients in the treatment group were treated with an isotopic phosphorus-32 dressing combined with diprospan and mucopolysaccharide polysulphate cream. The effectiveness, recurrence, and adverse reaction rates were observed in all three groups. RESULTS: The treatment group had the most significant decrease in the itching scores. The respective effectiveness, recurrence, and adverse reaction rates were 81.4%, 43.6%, and 74.1% in Control Group 1; 56%, 38.7%, and 64% in Control Group 2; and 96.7%, 11.2%, and 41% in the treatment group. The differences were statistically significant (p < 0.05). CONCLUSION: An isotope phosphorus-32 dressing combined with diprospan and mucopolysaccharide polysulphate cream keloid treatment delivers a fast onset, good effectiveness, and low recurrence and adverse effect rates.

Earth-affecting solar transients: a review of progresses in solar cycle 24.

This review article summarizes the advancement in the studies of Earth-affecting solar transients in the last decade that encompasses most of solar cycle 24. It is a part of the effort of the International Study of Earth-affecting Solar Transients (ISEST) project, sponsored by the SCOSTEP/VarSITI program (2014-2018). The Sun-Earth is an integrated physical system in which the space environment of the Earth sustains continuous influence from mass, magnetic field, and radiation energy output of the Sun in varying timescales from minutes to millennium. This article addresses short timescale events, from minutes to days that directly cause transient disturbances in the Earth's space environment and generate intense adverse effects on advanced technological systems of human society. Such transient events largely fall into the following four types: (1) solar flares, (2) coronal mass ejections (CMEs) including their interplanetary counterparts ICMEs, (3) solar energetic particle (SEP) events, and (4) stream interaction regions (SIRs) including corotating interaction regions (CIRs). In the last decade, the unprecedented multi-viewpoint observations of the Sun from space, enabled by STEREO Ahead/Behind spacecraft in combination with a suite of observatories along the Sun-Earth lines, have provided much more accurate and global measurements of the size, speed, propagation direction, and morphology of CMEs in both 3D and over a large volume in the heliosphere. Many CMEs, fast ones, in particular, can be clearly characterized as a two-front (shock front plus ejecta front) and three-part (bright ejecta front, dark cavity, and bright core) structure. Drag-based kinematic models of CMEs are developed to interpret CME propagation in the heliosphere and are applied to predict their arrival times at 1 AU in an efficient manner. Several advanced MHD models have been developed to simulate realistic CME events from the initiation on the Sun until their arrival at 1 AU. Much progress has been made on detailed kinematic and dynamic behaviors of CMEs, including non-radial motion, rotation and deformation of CMEs, CME-CME interaction, and stealth CMEs and problematic ICMEs. The knowledge about SEPs has also been significantly improved. An outlook of how to address critical issues related to Earth-affecting solar transients concludes this article.

The benefit of adjuvant radiotherapy on overall survival in resected stage I to II pancreatic cancer: A propensity-adjusted analysis.

BACKGROUND: The survival time of patients with early pancreatic cancer (PC) is still disappointing, even after surgical resection. PC has an extremely poor prognosis. Herein, we aimed to investigate the survival effect of postoperative radiotherapy (PORT) on resected stage I to II PC. MATERIAL AND METHODS: A large eligible sample of patients was identified from 2010 to 2015 from the Surveillance, Epidemiology, and End Results (SEER) registry. Survival analysis was conducted to evaluate the efficiency of PORT. Propensity score matching (PSM) analysis was used to reduce selection bias and to make the groups comparable. RESULTS: A total of 3219 patients with resected stage I to II PC was included after rigid screening. The median overall survival (OS) was 26 months with PORT (n = 1055) versus 21 months with non-PORT (n = 2164) before matching (p<0.001). By multivariable analysis, PORT remained a favorable prognostic predictor for OS. In PSM analysis, receiving PORT was associated with improved OS (median, 26 months vs. 23 months; at 2 years, 51.7% vs. 46.7%; at 5 years, 23.3% vs. 17.4% (P = 0.006). After further meticulous exploration, only the stage IIB subgroup benefited from PORT (p<0.001). This result was due to the positive lymph node state (N+), whose mortality risk was cut by 23.4% (p<0.001) by PORT. CONCLUSION: Addition of PORT to the treatment of patients with resected stage I to II PC conveys a survival benefit, particularly among those with N-positive or stage IIB disease.

Web- and Artificial Intelligence-Based Image Recognition For Sperm Motility Analysis: Verification Study.

BACKGROUND: Human sperm quality fluctuates over time. Therefore, it is crucial for couples preparing for natural pregnancy to monitor sperm motility. OBJECTIVE: This study verified the performance of an artificial intelligence-based image recognition and cloud computing sperm motility testing system (Bemaner, Createcare) composed of microscope and microfluidic modules and designed to adapt to different types of smartphones. METHODS: Sperm videos were captured and uploaded to the cloud with an app. Analysis of sperm motility was performed by an artificial intelligence-based image recognition algorithm then results were displayed. According to the number of motile sperm in the vision field, 47 (deidentified) videos of sperm were scored using 6 grades (0-5) by a male-fertility expert with 10 years of experience. Pearson product-moment correlation was calculated between the grades and the results (concentration of total sperm, concentration of motile sperm, and motility percentage) computed by the system. RESULTS: Good correlation was demonstrated between the grades and results computed by the system for concentration of total sperm (r=0.65, P<.001), concentration of motile sperm (r=0.84, P<.001), and motility percentage (r=0.90, P<.001). CONCLUSIONS: This smartphone-based sperm motility test (Bemaner) accurately measures motility-related parameters and could potentially be applied toward the following fields: male infertility detection, sperm quality test during preparation for pregnancy, and infertility treatment monitoring. With frequent at-home testing, more data can be collected to help make clinical decisions and to conduct epidemiological research.

Efficient light focusing through an MMF based on two-step phase shifting and parallel phase compensating.

Based on a parallel phase compensation scheme, we propose an efficient wavefront shaping method using a spatial light modulator (SLM) for quickly generating a series of focused spots through a multimode fiber (MMF). The compensated phase mask obtained by a two-step phase-shifting technique is loaded to the SLM for generating a focused spot at an arbitrary target position out of the fiber facet. Furthermore, the parallel algorithm we present makes it possible to obtain a series of compensated phase masks, which could be used to generate a series of focused spots at different locations. We experimentally obtained 100 tightly focused spots, with an average focused efficiency of 21.60% and an average focused diameter of 1.9240 µm, and only one-time parallel-compensated phase retrieval is required without multiple iteration optimization.

Glass stabilized ultra-stable dual-emitting Mn-doped cesium lead halide perovskite quantum dots for cryogenic temperature sensing.

Mn-Doped CsPb(Cl/Br)3 quantum dots possess multi-functional optical, electronic and magnetic characteristics. However, they usually suffer from decomposition in air, and Mn2+ dopants will be gradually expelled from the perovskite host due to a radius mismatch between Pb2+ and Mn2+. To solve these crucial issues, the synthesis of glass stabilized Mn-doped quantum dots via an appropriate glass composition design and in situ glass crystallization is reported. Mn2+ dopants act as nucleating agents to promote the nucleation/growth of CsPb(Cl/Br)3 from B-P-Zn-Cs-Pb based oxyhalide glass and partition into the perovskite host to produce dual-color luminescence via efficient exciton-to-dopant energy transfer. Benefitting from the effective protection of robust glass, Mn-doped CsPb(Cl/Br)3 quantum dots exhibit superior water resistance and thermal stability. Particularly, almost 100% luminescence is retained after immersing the composite in water for 30 days. Interestingly, rapid thermal quenching for exciton recombination relative to Mn2+ d-d transition at cryogenic temperatures enables its promising applications as a ratiometric temperature sensing medium.

Modular-Based Integrated Microsystem with Multiple Sample Preparation Modules for Automated Forensic DNA Typing from Reference to Challenging Samples.

The realization of an automated short tandem repeat (STR) analysis for forensic investigations is facing a unique challenge, that is DNA evidence with wide disparities in sample types, quality, and quantity. We developed a fully integrated microsystem in a modular-based architecture to accept and process various forensic samples in a "sample-in-answer-out" manner for forensic STR analysis. Two sample preparation modules (SPMs), the direct and the extraction SPM, were designed to be easily assembled with a capillary array electrophoresis (CAE) chip using a chip cartridge to efficiently achieve an adequate performance to different samples at a low cost. The direct SPM processed buccal swabs to produce STR profiles without DNA extraction in about 2 h. The extraction SPM analyzed more challenging blood samples based on chitosan-modified quartz filter paper for DNA extraction. This newly developed quartz filter provided a 90% DNA extraction efficiency and the "in situ" PCR capability, which enabled DNA extraction and PCR performed within a single chamber with all the DNA concentrated in the filter. We demonstrated that minute amounts of blood (0.25 µL), highly diluted blood (0.5 µL blood in 1 mL buffer), and latent bloodstains (5-µL bloodstain on cloth washed with detergent) can be automatically analyzed using our microsystem, reliably producing full STR profiles with a 100% calling of all the alleles. This modular-based microsystem with the capability of analyzing a wide range of samples should be able to play an increasing role in both urgent situations and routine forensic investigations, dramatically extending the applications and utility of automated DNA typing.

Changing expression profiles of mRNA, lncRNA, circRNA, and miRNA in lung tissue reveal the pathophysiological of bronchopulmonary dysplasia (BPD) in mouse model.

New perinatal care technologies have improved the survival rate of preterm neonates, but the prevalence of bronchopulmonary dysplasia (BPD), one of the most intractable problems in neonatal intensive care unit (NICU), remains unchanged. In present study, high-throughput sequencing (HTS) was performed to detect the expression profiles of long noncoding RNAs (lncRNAs), messenger RNAs (mRNAs), circular RNAs (circRNAs), and microRNAs (miRNAs) in hyperoxia-induced BPD mouse model. Significant differentially expressed RNAs were selected and clustered between the BPD group and the control group. The results revealed that expressions of 1778 lncRNAs, 1240 mRNAs, 97 circRNAs, and 201 miRNAs were significantly altered in the BPD group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to predict the potential functions of differentially expressed RNAs. lncRNA-mRNA and circRNA-miRNA coexpression networks were constructed to detect their association with the pathogenesis of BPD. Our study provides a systematic perspective on the potential function of RNAs during BPD.

The Association between Vitamin D Deficiency and Sleep Disorders: A Systematic Review and Meta-Analysis.

Epidemiology studies have investigated the association between vitamin D and the risk of sleep disorders, but the results remain controversial. Therefore, we conducted this meta-analysis with the goal of clarifying the association between vitamin D and sleep disorders risk. All relevant studies were searched using PubMed, EMBASE, and Web of Science from inception to January 2018. Pooled odds ratios (ORs) and 95% confidence interval (CIs) were calculated using a fixed-effect model A total of nine studies (6 cross-sectional, 2 case-control, and 1 cohort studies) involving 9397 participants were included. By comparing the lowest verse highest levels of serum vitamin D, we found that participants with vitamin D deficiency (VDD) had a significantly increased risk of sleep disorders (OR: 1.50, 95% CI: 1.31, 1.72). Subgroup analysis showed that VDD also was associated with poor sleep quality (OR: 1.59, 95% CI: 1.23, 2.05), short sleep duration (OR: 1.74, 95% CI: 1.30, 2.32), and sleepiness (OR: 1.36, 95% CI: 1.12, 1.65). Subgroup analyses further indicated that serum 25(OH)D <20 ng/mL could significantly increase the risk of unhealthy sleep. This meta-analysis suggest that vitamin D deficiency is associated with a higher risk of sleep disorders. More high-quality cohort studies and randomized controlled trials (RCTs) are needed to verify this association.

Differential Expression of CircRNAs in Embryonic Heart Tissue Associated with Ventricular Septal Defect.

Objectives: To explore and validate the differential expression of circRNAs in the myocardium of congenital ventricular septal defect (VSD) and to explore a new avenue of research regarding the pathological mechanisms of VSD. Methods: We detected circRNAs expression profiles in heart tissues taken from six aborted fetuses with VSD and normal group using circRNA microarray. Some differentially expressed circRNAs were studied by bioinformatics analysis. Finally, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to confirm these results. Results: This study found abundant circRNAs in the myocardium taken from individuals in the normal group and the VSD group. After that, totally 6234 differentially expressed circRNAs between the normal group and the VSD group were confirmed (Fold change ≥ 2.0; p < 0.05). Then, this research carried out bioinformatics analysis and predicted the potential biological functions of circRNAs. Finally, the over-expression of hsa_circRNA_002086 and under-expression of hsa_circRNA_007878, hsa_circRNA_100709, hsa_circRNA_101965, hsa_circRNA_402565 were further validated by qRT-PCR. Conclusions: There is a significant difference in expression of the circRNA in cardiac tissue from VSD group compared to the normal group. Combined with the microarray results and previous researches, circRNAs may contribute to the occurrence of VSD by acting as miRNA sponges or by binding proteins, these possible roles for circRNAs in VSD require elucidation in additional studies.

Peptidomic Analysis of Maternal Serum to Identify Biomarker Candidates for Prenatal Diagnosis of Tetralogy of Fallot.

Tetralogy of Fallot (TOF) is the most common form of cyanotic congenital heart disease. To identify endogenous peptides possibly involved in the progression of TOF, we performed comparative peptidomic profiling of maternal serum between normal fetuses and fetuses suffering from TOF. A total of 278 differentially expressed peptides, including 94 over-expressed peptides and 184 under-expressed peptides, originating from 227 protein precursors were identified by liquid chromatography/mass spectrometry (LC/MS) in maternal serum of fetuses with TOF compared to normal controls. Further, ingenuity pathway analysis (IPA) was used to identify putative roles for these peptides in cardiovascular development. Two peptides were derived from functional domains of proteins involved in heart development and associated with TOF; these may represent candidate bioactive peptides involved in TOF. These peptides may be related to the pathologic changes in the heart associated with TOF, and may be useful as novel biomarkers for prenatal diagnosis of TOF. J. Cell. Biochem. 119: 468-477, 2018. © 2017 Wiley Periodicals, Inc.

A fully integrated microchip system for automated forensic short tandem repeat analysis.

We have successfully developed an integrated microsystem that combines two plastic microchips for DNA extraction and PCR amplification with a glass capillary array electrophoresis chip together in a compact control and detection instrument for automated forensic short tandem repeat (STR) analysis. DNA extraction followed by an "in situ PCR" was conducted in a single reaction chamber of the microchip based on a filter paper-based extraction methodology. PCR products were then mixed with sizing standards by an injection electrode and injected into the electrophoresis chip for four-color confocal fluorescence detection. The entire STR analysis can be completed in about two hours without any human intervention. Since the 15-plex STR system has a more stringent requirement for PCR efficiency, we optimized the structure of the plastic DNA extraction and amplification chip, in which the reaction chamber was formed by sandwiching a hollow structure layer with two blank cover layers, to reduce the adsorption of PCR reagents to the surfaces. In addition, PCR additives, bovine serum albumin, poly(ethylene glycol), and more magnesium chloride were included into the on-chip multiplex STR system. The limit-of-detection study demonstrated that our microsystem was able to produce full 15-plex STR profiles from 3.75 ng standard K562 DNA. Buccal swab and whole blood samples were also successfully typed by our system, validating the feasibility of performing rapid DNA typing in a "sample-in-answer-out" manner for on-site forensic human identification.

The Optimization of Electrophoresis on a Glass Microfluidic Chip and its Application in Forensic Science.

Microfluidic chips offer significant speed, cost, and sensitivity advantages, but numerous parameters must be optimized to provide microchip electrophoresis detection. Experiments were conducted to study the factors, including sieving matrices (the concentration and type), surface modification, analysis temperature, and electric field strengths, which all impact the effectiveness of microchip electrophoresis detection of DNA samples. Our results showed that the best resolution for ssDNA was observed using 4.5% w/v (7 M urea) lab-fabricated LPA gel, dynamic wall coating of the microchannel, electrophoresis temperatures between 55 and 60°C, and electrical fields between 350 and 450 V/cm on the microchip-based capillary electrophoresis (µCE) system. One base-pair resolution could be achieved in the 19-cm-length microchannel. Furthermore, both 9947A standard genomic DNA and DNA extracted from blood spots were demonstrated to be successfully separated with well-resolved DNA peaks in 8 min. Therefore, the microchip electrophoresis system demonstrated good potential for rapid forensic DNA analysis.

A fully integrated and automated microsystem for rapid pharmacogenetic typing of multiple warfarin-related single-nucleotide polymorphisms.

A fully integrated and automated microsystem consisting of low-cost, disposable plastic chips for DNA extraction and PCR amplification combined with a reusable glass capillary array electrophoresis chip in a modular-based format was successfully developed for warfarin pharmacogenetic testing. DNA extraction was performed by adopting a filter paper-based method, followed by "in situ" PCR that was carried out directly in the same reaction chamber of the chip without elution. PCR products were then co-injected with sizing standards into separation channels for detection using a novel injection electrode. The entire process was automatically conducted on a custom-made compact control and detection instrument. The limit of detection of the microsystem for the singleplex amplification of amelogenin was determined to be 0.625 ng of standard K562 DNA and 0.3 µL of human whole blood. A two-color multiplex allele-specific PCR assay for detecting the warfarin-related single-nucleotide polymorphisms (SNPs) 6853 (-1639G>A) and 6484 (1173C>T) in the VKORC1 gene and the *3 SNP (1075A>C) in the CYP2C9 gene was developed and used for validation studies. The fully automated genetic analysis was completed in two hours with a minimum requirement of 0.5 µL of input blood. Samples from patients with different genotypes were all accurately analyzed. In addition, both dried bloodstains and oral swabs were successfully processed by the microsystem with a simple modification to the DNA extraction and amplification chip. The successful development and operation of this microsystem establish the feasibility of rapid warfarin pharmacogenetic testing in routine clinical practice.

[The Optics and Magnetic Properties of Cr Doped ZnO Thin Films].

The precursor solution is sent to the ultrasonic nozzle directly through a needle tube to prepare Zn1-xCrxO (x=0, 0.01, 0.03 and 0.05)films on quartz substratesby ultrasonic spray method. The structures, optical and magnetic properties of the films were measured by X-ray diffracmeter(XRD), scanning electron microscope(SEM), fluorescence spectrometer, ultraviolet-visible light detector, vibrating sample magnetometer (VSM) and so on. The experimental results indicate that, the undopedZnO thin films exhibit the hexagonal wurtzite crystalline structure with a preferential orientation of (002); the Cr doping restrains the preferred orientation of C axis; the average grain sizes of the samples increase withCr doping, and thesize attains the maximum(31.4 nm) when x=3%. The SEMimages show that the Zn1-xCrxO (x=0, 0.01, 0.03 and 0.05) films are grain-like particles. And it exhibits a long strip shape when x=5%. Moreover, the doping of Cr makes the photoluminescence (PL) spectra of Zn1-xCrxO films change evidently. The undoped sample shows an ultraviolet emission peak at 378 nm as well as a defect related green peak at around 550 nm. However, for the doping samples, there is only a wide range of emission peak from 350 to 550 nm. By gaussian fitting，it is found that VZn, Zni and V-Zn defects exist in the Cr doping films, and VZn is largest when x=3%. The band gap increases with the doping of Cr, and reaches the maximum when x=3%. The doping of Cr hasthe band gap of the samples increase, and the band gapreachs themaximum(3.37 eV) when x=3%. Magnetic measured results show that threedoping samples Zn1-xCrxO(x=1%, 3% and 5%) are ferromagnetic at room temperature, and the magnetization of Zn1-xCrxO (x=3%) is the largest, which is corresponding to the most VZn defect. The experimental results also prove the the oretical prediction that the substitutive Cr in the oxidation state of +3 and the neutral Zn vacancy in the ZnO∶Cr sample are the most favorable defect complex to maintain a high stability of ferromagnetic order.

Fully automated sample preparation microsystem for genetic testing of hereditary hearing loss using two-color multiplex allele-specific PCR.

A fully automated microsystem consisting of a disposable DNA extraction and PCR microchip, as well as a compact control instrument, has been successfully developed for genetic testing of hereditary hearing loss from human whole blood. DNA extraction and PCR were integrated into a single 15-µL reaction chamber, where a piece of filter paper was embedded for capturing genomic DNA, followed by in-situ PCR amplification without elution. Diaphragm microvalves actuated by external solenoids together with a "one-way" fluidic control strategy operated by a modular valve positioner and a syringe pump were employed to control the fluids and to seal the chamber during thermal cycling. Fully automated DNA extractions from as low as 0.3-µL human whole blood followed by amplifications of 59-bp ß-actin fragments can be completed on the microsystem in about 100 min. Negative control tests that were performed between blood sample analyses proved the successful elimination of any contamination or carryover in the system. To more critically test the microsystem, a two-color multiplex allele-specific PCR (ASPCR) assay for detecting c.176_191del16, c.235delC, and c.299_300delAT mutations in GJB2 gene that accounts for hereditary hearing loss was constructed. Two allele-specific primers, one labeled with TAMRA for wild type and the other with FAM for mutation, were designed for each locus. DNA extraction from blood and ASPCR were performed on the microsystem, followed by an electrophoretic analysis on a portable microchip capillary electrophoresis system. Blood samples from a healthy donor and five persons with genetic mutations were all accurately analyzed with only two steps in less than 2 h.

A filter paper-based microdevice for low-cost, rapid, and automated DNA extraction and amplification from diverse sample types.

A plastic microfluidic device that integrates a filter disc as a DNA capture phase was successfully developed for low-cost, rapid and automated DNA extraction and PCR amplification from various raw samples. The microdevice was constructed by sandwiching a piece of Fusion 5 filter, as well as a PDMS (polydimethylsiloxane) membrane, between two PMMA (poly(methyl methacrylate)) layers. An automated DNA extraction from 1 µL of human whole blood can be finished on the chip in 7 minutes by sequentially aspirating NaOH, HCl, and water through the filter. The filter disc containing extracted DNA was then taken out directly for PCR. On-chip DNA purification from 0.25-1 µL of human whole blood yielded 8.1-21.8 ng of DNA, higher than those obtained using QIAamp® DNA Micro kits. To realize DNA extraction from raw samples, an additional sample loading chamber containing a filter net with an 80 µm mesh size was designed in front of the extraction chamber to accommodate sample materials. Real-world samples, including whole blood, dried blood stains on Whatman® 903 paper, dried blood stains on FTA™ cards, buccal swabs, saliva, and cigarette butts, can all be processed in the system in 8 minutes. In addition, multiplex amplification of 15 STR (short tandem repeat) loci and Sanger-based DNA sequencing of the 520 bp GJB2 gene were accomplished from the filters that contained extracted DNA from blood. To further prove the feasibility of integrating this extraction method with downstream analyses, "in situ" PCR amplifications were successfully performed in the DNA extraction chamber following DNA purification from blood and blood stains without DNA elution. Using a modified protocol to bond the PDMS and PMMA, our plastic PDMS devices withstood the PCR process without any leakage. This study represents a significant step towards the practical application of on-chip DNA extraction methods, as well as the development of fully integrated genetic analytical systems.

[Expression and significance of tumor susceptibility gene 101 in hepatocellular carcinoma tissues].

AIM: To study the expression and clinical significance of tumor susceptibility gene 101 (TSG101) expression in hepatocellular carcinoma (HCC) tissues. METHODS: The expression of TSG101 in HCC and corresponding non-tumor tissues was detected by immunohistochemistry and Western blotting. Statistical analyses were conducted to test the relationships of TSG101 expression with clinical parameters such as age, gender, TNM stage, tumor metastasis, and so on. RESULTS: Immunohistochemistry and Western blotting showed that the expression of TSG101 in HCC was significantly higher than that in corresponding non-tumor tissues (P<0.05), and the expression rate was also higher in HCC (53/66, 80.3%) than in non-cancer tissues(18/66, 20.7%)(P<0.05). Higher TSG101 expression in HCC was significantly correlated with TNM stage (P<0.05) and metastasis (P<0.05), but not with age, gender and HBsAg (P>0.05). Multiple regression analysis indicated that TSG101 expression rate was significantly associated with TNM stage and metastasis (P<0.05), but not with gender, age and HBsAg (P>0.05). CONCLUSION: The expression of TSG101 in HCC is higher than that in corresponding non-cancer tissues and the expression level is closely correlated with TNM stage and metastasis of HCC.