Features High Throughput Research Articles

Advanced integrative molecular platform for high-throughput screening of drug-resistant tuberculosis

A real time-polymerase chain reaction-based test in lyophilized form, was developed to simultaneously identify Mycobacterium tuberculosis complex (MTC) by targeting IS6110, rrs as dual markers, as well as mutations causing rifampicin and isoniazid resistance. The test was evaluated for pulmonary and non-pulmonary specimens from sample isolation to PCR analysis. The test demonstrated limit of detection of 25 CFU/mL for MTB, 200 CFU/mL for rpoB and inhA/katG targets with >95 % CI. The specificity for MTC was supported by a comprehensive clinical validation (n = 100). This load-and-go molecular platform, with features of high throughput, long shelf-life, room temperature storage provides simultaneous detection of MTC and its drug-resistant mutations in minimal time. The test named “PathoDetect TM MTB-RIF and INH resistance detection kit” has been approved by Central Drugs Standard Control Organisation, Indian Council of Medical Research and would have implications for tuberculosis elimination programs.

Application of microfluidic technologies in forensic analysis.

The application of microfluidic technology in forensic medicine has steadily expanded over the last two decades due to the favorable features of low cost, rapidity, high throughput, user-friendliness, contamination-free, and minimum sample and reagent consumption. In this context, bibliometric methods were adopted to visualize the literature information contained in the Science Citation Index Expanded from 1989 to 2022, focusing on the co-occurrence analysis of forensic and microfluidic topics. A deep interpretation of the literature was conducted based on co-occurrence results, in which microfluidic technologies and their applications in forensic medicine, particularly forensic genetics, were elaborated. The purpose of this review is to provide an impartial evaluation of the utilization of microfluidic technology in forensic medicine. Additionally, the challenges and future trends of implementing microfluidic technology in forensic genetics are also addressed.

The design, validation, and utility of the “ZJU CottonSNP40K” liquid chip through genotyping by target sequencing

Genotyping platforms provide robust support for genomics, genetics, and molecular breeding. Given the economic importance of cotton, it is urgent to develop a cotton genotyping platform that features high throughput, precision, efficiency, and cost-effectiveness. Here we reported the development of a liquid SNP chip named “ZJU CottonSNP40K” through genotyping by target sequencing, along with its utility. Representative SNPs of this chip were selected by screening more than 3,828,396 SNPs extracted from the abundance of resequencing data available from nearly 1000 cotton accessions. After strict selection, the final array includes 40,071 core SNPs evenly distributed across 26 chromosomes, 27.64 % of which are within genic regions. Testing on 13 cotton accessions showed the chip has high genotyping efficiency and accuracy. The SNP array was further used to genotype 328 (TM-1 × latifolium)F2 individuals and a high-density genetic map was constructed for QTL mapping. The genetic map covered 2963.8 cM with an average distance of 0.57 cM, and yielded a total of 116 QTLs associated with ten agricultural traits. As an accurate high-throughput genotyping SNP array, ZJU CottonSNP40K will facilitate cotton functional studies and molecular breeding.

Polarity-Specific Profiling of Metabolites in Single Cells by Probe Electrophoresis Mass Spectrometry.

Sensitive analysis of metabolites in a single cell is of fundamental significance for the better understanding of biological variability, differential susceptibility in disease therapy, and cell-to-cell heterogeneity as well. Herein, polarity-specific profiling of metabolites in a single cell was implemented by probe electrophoresis mass spectrometry (PEMS), which combined electrophoresis sampling of metabolites from a single cell and nanoelectrospray ionization-mass spectrometry (nanoESI-MS) analysis of the sampled metabolites. Enhanced extraction of either negatively or positively charged metabolites from a single cell was achieved by applying a DC voltage offset of +2.0 and -2.0 V to the probe, respectively. The experimental data demonstrated that PEMS features high throughput (≥200 peaks) and high sensitivity (≥10-times signal enhancement for [Choline + H]+, [Glutamine + H]+, [Arginine + H]+, etc.) in comparison with direct nanoESI-MS analysis. The biological effects of CdSe quantum dots (QDs) and γ-radiation on Allium cepa cells were investigated by PEMS, which revealed that CdSe QDs lead to the increase of intracellular amines while γ-radiation causes the decrease of intracellular acids. Therefore, this work provides an alternative platform to probe novel insights of cells by sensitive analysis of polarity-specific metabolites in a single cell.

Application of the poly (POSS-octavinyl-co-N-methylacetamide-co-divinylbenzene) solid extraction column in analyzing preservatives.

In this study, a special poly solid-phase extraction (in-tube SPE) column consisting of poly (POSS-octavinyl-co-N-methylacetamide-co-divinylbenzene) [poly (POSS-OS-co-DVB-co-NMA)] was prepared based on the chemical structure of the preservatives, and was used as medium for extraction analysis in combination with UPLC. The composition of polymer SPE was optimized and characterized; good scanning electron microscopy (SEM) properties and satisfactory porosity were obtained with 30% monomer (POSS-OS:DVB:NMA = 2wt%:13wt%:15wt%) and 70wt% porogenic solvent (PEG20000:DMSO:ACN = 10wt%:50wt%:10wt%). The experimental parameters of the in-tube SPE-UPLC analysis were optimized systematically. Then, the in-tube SPE-UPLC method was applied for analyzing the beverage sample, and correlation coefficients (R2) > 0.99 were obtained for the linear relationship within limits of 0.1~5.0μgmL-1. Excellent extraction efficiency, good precision, and satisfactory limit of detection sensitivity between 0.03 and 0.10μgmL-1 were obtained. The recovery ranged from 71.5 to 88.0%, with RSD ≤ 6.1%. Furthermore, the proposed method has the features of simple sample pretreatment, high throughput, rapid analysis, cost-effectiveness, and satisfactory sensitivity. Hence, the developed in-tube SPE-UPLC method based on the poly (POSS-OS-co-DVB-co-NMA) SPE column can be potentially used for simple and sensitive detection of preservatives.

Effective practical removal of acetaldehyde by a sandwich-type plasma-in-honeycomb reactor under surrounding ambient conditions

The effective removal of acetaldehyde by humidified air plasma was investigated with a high throughput of contaminated gas in a sandwiched honeycomb catalyst reactor at surrounding ambient temperature. Here, acetaldehyde at the level of a few ppm was successfully oxidized by the honeycomb plasma discharge despite the harsh condition of large water content in the feed gas. The conversion rate of acetaldehyde increased significantly with the presence of catalysts coating on the surface channels. The increased conversion rate was also obtained with a high specific energy input (SEI) and total flow rate. Interestingly, the conversion changed negligibly under the acetaldehyde concentration range from 5 to 20 ppm. However, the conversion rate decreased toward increased water amount in the feed gas. Notably, about 60% of acetaldehyde in the feed was oxidized under SEI of 40 J/L at water amounts ≤ 2.5%, approximately 0.5 g/kWh for acetaldehyde removal. Also, the plasma-catalyst reaction was superior to the thermal reactive catalyst for acetaldehyde removal in airborne pollutants. In comparison with other plasma-catalyst sources for acetaldehyde removal, the energy efficiency under the condition is comparable. Moreover, the honeycomb plasma discharge features high throughput, avoiding pressure drop, and straightforward reactor configuration, suggesting potential practical applications.

Research advances in clinical biochemical analysis systems based on microfluidic driving and control technique

Microfluidic techniques have the features of high throughput, low consumption, as well as ease of miniaturization, integration, and automation, thus emerging as a feasible solution to cost-effective clinical biochemical analysis. Various clinical biochemical analysis systems based on the microfluidic technique, with different driving and control methods, are introduced in this article, in addition to commercially available microfluidic biochemical analysis instruments. The characteristics of these systems are comprehensively reviewed.

Microfluidics for Medical Additive Manufacturing

Additive manufacturing plays a vital role in the food, mechanical, pharmaceutical, and medical fields. Within these fields, medical additive manufacturing has led to especially obvious improvements in medical instruments, prostheses, implants, and so forth, based on the advantages of cost-effectiveness, customizability, and quick manufacturing. With the features of precise structural control, high throughput, and good component manipulation, microfluidic techniques present distinctive benefits in medical additive manufacturing and have been applied in the areas of drug discovery, tissue engineering, and organs on chips. Thus, a comprehensive review of microfluidic techniques for medical additive manufacturing is useful for scientists with various backgrounds. Herein, we review recent progress in the development of microfluidic techniques for medical additive manufacturing. We evaluate the distinctive benefits associated with microfluidic technologies for medical additive manufacturing with respect to the fabrication of droplet/fiber templates with different structures. Extensive applications of microfluidic techniques for medical additive manufacturing are emphasized, such as cell guidance, three-dimensional (3D) cell culture, tissue assembly, and cell-based therapy. Finally, we present challenges in and future perspectives on the development of microfluidics for medical additive manufacturing.

Integrated Array Chip for High-Throughput Screening of Species Differences in Metabolism.

Species differences in metabolism may produce failure prediction of drug efficacy/toxicity in humans. Integration of metabolic competence and cellular effect assays in vitro can provide insight into the species differences in metabolism; however, a co-culture platform with features of high throughput, operational simplicity, low sample consumption, and independent layouts is required for potential usage in industrial test settings. Herein, we developed an integrated array chip (IAC) to evaluate the species differences in metabolism through metabolism-induced anticancer bioactivity as a case. The IAC consisted of two functional parts: a micropillar chip for immobilization of liver microsomes and a microwell chip for three-dimensional (3D) tumor cell culture. First, optimized parameters of the micropillar chip for microsomal encapsulation were obtained by cross-shaped protrusions and a 2.5 μL volume of 3D agarose spots. Next, we examined factors influencing metabolism-induced anticancer bioactivity. Feasibility of the IAC was validated by four model prodrugs using image-based bioactivity detection and mass spectrometry (MS)-based metabolite analysis. Finally, a species-specific IAC was used for selection of animal species that best resembles metabolism-induced drug response to humans at throughputs. Overall, the IAC provides a promising co-culture platform for identifying species differences in metabolism and selection of animal models to accelerate drug discovery.

Identification of Spherical and Nonspherical Proteins by a Solid-State Nanopore.

The three-dimensional structure of a protein plays an important role in protein dynamics in the biological system of human. By now, it remains a challenge to characterize and quantify the shape of a protein at the single-molecule level. The nanopores, as a novel single-molecule sensor, has been widely applied in many fields such as DNA sequencing and human diseases diagnosis. In this paper, we investigated the translocation of spherelike con.A and the prolate bovine serum albumin (BSA) under an electric field by a solid-state nanopore. By analyzing the ionic current, the con.A and the BSA could be characterized and differentiated due to their intrinsic shape difference. Because the prolate BSA will have the preferred orientations for a higher electric field, when it is residing inside the nanopore, multiple ionic current blockade levels will be observed. While for the spherical con.A, there is only one ionic current blockade level. The method presented here will be potentially applied to fingerprint a single protein as a new method having the features of low cost and high throughput in the near future.

Virtual screening of eighteen million compounds against dengue virus: Combined molecular docking and molecular dynamics simulations study

Dengue virus is a major issue of tropical and sub-tropical regions. Dengue virus has been the cause behind the major alarming epidemics in the history with mass causalities from the decades. Unavailability of on-shelf drugs for the prevention of further proliferation of virus inside the human body results in immense number of deaths each year. This issue necessitates the design of novel anti-dengue drug. The protease enzyme pathway is the critical target for drug design due to its significance in the replication, survival and other cellular activities of dengue virus. Therefore, approximately eighteen million compounds from the ZINC database have been virtually screened against nonstructural protein 3 (NS3). The incremental construction algorithm of Glide docking program has been used with its features high throughput virtual screening (HTVS), standard precision (SP), extra precision (XP) and in combination of Prime module, induced fit docking (IFD) approach has also been applied. Five top-ranked compounds were then selected from the IFD results with better predicted binding energies with the catalytic triad residues (His51, Asp75, and Ser135) that may act as potential inhibitors for the underlying target protease enzyme. The top-ranked compounds ZINC95518765, ZINC44921800, ZINC71917414, ZINC39500661, ZINC36681949 have shown the predicted binding energies of −7.55, −7.36, −8.04, −8.41, −9.18kcal/mol, respectively, forming binding interactions with three catalytically important amino acids. Top-docking poses of compounds are then used in molecular dynamics (MD) simulations. In computational studies, our proposed compounds confirm promising results against all the four serotypes of dengue virus, strengthening the opportunity of these compounds to work as potential on-shelf drugs against dengue virus. Further experimentation on the proposed compounds can result in development of strong inhibitors.

Association between PNPLA3 polymorphisms (rs738409) and non-alcoholic fatty liver disease

Objective To develop a novel method for genotyping rs738409 (C>G) single nucleotide polymorphism(SNP), and explore the association between the rs738409 genotypes and non-alcoholic fatty liver disease (NAFLD). Methods Method establishment and analysis of genetic susceptibility. The principle of amplification refractory mutation system (ARMS) and combined a TaqMan fluorogenic probe as signal report were used, by monitoring the difference of cycle threshold (ΔCt=C allele-special primer Ct values minus G allele-special primer Ct values) between the two PCR reactions in a real-time PCR, the method for rs738409 genotyping was established (ARMS-TaqMan). 618 subjects (men: 401; women: 217), in an annual health check-up program from January 2011 to December 2014 in Aerospace Center Hospital, were performed for rs738409 genotyping by the ARMS-TaqMan assay. Fatty liver was diagnosed based on abdominal ultrasonography. The chi-square test and multiple logistic analyses were used to analyze the relationship of rs738409 genotypes and NAFLD. Results The ΔCt by ARMS-TaqMan for rs738409 genotyping were -13.1±1.4 of CC alleles (243 cases), 0.01±0.45 of CG alleles (282 cases), and 12.7±1.9 of GG alleles (93 cases), respectively. The GG alleles frequency of rs738409 were significantly higher in patients with NAFLD compared with subjects without NAFLD (21.5% vs 12.3%, χ2=8.677, P=0.003). In comparison to subjects with CC alleles, the OR (95% confidence interval) adjusted for age, gender, total cholesterol, triglyceride, high density lipoprotein-cholesterol, low density lipoprotein-cholesterol, fasting blood glucose and body mass index was 1.35 (0.91-2.00) in subjects with CG alleles and was 2.21 (1.32-3.71) in subjects with GG alleles (P=0.013). Variant rs738409 genotypes were associated with significant increased trend in alanine aminotransferase (ALT) level from CC alleles, CG alleles to GG alleles (F=8.980, P<0.001), and in aspartate aminotansferase (AST) between GG alleles and CC alleles (F=6.491, P<0.001). Conclusions The novel ARMS-TaqMan assay had the features of accuracy, one step and high-throughput for rs738409 genotyping. The G allele of rs738409 was a risk factor of NAFLD susceptibility and associated with higher level serum ALT and AST.(Chin J Lab Med, 2016, 39: 45-49) Key words: Polymorphism, Genetic; Non-alcoholic fatty liver disease; Real-time polymerase chain reaction

A Metropolis Criterion Based Fuzzy Q-Learning Flow Controller for High-Speed Networks

For the congestion problems in high-speed networks, a Metropolis criterion based fuzzy Q-learning flow controller is proposed. Because of the uncertainties and highly time-varying, it is not easy to accurately obtain the complete information. In this case, the Q-learning, which is independent of mathematic model, and prior-knowledge, has good performance. The fuzzy inference and Metropolis criterion are introduced in order to facilitate generalization in large state space and balance exploration and exploitation in action selection individually. Simulation results show that the controller can learn to take the best action to regulate source flow with the features of high throughput and low packet loss ratio, and can avoid the occurrence of congestion effectively.

Structural characterization of synthetic polymers using thermal-assisted atmospheric pressure glow discharge mass spectrometry

With the development of material science and the practical needs of the polymer industry, rapid characterization of synthetic polymers using mass spectrometry is of sustainable interest. Herein a new method for characterizing synthetic polymers using thermal-assisted atmospheric pressure glow discharge mass spectrometry (TA-APGD-MS) is established. After illustration of the mechanism of ion formation, typical polymer samples such as polystyrene (PS), polyoxymethylene (POM) and poly (butanediol succinate) (PBS) were directly characterized at the molecular level using TA-APGD-MS. The thermal degradation products of synthetic polymers including monomer units and/or other fragments were rapidly detected by tandem mass spectrometry, providing rich information about the chemical composition for the structural characterization of homo- and co-polymers. The result suggests that TA-APGD-MS allows direct and rapid analysis of both synthetic homo-polymers and co-polymers under ambient conditions without any sample pretreatment. This method features high throughput, high sensitivity and rich information, showing promising applications in polymer science.

Determination of trace metals in seawater by an automated flow injection ion chromatograph pretreatment system with ICPMS

A novel flow injection ion chromatograph (FI-IC) system has been developed to fully automate pretreatment procedures for multi-elemental analysis of trace metals in seawater by inductively coupled plasma mass spectrometer (ICPMS). By combining 10-port, 2 position and 3-way valves in the FI-IC manifold, the system effectively increase sample throughput by simultaneously processing three seawater samples online for: sample loading, injection, buffering, preconcentration, matrix removal, metal elution, and sample collection. Forty-two seawater samples can be continuously processed without any manual handing. Each sample pretreatment takes about 10 min by consuming 25 mL of seawater and producing 5 mL of processed concentrated samples for multi-elemental offline analysis by ICPMS. The offline analysis improve analytical precision and significantly increase total numbers of isotopes determined by ICPMS, which include the metals Al, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Ti, V, and Zn. The blank value and detection limits of trace metals using the system with ICPMS analysis all range from 0.1 to 10 parts per trillion (ppt), except Al, Fe, and Zn. The accuracy of the pretreatment system was validated by measuring open-ocean and coastal reference seawater, NASS-5 and CASS-4. Using the system with ICPMS analysis, we have obtained reliable trace metal concentrations in the water columns of the South China Sea. Possessing the features of full automation, high throughput, low blank, and low reagent volume used, the system automates and simplifies rigorous and complicated pretreatment procedures for multi-elemental analysis of trace metals in seawater and effectively enhances analytical capacity for trace metal analysis in environmental and seawater samples.

Continuous-Stage-Movement Blankingless Electron-Beam Lithography for Patterned Media Template Fabrication

A slider and spindle (r–θ) stage electron beam (e-beam) mastering system with continuous-stage-movement blankingless beam shift lithography (BLSL) and flyback e-beam lithography (CSFL) brings together the features of high resolution, high accuracy, and high throughput to meet the various requirements for fabricating master templates for discrete track media and bit patterned media (BPM) for the next generation of hard disk drives. With the CSFL capability, we achieve multiple-overlay bit writing with one, two, four, and eight overlays for a BPM bit array to markedly improve track-to-track phase alignment accuracy from 5.6 nm sigma in a single write to 1.04 nm sigma in an eight overlay write. CSFL, in conjunction with the high-frequency response of the e-beam deflector, achieves 25 MHz high-throughput BPM bit array write with track pitches and bit pitches of 120 nm in the entire write range from 20 to 140 mm in diameter. This write takes only 11.6 h under the conditions of a line velocity of 3,000 mm/s, a beam current of 150 nA, a maximum rotation speed of 2,865 rpm, and a constant line velocity (CLV) write mode.

A Genetic Based Fuzzy Q-Learning Flow Controller for High-Speed Networks

For the congestion problems in high-speed networks, a genetic based fuzzy Q-learning flow controller is proposed. Because of the uncertainties and highly time-varying, it is not easy to accurately obtain the complete information for high-speed networks. In this case, the Q-learning, which is independent of mathematic model, and prior-knowledge, has good performance. The fuzzy inference is introduced in order to facilitate generalization in large state space, and the genetic operators are used to obtain the consequent parts of fuzzy rules. Simulation results show that the proposed controller can learn to take the best action to regulate source flow with the features of high throughput and low packet loss ratio, and can avoid the occurrence of congestion effectively.

Construction of a series of vectors for high throughput cloning and expression screening of membrane proteins from Mycobacterium tuberculosis

BackgroundOne of the major challenges for membrane protein structural genomics is establishing high-throughput cloning and expression screening methods to obtain enough purified protein in a homogeneous preparation for structural and functional studies. Here a series of ligation independent cloning based vectors were constructed to address this challenge.ResultsThe feasibility of these vectors was tested with 41 putative membrane proteins from Mycobacterium tuberculosis. The efficiency for direct cloning of these target genes from PCR products was 95% (39/41). Over 40% of cloned genes were overexpressed in Escherichia coli BL21 (DE3)-RP codon plus strain in the first round of expression screening. For those proteins which showed no expression, three protein fusion partners were prepared and it was found that each of the target proteins could be overexpressed by at least one of these fusions, resulting in the overexpression of two thirds of the cloned genes.ConclusionThis expression platform features high throughput cloning, high flexibility for different constructs, and high efficiency for membrane protein overexpression, and is expected to be useful in membrane protein structural and functional studies.

Performance Evaluation of Ethernet Frame Burst Mode in EPON Downstream Link

We apply IEEE 802.3 frame burst mode (FBM) to the Ethernet passive optical network (EPON) downstream link and compare its performance with non-frame burst mode for various traffic patterns. Although in light traffic loads (ρ<0.5) the efficiency of the FBM mechanism is not significant, it does feature high throughput, small jitter, low queue occupancy, and short queuing delay in optical line terminals under various traffic loads with various numbers of optical network units (ONUs). The FBM performance always approaches that of full-duplex mode, especially under heavy traffic loads (ρ>0.5). Moreover, an increase in number of ONUs will decrease the burst performance. Our work shows that FBM scheme is very useful for EPON transmission and has low design complexity.

The research progress of tiling array technology and applications

Tiling array technology was improved from microarray technology. Over the past five years, tiling array has become an important tool for gathering genome information. Its features of high density and high throughput allow people to probe into life from the whole-genome level. This paper is a survey of tiling array technology and its applications. In addition, some typical algorithms for identifying expressed probe signals are described and compared.