Tweezer PCR: A Highly Specific Method for Accurate Identification of Low-Abundance Mutations.

Somatic mutation is a valuable biomarker for tracking tumor progression and migration due to its distinctive feature in various tumors and its wide distribution throughout body fluids. However, accurately detecting somatic mutations from the abundant DNA of noncancerous origins remains a practical challenge in the clinic. Herein, we developed an ultraspecific method, called tweezer PCR, for detecting low-abundance mutations inspired by the design of DNA origami. The high specificity of tweezer PCR relies on a tweezer-shaped primer containing six basic functional units: a primer, a hairpin, a linker, a blocker, a spacer, and a toehold. After optimizing the structure of the tweezer-shaped primer and enhancing its specificity by adding additional Mg2+ and Na+, tweezer PCR distinguished as low as 20 copies of mutations from 2 million copies of wild-type templates per test. By testing synthesized plasmids and plasma samples gathered from nonsmall-cell lung cancer patients, tweezer PCR showed higher specificity and robustness for detecting low-copy-number mutations in contrast with digital droplet PCR. Additionally, the need for conventional instruments makes tweezer PCR a practically accessible method for testing low-abundance mutations. Because of its numerous advantages, we believe that tweezer PCR offers a precise, robust, and pragmatic tool for cancer screening, prognosis, and genotyping in the clinic.

Assessing airborne transmission risks in COVID-19 hospitals by systematically monitoring SARS-CoV-2 in the air.

IMPORTANCE: Risk management and control of airborne transmission in hospitals is crucial in response to a respiratory virus pandemic. However, the formulation of these infection control measures is often based on epidemiological investigations, which are an indirect way of analyzing the transmission route of viruses. This can lead to careless omissions in infection prevention and control or excessively restrictive measures that increase the burden on healthcare workers. The study provides a starting point for standardizing transmission risk management in designated hospitals by systemically monitoring viruses in the air of typical spaces in COVID-19 hospitals. The negative results of 359 air samples in the clean and emergency zones demonstrated the existing measures to interrupt airborne transmission in a designated COVID-19 hospital. Some positive cases in the corridor of the contaminant zone during rounds and meal delivery highlighted the importance of monitoring airborne viruses for interrupting nosocomial infection.

LINC01354 enhances the metastatic ability of gastric cancer cells by adjusting miR-153-5p/CADM2 expression.

LINC01354 is a long non-coding RNA (lncRNA) highly expressed in gastric cancer (GC). However, studies have shown that it plays a critical role in the progression of other tumors. This study attempts to uncover the role of LINC01354 in GC. LINC01354 expression in GC tissues and cell lines was assessed using qRT-PCR. Subsequently, LINC01354 knockdown and overexpression were induced in GC cells, and epithelial-mesenchymal transition (EMT) progression was detected. A dual-luciferase reporter assay was used to assess the relation between LINC01354, miR-153-5p, and CADM2. Finally, the metastatic ability of GC cells was assessed by Transwell and wound healing assays. LINC01354 expression was abnormally elevated in cancerous tissues and GC cells, and LINC01354 knockdown suppressed EMT progression, migration, and invasion of GC cells. Transfection of miR-153-5p mimics inhibited the expression of CADM2 by banding to the 3'UTR region, while LINC01354 promoted CADM2 expression by blocking miR-153-5p. The fluorescence experiment indicated that CADM2 is directly regulated by LINC01354/miR-153-5p. Overexpression of LINC01354 promoted EMT progression, migration, and invasion of GC cells, which could be absolutely reversed by co-expression of miR-153-5p. Our research demonstrates that LINC01354 has an important function in the EMT progression of GC cells. LINC01354 promotes GC cell migration and invasion by adjusting miR-153-5p/CADM2 expression.

Effect of biochar and bioorganic fertilizer on the microbial diversity in the rhizosphere soil of Sesbania cannabina in saline-alkaline soil.

Introduction: Biochar and bioorganic fertilizer (BOF) application in agriculture has garnered increasing interest recently. However, the effects of biochar and BOF on rhizosphere soil microecology, especially in a region with saline-alkaline soil, remain largely unexplored. Methods: In this study, we performed Illumina-based 16S rRNA sequencing to investigate the effects of biochar with or without BOF addition, as well as at different addition rates and particles sizes, on the microecology of saline-alkaline rhizosphere soil. Results: In the field experiment, biochar and BOF application altered the rhizosphere soil microecology. Actinobacteriota, Proteobacteria, and Chloroflexi accounted for >60% of the total bacterial population in each treatment. In the different treatments, Actinobacteria and Alphaproteobacteria were the predominant classes; Micromonosporales and Vicinamibacterales were the dominant orders; norank_f__Geminicoccaceae and Micromonosporaceae were the most abundant families; and Micromonospora and norank_f_Geminicoccaceae were the predominant genera. Application of biochar with or without BOF decreased soil electrical conductivity (EC) by 7% -11.58% only at the depth of 10 cm below the surface, again, soil EC can be significantly reduced by an average of 4% at 10 cm depth soil after planting Sesbania cannabina. Soil organic carbon, organic matter, available potassium, and available phosphorus contents had significant effects on the soil bacterial community structure. Conclusion: Co-application of biochar and BOF resulted in the greatest improvement of rhizosphere soil microecology, either by promoting plant growth or improving the nutrition and physicochemical properties of soil, followed by BOF alone and biochar alone. Additionally, higher application rate of biochar was better than lower application rate, and fine biochar had a stronger effect than coarse biochar. These results provide guidance for the development of new saline-alkaline soil remediation strategies.

One-Stop Extraction and In Situ RT-qPCR for Ultrasensitive Detection of Highly Diluted SARS-CoV-2 in Large-Volume Samples from Aquatic Environments.

Surveillance of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in aquatic environments attracted attention due to its considerable impacts on human health and ecology, especially in countries with poor sanitation standards. Based on a strategy of one-stop extraction and in situ amplification, we developed an ultrasensitive method that uses a polyacrylamide derivative-modified filter disc (PAD-FD), in which highly diluted RNA can be efficiently concentrated onto the filter disc and directly used for amplification. A newly designed spin column with a cup-like filter base facilitated the non-contact transfer of the affinity filter disc from the column to a PCR tube. The limit of detection of the PAD-FD coupled with RT-qPCR is 10 copies/mL. Using 32 suspected SARS-CoV-2 samples, we demonstrated that the detection rate of our method (62.5%, 20/32) was triple the rate of the commercial kit (18.8%, 6/32). Using a PAD-FD, 56.3% (18/32) and 40.6% (13/32) of the 10-fold-dilution samples with river and tap water, respectively, were detected. Even when diluted 100-fold, 28.1% (9/32) and 37.5% (12/32) were still detected in river and tap water, respectively. We believe that the PAD-FD method offers an accurate testing tool for monitoring viral RNA in aquatic environments, contributing to the forewarning of the SARS-CoV-2 outbreak and the breaking of the transmission chain.

An ultrasensitive and rapid "sample-to-answer" microsystem for on-site monitoring of SARS-CoV-2 in aerosols using "in situ" tetra-primer recombinase polymerase amplification.

Airborne transmissibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the urgent need for aerosol monitoring of SARS-CoV-2 to prevent sporadic outbreaks of COVID-19. The inadequate sensitivity of conventional methods and the lack of an on-site detection system limited the practical SARS-CoV-2 monitoring of aerosols in public spaces. We have developed a novel SARS-CoV-2-in-aerosol monitoring system (SIAMs) which consists of multiple portable cyclone samplers for collecting aerosols from several venues and a sensitive "sample-to-answer" microsystem employing an integrated cartridge for the analysis of SARS-CoV-2 in aerosols (iCASA) near the sampling site. By seamlessly combining viral RNA extraction based on a chitosan-modified quartz filter and "in situ" tetra-primer recombinase polymerase amplification (tpRPA) into an integrated microfluidic cartridge, iCASA can provide an ultra-high sensitivity of 20 copies/mL, which is nearly one order of magnitude greater than that of the commercial kit, and a short turnaround time of 25 min. By testing various clinical samples of nasopharyngeal swabs, saliva, and exhaled breath condensates obtained from 23 COVID-19 patients, we demonstrate that the positive rate of our system was 3.3 times higher than those of the conventional method. Combining with multiple portable cyclone samplers, we detected 52.2% (12/23) of the aerosol samples, six times higher than that of the commercial kit, collected from the isolation wards of COVID-19 patients, demonstrating the excellent performance of our system for SARS-CoV-2-in-aerosol monitoring. We envision the broad application of our microsystem in aerosol monitoring for fighting the COVID-19 pandemic.

Tumor Cell-Intrinsic BTLA Receptor Inhibits the Proliferation of Tumor Cells via ERK1/2.

B and T lymphocyte attenuator (BTLA) is an immune checkpoint molecule that mediates the escape of tumor cells from immunosurveillance. Consequently, BTLA and its ligand herpesvirus entry mediator (HVEM) are potentially immunotherapeutic targets. However, the potential effects of BTLA on tumor cells remain incompletely unknown. Here, we show that BTLA is expressed across a broad range of tumor cells. The depletion of BTLA or HVEM promotes cell proliferation and colony formation, which is reversed by the overexpression of BTLA in BTLA knockout cells. In contrast, overexpression of BTLA or HVEM inhibits tumor cell proliferation and colony formation. Furthermore, the proliferation of a subpopulation with high BTLA was also significantly slower than that of the low BTLA subpopulation. Mechanistically, the coordination of BTLA and HVEM inhibits its major downstream extracellular regulated protein kinase (ERK1/2) signaling pathway, thus preventing tumor cell growth. This study demonstrates that tumor cell-intrinsic BTLA/HVEM is a potential tumor suppressor and is likely to have a potential antagonist for immunotherapy, thus representing a potential biomarker for the optimal cancer immunotherapeutic treatment.

Impact of Biochar and Bioorganic Fertilizer on Rhizosphere Bacteria in Saline-Alkali Soil.

Biochar and bioorganic fertilizers (BOF) that are used in agriculture can, both directly and indirectly, impact rhizosphere soil microorganisms. However, changes to the halophyte rhizosphere bacterial community after applying biochar and BOF to saline−alkali soil have not been thoroughly described. This study has investigated the bacterial communities of halophytes in saline−alkali soil through the addition of different biochar and BOF formulas using Illumina-based sequencing of the 16S rRNA gene fragment. B_BOF (biochar and BOF combined application) had the best effect, either by promoting the plant growth or by improving the physical and chemical properties of the soil. The concentration of the rhizosphere bacterial communities correlated with the changes in soil organic matter (OM) and organic carbon (OC). Proteobacteria, Actinobacteria, Chloroflexi, and Acidobacteria accounted for >80% of the total bacteria in each treatment. In addition, the abundance of Micromonospora was much higher in response to B_BOF than to the other treatments. BOF, with or without biochar, significantly influenced the bacterial community composition in the saline−alkali soil. The OC, OM, total nitrogen, and the available phosphorus had significant effects on the bacterial structure of this soil. The complex correlation of the bacterial communities between CK and B_BOF was higher compared to that between CK and FB or between CK and BOF. These findings suggested that the plant growth, the soil characteristics, and the diversity or community composition of the rhizosphere bacteria in saline−alkali soil were significantly influenced by B_BOF, followed by BOF, and then biochar; fine biochar had a stronger effect than medium or coarse biochar. This study provides an insight into the complex microbial compositions that emerge in response to biochar and BOF.

A Multi-Segmented Human Bioheat Model for Asymmetric High Temperature Environments.

In workplaces such as steel, power grids, and construction, firefighters and other workers often encounter non-uniform high-temperature environments, which significantly increase the risk of local heat stress and local heat discomfort for the workers. In this paper, a multi-segment human bioheat model is developed to predict the human thermal response in asymmetric high-temperature environments by considering the sensitivity of the modeling to angular changes in skin temperature and the effects of high temperatures on human thermoregulatory and physiological responses simultaneously. The extended model for asymmetric high-temperature environments is validated with the current model results and experimental data. The result shows that the extended model predicts the human skin temperature more accurately. Under non-uniform high-temperature conditions, the local skin temperature predictions are highly consistent with the experimental data, with a maximum difference of 2 °C. In summary, the proposed model can accurately predict the temperature of the human core and skin layers. It has the potential to estimate human physiological and thermoregulatory responses under uniform and non-uniform high-temperature environments, providing technical support for local heat stress and local thermal discomfort protection.

LncRNA MEG3-TRPV1 signaling regulates chronic inflammatory pain in rats.

Osteoarthritis (OA) is a common osteoarthropathy with chronic inflammatory pain as the core symptom in middle-aged and elderly people. LncRNA MEG3 (Maternally expressed gene 3) is involved in the development of OA via regulation of angiogenesis, which causes the activation and overexpression of transient receptor potential vanilloid type-1 (TRPV1). In this study, we investigated the mechanism of MEG3-TRPV1 signaling in chronic inflammatory pain (CIP) of rat model. Chronic inflammatory pain was modeled using subcutaneous microinjection of complete Freund's adjuvant (CFA) into the left hind paw of rats. We showed that TRPV1 mRNA and protein were significantly increased, while MEG3 mRNA was significantly decreased, in the DRG and SDH of CFA-induced rats. In addition, intrathecal injection of MEG3-overexpressing lentivirus significantly downregulated TRPV1 expression and alleviated chronic inflammatory pain in CFA-induced rats. Treatment with a TRPV1 antagonist also significantly relieved chronic inflammatory pain in CFA-induced rats. In general, our results reveal that MEG3 alleviates chronic inflammatory pain by downregulating TRPV1 expression. These findings may provide new therapeutic targets in the treatment of patients with OA.

Diversity of endophytic bacteria of mulberry (Morus L.) under cold conditions.

Endophytic bacteria are known to impact the growth and fitness of agriculturally relevant plants. However, there are limited reports describing endophytic bacteria related to mulberry (Morus L.). The present study used Illumina-based 16S rRNA gene sequencing to investigate the endophytic bacterial communities of two mulberry cultivars with differing resistance to low temperature, under cold conditions. In most cases, the bacterial communities of endophytes in the root exhibited higher richness compared with those in the stem, and the communities in resistant cultivar X792 exhibited higher richness compared with those of the sensitive cultivar "Da Shi" (DS). The difference in the proportion of unique operational taxonomic units showed the same trend. The number of genera with significant differences in abundance was greater between organs than between months, and greater between months than between cultivars. Microbial diversity analysis showed that Proteobacteria and Actinobacteria were the dominant phyla in all samples, while Pseudomonas, Steroidobacter, and Rhodococcus were the dominant genera in different samples. There were significant differences between cultivars DS and X792 in the relative abundance of Pseudomonas, Acidibacter, Frigoribacterium, Gaiella, and Pseudokineococcus. PICRUSt predictions indicated that the relative abundances of endophytic bacteria in membrane transport and signal transduction were significantly higher in the stem of resistant cultivar X792 in January compared with that of sensitive cultivar DS. Analysis of ß-Diversity also revealed distinct differences in endophytic bacterial communities of stem and root, and communities of the stem in January and February. The complex correlation of the endophytic communities was higher in sensitive mulberry cultivar DS compared with resistant cultivar X792, in the stem compared with the root, and in January compared with February. Overall, findings from this study suggested that the diversity and community structure of endophytic bacteria in mulberry were significantly influenced by organs and months, followed by the host cultivar. The study provides insight into the complex microbial diversity of mulberry under cold conditions.

A plasmon modulator by directly controlling the couple of photon and electron.

The manipulation of surface plasmon polaritons plays a pivotal role in plasmonic science and technology, however, the modulation efficiency of the traditional method suffers from the weak light-matter interaction. Herein, we propose a new method to overcome this obstacle by directly controlling the couple of photon and electron. In this paper, a hybrid graphene-dielectric- interdigital electrode structure is numerically and experimentally investigated. The plasmon is excited due to the confined carrier which is regulated by the potential wells. The frequency of plasmon can be tuned over a range of ~ 33 cm-1, and the obtained maximum extinction ratio is 8% via changing the confined area and the density of carrier. These findings may open up a new path to design the high efficiency all-optical modulator because the electrons can also be driven optically.

Human physiological responses of exposure to extremely cold environments.

Extremely cold events have occurred more frequently in the past few years. People exposed to extremely cold exposure could suffer the threats of human health and safety like cold stress and injury. This study aims to investigate human physiological responses of exposure to extremely cold environments and the moment of temperature step. The experiments of 12 subjects exposed to three different cold exposure conditions (-5 °C, -10 °C, -15 °C) were carried out in a climate chamber. Most critical physiological parameters, including the core temperature, local skin temperature, blood pressure, heart rate, respiration rate and blood oxygen saturation, were measured to evaluate human physiological responses. In the particular short term study, the results show that the local skin temperature and blood pressure are the most significant indexes for evaluating the risk of cold strain in extremely cold environment. The finger temperature is a critical index of hand and finger flexibility, and it will lead to serious injuries and reduced manual performance when exposed to below -5 °C for more than 20 min. The high physiological strain at the very beginning moment of cold exposure can significantly affect the ability to make correct judgment and action, and it is suggested that the personnel adapt for 3 min after entering into the extremely cold environment to stabilize physiological parameters and thus enhancing the safety and occupational performance. The experimental data of this study is also of great significance for the development and validation of thermophysiological models.

Gene Cloning and Bioinformatics Analysis of Limonene-3-hydroxylase from Schizonepeta tenuifolia / 中国实验方剂学杂志

Objective:This paper aims to clone the cDNA sequence of<italic> limonene</italic>-3-<italic>hydroxylase</italic>（<italic>StL</italic>3<italic>OH</italic>） in <italic>Schizonepeta tenuifolia</italic> and analyze its sequence by bioinformatics. Method:Specific primers were designed based on sequences of<italic> StL</italic>3<italic>OH </italic>gene screened from transcriptome sequencing data of <italic>S. tenuifolia</italic> and the cDNA sequence of <italic>StL</italic>3<italic>OH </italic>gene was cloned by reverse transcription polymerase chain reaction （RT-PCR） and analyzed for its bioinformatics. Result:The <italic>StL3OH</italic> gene cDNA sequence length was 1 598 bp，containing a 1 497 bp long complete open reading frame which encoded 498 amino acids. StL3OH protein had a theoretical relative molecular mass of 56.40 kDa，with a hydrophilic and unstable nature. Bioinformatics analysis showed that StL3OH protein had no signal peptide but had a transmembrane domain which might be located in endoplasmic reticulum. Multiple sequence alignment and cluster analysis showed that the amino acid sequence of MsL3OH protein had a high similarity with StL3OH protein，both of which contained cytochrome P450 heme binding region，belonging to the D subfamily of cytochrome CYP71 family. Codon bias analysis showed that <italic>StL</italic>3<italic>OH</italic> gene preferred guanine/cytosine（G/C） ending codon，with 27 skewed codons， and Nicotiana benthamiana was proven to be the most suitable host for exogenous expression of <italic>StL</italic>3<italic>OH</italic> gene. Conclusion:The cDNA sequence of<italic> StL3OH</italic> gene was cloned from <italic>S. tenuifolia</italic> for the first time，which will provide a basis for further study on the structure and function of StL3OH protein and the regulation mechanism of <italic>StL3OH </italic>gene in the accumulation and biosynthesis of monoterpenes in<italic> S. tenuifolia</italic>.

TRIM52 positively mediates NF-κB to promote the growth of human benign prostatic hyperplasia cells through affecting TRAF2 ubiquitination.

AIMS: Benign prostatic hyperplasia (BPH) is a progressive disease, which severely affects men's health. Here, we sought to analyze the functions and mechanism of action of the tripartite motif protein 52 (TRIM52), a novel prostate basal cell biomarker in BPH. MATERIALS AND METHODS: Immunohistochemistry assay was performed in sectioned human BPH tissues, BPH-1 cells, and prostate RWPE-1 cells, to detect the expressions of TRIM52 and NF-κB. Western blotting and qRT-PCR analyses were conducted to measure the relative protein and mRNA expression levels, respectively. Further, lentiviral transfection was performed in BPH-1 and RWPE-1 cells to study the overexpression and siRNA knockdown of TRIM52. Dual-luciferase reporter assay was applied to evaluate the relationship between NF-κB and TRIM52. Furthermore, CCK-8 assay and flow cytometry were employed to analyze cell proliferation and apoptosis. KEY FINDINGS: TRIM52 and NF-κB levels were elevated in BPH tissues, and TRIM52 expression positively correlated with NF-κB expression. TRIM52 silencing suppressed the growth of BPH-1 cells and decreased the promoter activity of NF-κB. Moreover, the NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), suppressed TRIM52-induced proliferation of RWPE-1 cells and inhibited NF-κB promoter activity in oeTRIM52 transfected RWPE-1 cells. Silencing TRIM52 also inhibited TRAF2 ubiquitination in BPH-1 cells. Further, NF-κB promoter activity in siNC transfected cells was enhanced by the recombinant protein TNF-α and inhibited by siTRIM52. SIGNIFICANCE: TRIM52 accelerated the growth of BPH-1 cells by upregulating NF-κB, and TRIM52 could promote TRAF2 ubiquitination. These findings might contribute to the understanding of the biological functions and action mechanisms of TRIM52 in BPH.

A Fully Integrated In Vitro Diagnostic Microsystem for Pathogen Detection Developed Using a "3D Extensible" Microfluidic Design Paradigm.

Microfluidics is facing critical challenges in the quest of miniaturizing, integrating, and automating in vitro diagnostics, including the increasing complexity of assays, the gap between the macroscale world and the microscale devices, and the diverse throughput demands in various clinical settings. Here, a "3D extensible" microfluidic design paradigm that consists of a set of basic structures and unit operations was developed for constructing any application-specific assay. Four basic structures-check valve (in), check valve (out), double-check valve (in and out), and on-off valve-were designed to mimic basic acts in biochemical assays. By combining these structures linearly, a series of unit operations can be readily formed. We then proposed a "3D extensible" architecture to fulfill the needs of the function integration, the adaptive "world-to-chip" interface, and the adjustable throughput in the X, Y, and Z directions, respectively. To verify this design paradigm, we developed a fully integrated loop-mediated isothermal amplification microsystem that can directly accept swab samples and detect Chlamydia trachomatis automatically with a sensitivity one order higher than that of the conventional kit. This demonstration validated the feasibility of using this paradigm to develop integrated and automated microsystems in a less risky and more consistent manner.

How Lifestyle Factors Affect Cognitive and Executive Function and the Ability to Learn in Children.

In today's research environment, children's diet, physical activity, and other lifestyle factors are commonly studied in the context of health, independent of their effect on cognition and learning. Moreover, there is little overlap between the two literatures, although it is reasonable to expect that the lifestyle factors explored in the health-focused research are intertwined with cognition and learning processes. This thematic review provides an overview of knowledge connecting the selected lifestyle factors of diet, physical activity, and sleep hygiene to children's cognition and learning. Research from studies of diet and nutrition, physical activity and fitness, sleep, and broader influences of cultural and socioeconomic factors related to health and learning, were summarized to offer examples of research that integrate lifestyle factors and cognition with learning. The literature review demonstrates that the associations and causal relationships between these factors are vastly understudied. As a result, current knowledge on predictors of optimal cognition and learning is incomplete, and likely lacks understanding of many critical facts and relationships, their interactions, and the nature of their relationships, such as there being mediating or confounding factors that could provide important knowledge to increase the efficacy of learning-focused interventions. This review provides information focused on studies in children. Although basic research in cells or animal studies are available and indicate a number of possible physiological pathways, inclusion of those data would distract from the fact that there is a significant gap in knowledge on lifestyle factors and optimal learning in children. In a climate where childcare and school feeding policies are continuously discussed, this thematic review aims to provide an impulse for discussion and a call for more holistic approaches to support child development.

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.