A Wax Interface-Enabled One-Pot Multiplexed Nucleic Acid Testing Platform for Rapid and Sensitive Detection of Viruses and Variants.

High-quality, low-cost, and rapid detection is essential for the society to reopen the economy during the critical period of transition from Coronavirus Disease 2019 (COVID-19) pandemic response to pandemic control. In addition to performing sustainable and target-driven tracking of SARS-CoV-2, conducting comprehensive surveillance of variants and multiple respiratory pathogens is also critical due to the frequency of reinfections, mutation immune escape, and the growing prevalence of the cocirculation of multiple viruses. By utilizing a 0.05 cents wax interface, a Stable Interface assisted Multiplex Pathogenesis Locating Estimation in Onepot (SIMPLEone) using nested RPA and CRISPR/Cas12a enzymatic reporting system is successfully developed. This smartphone-based SIMPLEone system achieves highly sensitive one-pot detection of SARS-CoV-2 and its variants, or multiple respiratory viruses, in 40 min. A total of 89 clinical samples, 14 environmental samples, and 20 cat swab samples are analyzed by SIMPLEone, demonstrating its excellent sensitivity (3-6 copies/reaction for non-extraction detection of swab and 100-150 copies/mL for RNA extraction-based assay), accuracy (>97.7%), and specificity (100%). Furthermore, a high percentage (44.2%) of co-infection cases are detected in SARS-CoV-2-infected patients using SIMPLEone's multiplex detection capability.

Species-specific FMRP regulation of RACK1 is critical for prenatal cortical development.

Fragile X messenger ribonucleoprotein 1 protein (FMRP) deficiency leads to fragile X syndrome (FXS), an autism spectrum disorder. The role of FMRP in prenatal human brain development remains unclear. Here, we show that FMRP is important for human and macaque prenatal brain development. Both FMRP-deficient neurons in human fetal cortical slices and FXS patient stem cell-derived neurons exhibit mitochondrial dysfunctions and hyperexcitability. Using multiomics analyses, we have identified both FMRP-bound mRNAs and FMRP-interacting proteins in human neurons and unveiled a previously unknown role of FMRP in regulating essential genes during human prenatal development. We demonstrate that FMRP interaction with CNOT1 maintains the levels of receptor for activated C kinase 1 (RACK1), a species-specific FMRP target. Genetic reduction of RACK1 leads to both mitochondrial dysfunctions and hyperexcitability, resembling FXS neurons. Finally, enhancing mitochondrial functions rescues deficits of FMRP-deficient cortical neurons during prenatal development, demonstrating targeting mitochondrial dysfunction as a potential treatment.

Elevated levels of FMRP-target MAP1B impair human and mouse neuronal development and mouse social behaviors via autophagy pathway.

Fragile X messenger ribonucleoprotein 1 protein (FMRP) binds many mRNA targets in the brain. The contribution of these targets to fragile X syndrome (FXS) and related autism spectrum disorder (ASD) remains unclear. Here, we show that FMRP deficiency leads to elevated microtubule-associated protein 1B (MAP1B) in developing human and non-human primate cortical neurons. Targeted MAP1B gene activation in healthy human neurons or MAP1B gene triplication in ASD patient-derived neurons inhibit morphological and physiological maturation. Activation of Map1b in adult male mouse prefrontal cortex excitatory neurons impairs social behaviors. We show that elevated MAP1B sequesters components of autophagy and reduces autophagosome formation. Both MAP1B knockdown and autophagy activation rescue deficits of both ASD and FXS patients' neurons and FMRP-deficient neurons in ex vivo human brain tissue. Our study demonstrates conserved FMRP regulation of MAP1B in primate neurons and establishes a causal link between MAP1B elevation and deficits of FXS and ASD.

Cosbin: cosine score-based iterative normalization of biologically diverse samples.

Motivation: Data normalization is essential to ensure accurate inference and comparability of gene expression measures across samples or conditions. Ideally, gene expression data should be rescaled based on consistently expressed reference genes. However, to normalize biologically diverse samples, the most commonly used reference genes exhibit striking expression variability and size-factor or distribution-based normalization methods can be problematic when the amount of asymmetry in differential expression is significant. Results: We report an efficient and accurate data-driven method-Cosine score-based iterative normalization (Cosbin)-to normalize biologically diverse samples. Based on the Cosine scores of cross-condition expression patterns, the Cosbin pipeline iteratively eliminates asymmetric differentially expressed genes, identifies consistently expressed genes, and calculates sample-wise normalization factors. We demonstrate the superior performance and enhanced utility of Cosbin compared with six representative peer methods using both simulation and real multi-omics expression datasets. Implemented in open-source R scripts and specifically designed to address normalization bias due to significant asymmetry in differential expression across multiple conditions, the Cosbin tool complements rather than replaces the existing methods and will allow biologists to more accurately detect true molecular signals among diverse phenotypic groups. Availability and implementation: The R scripts of Cosbin pipeline are freely available at https://github.com/MinjieSh/Cosbin. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Sensitive and Rapid Diagnosis of Respiratory Virus Coinfection Using a Microfluidic Chip-Powered CRISPR/Cas12a System.

The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 is profoundly influencing the global healthcare system and people's daily lives. The high resource consumption of coronavirus disease 2019 (COVID-19) is resulting in insufficient surveillance of coinfection or resurgence of other critical respiratory epidemics, which is of public concern. To facilitate evaluation of the current coinfection situation, a microfluidic system (MAPnavi) is developed for the rapid (<40 min) and sensitive diagnosis of multiple respiratory viruses from swab samples in a fully sealed and automated manner, in which a nested-recombinase polymerase amplification and the CRISPR-based amplification system is first proposed to ensure the sensitivity and specificity. This novel system has a remarkably low limit of detection (50-200 copies mL-1 ) and is successfully applied to detect 171 clinical samples (98.5% positive predictive agreement; 100% negative predictive agreement), and the results identify 45.6% coinfection among clinical samples from patients with COVID-19. This approach has the potential to shift diagnostic and surveillance efforts from targeted testing for a high-priority virus to comprehensive testing of multiple virus sets and to greatly benefit the implementation of decentralized testing.

Comparative assessment and novel strategy on methods for imputing proteomics data.

Missing values are a major issue in quantitative proteomics analysis. While many methods have been developed for imputing missing values in high-throughput proteomics data, a comparative assessment of imputation accuracy remains inconclusive, mainly because mechanisms contributing to true missing values are complex and existing evaluation methodologies are imperfect. Moreover, few studies have provided an outlook of future methodological development. We first re-evaluate the performance of eight representative methods targeting three typical missing mechanisms. These methods are compared on both simulated and masked missing values embedded within real proteomics datasets, and performance is evaluated using three quantitative measures. We then introduce fused regularization matrix factorization, a low-rank global matrix factorization framework, capable of integrating local similarity derived from additional data types. We also explore a biologically-inspired latent variable modeling strategy-convex analysis of mixtures-for missing value imputation and present preliminary experimental results. While some winners emerged from our comparative assessment, the evaluation is intrinsically imperfect because performance is evaluated indirectly on artificial missing or masked values not authentic missing values. Nevertheless, we show that our fused regularization matrix factorization provides a novel incorporation of external and local information, and the exploratory implementation of convex analysis of mixtures presents a biologically plausible new approach.

A fully integrated SNP genotyping system for hereditary hearing-loss detection.

Hereditary hearing loss is one of the most common human neurosensory disorders, and there is a great need for early intervention methods such as genetically screening newborns. Single nucleotide polymorphisms (SNPs) are the major genetic targets for hearing-loss screening. In this study, a fully integrated SNP genotyping system was constructed to identify hereditary hearing loss-related genetic markers from human whole blood. The entire detection process, including blood cell lysis, nucleic acid extraction, the reaction mixture distribution, the chambers sealing and the two-colour multiplex competitive allele-specific polymerase chain reaction (KASP), can be automatically conducted in a self-contained cassette within 3 hours. To critically evaluate the performance of the system, its specificity, sensitivity and stability were assessed. Then, 13 clinical samples were genotyped with this fluidic cassette system to detect seven hotspot deafness-associated mutations in three genes (MT-RNR1, GJB2 and SLC26A4). The detection results of the cassette system were 100% concordant with those obtained by Sanger sequencing, proving its accuracy in the genetic screening of inherited hearing loss.

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-CoV-2 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 800 µL 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.

FXR1 regulation of parvalbumin interneurons in the prefrontal cortex is critical for schizophrenia-like behaviors.

Parvalbumin interneurons (PVIs) are affected in many psychiatric disorders including schizophrenia (SCZ), however the mechanism remains unclear. FXR1, a high confident risk gene for SCZ, is indispensable but its role in the brain is largely unknown. We show that deleting FXR1 from PVIs of medial prefrontal cortex (mPFC) leads to reduced PVI excitability, impaired mPFC gamma oscillation, and SCZ-like behaviors. PVI-specific translational profiling reveals that FXR1 regulates the expression of Cacna1h/Cav3.2 a T-type calcium channel implicated in autism and epilepsy. Inhibition of Cav3.2 in PVIs of mPFC phenocopies whereas elevation of Cav3.2 in PVIs of mPFC rescues behavioral deficits resulted from FXR1 deficiency. Stimulation of PVIs using a gamma oscillation-enhancing light flicker rescues behavioral abnormalities caused by FXR1 deficiency in PVIs. This work unveils the function of a newly identified SCZ risk gene in SCZ-relevant neurons and identifies a therapeutic target and a potential noninvasive treatment for psychiatric disorders.

A Portable Microfluidic System for Point-of-Care Detection of Multiple Protein Biomarkers.

Protein biomarkers are indicators of many diseases and are commonly used for disease diagnosis and prognosis prediction in the clinic. The urgent need for point-of-care (POC) detection of protein biomarkers has promoted the development of automated and fully sealed immunoassay platforms. In this study, a portable microfluidic system was established for the POC detection of multiple protein biomarkers by combining a protein microarray for a multiplex immunoassay and a microfluidic cassette for reagent storage and liquid manipulation. The entire procedure for the immunoassay was automatically conducted, which included the antibody-antigen reaction, washing and detection. Alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and carcinoma antigen 125 (CA125) were simultaneously detected in this system within 40 min with limits of detection of 0.303 ng/mL, 1.870 ng/mL, and 18.617 U/mL, respectively. Five clinical samples were collected and tested, and the results show good correlations compared to those measured by the commercial instrument in the hospital. The immunoassay cassette system can function as a versatile platform for the rapid and sensitive multiplexed detection of biomarkers; therefore, it has great potential for POC diagnostics.

Missed diagnosis of femoral deep artery rupture after femoral shaft fracture: A case report.

BACKGROUND: Vascular injury is a rare complication of femoral shaft fractures, and rupture of the deep femoral artery is more difficult to diagnose because of its anatomical location and symptoms. Despite its low incidence, deep femoral artery rupture can lead to life-threatening outcomes, such as compartment syndrome, making early identification and diagnosis critical. CASE SUMMARY: A 45-year-old male patient was admitted to our hospital due to right lower limb trauma in a car accident, with complaints of severe pain and swelling on his right thigh. X-ray demonstrated a right femoral shaft fracture. During preparation for emergency surgery, his blood pressure and blood oxygen saturation dropped, and sensorimotor function was lost. Computed tomography angiography was performed immediately to confirm the diagnosis of rupture of the deep femoral artery and compartment syndrome, so fasciotomy and vacuum-assisted closure were performed. Rhabdomyolysis took place after the operation and the patient was treated with appropriate electrolyte correction and diuretic therapy. Twenty days after the fasciotomy, treatment with the Hoffman Type II External Fixation System was planned, but it was unable to be immobilized internally based on a new esophageal cancer diagnosis. We kept the external fixation for 1 year, and 3 years of follow-up showed improvement of the patient's overall conditions and muscle strength. CONCLUSION: For patients with thigh swelling, pain, anemia, and unstable vital signs, anterior femoral artery injury should be highly suspected. Once diagnosed, surgical treatment should be performed immediately and complications of artery rupture must be suspected and addressed in time.

RGS6 Mediates Effects of Voluntary Running on Adult Hippocampal Neurogenesis.

Voluntary running enhances adult hippocampal neurogenesis, with consequences for hippocampal-dependent learning ability and mood regulation. However, the underlying mechanism remains unclear. Here, we show that voluntary running induces unique and dynamic gene expression changes specifically within the adult-born hippocampal neurons, with significant impact on genes involved in neuronal maturation and human diseases. We identify the regulator of G protein signaling 6 (RGS6) as a key factor that mediates running impact on adult-born neurons. RGS6 overexpression mimics the positive effects of voluntary running on morphological and physiological maturation of adult new neurons and reduced sensitivity of adult-born neurons to the inhibitory effect of GABAB (γ-Aminobutyric acid B) receptor activation. Knocking down RGS6 abolishes running-enhanced neuronal maturation and hippocampal neurogenesis-dependent learning and anxiolytic effect. Our study provides a data resource showing genome-wide intrinsic molecular changes in adult-born hippocampal neurons that contribute to voluntary running-induced neurogenesis.

An enhanced centrifugation-assisted lateral flow immunoassay for the point-of-care detection of protein biomarkers.

Protein biomarkers are widely used for disease diagnosis, but the current detection methods utilized in centralized laboratories are mainly based on enzyme-linked immunosorbent assay (ELISA)-derived sandwich-type immunoassays such as chemiluminescent or electrochemiluminescent immunoassays, which suffer from long detection times and cumbersome instruments. For the point-of-care (POC) detection of protein biomarkers, various test strips for lateral flow immunoassay (LFIA) have been manufactured, but their detection sensitivities and capabilities for raw samples are limited. In this study, an enhanced centrifugation-assisted lateral flow immunoassay (ECLFIA) was established to rapidly detect protein biomarkers in whole blood with a higher sensitivity than LFIA. By inserting a nitrocellulose membrane into a centrifugal disc, fully automated operations, including sample preparation, active lateral flow actuation, washing, and signal amplification, which could hardly be performed in conventional LFIA, were enabled on the centrifugal platform for ECLFIA. The entire process for detecting human prostate specific antigen (PSA) in a drop of blood (20 µL) could be completed in 15 min. The limit of detection for our ECLFIA system was 0.028 ng mL-1, showing a 21.4-fold improvement compared to that of LFIA. Moreover, this system was utilized to detect PSA in 34 clinical samples. The results were compared to those measured using a commercial instrument used in the hospital, and a good correlation coefficient of 0.986 was obtained, demonstrating the practicality of this ECLFIA system. In summary, the ECLFIA system established in this study can be an efficient tool for the POC detection of protein biomarkers with comprehensive advantages in sensitivity, simplicity and speed.

Hydrolysis and Solvolysis as Benign Routes for the End-of-Life Management of Thermoset Polymer Waste.

The production of thermoset polymers is increasing globally owing to their advantageous properties, particularly when applied as composite materials. Though these materials are traditionally used in more durable, longer-lasting applications, ultimately, they become waste at the end of their usable lifetimes. Current recycling practices are not applicable to traditional thermoset waste, owing to their network structures and lack of processability. Recently, researchers have been developing thermoset polymers with the right functionalities to be chemically degraded under relatively benign conditions postuse, providing a route to future management of thermoset waste. This review presents thermosets containing hydrolytically or solvolytically cleavable bonds, such as esters and acetals. Hydrolysis and solvolysis mechanisms are discussed, and various factors that influence the degradation rates are examined. Degradable thermosets with impressive mechanical, thermal, and adhesion behavior are discussed, illustrating that the design of material end-of-life need not limit material performance.

The Influence of Diabetes Mellitus on Patients Undergoing Primary Total Lower Extremity Arthroplasty: A Systematic Review and Meta-Analysis.

BACKGROUND: Diabetes mellitus (DM) is a common disease that has an adverse impact on most orthopedic surgeries, and its prevalence has gradually increased in recent years. We aim to investigate the influence of DM on comorbidities and complications of patients undergoing primary total lower extremity arthroplasty. METHODS: PubMed, Embase, Cochrane Library, Medline, and Web of Science were systematically searched for relevant studies published before December 2019. Demographic data, comorbidities, and postoperative complications after primary total hip arthroplasties (THA) or primary total knee arthroplasties (TKA) were assessed between DM and non-DM patients. Meta-analysis was conducted using Review Manager 5.3, and forest plots were drawn for each variable. RESULTS: A total of 1,560,461 patients (215,916 patients with DM and 1,344,545 patients without DM) from 23 studies were included in this meta-analysis. The incidences of several preoperative comorbidities (hypertension (HTN), kidney disease, cardiac and cerebrovascular disease) were generally higher in patients with DM. Moreover, DM patients had a higher rate of postoperative complications (superficial and deep infection, deep vein thrombosis (DVT), and in-hospital mortality) compared to non-DM patients. CONCLUSIONS: DM patients were more likely to suffer from comorbidities and had a higher risk of complications in total lower extremity arthroplasty compared to non-DM patients. It is necessary to identify DM and control hyperglycemia in the perioperative period to prevent postoperative complications in patients with DM.

Biodegradable cell-laden starch foams for the rapid fabrication of 3D tissue constructs and the application in neural tissue engineering.

Cells encapsulation by biomaterials has been widely studied as a strategy of building tissue construct in tissue engineering. Conventional encapsulation of cells using hydrogels often needs the polymerization process or relatively complex molding process. In this study, we developed a facile strategy for the in situ fabrication of biodegradable cell-laden starch foams. By utilizing the unique gelatinization property of starch, cell-laden starch foams with tunable architecture were rapidly prepared in a green and biological-friendly process. The bubble size and stiffness of starch foams could be tuned by controlling the content of premixed starch in the cell culture medium. Cells were encapsulated in situ during the foaming process, and the resultant starch foams could be used as building blocks to fabricate three-dimensional tissue construct. The potential application of the cell-laden starch foams in neural tissue engineering was also validated. RSC96 Schwann cells were encapsulated in the starch foams and revealed good viability. Due to the serum-induced degradation of the starch, RSC96 Schwann cells could be released from the starch foams in a controlled manner while remaining high viability. Dorsal root ganglion (DRG) neurons co-cultured with the cell-laden starch foams extended significantly longer neurites compared with neurons cultured in minimum Eagle's medium (664.88 ± 190.39 µm vs. 311.19 ± 105.25 µm). DRG neurons retained high viability even after encapsulation in the starch foams for 3 days. This facile strategy of rapidly fabricating cell-laden starch foams can be further extended to construct centimeter-scale micro-tissue for tissue engineering applications. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:104-116, 2020.

Rapid and Automated Detection of Six Contaminants in Milk Using a Centrifugal Microfluidic Platform with Two Rotation Axes.

Antibiotic residues and illegal additives are among the most common contaminants in milk and other dairy products, and they have become essential public health concerns. To ensure the safety of milk, rapid and convenient screening methods are highly desired. Here, we integrated microarray technology into a microfluidic device to achieve rapid, sensitive, and fully automated detection of chloramphenicol, tetracyclines, enrofloxacin, cephalexin, sulfonamides, and melamine in milk on a centrifugal microfluidic platform with two rotation axes. All the liquid reagent for the immunoassay was prestored in the reagent chambers of the microdevice and can be released on demand. The whole detection can be automatically accomplished within 17 min, and the limits of detection were defined as 0.92, 1.01, 1.83, 1.14, 1.96, and 7.80 µg/kg for chloramphenicol, tetracycline (a typical drug of tetracyclines), enrofloxacin, cephalexin, sulfadiazine (a typical drug of sulfonamides), and melamine, respectively, satisfying the national standards for maximum residue limits in China. Raw milk samples were used to test the performance of the current immunoassay system, and the recovery rates in the repeatability tests ranged from 80 to 111%, showing a good performance. In summary, the immunoassay system established in this study can simultaneously detect six contaminants of four samples in a fully automated, cost-effective, and easy-to-use manner and thus has great promise as a screening tool for food safety testing.

A novel electromagnet-triggered pillar valve and its application in immunoassay on a centrifugal platform.

The lab-on-a-disc is a powerful microfluidic platform that skillfully takes advantage of centrifugal force to controllably drive liquids with the assistance of passive or active valves. However, the passive valves are mainly triggered by the rotation speed and can be easily influenced by the surface chemistry of the channel, while the active valves usually require a complicated fabrication or actuation procedure. In this study, a novel active valve that can be easily triggered by an electromagnet was proposed and applied on the centrifugation platform. This valve, named the electromagnet-triggered pillar (ETP) valve, consisted of a metal pin and pressure sensitive adhesive (PSA) tape, and is closed until the pin is lifted up by an electromagnet to partially separate the PSA tape from the substrate. As a typical application, this valve is utilized to construct a centrifugal chip for mycotoxin detection. With four ETP valves in a unit, the sample and liquid reagents can be sequentially released into the reaction chamber that was spotted with mycotoxin conjugates to accomplish the whole immunoassay. Four mycotoxins (aflatoxin B1, ochratoxin A, T-2 toxin, and zearalenone) were simultaneously detected on this chip with limits of detection lower than the permissible limits set by the regulatory agencies of China, demonstrating the practicability of this easy-to-use active valve.

Enhancing the Sensitivity of Lateral Flow Immunoassay by Centrifugation-Assisted Flow Control.

Lateral flow immunoassay (LFIA) is widely used but is limited by its sensitivity. In this study, a novel centrifugation-assisted lateral flow immunoassay (CLFIA) was proposed that had enhanced sensitivity compared to traditional LFIA based on test strips. For CLFIA, a vaulted piece of nitrocellulose membrane was prepared and inserted into a centrifugal disc. Powered by the centrifugal force, the sample volume on the disc was not limited and the flow rate of the reaction fluid was steady and adjustable at different rotation speeds. It was found that lower rotation speeds and larger sample volumes resulted in greater signal intensity in the nitrocellulose membrane as well as higher sensitivity, indicating that the actively controlled flow on the disc allowed for sensitivity enhancement of CLFIA. To operate CLFIA on the centrifugal disc, a portable and cost-effective operating device was constructed to rotate the disc with a stepper motor and collect the results with a smartphone. The proposed method was successfully applied to detect prostate specific antigen (PSA) in human serum. Standard curves were established for CLFIA and LFIA, and both had correlation coefficients of up to 0.99. Under optimal conditions (1500 rpm rotation speed, 120 µL sample volume), the detection limit of CLFIA reached 0.067 ng/mL, showing a 6.2-fold improvement in sensitivity compared to that of LFIA. With clinical serum samples, a good correlation was observed between PSA concentrations measured by CLFIA and by a bulky commercial instrument in hospital. In summary, this portable, cost-effective, and easy-to-use system holds great promise for biomarker detection with enhanced sensitivity compared to traditional LFIA.