HCR-Assisted RTF-EXPAR-Based Lateral Flow Analysis for Sensitive Detection of H1N1 Influenza Virus.

The H1N1 influenza virus is a significant pathogen responsible for seasonal influenza, and its frequent outbreaks pose substantial challenges to global public health. The present study successfully developed a lateral flow analysis platform that integrates reverse transcription-free exponential amplification reaction (RTF-EXPAR) and hybridization chain reaction (HCR) processes with functionalized quantum dots for the direct detection of H1N1 influenza virus RNA, eliminating the need for reverse transcription. The fluorescence signal on the band recorded with a smartphone can be utilized for the quantitative determination of the target. Interestingly, the dual signal amplification strategy exhibits high sensitivity with a remarkably low detection limit of 10 aM. Moreover, this platform exhibits excellent flexibility and universality, where the various pathogens can be determined by replacing the specific nucleic acid fragments in RTF-EXPAR. The aforementioned advantages reveal its huge potential in the early diagnosis of H1N1 influenza virus infection and developing point-of-care testing (POCT) equipment for nucleic acid analysis.

Isotope-dilution liquid chromatography-tandem mass spectrometry for quantification of human retinol binding protein 4 in serum.

Serum retinol binding protein 4 levels play critical roles in the early diagnosis and therapeutic monitoring of diabetic kidney disease. In this paper, an liquid chromatography-tandem mass spectrometry approach for the absolute quantification of serum RBP4 with high sensitivity and specificity was presented. Following series of procedures including denaturation, reduction, alkylation and trypsin digestion, the signature peptides of RBP4 were separated on the HPLC system by gradient elution. Quantitative analysis was achieved by a triple quadrupole mass spectrometer under multiple reaction monitoring in the positive ion (ESI+) mode. The calibration range was from 6.25 to 125 mg/L (R2 > 0.993), the lower limit of quantification was 2.50 mg/L, and the lower limit of detection reached 0.0150 mg/L. In the study, the accuracy ranged from 94.6% to 107%, and the relative standard deviation of intra-assay and inter-assay imprecision was less than 5%. The method was demonstrated to realize sensitive and reliable absolute quantification of serum RBP4 conforming to guidelines for bioanalytical method validation.

Research progress of microfluidics-based food safety detection.

High demands for food safety detection and analysis have been advocated with people's increasing living standards. Even though numerous analytical testing techniques have been proposed, their widespread adoption is still constrained by the high limit of detection, narrow detection ranges, and high implementation costs. Due to their advantages, such as reduced sample and reagent consumption, high sensitivity, automation, low cost, and portability, using microfluidic devices for food safety monitoring has generated significant interest. This review provides a comprehensive overview of the latest microfluidic detection platforms (published in recent 4 years) and their applications in food safety, aiming to provide references for developing efficient research strategies for food contaminant detection and facilitating the transition of these platforms from laboratory research to practical field use.

Facile prepared microfluidic chip for multiplexed digital RT-qPCR test.

The chip-based digital polymerase chain reaction (PCR) is an indispensable technique for amplifying and quantifying nucleic acids, which has been widely employed in molecular diagnostics at both fundamental and clinical levels. However, the previous designs have yet to achieve widespread application due to limitations in complex chip fabrication, pretreatment procedures, special surface properties, and low throughput. This study presents a facile digital microfluidic chip driven by centrifugal force for digital PCR analysis. Interestingly, regardless of the hydrophilicity or hydrophobicity of the inner chip surface, an efficient digitization process can be achieved. PCR reagents introduced into the inlet can be allocated to 9600 microchambers and subsequently isolated by the immiscible phase (silicone oil). The centrifugal priming approach offers a facile means to achieve high-throughput analysis. The design was further employed for the quantification of nucleic acids using digital PCR. The calculated result exhibited a strong correlation with the measured value at the concentrations from 1 copy/µL to 1000 copies/µL (R2  = 0.99). Additionally, the chip also allowed digital multiplexed analysis, thereby indicating its potential for multi-target detection applications.

Advancements of CRISPR technology in public health-related analysis.

Pathogens and contaminants in food and the environment present significant challenges to human health, necessitating highly sensitive and specific diagnostic methods. Traditional approaches often struggle to meet these requirements. However, the emergence of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system has revolutionized nucleic acid diagnostics. The present review provides a comprehensive overview of the biological sensing technology based on the CRISPR/Cas system and its potential applications in public health-related analysis. Additionally, it explores the enzymatic cleavage capabilities mediated by Cas proteins, highlighting the promising prospects of CRISPR technology in addressing bioanalysis challenges. We discuss commonly used CRISPR-Cas proteins and elaborate on their application in detecting foodborne bacteria, viruses, toxins, other chemical pollution, and drug-resistant bacteria. Furthermore, we highlight the advantages of CRISPR-based sensors in the field of public health-related analysis and propose that integrating CRISPR-Cas biosensing technology with other technologies could facilitate the development of more diverse detection platforms, thereby indicating promising prospects in this field.

A portable CRISPR-Cas12a triggered photothermal biosensor for sensitive and visual detection of Staphylococcus aureus and Listeria monocytogenes.

The detection of foodborne pathogens is crucial for ensuring the maintenance of food safety. In the present study, a portable CRISPR-Cas12a triggered photothermal biosensor integrating branch hybrid chain reaction (bHCR) and DNA metallization strategy for sensitive and visual detection of foodborne pathogens was proposed. The sheared probes were utilized to block the locker probes, which enabled preventing the assembly of bHCR in the absence of target bacteria, while target bacteria can activate the cleavage of sheared probes through CRISPR-Cas12a. Therefore, the locker probes functioned as initiating chains, triggering the formation of the branching double-stranded DNA consisting of H1, H2, and H3. The silver particles, which were in situ deposited on the DNA structure, functioned as a signal factor for conducting photothermal detection. Staphylococcus aureus and Listeria monocytogenes were selected as the foodborne pathogens to verify the analytical performance of this CRISPR-Cas12a triggered photothermal sensor platform. The sensor exhibited a sensitive detection with a low detection limit of 1 CFU/mL, while the concentration ranged from 100 to 108 CFU/mL. Furthermore, this method could efficiently detect target bacteria in multiple food samples. The findings demonstrate that this strategy can serve as a valuable reference for the development of a portable platform enabling quantitative analysis, visualization, and highly sensitive detection of foodborne bacteria.

Microfluidic-based platforms for cell-to-cell communication studies.

Intercellular communication is critical to the understanding of human health and disease progression. However, compared to traditional methods with inefficient analysis, microfluidic co-culture technologies developed for cell-cell communication research can reliably analyze crucial biological processes, such as cell signaling, and monitor dynamic intercellular interactions under reproducible physiological cell co-culture conditions. Moreover, microfluidic-based technologies can achieve precise spatial control of two cell types at the single-cell level with high throughput. Herein, this review focuses on recent advances in microfluidic-based 2D and 3D devices developed to confine two or more heterogeneous cells in the study of intercellular communication and decipher the advantages and limitations of these models in specific cellular research scenarios. This review will stimulate the development of more functionalized microfluidic platforms for biomedical research, inspiring broader interests across various disciplines to better comprehend cell-cell communication and other fields, such as tumor heterogeneity and drug screening.

Mechanical stimuli-induced CCL2 restores adult mouse cells to regenerate hair follicles.

Aged cells have declined regenerative ability when subjected to environmental insult. Here we elucidate the mechanism by which mechanical stimulus induces hair regeneration at the microenvironmental regulation level using the hair plucking and organoid culture models. We observed that the skin cells harvested from post-plucking day 3 (PPD3) have the best self-organizing ability during skin organoid culture and have the highest hair regeneration upon transplantation. By bulk RNA sequencing (RNA-seq) and single-cell RNA-seq analysis and in situ hybridization, we identified that the chemokine signaling pathway genes including CCL2 are significantly increased in the skin at PPD3 and in skin organoid cultures. Immunostaining shows that the PPD3 skin epithelial cells have increased multipotency, which is verified by the ability to self-organize to form epidermal aggregates during organoid culture. By adding CCL2 recombinant protein to the organoid culture using an environmental reprogramming protocol, we observed the PPD0 adult skin cells, which lose their regenerative ability can self-organize in organoid culture and regenerate hair follicles robustly upon transplantation. Our study demonstrates that CCL2 functions in immune regulation of hair regeneration under mechanical stimulus, and enhances cell multipotency during organoid culture. This provides a therapeutic potential for future clinical application.

COX2-ATP Synthase Regulates Spine Follicle Size in Hedgehogs.

Skin evolves essential appendages with adaptive patterns that synergistically insulate the body from environmental insults. How similar appendages in different animals generate diversely-sized appendages remain elusive. Here we used hedgehog spine follicles and mouse hair follicles as models to investigate how similar follicles form in different sizes postnatally. Histology and immunostaining show that the spine follicles have a significantly greater size than the hair follicles. By RNA-sequencing analysis, we found that ATP synthases are highly expressed in hedgehog skin compared to mouse skin. Inhibition of ATP synthase resulted in smaller spine follicle formation during regeneration. We also identified that the mitochondrial gene COX2 functions upstream of ATP synthase that influences energy metabolism and cell proliferation to control the size of the spine follicles. Our study identified molecules that function differently in forming diversely-sized skin appendages across different animals, allowing them to adapt to the living environment and benefit from self-protection.

Precisely controlled and deeply penetrated micro-nano hybrid multifunctional motors with enhanced antibacterial activity against refractory biofilm infections.

The biofilm resistance of microorganisms has severe economic and environmental implications, especially the contamination of facilities associated with human life, including medical implants, air-conditioning systems, water supply systems, and food-processing equipment, resulting in the prevalence of infectious diseases. Once bacteria form biofilms, their antibiotic resistance can increase by 10-1,000-fold, posing a great challenge to the treatment of related diseases. In order to overcome the contamination of bacterial biofilm, destroying the biofilm's matrix so as to solve the penetration depth dilemma of antibacterial agents is the most effective way. Here, a magnetically controlled multifunctional micromotor was developed by using H2O2 as the fuel and MnO2 as the catalyst to treat bacterial biofilm infection. In the presence of H2O2, the as-prepared motors could be self-propelled by the generated oxygen microbubbles. Thereby, the remotely controlled motors could drill into the EPS of biofilm and disrupt them completely with the help of bubbles. Finally, the generated highly toxic •OH could efficiently kill the unprotected bacteria. This strategy combined the mechanical damage, highly toxic •OH, and precise magnetic guidance in one system, which could effectively eliminate biologically infectious fouling in microchannels within 10 min, possessing a wide range of practical application prospects especially in large scale and complex infection sites.

Signaligner Pro: A Tool to Explore and Annotate Multi-day Raw Accelerometer Data.

Human activity recognition using wearable accelerometers can enable in-situ detection of physical activities to support novel human-computer interfaces. Many of the machine-learning-based activity recognition algorithms require multi-person, multi-day, carefully annotated training data with precisely marked start and end times of the activities of interest. To date, there is a dearth of usable tools that enable researchers to conveniently visualize and annotate multiple days of high-sampling-rate raw accelerometer data. Thus, we developed Signaligner Pro, an interactive tool to enable researchers to conveniently explore and annotate multi-day high-sampling rate raw accelerometer data. The tool visualizes high-sampling-rate raw data and time-stamped annotations generated by existing activity recognition algorithms and human annotators; the annotations can then be directly modified by the researchers to create their own, improved, annotated datasets. In this paper, we describe the tool's features and implementation that facilitate convenient exploration and annotation of multi-day data and demonstrate its use in generating activity annotations.

Establishing functional lentiviral vector production in a stirred bioreactor for CAR-T cell therapy.

As gene delivery tools, lentiviral vectors (LV) have broad applications in chimeric antigen receptor therapy (CAR-T). Large-scale production of functional LV is limited by the adherent, serum-dependent nature of HEK293T cells used in the manufacturing. HEK293T adherent cells were adapted to suspension cells in a serum-free medium to establish large-scale processes for functional LV production in a stirred bioreactor without micro-carriers. The results showed that 293 T suspension was successfully cultivated in F media (293 CD05 medium and SMM293-TII with 1:1 volume ratio), and the cells retained the capacity for LV production. After cultivation in a 5.5 L bioreactor for 4 days, the cells produced 1.5 ± 0.3 × 107 TU/mL raw LV, and the lentiviral transduction efficiency was 48.6 ± 2.8% in T Cells. The yield of LV equaled to the previous shake flask. The critical process steps were completed to enable a large-scale LV production process. Besides, a cryopreservation solution was developed to reduce protein involvement, avoid cell grafting and reduce process cost. The process is cost-effective and easy to scale up production, which is expected to be highly competitive.

An Open-Source Monitor-Independent Movement Summary for Accelerometer Data Processing.

BACKGROUND: Physical behavior researchers using motion sensors often use acceleration summaries to visualize, clean, and interpret data. Such output is dependent on device specifications (e.g., dynamic range, sampling rate) and/or are proprietary, which invalidate cross-study comparison of findings when using different devices. This limits flexibility in selecting devices to measure physical activity, sedentary behavior, and sleep. PURPOSE: Develop an open-source, universal acceleration summary metric that accounts for discrepancies in raw data among research and consumer devices. METHODS: We used signal processing techniques to generate a Monitor-Independent Movement Summary unit (MIMS-unit) optimized to capture normal human motion. Methodological steps included raw signal harmonization to eliminate inter-device variability (e.g., dynamic g-range, sampling rate), bandpass filtering (0.2-5.0 Hz) to eliminate non-human movement, and signal aggregation to reduce data to simplify visualization and summarization. We examined the consistency of MIMS-units using orbital shaker testing on eight accelerometers with varying dynamic range (±2 to ±8 g) and sampling rates (20-100 Hz), and human data (N = 60) from an ActiGraph GT9X. RESULTS: During shaker testing, MIMS-units yielded lower between-device coefficient of variations than proprietary ActiGraph and ENMO acceleration summaries. Unlike the widely used ActiGraph activity counts, MIMS-units were sensitive in detecting subtle wrist movements during sedentary behaviors. CONCLUSIONS: Open-source MIMS-units may provide a means to summarize high-resolution raw data in a device-independent manner, thereby increasing standardization of data cleaning and analytical procedures to estimate selected attributes of physical behavior across studies.

Investigating Health Risk Environments in Housing Programs for Young Adults: Protocol for a Geographically Explicit Ecological Momentary Assessment Study.

BACKGROUND: Young adults who experience homelessness are exposed to environments that contribute to risk behavior. However, few studies have examined how access to housing may affect the health risk behaviors of young adults experiencing homelessness. OBJECTIVE: This paper describes the Log My Life study that uses an innovative, mixed-methods approach based on geographically explicit ecological momentary assessment (EMA) through cell phone technology to understand the risk environment of young adults who have either enrolled in housing programs or are currently homeless. METHODS: For the quantitative arm, study participants age 18-27 respond to momentary surveys via a smartphone app that collects geospatial information repeatedly during a 1-week period. Both EMAs (up to 8 per day) and daily diaries are prompted to explore within-day and daily variations in emotional affect, context, and health risk behavior, while also capturing infrequent risk behaviors such as sex in exchange for goods or services. For the qualitative arm, a purposive subsample of participants who indicated engaging in risky behaviors are asked to complete an in-depth qualitative interview using an interactive, personalized geospatial map rendering of EMA responses. RESULTS: Recruitment began in June of 2017. To date, 170 participants enrolled in the study. Compliance with EMA and daily diary surveys was generally high. In-depth qualitative follow-ups have been conducted with 15 participants. We expect to recruit 50 additional participants and complete analyses by September of 2019. CONCLUSIONS: Mixing the quantitative and qualitative arms in this study will provide a more complete understanding of differences in risk environments between homeless and housed young adults. Furthermore, this approach can improve recall bias and enhance ecological validity. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/12112.

A Pilot Study of Perceptual-Motor Training for Peripheral Prisms.

PURPOSE: Peripheral prisms (p-prisms) shift peripheral portions of the visual field of one eye, providing visual field expansion for patients with hemianopia. However, patients rarely show adaption to the shift, incorrectly localizing objects viewed within the p-prisms. A pilot evaluation of a novel computerized perceptual-motor training program aiming to promote p-prism adaption was conducted. METHODS: Thirteen patients with hemianopia fitted with 57Δ oblique p-prisms completed the training protocol. They attended six 1-hour visits reaching and touching peripheral checkerboard stimuli presented over videos of driving scenes while fixating a central target. Performance was measured at each visit and after 3 months. RESULTS: There was a significant reduction in touch error (P = 0.01) for p-prism zone stimuli from pretraining median of 16.6° (IQR 12.1°-19.6°) to 2.7° ( IQR 1.0°-8.5°) at the end of training. P-prism zone reaction times did not change significantly with training (P > 0.05). P-prism zone detection improved significantly (P = 0.01) from a pretraining median 70% (IQR 50%-88%) to 95% at the end of training (IQR 73%-98%). Three months after training improvements had regressed but performance was still better than pretraining. CONCLUSIONS: Improved pointing accuracy for stimuli detected in prism-expanded vision of patients with hemianopia wearing 57Δ oblique p-prisms is possible and training appears to further improve detection. TRANSLATIONAL RELEVANCE: This is the first use of this novel software to train adaptation of visual direction in patients with hemianopia wearing peripheral prisms.

[Clinical observation on therapeutic effect of electroacupuncture on chronic prostatitis and detection of urethral sphincter EMG].

OBJECTIVE: To observe the clinical therapeutic effect of electroacupuncture for chronic prostatitis and investigate its mechanism. METHODS: Forty-eight cases were randomly divided into an electroacupuncture group and a western medicine group, 24 cases in each group. The electroacupuncture group was treated by electroacupuncture and Guanyuan (CV 4), Zhongji (CV 3). Ciliao (BL 32) and Huiyang (BL 35) were selected as main acupoints. The western medicine group was treated by oral administration of Sparfloxacin tablets and Prostat tablets. The scores of NIH-CPSI, changes in uroflow rate-urethral sphincter EMG and their therapeutic effects were observed. RESULTS: The total effective rate was 87.5 % in the electroacupuncture group which, was better than 62. 5% in the western medicine group (P(<0. 05). There were significant differences in the scores of NIH-CPSI and Q(max), Q(ave), TL value before and after treatment in the electroacupuncture group (all P<0.05), and with a significant difference in the scores of NIH-CPSI and Q(max). Q(ave), TL value after treatment between the two groups (all P<0.05). CONCLUSION: Electroacupuncture based on syndrome differentiation has better therapeutic effect on chronic prostatitis than that of routine clinical medicine.