Well-Paired-Seq2: High-Throughput and High-Sensitivity Strategy for Characterizing Low RNA-Content Cell/Nucleus Transcriptomes.

Single-cell RNA sequencing (scRNA-seq) is a transformative technology that unravels the intricate cellular state heterogeneity. However, the Poisson-dependent cell capture and low sensitivity in scRNA-seq methods pose challenges for throughput and samples with a low RNA-content. Herein, to address these challenges, we present Well-Paired-Seq2 (WPS2), harnessing size-exclusion and quasi-static hydrodynamics for efficient cell capture. WPS2 exploits molecular crowding effect, tailing activity enhancement in reverse transcription, and homogeneous enzymatic reaction in the initial bead-based amplification to achieve 3116 genes and 8447 transcripts with an average of ∼20000 reads per cell. WPS2 detected 1420 more genes and 4864 more transcripts than our previous Well-Paired-Seq. It sensitively characterizes transcriptomes of low RNA-content single cells and nuclei, overcoming the Poisson limit for cell and barcoded bead capture. WPS2 also profiles transcriptomes from frozen clinical samples, revealing heterogeneous tumor copy number variations and intercellular crosstalk in clear cell renal cell carcinomas. Additionally, we provide the first single-cell-level characterization of rare metanephric adenoma (MA) and uncover potential specific markers. With the advantages of high sensitivity and high throughput, WPS2 holds promise for diverse basic and clinical research.

The dynamics and removal efficiency of antibiotic resistance genes by UV-LED treatment: An integrated research on single- or dual-wavelength irradiation.

Antimicrobial resistance has gained increasing attention, because of the awareness of its potential health risks. Strategies for the removal of antibiotic resistance genes (ARGs) are urgently required. In the present study, UV-LEDs at wavelength of 265 and 285 nm were integrated at five conditions, including single 265 nm UV-LED, single 285 nm UV-LED, and combined 265 nm and 285 nm UV-LED at different intensities, to remove tet A, cat 1, and amp C. The ARGs removal efficiency, gene behavior, and possible cellular mechanism were analyzed using real-time quantitative PCR, flow cytometry, and transmission electron microscopy (TEM). The 265 nm UV-LED is more effective than the 285 nm UV-LED and their combinations in terms of ARGs control, in which 1.91, 1.71, and 1.45 log were removed for tet A, cat 1, and amp C, respectively, at a UV dosage of 500 mJ/cm2. The intracellular gene leakage was detected in all five UV-LED experiment scenarios even when the cell membrane damage was insignificant with the highest increase of 0.69 log ARGs. ROS was generated during the irradiation, and the ROS was strongly negative correlated with intracellular ARGs, which could promote the degradation and removal of ARGs. This study provides a new insight of intracellular ARGs removal, because direct irradiation, ROS oxidation, and leakage to the extracellular serve as the three main pathways under high-dosage UV-LED irradiation. Further research should be focused on the mechanism and optimization of UV technology with 265 nm UV-LED for ARG control.

Removal of tetracycline-resistant Escherichia coli and its genes through ultrasound treatment combined with ultraviolet light emitting diodes.

Antibiotic resistance has gained increasing attention worldwide, and wastewater treatment plants have been regarded as hotspots for antibiotic-resistant bacteria and antibiotic-resistant genes (ARGs). In this study, we evaluated the removal of tetracycline-resistant Escherichia coli and its related genes through ultrasound (US) treatment with different input levels of US-specific energy combined with ultraviolet light emitting diodes (UV-LEDs). Simultaneous US with UV-LEDs effectively eliminated tetracycline-resistant E. coli with the normal suggested UV-LEDs dosage (below 30 mJ/cm2). The removal efficiency increased with the addition of US (specific input energy of 8-16 kJ/L), and simultaneous US treatment with UV-LEDs was relatively more effective than US pretreatment. Analyses of cell damage by K+ leakage and flow cytometry showed that the cell wall kept its integrity during the applied treatment conditions. Consequently, the removal efficiencies of 16 S rRNA, tet M, and tet Q were unsatisfactory because less than 1 log reduction was achieved. Increasing the US energy remarkably damaged the cell wall and potentially promoted the reaction. The removal of ARGs increased four times when using US-specific input energy at 330 kJ/L with 5 mJ/cm2 compared with UV-LEDs alone. The US treatment combined with UV-LEDs is a novel process that does not require chemicals. Results of this research can provide theoretical support for the removal of ARGs.

Borderline Nerve Conduction Velocities for Median Neuropathy at the Carpal Tunnel.

PURPOSE: Patient knowledge of the frequency with which electrodiagnostic testing (EDx) for suspected median neuropathy at the carpal tunnel addresses nuance in the distinction between normal and abnormal neurophysiology might help them make an informed decision about whether or not to have this test. We reviewed a large set of consecutive EDx for possible carpal tunnel syndrome (CTS) and associated medical records to determine (1) the percentage of EDx measurements within 10% of threshold values; (2) discordance between clinician and EDx diagnosis of CTS using diagnostic performance characteristics; and (3) demographic and disease characteristics independently associated with EDx diagnosis of median neuropathy at the carpal tunnel. METHODS: We retrospectively reviewed nerve conduction study (NCS) results of 537 consecutive patients evaluated for possible idiopathic median neuropathy at the carpal tunnel. We measured the number of patients within 10% of 3 NCS diagnostic thresholds; the diagnostic performance characteristics comparing clinician and EDx diagnosis; and patient and disease characteristics associated with EDx diagnosis of CTS. RESULTS: The 3 NCS parameters were within 10% of the threshold for diagnosis of median neuropathy at the carpal tunnel in 2.6% to 33% of patients. Overall, 76% of EDx results were interpreted as median neuropathy at the carpal tunnel, 19% as normal, and 5% as another diagnosis (eg, cervical radiculopathy). Patients with normal EDx were significantly younger, more likely not to report paresthesias/numbness, more likely to have prior normal EDx, and less likely to have had a previous contralateral carpal tunnel release. CONCLUSIONS: This data set reflecting management strategies for suspected CTS at a large institution confirms inherent diagnostic uncertainty, relatively strong concordance between clinician and EDx diagnosis, and the importance of focusing on paresthesia rather than pain. These findings support the use of clinical prediction rules and may help inform a patient's decision regarding whether or not to have EDx. TYPE OF STUDY/LEVEL OF EVIDENCE: Diagnostic III.

Electrodiagnostic test results in people with a working diagnosis of cubital tunnel syndrome.

Background: Electrodiagnostic tests (EDx) can determine when symptoms and signs suggestive of idiopathic ulnar neuropathy at the elbow (cubital tunnel syndrome; CubTS) is due to measurable ulnar neuropathy at the elbow (UNE), cervical radiculopathy, or median neuropathy at the carpal tunnel, and when there is no measurable neuropathology associated with the symptoms. The role of EDx in management of CubTS is debated. Questions: (1) What is the percentage of patients with CubTS (both including and excluding patients with a previous electrodiagnosis of idiopathic UNE) that have EDx results consistent with idiopathic UNE, other neuropathology, and no detectable neuropathology (2) What factors (e.g. age and gender); are independently associated with electrodiagnosis of UNE. Methods: We retrospectively reviewed all medical records of 133 patients with a working diagnosis of CubTS sent for EDx over a 5-year period in one large urban medical center. We recorded data on patient demographics, comorbidities, non-specialist or specialist referring physician, and EDx results. Results: Among 133 patients, 61% (N = 81) of EDx identified idiopathic UNE, 14% (N = 18) identified other neuropathology, and for 26% (N = 34) there was no measurable neuropathology. Among the 14 patients with a previous ipsilateral or contralateral electrodiagnosis of UNE, all 14 had electrodiagnosis of UNE. Older age and men were independently associated with an increased likelihood of UNE. Conclusions: The observation that people diagnosed with CubTS often do not have UNE, particularly if they are relatively young, suggests that the diagnosis of CubTS may benefit from a more stringent clinical prediction rule. Level of Evidence: Diagnostic; Retrospective cohort study; Level III.

Contamination and Health Risk Assessment of Multiple Mycotoxins in Edible and Medicinal Plants.

Edible and medicinal plants (EMPs) are widely used but are easily infected by harmful fungi which produce mycotoxins. Herein, 127 samples from 11 provinces were collected to investigate 15 mycotoxins based on geographic, demographic, processing, and risk characteristics. A total of 13 mycotoxins were detected, and aflatoxin B1 (0.56~97.00 µg/kg), deoxynivalenol (9.41~1570.35 µg/kg), fumonisin B1 (8.25~1875.77 µg/kg), fumonisin B2 (2.74~543.01 µg/kg), ochratoxin A (0.62~19.30 µg/kg), and zearalenone (1.64~2376.58 µg/kg) occurred more frequently. Mycotoxin levels and species were significantly different by region, types of EMPs, and method of processing. The margin of exposure (MOE) values was well below the safe MOE (10,000). AFB1 exposure from Coix seed and malt consumption in China was of high health concern. The hazard Index (HI) method showed the range of 113.15~130.73% for malt, indicating a public health concern. In conclusion, EMPs should be concerned because of the cumulative effects of co-occurred mycotoxins, and safety management strategies should be developed in follow-up studies.

Mechanism of melanogenesis inhibition by Keggin-type polyoxometalates.

Abnormal melanin overproduction can result in hyperpigmentation syndrome in human skin diseases and enzymatic browning of fruits and vegetables. Recently, our group found that Keggin-type polyoxometalates (POMs) can efficiently inhibit tyrosinase activity. However, it remains unclear whether Keggin-type POMs exhibit optimal effects in vivo. Additionally, the inhibitory effect and mechanism of action of POMs on cellular tyrosinase activity and melanogenesis have been rarely reported. Here we demonstrate that our screened and synthesised PMo11Zn and GaMo12 show superior inhibitory effects on melanin formation as well as inhibition of cellular tyrosinase activity compared to other Keggin-type POMs. Intriguingly, we reveal that Keggin-type POMs competitively bind to tyrosinase mainly through more interactions with Cu2+ ions and the amino acid residue is capable of forming van der Waals, cation-π and hydrogen bonds, resulting in a reversible non-covalent complex formation. Our findings provide valuable insights into the design, synthesis and screening of polyoxometalates as multifunctional metallodrugs and food preservatives against hyperpigmentation.

Synthesis of acylated derivatives of chitosan oligosaccharide and evaluation of their potential antifungal agents on Fusarium oxysporum.

Chitosan oligosaccharide (COS) is an important carbohydrate-based biomaterial for synthesizing candidate drugs and biological agents. This study synthesized COS derivatives by grafting acyl chlorides of different alkyl chain lengths (C8, C10, and C12) onto COS molecules and further investigated their physicochemical properties and antimicrobial activity. The COS acylated derivatives were characterized using Fourier transform infrared spectroscopy, 1H nuclear magnetic resonance spectroscopy, X-ray diffraction, and thermogravimetric analysis. COS acylated derivatives were successfully synthesized and possessed high solubility and thermal stability. As for the evaluation of antibacterial activity, COS acylated derivatives did not significantly inhibit Escherichia coli and Staphylococcus aureus, but they significantly inhibited Fusarium oxysporum, which was superior to that of COS. Transcriptomic analysis revealed that COS acylated derivatives exerted antifungal activity mainly by downregulating the expression of efflux pumps, disrupting cell wall integrity, and impeding normal cell metabolism. Our findings provided a fundamental theory for the development of environmentally friendly antifungal agents.

Comparison of two commercial methods with a UHPLC-MS/MS method for the determination of multiple mycotoxins in cereals.

Immunoassay-based techniques are important on-site screening tools for the detection of mycotoxins in cereals. This study aims to evaluate the trueness, precision, repeatability and cross-reactivity of commercially available test strips, ELISA kits and UHPLC-MS/MS on analyzing zearalenone, ochratoxin A, deoxynivalenol, T-2 toxin and fumonisin B1. The results showed that false negative rate (25.7 %-37.4 %) of all tested mycotoxins by test strips was higher than the false positive rate (0 %-31.0 %). The repeatability of ELISA kits at the declared LOD dispersed from -85.7 % to +98.4 %. ELISA kits were more accurate at 50 % of the maximum residue limit (MRL) of mycotoxins than 150 % and 200 %. All the tested deoxynivalenol/zearalenone derivatives had cross-reactivity with different level, and sample matrix could reinforce this overestimation of target mycotoxin. This study emphasized that higher-quality antibody screening and more analytical performance investigations are need to address for on-site detection of mycotoxins in the future.

Understanding Individual Differences in Metacognitive Strategy Use, Task Demand, and Performance in Integrated L2 Speaking Assessment Tasks.

This study investigated the concept of individual differences (IDs) in the use of metacognitive strategies (planning, problem-solving, monitoring, and evaluating) and its relationship with task demand and learner performance within Kormos' Bilingual Speech Production Model from the lens of Chinese English-as-foreign-language (EFL) learners in the context of integrated L2 speaking assessment. To measure metacognitive strategies, we administered an inventory on 134 Chinese EFL learners after they completed four integrated L2 speaking assessment tasks. Descriptive analysis and multiple linear regression were adopted for data analysis, and results show that: (a) IDs displayed variance in Chinese EFL learners' metacognitive strategy use; (b) among the four metacognitive strategies under investigation, problem-solving was reported to be used the most frequently in sharp contrast to monitoring, which had the lowest frequency; (c) metacognitive strategies worked interactively, responding to task demands involved in the four integrated L2 speaking assessment tasks; and (d) Chinese EFL learners' use of metacognitive strategies, in individual and interactive working modes, had no relationship with their speaking performance. These results are expected to present some insights into the role of IDs in metacognitive strategy use during L2 speech production under assessment conditions, which will add robust evidence to the existing literature on L2 speaking, in particular on metacognitive strategy use in L2 speaking assessment. In the meantime, the findings will provide some empirical validation support for Kormos' model, which will further provide some implications for L2 speaking instruction and L2 assessment.