Deep learning enhancing guide RNA design for CRISPR/Cas12a-based diagnostics.

Rapid and accurate diagnostic tests are fundamental for improving patient outcomes and combating infectious diseases. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas12a-based detection system has emerged as a promising solution for on-site nucleic acid testing. Nonetheless, the effective design of CRISPR RNA (crRNA) for Cas12a-based detection remains challenging and time-consuming. In this study, we propose an enhanced crRNA design system with deep learning for Cas12a-mediated diagnostics, referred to as EasyDesign. This system employs an optimized convolutional neural network (CNN) prediction model, trained on a comprehensive data set comprising 11,496 experimentally validated Cas12a-based detection cases, encompassing a wide spectrum of prevalent pathogens, achieving Spearman's ρ = 0.812. We further assessed the model performance in crRNA design for four pathogens not included in the training data: Monkeypox Virus, Enterovirus 71, Coxsackievirus A16, and Listeria monocytogenes. The results demonstrated superior prediction performance compared to the traditional experiment screening. Furthermore, we have developed an interactive web server (https://crispr.zhejianglab.com/) that integrates EasyDesign with recombinase polymerase amplification (RPA) primer design, enhancing user accessibility. Through this web-based platform, we successfully designed optimal Cas12a crRNAs for six human papillomavirus (HPV) subtypes. Remarkably, all the top five predicted crRNAs for each HPV subtype exhibited robust fluorescent signals in CRISPR assays, thereby suggesting that the platform could effectively facilitate clinical sample testing. In conclusion, EasyDesign offers a rapid and reliable solution for crRNA design in Cas12a-based detection, which could serve as a valuable tool for clinical diagnostics and research applications.

The meniscotibial ligament does exist: An anatomic and histological description.

Purpose: To describe the anatomical and histological characteristics of the human MTL (meniscotibial ligament) that keeps the meniscus stable and are rarely discussed. Study design: Descriptive laboratory study. Methods: In total, six fresh-frozen adult cadaver knees were dissected, and the dissection protocol were designed by two experienced anatomy professors. The anatomical morphology of MTL was observed. The main anatomical specimens included meniscus, tibial plateau, MTL. The osteotome was used to excise the portion of the tibial plateau, which could obtain the complex including partial meniscus, MTL, and a tibial fragment. A histopathologic study was performed by two experienced pathologists. Results: Macroscopically, the MTL could be divided into two parts: medial meniscotibial ligament (MMTL)and lateral meniscotibial ligament (LMTL). The MMTL is distributed continuously, whereas the LMTL is discontinuous on the tibial plateau. The average length from the tibial attachment of the LMTL to the articular surface was 19 ± 1.0mm (mean ± SD). The average length from the tibial attachment of the MMTL to the articular surface was 10 ± 1.2 mm (mean ± SD). Microscopy of the MTL showed that the MTL is a ligamentous tissue, composed of a network of oriented collagenous fibers. Conclusions: In all knees, the MTL was inserted on the outer edge of the meniscus, attaching to the tibia below the level of articular cartilage, which was key to maintaining the rotational stability of knee and the meniscus in the physiological position on the tibial plateau. Histological analysis of this ligament demonstrated that the MTL is a veritable ligamentous structure, which is made up of collagen type I-expressing fibroblasts. Clinical relevance: This article contributes to the understanding of the anatomical and histological characteristics of the MTL. It is beneficial to promote the development of relevant surgical techniques for the MTL lesion.

Transcending antibiotic resistance: The potential of mass Galla chinensis et camelliae Fermentata to Dismantle Helicobacter pylori biofilms and enhance anti-biotic activity.

ETHNOPHARMACOLOGICAL RELEVANCE: Helicobacter pylori (H. pylori) infections are on the rise, presenting a significant global health challenge. Mass Galla chinesis et camelliae Fermentata (Chinese gall leaven, CGL), a traditional Chinese medicinal product made from the fermentation of Rhus chinensis Mill., is frequently employed to address digestive system ailments. Contemporary pharmacological research reveals that CGL exhibits anti-inflammatory, anti-diarrheal, and enzyme-inhibitory activities and holds potential as a treatment for H. pylori infections. However, the precise mechanisms underlying CGL's efficacy against H. pylori remain to be fully elucidated. AIM: The objective of the study is to evaluate CGL's ability to disrupt the H. pylori biofilm and to explore its synergistic potential with antibiotics in targeting the biofilm-efflux pump system, a mechanism implicated in bacterial resistance. METHORDS: The study determined the Minimum Inhibitory Concentration (MIC) of CGL and metronidazole against H. pylori and evaluated their effects on H. pylori biofilms using an in vitro model. Structural changes induced by drug interventions were compared to those in untreated and antibiotic-treated models through scanning electron microscopy and laser confocal microscopy. The accumulation of H33342 dye in planktonic and biofilm H. pylori before and after drug treatment was assessed to evaluate cell viability and biofilm disruption. The study also involved adding experimental drugs to a biofilm H. pylori medium containing D-glucose, measuring glucose concentrations post-intervention using the glucose oxidase method, and calculating changes in glucose uptake. Finally, the relative expression levels of several genes in planktonic and biofilm H. pylori treated with CGL alone or in combination with antibiotics were measured to understand the impact on the biofilm-efflux pump system. RESULTS: Both CGL alone and in combination with metronidazole demonstrated effective disruption of H. pylori biofilms. The combination therapy was particularly effective in reducing the biofilm transfer-enhancing effect of metronidazole and decreasing SpoT expression in the 'SpoT-(p)ppGpp' pathway, especially in biofilms. It showed a greater inhibition of the 'σ54-gluP-sugar uptake' pathway, with significant reductions in rpoN and gluP expression under biofilm conditions compared to CGL or metronidazole alone. The treatment also suppressed H. pylori proliferation and may have altered glucose uptake mechanisms. Moreover, it significantly inhibited the 'hp0939/hp0497/hp0471-RND efflux pump' pathway, with a notable reduction in gene expression compared to the 1/2 MIC metronidazole treatment. CONCLUSION: This study demonstrates that CGL effectively hinders the development of drug resistance in H. pylori by targeting biofilm formation and critical molecular pathways associated with antibiotic resistance. The synergistic effect of combining CGL with metronidazole notably enhances biofilm disruption and inhibits the bacterium's metabolic and reparative mechanisms. Further in vivo studies are needed to confirm these results and to investigate additional mechanisms of CGL's action.

CoHIT: a one-pot ultrasensitive ERA-CRISPR system for detecting multiple same-site indels.

Genetic testing is crucial for precision cancer medicine. However, detecting multiple same-site insertions or deletions (indels) is challenging. Here, we introduce CoHIT (Cas12a-based One-for-all High-speed Isothermal Test), a one-pot CRISPR-based assay for indel detection. Leveraging an engineered AsCas12a protein variant with high mismatch tolerance and broad PAM scope, CoHIT can use a single crRNA to detect multiple NPM1 gene c.863_864 4-bp insertions in acute myeloid leukemia (AML). After optimizing multiple parameters, CoHIT achieves a detection limit of 0.01% and rapid results within 30 minutes, without wild-type cross-reactivity. It successfully identifies NPM1 mutations in 30 out of 108 AML patients and demonstrates potential in monitoring minimal residual disease (MRD) through continuous sample analysis from three patients. The CoHIT method is also competent for detecting indels of KIT, BRAF, and EGFR genes. Integration with lateral flow test strips and microfluidic chips highlights CoHIT's adaptability and multiplexing capability, promising significant advancements in clinical cancer diagnostics.

Potential for Recycling Metakaolin/Slag-Based Geopolymer Concrete of Various Strength Levels in Freeze-Thaw Conditions.

Geopolymer concrete (GPC) represents an innovative green and low-carbon construction material, offering a viable alternative to ordinary Portland cement concrete (OPC) in building applications. However, existing studies tend to overlook the recyclability aspect of GPC for future use. Various structural applications necessitate the use of concrete with distinct strength characteristics. The recyclability of the parent concrete is influenced by these varying strengths. This study examined the recycling potential of GPC across a spectrum of strength grades (40, 60, 80, and 100 MPa, marked as C40, C60, C80, and C100) when subjected to freeze-thaw conditions. Recycling 5-16 mm recycled geopolymer coarse aggregate (RGAs) from GPC prepared from 5 to 16 mm natural coarse aggregates (NAs). The cementitious material comprised 60% metakaolin and 40% slag, with natural gravel serving as the NAs, and the alkali activator consisting of sodium hydroxide solution and sodium silicate solution. The strength of the GPC was modulated by altering the Na/Al ratio. After 350 freeze-thaw cycles, the GPC specimens underwent crushing, washing, and sieving to produce RGAs. Subsequently, their physical properties (apparent density, water absorption, crushing index, and attached mortar content and microstructure (microhardness, SEM, and XRD) were thoroughly examined. The findings indicated that GPC with strength grades of C100, C80, and C60 were capable of enduring 350 freeze-thaw cycles, in contrast to C40, which did not withstand these conditions. RGAs derived from GPC of strength grades C100 and C80 complied with the criteria for Class II recycled aggregates, whereas RGAs produced from GPC of strength grade C60 aligned with the Class III level. A higher-strength grade in the parent concrete correlated with enhanced performance characteristics in the resulting recycled aggregates.

Differentiating Microplastics from Natural Particles in Aqueous Suspensions Using Flow Cytometry with Machine Learning.

Microplastics (MPs) in natural waters are heterogeneously mixed with other natural particles including algal cells and suspended sediments. An easy-to-use and rapid method for directly measuring and distinguishing MPs from other naturally present colloids in the environment would expedite analytical workflows. Here, we established a database of MP scattering and fluorescence properties, either alone or in mixtures with natural particles, by stain-free flow cytometry. The resulting high-dimensional data were analyzed using machine learning approaches, either unsupervised (e.g., viSNE) or supervised (e.g., random forest algorithms). We assessed our approach in identifying and quantifying model MPs of diverse sizes, morphologies, and polymer compositions in various suspensions including phototrophic microorganisms, suspended biofilms, mineral particles, and sediment. We could precisely quantify MPs in microbial phototrophs and natural sediments with high organic carbon by both machine learning models (identification accuracies over 93%), although it was not possible to distinguish between different MP sizes or polymer compositions. By testing the resulting method in environmental samples through spiking MPs into freshwater samples, we further highlight the applicability of the method to be used as a rapid screening tool for MPs. Collectively, this workflow can be easily applied to a diverse set of samples to assess the presence of MPs in a time-efficient manner.

Simultaneous removal of calcium, phosphorus, and bisphenol A from industrial wastewater by Stutzerimonas sp. ZW5 via microbially induced calcium precipitation (MICP): Kinetics, mechanism, and stress response.

The biological treatment of complex industrial wastewater has always been a research hotspot. In this experiment, a salt-tolerant strain Stutzerimonas sp. ZW5 with aerobic denitrification and biomineralization ability was screened, and the optimum conditions of ZW5 were explored by kinetics. The removal efficiencies of nitrate (NO3--N), bisphenol A (BPA), phosphorus (PO43--P), and calcium (Ca2+) were 94.47 %, 100 %, 98.87 %, and 83.04 %, respectively. The removal mechanism of BPA was the adsorption of microbial induced calcium precipitation (MICP) and extracellular polymeric substances (EPS). Moreover, BPA could weaken the electron transfer ability and growth metabolism of microorganisms and affect the structure of biominerals. At the same time, the stress response of microorganisms would increase the secretion of EPS to promote the process of biomineralization. Through nitrogen balance experiments, it was found that the addition of BPA would lead to a decrease in the proportion of gaseous nitrogen. This experiment offers novel perspectives on the treatment of industrial effluents and microbial stress response.