Environmental parameters factors exploration on lettuce seed germination with hydrogel.

Lettuce (Lactuca sativa) germination is sensitive to environmental conditions. Recently, hydrogel has received increased attention as an alternative media to soil for seed germination. Compared to soil seeding, hydrogel-aided germination provides more controlled seeding environments. However, there are still challenges preventing hydrogel-aided seed germination from being widely used in industry production or academic studies, such as hydrogel formulation variations, seeding operation standardization, and germination evaluation. In this study, we tested how the combination of multiple environmental conditions affect lettuce seed germination time, which is measured as the time needed for the first pair of leaves to appear (leaf emergence) or, alternatively, the third leaf to appear (leaf development). We found that germination time and success rate of two lettuce varieties (Iceberg A and Butter Crunch) showed different sensitivities to pH, Hoagland formulations and concentrations, light intensity, and hydrogel content. We have conducted statistical analysis on the correlation between germination time and these environmental conditions.

A data-driven crop model for maize yield prediction.

Accurate estimation of crop yield predictions is of great importance for food security under the impact of climate change. We propose a data-driven crop model that combines the knowledge advantage of process-based modeling and the computational advantage of data-driven modeling. The proposed model tracks the daily biomass accumulation process during the maize growing season and uses daily produced biomass to estimate the final grain yield. Computational studies using crop yield, field location, genotype and corresponding environmental data were conducted in the US Corn Belt region from 1981 to 2020. The results suggest that the proposed model can achieve an accurate prediction performance with a 7.16% relative root-mean-square-error of average yield in 2020 and provide scientifically explainable results. The model also demonstrates its ability to detect and separate interactions between genotypic parameters and environmental variables. Additionally, this study demonstrates the potential value of the proposed model in helping farmers achieve higher yields by optimizing seed selection.

Two CRISPR/Cas12a-based methods for fast and accurate detection of single-base mutations.

Current single-base mutation detection approaches are time-consuming, labor-intensive, and costly. This highlights the critical need for speedy and accurate technology capable of detecting single-base alterations. Using clustered regularly interspaced short palindromic repeats/associated protein 12a (CRISPR/Cas12a), two fundamental approaches for getting 100% differentiation of single-base mutations have been established, by which fluorescence signals could be detected for variants but not for wild strains. The first method required both polymerase chain reaction (PCR) and CRISPR/Cas12a cleavage: By introducing a mismatched base at the 3' end of the primers and adjusting the PCR settings, the wild strain strand amplifications were completely blocked prior to CRISPR/Cas12a cleavage. The parameters for Method 1 (PCR + CRISPR/Cas12a) could be easily controlled and adjusted to attain a sensitivity of one copy (about 6 copies µL-1). The second method included isothermal recombinase polymerase amplification (RPA) and CRISPR/Cas12a cleavage: By introducing an extra mismatched base adjacent to the single-base mutant site by RPA (IMAS-RPA), the RPA products from the wild strains were rendered incapable of triggering the cleavage activity of CRISPR/Cas12a. Method 2 (IMAS-RPA) was rapid and easy to implement (can be finished within 1 h). Because each method has its own set of advantages, the laboratory environment-appropriate methods can be selected independently. Both approaches are expected to aid in clinical diagnosis to some extent in the near future.

Cas12a/Guide RNA-Based Platforms for Rapidly and Accurately Identifying Staphylococcus aureus and Methicillin-Resistant S. aureus.

In order to ensure the prevention and control of methicillin-resistant Staphylococcus aureus (MRSA) infection, rapid and accurate detection of pathogens and their resistance phenotypes is a must. Therefore, this study aimed to develop a fast and precise nucleic acid detection platform for identifying S. aureus and MRSA. We initially constructed a CRISPR-Cas12a detection system by designing single guide RNAs (sgRNAs) specifically targeting the thermonuclease (nuc) and mecA genes. To increase the sensitivity of the CRISPR-Cas12a system, we incorporated PCR, loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA). Subsequently, we compared the sensitivity and specificity of the three amplification methods paired with the CRISPR-Cas12a system. Finally, the clinical performance of the methods was tested by analyzing the fluorescence readout of 111 clinical isolates. In order to visualize the results, lateral-flow test strip technology, which enables point-of-care testing, was also utilized. After comparing the sensitivity and specificity of three different methods, we determined that the nuc-LAMP-Cas12a and mecA-LAMP-Cas12a methods were the optimal detection methods. The nuc-LAMP-Cas12a platform showed a limit of detection (LOD) of 10 aM (~6 copies µL-1), while the mecA-LAMP-Cas12a platform demonstrated a LOD of 1 aM (~1 copy µL-1). The LOD of both platforms reached 4 × 103 fg/µL of genomic DNA. Critical evaluation of their efficiencies on 111 clinical bacterial isolates showed that they were 100% specific and 100% sensitive with both the fluorescence readout and the lateral-flow readout. Total detection time for the present assay was approximately 80 min (based on fluorescence readout) or 85 min (based on strip readout). These results indicated that the nuc-LAMP-Cas12a and mecA-LAMP-Cas12a platforms are promising tools for the rapid and accurate identification of S. aureus and MRSA. IMPORTANCE The spread of methicillin-resistant Staphylococcus aureus (MRSA) poses a major threat to global health. Isothermal amplification combined with the trans-cleavage activity of Cas12a has been exploited to generate diagnostic platforms for pathogen detection. Here, we describe the design and clinical evaluation of two highly sensitive and specific platforms, nuc-LAMP-Cas12a and mecA-LAMP-Cas12a, for the detection of S. aureus and MRSA in 111 clinical bacterial isolates. With a limit of detection (LOD) of 4 × 103 fg/µL of genomic DNA and a turnaround time of 80 to 85 min, the present assay was 100% specific and 100% sensitive using either fluorescence or the lateral-flow readout. The present assay promises clinical application for rapid and accurate identification of S. aureus and MRSA in limited-resource settings or at the point of care. Beyond S. aureus and MRSA, similar CRISPR diagnostic platforms will find widespread use in the detection of various infectious diseases, malignancies, pharmacogenetics, food contamination, and gene mutations.

Identification and Expression Profiles of Putative Soluble Chemoreception Proteins from Lasioderma serricorne (Coleoptera: Anobiidae) Antennal Transcriptome.

The cigarette beetle, Lasioderma serricorne (Fabricius) (Coleoptera: Anobiidae), is a destructive stored product pest worldwide. Adult cigarette beetles are known to rely on host volatiles and pheromones to locate suitable habitats for oviposition and mating, respectively. However, little is known about the chemosensory mechanisms of these pests. Soluble chemoreception proteins are believed to initiate olfactory signal transduction in insects, which play important roles in host searching and mating behaviors. In this study, we sequenced the antennal transcriptome of L. serricorne and identified 14 odorant-binding proteins (OBPs), 5 chemosensory proteins (CSPs), and 2 Niemann-Pick C2 proteins (NPC2). Quantitative realtime PCR (qPCR) results revealed that several genes (LserOBP2, 3, 6, and 14) were predominantly expressed in females, which might be involved in specific functions in this gender. The five LserOBPs (LserOBP1, 4, 8, 10, and 12) that were highly expressed in the male antennae might encode proteins involved in specific functions in males. These findings will contribute to a better understanding of the olfactory system in this stored product pest and will assist in the development of efficient and environmentally friendly strategies for controlling L. serricorne.

Design of Rapid Detection System for Five Major Carbapenemase Families (bla KPC, bla NDM, bla VIM, bla IMP and bla OXA-48-Like) by Colorimetric Loop-Mediated Isothermal Amplification.

PURPOSE: Carbapenemase-producing Enterobacteriaceae (CPE) infection constitutes a public health threat. Timely and efficient diagnosis is of paramount importance for prompt and effective therapy. In order to quickly and comprehensively detect the five major families of carbapenemases (bla KPC, bla NDM, bla VIM, bla IMP, and bla OXA-48-like), colorimetric loop-mediated isothermal amplification (LAMP) was employed. MATERIALS AND METHODS: Five sets of LAMP primers were designed, each of which can, respectively, amplify all the carbapenemase subtypes described in this work. Twenty whole genome sequencing-verified-"standard strains", including 1 bla NDM-1, 1 bla NDM-5, 1 bla NDM-6, 1 bla NDM-7, 2 bla IMP-4, 1 bla IMP-8, 2 bla KPC-2, 1 bla KPC-3, 1 bla KPC-4, 1 bla KPC-5, 1 bla KPC-6, 1 bla KPC-7, 1 bla OXA-48 and 1 bla OXA-181 carrier, and 1 bla VIM and bla OXA-244, 1 bla KPC-2 and bla IMP-4, 1 bla KPC-2 and bla VIM-1 and 1 bla KPC-2 and bla NDM-1-co-carriers, were used to establish a 25-microliter visual LAMP reaction system (kept at 65°C for 30 minutes in water bath). Color change from bright pink to yellow indicated positive amplification. In addition, 126 pre-verified clinical carbapenem-resistant Enterobacteriaceae (CRE) isolates, including 65 CPE (23 bla NDM, 2 bla OXA-48-like, 1 bla KPC and bla VIM, 2 bla IMP, and 37 bla KPC carriers) and 61 non-CPE, were also detected. RESULTS: With the lowest detection limit of 10 colony forming units (CFU) per reaction for LAMP and 103 CFU per reaction for PCR, the LAMP system demonstrated dramatically higher sensitivity while retaining the same specificity. Furthermore, we demonstrated concordant results between the two methods for the 126 clinical isolates. CONCLUSION: Therefore, LAMP could be used for rapid identification of the five major carbapenemase gene families in routine clinical laboratories.

MBLs, Rather Than Efflux Pumps, Led to Carbapenem Resistance in Fosfomycin and Aztreonam/Avibactam Resistant Elizabethkingia anophelis.

OBJECTIVE: To assess the risk factors associated with infections and in-hospital mortality, antimicrobial susceptibility patterns and carbapenem resistance mechanisms in E. anophelis. METHODS: This retrospective case-control study was conducted to reveal the risk factors associated with Elizabethkingia anophelis (E. anophelis) infection and in-hospital mortality in a university tertiary hospital in southwest China, using multivariable logistic-regression analyses. Complete 16S rRNA gene sequencing was used to reconfirm the identity of all isolates. We employed the broth microdilution method to investigate the antimicrobial susceptibility profiles. The presence of resistance genes was confirmed by polymerase chain reaction and DNA sequencing. Full-length resistance genes were cloned into the pET-28a vector for further functional studies. RESULTS: Our multivariate analysis indicated that coronary artery disease, chronic obstructive pulmonary disease, surgery in the past 6 months, anemia and systemic steroid use were independent risk factors for the acquisition of E. anophelis. Additionally, anemia was the only independent risk factor associated with in-hospital mortality in patients with E. anophelis infections. E. anophelis isolates showed high in-vitro susceptibility towards minocycline (100%) and piperacillin/tazobactam (71.8%), but were resistant to colistin, fosfomycin, ceftazidime/avibactam and aztreonam/avibactam. The PCR revealed the presence of blaGOB and blaBlaB in 37 isolates, and blaCME ß-lactamase genes in 36 isolates out of 39 E. anophelis isolates. Additionally, we showed that two metallo-ß-lactamases (MBLs) BlaB and GOB, were responsible for carbapenem resistance and the serine-ß-lactamase, CME, was functionally involved in resistance to cephalosporins and monobactams. Interestingly, the various putative efflux pumps in E. anophelis were not responsible for resistance. CONCLUSION: Our findings will help clinicians to identify high-risk patients and suggests that minocycline should be considered as a therapeutic option for E. anophelis infections. Additionally, carbapenem resistance in E. anophelis is mainly associated with the MBLs, BlaB and GOB, rather than various putative efflux pumps.

Establishment and validation of a mathematical diagnosis model to distinguish benign pulmonary nodules from early non-small cell lung cancer in Chinese people.

BACKGROUND: In this study, we aimed to establish and validate a mathematical diagnosis model to distinguish benign pulmonary nodules (BPNs) from early non-small cell lung cancer (eNSCLC) based on clinical characteristics, radiomics features, and hematological biomarkers. METHODS: Medical records from 81 patients (27 BPNs, 54 eNSCLC) were used to establish a novel mathematical diagnosis model and an additional 61 patients (21 BPNs, 40 eNSCLC) were used to validate this new model. To establish a clinical diagnosis model, a least absolute shrinkage and selection operator (LASSO) regression was applied to select predictors for eNSCLC, then multivariate logistic regression analysis was performed to determine independent predictors of the probability of eNSCLC, and to establish a clinical diagnosis model. The diagnostic accuracy and discriminative ability of our model were compared with the PKUPH and Mayo models using the following 4 indices: area under the receiver-operating characteristics curve (ROC), net reclassification improvement index (NRI), integrated discrimination improvement index (IDI), and decision curve analysis (DCA). RESULTS: Multivariate logistic regression analysis identified age, border, and albumin (ALB) as independent diagnostic markers of eNSCLC. In the training cohort, the AUC of our model was 0.740, which was larger than the AUCs for the PKUPH model (0.717, P=0.755) and the Mayo model (0.652, P=0.275). Compared with the PKUPH and Mayo models, the NRI of our model increased by 3.7% (P=0.731) and 27.78% (P=0.008), respectively, while the IDI changed -4.77% (P=0.437) and 11.67% (P=0.015), respectively. Moreover, the DCA demonstrated that our model had a higher overall net benefit compared to previously published models. Importantly, similar findings were confirmed in the validation cohort. CONCLUSIONS: Age, border, and serum ALB levels were independent diagnostic markers of eNSCLC. Thus, our model could more accurately distinguish BPNs from eNSCLC and outperformed previously published models.