Isolation Procedure Research Articles

A biofilm-tropic Pseudomonas aeruginosa bacteriophage uses the exopolysaccharide Psl as receptor.

Bacteria in nature can exist in multicellular communities called biofilms. Biofilms also form in the course of many infections. Pseudomonas aeruginosa infections frequently involve biofilms, which contribute materially to the difficulty to treat these infections with antibiotic therapy. Many biofilm-related characteristics are controlled by the second messenger, cyclic-di-GMP, which is upregulated on surface contact. Among these factors is the exopolysaccharide Psl, which is a critically important component of the biofilm matrix. Here we describe the discovery of a P. aeruginosa bacteriophage, which we have called Clew-1, that directly binds to and uses Psl as a receptor. While this phage does not efficiently infect planktonically growing bacteria, it can disrupt P. aeruginosa biofilms and replicate in biofilm bacteria. We further demonstrate that the Clew-1 can reduce the bacterial burden in a mouse model of P. aeruginosa keratitis, which is characterized by the formation of a biofilm on the cornea. Due to its reliance on Psl for infection, Clew-1 does not actually form plaques on wild-type bacteria under standard in vitro conditions. This argues that our standard isolation procedures likely exclude bacteriophage that are adapted to using biofilm markers for infection. Importantly, the manner in which we isolated Clew-1 can be easily extended to other strains of P. aeruginosa and indeed other bacterial species, which will fuel the discovery of other biofilm-tropic bacteriophage and expand their therapeutic use.

The Role of -OEt Substituents in Molybdenum-Assisted Pentathiepine Formation-Access to Diversely Functionalized Azines.

1,2,3,4,5-pentathiepines (PTEs) are naturally occurring polysulfides of increasing scientific interest based on their identified pharmacological activities. Artificial PTEs with N-heterocyclic backbones are efficiently synthesized via mediation by a molybdenum-oxo-bistetrasulfido complex. A common feature of all precursor alkynes successfully used to date in this reaction is the presence of a -CH(OEt)2 group since the previously postulated mechanism requires the presence of one OEt- as the leaving group, and the second must become a transient ethoxonium moiety. This raised the question of whether there really is a need for two, maybe only one, or possibly even zero ethoxy substituents. This research problem was systematically addressed by respective variations in the precursor-alkyne derivatives and by employing one related allene species. It was found that the total absence of ethoxy substituents prevents the formation of PTEs entirely, while the presence of a single ethoxy group results in the possibility to distinctly functionalize the position on the resulting N-heterocyclic pyrrole five ring in the target compound. This position was previously exclusively occupied by an -OEt for all products of the molybdenum-mediated reaction. The allene was applied with similar success as precursor as with the related alkyne. The now-employable significant change in precursor composition gives access to a whole new PTE subfamily, allowing further modulation of (physico)-chemical properties such as solubility, and provides additional insight into the mechanism of PTE formation; it comprises a merely partial validation of the previous hypothesis. The new alkyne precursors and pentathiepines were characterized by a variety of instrumental analyses (NMR, mass spec, UV-vis) and in six cases (one alkyne precursor, one unexpected side product, and four PTEs) by single-crystal X-ray diffraction. Syntheses, isolation procedures, analytical data, and the impact of the findings on the previously proposed mechanism are described in detail herein.

Detection of soil-transmitted helminths and Schistosoma spp. by nucleic acid amplification test: Results of the first 5 years of the only international external quality assessment scheme.

Infections with soil-transmitted helminths (STH) and schistosomiasis (SCH) result in a significant global health burden, particularly in rural communities in low and middle-income countries. While microscopy remains the primary diagnostic method for STH and SCH in resource-limited settings, nucleic acid amplification tests (NAATs) are gaining prominence as tools for evaluation of public health control programs in endemic countries, and individual diagnosis in high-income countries. Despite the high sensitivity and specificity of NAATs, previous research has highlighted inter-laboratory variations, both in technical and clinical performance, justifying the need for continuous proficiency testing. Results from 5 rounds over a 5-year period of the so far only longitudinal international Helminth External Molecular Quality Assessment Scheme (HEMQAS), coordinated by the Dutch Foundation for Quality Assessment in Medical Laboratories (SKML), were examined in order to (i) assess the diagnostic proficiency of laboratories in detecting helminths in stool and (ii) identify potential factors contributing to variations in performance. Thirty-six laboratories, from 18 countries and 5 continents, participated in HEMQAS. The overall diagnostic performances were satisfying, with remarkably low numbers (<2%) of false-positive results. False-negative results were more often reported for stool (15%) than for DNA (5%) samples. False-negative results varied largely between targets (the highest number (29%) for Trichuris trichiura). Twenty-five laboratories provided a sufficient number of results for a robust comparison between participating laboratories, which confirmed substantial inter-laboratory variability in quantitative NAAT results (Cq-values). This variability likely arises from differences in pre-treatment, DNA isolation and DNA-target amplification procedures. This study emphasizes the complexity of molecular diagnosis for STH and SCH, highlighting the critical role of proper stool preparation and DNA isolation methods. The results underscore the necessity for laboratory professionals and public health decision-makers to recognize these complexities and continuously undertake external quality assessment schemes to ensure accurate and reliable performance in molecular diagnosis.

High-resolution melting curve analysis: A detection assay for Ceratocystis eucalypticola and C. manginecans in infected Eucalyptus

Eucalyptus spp. in plantations are negatively affected by canker and wilt diseases caused by several species of Ceratocystis, particularly those in the Latin American Clade (LAC). Ceratocystis eucalypticola and Ceratocystis manginecans are of particular concern where disease epidemics are reported globally, with recent outbreaks emerging in South African and Indonesian Eucalyptus plantations. Consequently, a rapid screening protocol is required for these pathogens. In this study, a high-resolution melting curve analysis (HRMA) was developed to detect C. eucalypticola and C. manginecans that bypasses time-consuming isolation and post-PCR procedures. Primers targeting a 172 bp region of the cerato-platanin (CP) gene were designed. Using these primers, the accuracy of HRMA to detect and distinguish between these two LAC species was assessed using pure fungal DNA, and DNA extracted directly from Eucalyptus samples naturally infected with C. eucalypticola. The assay accurately detected the presence of C. eucalypticola and C. manginecans and quantifies their DNA, both from cultures, and directly from wood samples. HRMA further differentiated these two species from all other tested LAC individuals. This assay was also able to detect the presence of all the tested LAC species and distinguish seven of these, including C. fimbriata, to species level. Ceratocystis polyconidia was the only non-LAC off-target species detected. Based on these results, the developed assay can be used to rapidly identify C. eucalypticola and C. manginecans directly from infected plant material or fungal cultures, with the potential to also screen for several other LAC species.

Basic Science and Principles of Stem Cells, Platelet‐Rich Plasma, and Exosomes

ABSTRACTBackgroundRegenerative medicine seeks to harness the natural healing abilities of the body and has scope for use in the treatment of dermatologic conditions. Stem cell therapy, platelet‐rich plasma (PRP), and exosomes are emerging as key components in regenerative medicine, particularly in skin rejuvenation and repair.ObjectivesThe goal of this article is to review the basic science behind the function of stem cells, PRP, and exosomes in regenerative medicine.MethodsA literature review was conducted using PubMed and Google Scholar, focusing on basic science literature regarding the structure, origin, mechanisms of action, and isolation of stem cells, PRP, and exosomes.ResultsThe evaluation found that stem cells facilitate skin regeneration by modulating the inflammatory milieu. PRP was shown to have positive effects on repairing skin elasticity and improving scars through the release of growth factors. Exosomes were found to enhance fibroblast proliferation and collagen synthesis.ConclusionsStem cell therapy, PRP, and exosomes each show mechanisms that render them useful in regenerative medicine. Future research is needed to further elucidate their mechanisms of action, standardize their isolation procedures, and optimize their potential for clinical application.

Single-mitochondrion sequencing uncovers distinct mutational patterns and heteroplasmy landscape in mouse astrocytes and neurons

BackgroundMitochondrial (mt) heteroplasmy can cause adverse biological consequences when deleterious mtDNA mutations accumulate disrupting “normal” mt-driven processes and cellular functions. To investigate the heteroplasmy of such mtDNA changes, we developed a moderate throughput mt isolation procedure to quantify the mt single-nucleotide variant (SNV) landscape in individual mouse neurons and astrocytes. In this study, we amplified mt-genomes from 1645 single mitochondria isolated from mouse single astrocytes and neurons to (1) determine the distribution and proportion of mt-SNVs as well as mutation pattern in specific target regions across the mt-genome, (2) assess differences in mtDNA SNVs between neurons and astrocytes, and (3) study co-segregation of variants in the mouse mtDNA.Results(1) The data show that specific sites of the mt-genome are permissive to SNV presentation while others appear to be under stringent purifying selection. Nested hierarchical analysis at the levels of mitochondrion, cell, and mouse reveals distinct patterns of inter- and intra-cellular variation for mt-SNVs at different sites. (2) Further, differences in the SNV incidence were observed between mouse neurons and astrocytes for two mt-SNV 9027:G > A and 9419:C > T showing variation in the mutational propensity between these cell types. Purifying selection was observed in neurons as shown by the Ka/Ks statistic, suggesting that neurons are under stronger evolutionary constraint as compared to astrocytes. (3) Intriguingly, these data show strong linkage between the SNV sites at nucleotide positions 9027 and 9461.ConclusionsThis study suggests that segregation as well as clonal expansion of mt-SNVs is specific to individual genomic loci, which is important foundational data in understanding of heteroplasmy and disease thresholds for mutation of pathogenic variants.

Advancing Recyclable Thermosets through C═C/C═N Dynamic Covalent Metathesis Chemistry.

Thermoset polymers have become integral to our daily lives due to their exceptional durability, making them feasible for a myriad of applications; however, this ubiquity also raises serious environmental concerns. Covalent adaptable networks (CANs) with dynamic covalent linkages that impart efficient reprocessability and recyclability to thermosets have garnered increasing attention. While various dynamic exchange reactions have been explored in CANs, many rely on the stimuli of active nucleophilic groups and/or catalysts, introducing performance instability and escalating the initial investment. Herein, we propose a new direct and catalyst-free C═C/C═N metathesis reaction between α-cyanocinnamate and aldimine as a novel dynamic covalent motif for constructing recyclable thermosets. This chemistry offers mild reaction conditions (room temperature and catalyst-free), ensuring high yields and simple isolation procedures. By incorporating dynamic C═C/C═N linkages into covalently cross-linked polymer networks, we obtained dynamic thermosets that exhibit both malleability and reconfigurability. The resulting tunable dynamic properties, coupled with the high thermal stability and recyclability of the C═C/C═N linkage-based networks, enrich the toolbox of dynamic covalent chemistry.

Identification of differentially expressed genes controlling the expression of flowering in Bambara groundnut (Vigna subterranea [L.] Verdc.)

Bambara groundnut flowering is a crucial developmental stage in the vegetative to reproductive period. The earliness to lateness of flowering is regulated by various interconnected genetic pathways encoded by genes. Deoxyribonucleic acid (DNA) of the selected accessions was extracted through leaf samples at 3 weeks old, using Dellaporta Miniprep for Plant DNA Isolation procedure. The high-quality DNA was sequenced using Diversity Arrays Technology (DArT) markers and single nucleotide polymorphisms (SNP’s) associated with flowering was identified. There is need to investigate the genetic make-up of the cleistogamous flower of Bambara through associated genes for improvement. This research work identified four markers associated with the flowering of Vigna subterranea and the role of variant identified genes in flowering. The identified markers from the sequence and the selected amino acid sequence were used as a query to search the legume protein database in Vigna radiata. The four markers with adequate information associated with flowering in the sequence were 24385352|F|0–28:T > C-28:T > C; 27641816|F|0–17:C > T-17:C > T; 24384204|F|0–24:C > T-24:C > T and 24346601|F|0–67:T > C-67:T > C and significant at P < 1.68 × 10−4 at chromosomes 7, 11, 4, and 5. The identified genes including histones, Polyketide, cyclase/dehydrase, Transcription factor MYC/MYB N-terminal, Rhamnogalacturonate lyase, DHHC-type zinc finger protein, Putative S-adenosyl-L-methionine-dependent methyltransferase, Ribosomal protein L2, D-galactoside/L-rhamnose binding SUEL lectin domain, Lipase GDSL, Histone deacetylase superfamily, Basic-leucine zipper domain, TUP1-like enhancer of split, Zinc finger ZZ-type, Homeodomain-like, Phosphatidylethanolamine-binding protein PEBP, Leucine-rich repeat which are tools in controlling flowering in Bambara groundnut. This study revealed that Bambara groundnut flowering is controlled by the interplay of genes.

Ablation apprentices and their first experience of pulmonary vein isolation procedure on paroxysmal atrial fibrillation with different sheaths

ObjectivesThis study aimed at exploring how using different kinds of sheaths will affect the very first ablation procedure of apprentices.Methods15 patients with paroxysmal atrial fibrillation were randomized to used fixed-curve, conventional steerable or visualized steerable sheath, and received complete isolation of pulmonary veins. All ablations were the very first procedure performed by 15 ablation apprentices. The use of fluoroscopy and catheter stability during the PVI were analyzed.ResultsProcedure duration was much longer in the fixed-curve group (116.8 ± 27 vs. 62.2 ± 17 vs. 60.4 ± 17, p < 0.001). X-ray exposure was lowest with visualized sheath (17.6 ± 5 vs. 18.6 ± 6 vs. 5.2 ± 6, p < 0.001). CF SD differed significantly, especially at the anterior aspect of LSPV (7.90 ± 2.90 vs. 5.04 ± 2.18 vs. 4.52 ± 2.40, p < 0.001) and posterior aspect of RSPV (6.84 ± 2.79 vs. 3.42 ± 2.04 vs. 3.50 ± 2.30, p < 0.001) in the fixed-curve group. Impedance drop was significantly smaller in the fixed-curve group at the anterior aspect of LSPV (8.74 ± 3.02 vs. 11.49 ± 5.48 vs. 12.57 ± 5.96, p = 0.005).ConclusionEven for the very first ablation procedure of an ablation apprentice, the use of steerable sheaths will significantly reduce the procedure duration and improve the catheter stability, but only visualized steerable sheath can reduce fluoroscopic time.

Isolation methods and characterization of primary rat neurovascular cells

BackgroundThere is significant interest in isolating cells of the blood-brain barrier (BBB) for use in in vitro screening of therapeutics and analyzing cell specific roles in neurovascular pathology. Primary brain cells play an advantageous role in BBB models; however, isolation procedures often do not produce cells at high enough yields for experiments. In addition, although numerous reports provide primary cell isolation methods, the field is lacking in documentation and detail of expected morphological changes that occur throughout culturing and there are minimal troubleshooting resources. Here, we present simplified, robust, and reproducible methodology for isolating astrocytes, pericytes, and endothelial cells, and demonstrate several morphological benchmarks for each cell type throughout the process and culture timeframe. We also analyze common considerations for developing neurovascular cell isolation procedures and recommend solutions for troubleshooting.ResultsThe presented methodology isolated astrocytes, pericytes, and endothelial cells and enabled cell attachment, maturation, and cell viability. We characterized milestones in cell maturation over 12 days in culture, a common timeline for applications of these cell types in BBB models. Phase contrast microscopy was used to show initial cell plating, attachment, and daily growth of isolated cells. Confocal microscopy images were analyzed to determine the identity of cell types and changes to cell morphology. Nuclear staining was also used to show the viability and proliferation of glial cells at four time points. Astrocyte branches became numerous and complex with increased culture time. Microglia, oligodendrocytes, and neurons were present in mixed glial cultures for 12 days, though the percentage of microglia and neurons expectedly decreased after passaging, with microglia demonstrating a less branched morphology.ConclusionsNeurovascular cells can be isolated through our optimized protocols that minimize cell loss and encourage the adhesion and proliferation of isolated cells. By identifying timepoints of viable glia and neurons within an astrocyte-dominant mixed culture, these cells can be used to evaluate drug targeting, uptake studies, and response to pathological stimulus in the neurovascular unit.

Isolation Procedure for Rat Pancreatic Ductal Cells.

Isolating pancreatic ductal cells from rats is a critical procedure in pancreatic research, offering valuable insights into pancreatic function, pathology, and potential treatments. The process involves several key steps, beginning with the proper removal of the rat's pancreas, followed by the initiation of the ductal cell isolation procedure. This aims to obtain pure and viable ductal cell populations for further experimentation and analysis.

Sealing efficacy of various rubber dam barrier materials used for isolation procedures: A randomized controlled trial

ABSTRACT Aim: The aim of the study was to assess the sealing efficacy of three dam seal materials. Methods: Ninety participants were enrolled after obtaining consent. After the placement of rubber dam (RD), a stream of air was applied at the interface area and visualized at ××6 magnification to identify sealing ability. Dam seal materials were divided into Group A: Liquid Dam, Group B: Kool-Dam, and Group C: OraSeal caulking putty substance. Leakage was assessed using self-designed criteria such as sealing ability, retention, ease of adaptability, and removal. The data were tabulated and analyzed using IBM SPSS 20 software. Results: Kool-Dam (Group B) demonstrated superior effectiveness in controlling saliva seepage around the clamp and teeth (P = 0.039) compared to other groups. In addition, it was evident that Kool-Dam (Group B) had greater effectiveness as compared to Liquid Dam and OraSeal (Group C) with relation to the retention of dam seal material (P = 0.039). There was no statistically significant difference (P &lt; 0.05) in the ease of adaptation or removal of dam seal material. Conclusion: This study highlights the importance of dam seal materials as an adjunct material to establish a tight seal over a RD placement. From the present study, it was evident that Kool-Dam exhibited superior performance over Liquid Dam and OraSeal dam seal material in terms of better sealing ability and retention of the dam seal materials.

Minimizing Exposure to Ionizing Radiation in Catheter-Based Treatment of Atrial Fibrillation: Safety and Efficacy

Pulmonary vein isolation using radiofrequency ablation is the most common method of catheter treatment for atrial fibrillation. Performing this procedure involves the use of X-ray radiation, which at certain stages of the procedure is an indispensable method for navigation and visualization of intracardiac structures. Approaches to minimize X-ray exposure during catheter interventions are attracting increased attention of the electrophysiology community due to the potential risks of adverse effects of ionizing radiation on patients and medical personnel. The aim. To evaluate the effectiveness and safety of performing the pulmonary vein isolation procedure with the intention to minimize X-ray exposure of patients and medical personnel. Materials and methods. Forty-three patients with various forms of atrial fibrillation were selected for the study: 28(65%) with paroxysmal and 15 (35%) with persistent forms. All the patients underwent pulmonary vein isolation usingcatheter radiofrequency ablation with the intention to minimize X-ray exposure at the National Amosov Institute of Cardiovascular Surgery in the period from June 2023 to April 2024. Results. In all 43 patients it was possible to electrically isolate all the veins. Catheter ablation of the cavotricuspidisthmus was performed in 5 (31.2%) patients from group A (“Zero-fluoro”), 7 (33.3%) from group B (“Near Zero-fluoro”) and 3 (50%) from group C (“Non Zero-fluoro”) (p = 0.696). Intraoperative reconnection of the pulmonary veins with the left atrium was observed in 6 (37.5%) patients of group A, 11 (52.3%) of group B, and 4 (66%) of group C (p = 0.429). X-ray time for transseptal puncture did not differ between groups A and B (3.56 ± 2.37 vs. 3.69 ± 2.3 min., p = 0.532), however, X-ray time for pulmonary vein isolation in group A was significantly less than that in group B (0.27 ± 0.13 vs. 1.96 ± 1.21 min., p &lt; 0.001). The total time in the left atrium was not statistically different between these two groups (81.33 ± 22.32 vs. 98.6 ± 29.83 min., p = 0.106). In group C (“Non Zero-fluoro”), the aforementioned parameters were not recorded. Total dose area product (DAP) and total radiation dose, as well as total X-ray time and total procedure time, were significantly lower when using the ionizing exposure minimization approach. DAP and total radiation dose in group A were the lowest (7.29 ± 5.16 Gy/cm2 and 76.62 ± 70.82 mGy, respectively) and significantly differed from those in group C (107.67 ± 97.59 Gy/cm2 and 882.32 ± 868.62 mGy, p &lt; 0.001 and p = 0.001, respectively). No intraoperative complications were observed in the studied sample. Conclusions. Minimizing X-ray exposure using modern navigation systems during pulmonary vein isolation using catheter radiofrequency ablation is not associated with increased intraprocedural complications in patients with paroxysmal and persistent atrial fibrillation. This approach allows you to significantly lower total DAP and the radiation dosewithout increasing the time of performing the pulmonary vein isolation procedure.

Green purification of Acmella oleracea extract by wiped-film short path molecular distillation boosts the insecticidal activity on mosquito larvae

The development of insecticide resistance by mosquito vectors highlighted an urgent need for effective and sustainable botanical insecticides. Acmella oleracea R.K. Jansen (Asteraceae)-derived extracts and the major constituents N-alkylamides have already demonstrated their potential as insecticidal and acaricidal agents. Nowadays, the development of scalable green extraction and purification processes in the manufacture of biopesticides is a topic of great interest. In this context, supercritical fluid extraction and wiped-film short path molecular (WSM) distillation techniques were combined for the first time to obtain an eco-friendly N-alkylamides-enriched fraction (AEF) from A. oleracea. This was characterized by a notably higher content of spilanthol and N-alkylamides than the supercritical fluid extract (SFE), as determined by HPLC-DAD-MS analysis. The AEF also contained free fatty acids (e.g., palmitic, linoleic, and linolenic acids) as revealed by GC-FID analysis. The mosquito larvicidal activity of SFE, AEF, and pure spilanthol was assessed against 3rd instar larvae of Aedes albopictus Skuse, Anopheles stephensi Liston, and An. gambiae Giles. Results showed that AEF exhibited an insecticidal efficacy comparable with that of spilanthol, and higher than that of SFE. These products were tested against human keratinocytes (HaCaT) confirming their dermal safety. The present study paves the way for the utilization of WSM distillation in the agrochemical industry for the manufacture of botanical insecticides. Indeed, this technique allows to avoid the use of organic solvents related to conventional extraction and spilanthol isolation procedures and gives a product devoid of undesired pigments appealing to other industrial application as well.

Isolation of a persistently quiescent muscle satellite cell population.

Although studies have identified characteristics of quiescent satellite cells (SCs), their isolation has been hampered by the fact that the isolation procedures result in the activation of these cells into their rapidly proliferating progeny (myoblasts). Thus, the use of myoblasts for therapeutic (regenerative medicine) or industrial applications (cellular agriculture) has been impeded by the limited proliferative and differentiative capacity of these myogenic progenitors. Here we identify a subpopulation of satellite cells isolated from mouse skeletal muscle using flow cytometry that is highly Pax7-positive, exhibit a very slow proliferation rate (7.7 ± 1.2 days/doubling), and are capable of being maintained in culture for at least 3 mo without a change in phenotype. These cells can be activated from quiescence using a p38 inhibitor or by exposure to freeze-thaw cycles. Once activated, these cells proliferate rapidly (22.7 ± 0.2 h/doubling), have reduced Pax7 expression (threefold decrease in Pax7 fluorescence vs. quiescence), and differentiate into myotubes with a high efficiency. Furthermore, these cells withstand freeze-thawing readily without a significant loss of viability (83.1 ± 2.1% live). The results presented here provide researchers with a method to isolate quiescent satellite cells, allowing for more detailed examinations of the factors affecting satellite cell quiescence/activation and providing a cell source that has a unique potential in the regenerative medicine and cellular agriculture fields.NEW & NOTEWORTHY We provide a method to isolate quiescent satellite cells from skeletal muscle. These cells are highly Pax7-positive, exhibit a very slow proliferation rate, and are capable of being maintained in culture for months without a change in phenotype. The use of these cells by muscle researchers will allow for more detailed examinations of the factors affecting satellite cell quiescence/activation and provide a novel cell source for the regenerative medicine and cellular agriculture fields.

Role of sodium sulfate in electrical conductivity and structure of lignin-derived carbons

Lignin is a promising renewable alternative to fossil fuels for producing carbon materials such as carbon fibers, activated carbons, or carbon black. Despite extensive research, lignin-derived carbon materials show limited graphitization relative to comparable petroleum-derived carbons. Further, lignin-derived carbons show high variation in graphitization and electrical conductivity depending on the source of the lignin. Herein, nine lignins, derived from various feedstocks and isolation procedures, are pyrolyzed to produce biochar at 1100∘C. These lignins have a range of chemical compositions, carbon structures, and particle sizes. As a result, the pyrolysis behavior of these lignins varies, with powdered, clumped powder, and “foam” biochar morphologies resulting from finely powdered lignin. The produced biochars vary widely in both electrical conductivity, from 0.19 to 19 S/cm, and in-plane graphitic crystallite size, from 3.4 to 41.2Å. A significant decrease in electrical conductivity is identified when Na2SO4 is removed from lignin, accompanied by an increase in graphitic crystallite size. Based on this finding, a quadratic relationship between biochar graphitic crystallite aspect ratio and electrical conductivity is proposed that builds on established quasi-percolation models for biochar electrical conductivity.

Demographic, Mental Health, Medical, and Social Characteristics of COVID-19 Patients who Underwent Home Isolation Treatment at the King Chulalongkorn Memorial Hospital, Thailand

Background: The SARS-CoV-2 virus began spreading worldwide in 2019. This new pandemic was a challenge to public health measures worldwide. Coronavirus disease affects physical health, mental well-being, and way of life. The disease itself, the isolation measures, and the economic impact interplayed in worsening the situation. Psychosocial and mental health factors should not be overlooked to improve care for patients. Objective: To explore the demographic, clinical, and social characteristics of COVID-19 patients who underwent home isolation treatment. The authors also investigated the prevalence of mental health problems, which are depression, anxiety, problematic alcohol use, nicotine use, and other substance use. Materials and Methods: The present study is a cross-sectional descriptive study that includes 150 patients with COVID-19 infection treated at King Chulalongkorn Memorial Hospital (KCMH). One hundred and twenty-two of them received home-isolation treatment, and 38 were admitted to the field hospital, where they were later isolated at home. Demographic data, psychosocial factors, substance use habits, clinical characteristics, and adherence to the isolation policy were collected. The authors conducted screenings for mental health problems at that time through psychiatric interviews carried out by either psychiatrists or psychiatric residents. Results: The prevalence of mental health problem among home-isolated patients for COVID-19 infection was 20%. The prevalence of anxiety and depression were 4% and 2.7%, respectively. In terms of substance use, 12% of the participants were active smokers, and 5.3% had problematic alcohol use. The participants who underwent home isolation procedures were more likely to have limited space for isolation at home. However, they had adequate and appropriate individual knowledge and basic social support during the quarantine. Conclusion: Mental health problems should be addressed during COVID-19 pandemic, in which substance use was the most prominent problem. Most residences in Bangkok had limited separate spaces for home isolation. However, the social support was adequate in facilitating their daily routine at home during home isolation.

Comparative analysis of commercially available kits for optimal DNA extraction from bovine fecal samples.

In the field of metagenomic research, the choice of DNA extraction methods plays a pivotal yet often underestimated role in shaping the reliability and interpretability of microbial community data. This study delves into the impact of five commercially available DNA extraction kits on the analysis of bovine fecal microbiota. Recognizing the importanceof accurate DNA extraction in elucidating microbial community dynamics, we systematically assessed DNA yield, quality, and microbial composition across these kits using 16S rRNA gene sequencing. Notably, the FastDNA spin soil kit yielded the highest DNA concentration, while significant variations in quality were observed across kits. Furthermore, differential abundance analysis revealed kit-specific biasesthat impacted taxa representation. Microbial richness and diversity were significantly influenced by the choice of extraction kit, with QIAamp DNA stool minikit, QIAamp Power Pro, and DNeasy PowerSoil outperforming the Stool DNA Kit. Principal-coordinate analysis revealed distinct clustering based on DNA isolation procedures, particularly highlighting the unique microbial community composition derived from the Stool DNA Kit. This study also addressed practical implications, demonstrating how kit selection influences the concentration of Gram-positive and Gram-negative bacterial taxa in samples. This research highlights the need for consideration of DNA extraction kits in metagenomic studies, offering valuable insights for researchers striving to advance the precision and depth of microbiota analyses in ruminants.

LC/MS-guided isolation and GNPS procedures to identify flavonoid of HCoV-OC43 inhibitors

The screening of based target compounds supported by LC/MS, MS/MS and Global Natural Products Social (GNPS) used to identify the compounds 1–10 of Butea monsperma. They were evaluated in human malignant embryonic rhabdomyoma cells (RD cells) infected with Human coronavirus OC43 (HCoV-OC43) and showed significant inhibitory activity. Target inhibition tests showed that compounds 6 and 8 inhibited the proteolytic enzyme 3CLpro, which is widely present in coronavirus and plays an important role in the replication process, with an effective IC50 value. The study confirmed that dioxymethylene of compound 8 may be a key active fragment in inhibiting coronavirus (EC50 7.2 μM, SI > 139.1). The results have led to identifying natural bioactive compounds for possible inhibiting HCoV-OC43 and developing drug for Traditional Chinese Medicine (TCM).

Single-Mitochondrion Sequencing Uncovers Distinct Mutational Patterns and Heteroplasmy Landscape in Mouse Astrocytes and Neurons.

Mitochondrial (mt) heteroplasmy can cause adverse biological consequences when deleterious mtDNA mutations accumulate disrupting 'normal' mt-driven processes and cellular functions. To investigate the heteroplasmy of such mtDNA changes we developed a moderate throughput mt isolation procedure to quantify the mt single-nucleotide variant (SNV) landscape in individual mouse neurons and astrocytes In this study we amplified mt-genomes from 1,645 single mitochondria (mts) isolated from mouse single astrocytes and neurons to 1. determine the distribution and proportion of mt-SNVs as well as mutation pattern in specific target regions across the mt-genome, 2. assess differences in mtDNA SNVs between neurons and astrocytes, and 3. Study cosegregation of variants in the mouse mtDNA. 1. The data show that specific sites of the mt-genome are permissive to SNV presentation while others appear to be under stringent purifying selection. Nested hierarchical analysis at the levels of mitochondrion, cell, and mouse reveals distinct patterns of inter- and intra-cellular variation for mt-SNVs at different sites. 2. Further, differences in the SNV incidence were observed between mouse neurons and astrocytes for two mt-SNV 9027:G>A and 9419:C>T showing variation in the mutational propensity between these cell types. Purifying selection was observed in neurons as shown by the Ka/Ks statistic, suggesting that neurons are under stronger evolutionary constraint as compared to astrocytes. 3. Intriguingly, these data show strong linkage between the SNV sites at nucleotide positions 9027 and 9461. This study suggests that segregation as well as clonal expansion of mt-SNVs is specific to individual genomic loci, which is important foundational data in understanding of heteroplasmy and disease thresholds for mutation of pathogenic variants.