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Microorganisms Apr 2024Compared with typical Earth soil, Martian soil and Mars simulant soils have distinct properties, including pH > 8.0 and high contents of silicates, iron-rich minerals,...
Compared with typical Earth soil, Martian soil and Mars simulant soils have distinct properties, including pH > 8.0 and high contents of silicates, iron-rich minerals, sulfates, and metal oxides. This unique soil matrix poses a major challenge for extracting microbial DNA. In particular, mineral adsorption and the generation of destructive hydroxyl radicals through cationic redox cycling may interfere with DNA extraction. This study evaluated different protocols for extracting microbial DNA from Mars Global Simulant (MGS-1), a Mars simulant soil. Two commercial kits were tested: the FastDNA SPIN Kit for soil ("MP kit") and the DNeasy PowerSoil Pro Kit ("PowerSoil kit"). MGS-1 was incubated with living soil for five weeks, and DNA was extracted from aliquots using the kits. After extraction, the DNA was quantified with a NanoDrop spectrophotometer and used as the template for 16S rRNA gene amplicon sequencing and qPCR. The MP kit was the most efficient, yielding approximately four times more DNA than the PowerSoil kit. DNA extracted using the MP kit with 0.5 g soil resulted in 28,642-37,805 16S rRNA gene sequence reads and 30,380-42,070 16S rRNA gene copies, whereas the 16S rRNA gene could not be amplified from DNA extracted using the PowerSoil kit. We suggest that the FastDNA SPIN Kit is the best option for studying microbial communities in Mars simulant soils.
PubMed: 38674704
DOI: 10.3390/microorganisms12040760 -
Archives of Pathology & Laboratory... Apr 2024The National Institutes of Health (NIH) Genotype-Tissue Expression (GTEx) project was designed to evaluate how genetic variation and epigenetic effects influence gene...
CONTEXT.—
The National Institutes of Health (NIH) Genotype-Tissue Expression (GTEx) project was designed to evaluate how genetic variation and epigenetic effects influence gene expression in normal tissue.
OBJECTIVE.—
To ensure that the grossly normal-appearing tissues collected were free from disease, each specimen underwent histologic evaluation.
DESIGN.—
In total, nearly 30 000 tissue aliquots collected from almost 1000 postmortem donors underwent histologic review by project pathologists, and detailed observations of any abnormalities or lesions present were recorded.
RESULTS.—
Despite sampling of normal-appearing tissue, in-depth review revealed incidental findings among GTEx samples that included neoplastic, autoimmune, and genetic conditions; the incidence of some of these conditions among GTEx donors differed from those previously reported for other populations. A number of age-related abnormalities observed during histologic review of tissue specimens are also described.
CONCLUSIONS.—
Histologic findings from the GTEx project may serve to improve populational awareness of several conditions and present a unique opportunity for others to explore age- and gender-influenced conditions. Resources from the study, including histologic image and sequencing data, are publicly available for research.
PubMed: 38670546
DOI: 10.5858/arpa.2023-0468-OA -
Biochemia Medica Jun 2024Clinical laboratories should guarantee sample stability in specific storage conditions for further analysis. The aim of this study is to evaluate the stability of plasma...
INTRODUCTION
Clinical laboratories should guarantee sample stability in specific storage conditions for further analysis. The aim of this study is to evaluate the stability of plasma samples under refrigeration for 29 common biochemical analytes usually ordered within an emergency context, in order to determine the maximum allowable period for conducting add-on testing.
MATERIALS AND METHODS
A total of 20 patient samples were collected in lithium heparin tubes without gel separator. All analyses were performed using Alinity systems (Abbott Laboratories, Abbott Park, USA) and samples were stored at 2-8 °C. Measurements were conducted in primary plasma tubes at specific time points up to 48 hours, with an additional stability study in plasma aliquots extending the time storage up to 96 hours. The stability limit was estimated considering the total limit of change criteria.
RESULTS
Of the 29 studied parameters, 24 demonstrated stabilities within a 48-hour storage period in primary plasma tubes. However, five analytes: aspartate aminotransferase, glucose, lactate dehydrogenase, inorganic phosphate and potassium evidenced instability at different time points (7.9 hours, 2.7 hours, 2.9 hours, 6.2 hours and 4.7 hours, respectively). The stability study in plasma aliquots showed that all parameters remained stable for 96 hours, except lactate dehydrogenase, with a stability limit of 63 hours.
CONCLUSIONS
A reduced stability of primary plasma samples was observed for five common biochemical analytes ordered in an emergency context. To ensure the quality of add-on testing for these samples, plasma aliquots provide stability for a longer period.
Topics: Humans; Blood Specimen Collection; Blood Chemical Analysis; Quality Control; Quality Assurance, Health Care; Aspartate Aminotransferases; L-Lactate Dehydrogenase; Plasma; Specimen Handling
PubMed: 38665870
DOI: 10.11613/BM.2024.020704 -
BMC Biotechnology Apr 2024The advancement of AAV vectors into clinical testing has accelerated rapidly over the past two decades. While many of the AAV vectors being utilized in clinical trials...
BACKGROUND
The advancement of AAV vectors into clinical testing has accelerated rapidly over the past two decades. While many of the AAV vectors being utilized in clinical trials are derived from natural serotypes, engineered serotypes are progressing toward clinical translation due to their enhanced tissue tropism and immune evasive properties. However, novel AAV vectors require formulation and stability testing to determine optimal storage conditions prior to their use in a clinical setting.
RESULTS
Here, we evaluated the thermal stability of AAV6.2FF, a rationally engineered capsid with strong tropism for lung and muscle, in two different buffer formulations; phosphate buffered saline (PBS), or PBS supplemented with 0.001% non-ionic surfactant Pluronic F68 (PF-68). Aliquots of AAV6.2FF vector encoding the firefly luciferase reporter gene (AAV6.2FF-ffLuc) were incubated at temperatures ranging from -20°C to 55°C for varying periods of time and the impact on infectivity and particle integrity evaluated. Additionally, the impact of several rounds of freeze-thaw treatments on the infectivity of AAV6.2FF was investigated. Vector infectivity was measured by quantifying firefly luciferase expression in HEK 293 cells and AAV particle integrity was measured by qPCR quantification of encapsidated viral DNA.
CONCLUSIONS
Our data demonstrate that formulating AAV6.2FF in PBS containing 0.001% PF-68 leads to increased stability and particle integrity at temperatures between -20℃ to 21℃ and protection against the destructive effects of freeze-thaw. Finally, AAV6.2FF-GFP formulated in PBS supplemented with 0.001% PF-68 displayed higher transduction efficiency in vivo in murine lung epithelial cells following intranasal administration than vector buffered in PBS alone further demonstrating the beneficial properties of PF-68.
Topics: Animals; Humans; HEK293 Cells; Poloxamer; Mice; Dependovirus; Genetic Vectors; Luciferases, Firefly; Temperature; Genes, Reporter
PubMed: 38664752
DOI: 10.1186/s12896-024-00853-6 -
Frontiers in Microbiology 2024Acute febrile illnesses (AFI) in developing tropical and sub-tropical nations are challenging to diagnose due to the numerous causes and non-specific symptoms. The...
INTRODUCTION
Acute febrile illnesses (AFI) in developing tropical and sub-tropical nations are challenging to diagnose due to the numerous causes and non-specific symptoms. The proliferation of rapid diagnostic testing and successful control campaigns against malaria have revealed that non- pathogens still contribute significantly to AFI burden. Thus, a more complete understanding of local trends and potential causes is important for selecting the correct treatment course, which in turn will reduce morbidity and mortality. Next-generation sequencing (NGS) in a laboratory setting can be used to identify known and novel pathogens in individuals with AFI.
METHODS
In this study, plasma was collected from 228 febrile patients tested negative for malaria at clinics across Senegal from 2020-2022. Total nucleic acids were extracted and converted to metagenomic NGS libraries. To identify viral pathogens, especially those present at low concentration, an aliquot of each library was processed with a viral enrichment panel and sequenced. Corresponding metagenomic libraries were also sequenced to identify non-viral pathogens.
RESULTS AND DISCUSSION
Sequencing reads for pathogens with a possible link to febrile illness were identified in 51/228 specimens, including (but not limited to): (N = 7), West Nile virus (N = 3), (N = 2), (N = 1), human herpesvirus 8 (N = 1), and Saffold virus (N = 1). Reads corresponding to were detected in 19 specimens, though their presence in the cohort was likely due to user error of rapid diagnostic testing or incorrect specimen segregation at the clinics. Mosquito-borne pathogens were typically detected just after the conclusion of the rainy season, while tick-borne pathogens were mostly detected before the rainy season. The three West Nile virus strains were phylogenetically characterized and shown to be related to both European and North American clades. Surveys such as this will increase the understanding of the potential causes of non-malarial AFI, which may help inform diagnostic and treatment options for clinicians who provide care to patients in Senegal.
PubMed: 38655084
DOI: 10.3389/fmicb.2024.1362714 -
BioRxiv : the Preprint Server For... Apr 2024Genomic scientists have long been promised cheaper DNA sequencing, but deep whole genomes are still costly, especially when considered for large cohorts in...
Genomic scientists have long been promised cheaper DNA sequencing, but deep whole genomes are still costly, especially when considered for large cohorts in population-level studies. More affordable options include microarrays + imputation, whole exome sequencing (WES), or low-pass whole genome sequencing (WGS) + imputation. WES + array + imputation has recently been shown to yield 99% of association signals detected by WGS. However, a method free from ascertainment biases of arrays or the need for merging different data types that still benefits from deeper exome coverage to enhance novel coding variant detection does not exist. We developed a new, combined, "Blended Genome Exome" (BGE) in which a whole genome library is generated, an aliquot of that genome is amplified by PCR, the exome regions are selected and enriched, and the genome and exome libraries are combined back into a single tube for sequencing (33% exome, 67% genome). This creates a single CRAM with a low-coverage whole genome (2-3x) combined with a higher coverage exome (30-40x). This BGE can be used for imputing common variants throughout the genome as well as for calling rare coding variants. We tested this new method and observed >99% r concordance between imputed BGE data and existing 30x WGS data for exome and genome variants. BGE can serve as a useful and cost-efficient alternative sequencing product for genomic researchers, requiring ten-fold less sequencing compared to 30x WGS without the need for complicated harmonization of array and sequencing data.
PubMed: 38645052
DOI: 10.1101/2024.04.03.587209 -
Journal of Food Protection Jun 2024A simple new, rapid, sensitive, and cost-effective HPLC-PDA method was developed and validated for the determination of tetracycline (TC), oxytetracycline (OTC), and...
A simple new, rapid, sensitive, and cost-effective HPLC-PDA method was developed and validated for the determination of tetracycline (TC), oxytetracycline (OTC), and ciprofloxacin (CIP) simultaneously. Chromatographic separations were carried out using a reversed-phase Shim-pack GIS C18 column (4.60 × 250.00 mm; 5.00 µm) at 30°C. Oxalic acid (0.05 M), acetonitrile, and methanol were used as mobile phase under gradient elution conditions at the flow rate of 1.50 mL min. Detection wavelength was set at 330 nm. An aliquot of 20.00 µL solution was injected, and three drugs were eluted within 7.39 ± 0.05 min. As per ICH guidelines linearity, recovery, accuracy, precision, selectivity, specificity, sensitivity, stability, column efficiency, system suitability, and robustness were determined for the validation of the proposed method. Calibration curves were linear over a studied concentration range of 8.00 µg mL with a correlation coefficient (r) of 0.999 for all drugs. Relative standard deviation (RSD) for intra- and interday precision was found less than 2.87% and 3.22%, respectively, indicating the method to be reproducible. The proposed method has been suitably applied for the estimation of TC, OTC, and CIP in pharmaceutical formulation and milk samples collected from local market in Bangladesh. Among 15 milk samples analyzed, most of the cases (more than 50%) TC, OTC, and CIP were detected above maximum residue levels (MRLs) though no significant toxicological effect on the health of consumers in the study area was identified.
Topics: Milk; Chromatography, High Pressure Liquid; Animals; Anti-Bacterial Agents; Cattle; Risk Assessment; Food Contamination; Humans; Reproducibility of Results; Oxytetracycline
PubMed: 38631421
DOI: 10.1016/j.jfp.2024.100279 -
Italian Journal of Food Safety Feb 2024The majority of human diseases attributed to seafood are caused by spp., and the most commonly reported species are , , and . The conventional methods for the detection...
Detection of pathogenic spp. in foods: polymerase chain reaction-based screening strategy to rapidly detect pathogenic , , and in bivalve mollusks and preliminary results.
The majority of human diseases attributed to seafood are caused by spp., and the most commonly reported species are , , and . The conventional methods for the detection of species involve the use of selective media, which are inexpensive and simple but time-consuming. The present work aimed to develop a rapid method based on the use of multiplex real-time polymerase chain reaction (PCR) to detect , , and in bivalve mollusks. 30 aliquots of bivalve mollusks () were experimentally inoculated with two levels of , , and . ISO 21872-1:2017 was used in parallel for qualitative analysis. The limit of detection of 50% was 7.67 CFU/g for , 0.024 CFU/g for , and 1.36 CFU/g for . For and , the real-time PCR protocol was demonstrated to amplify the pathogens in samples seeded with the lowest and highest levels. The molecular method evaluated showed a concordance rate of 100% with the reference microbiological method. was never detected in samples contaminated with the lowest level, and it was detected in 14 samples (93.33%) seeded with the highest concentration. In conclusion, the developed multiplex real-time PCR proved to be reliable for and Results for are promising, but further analysis is needed. The proposed method could represent a quick monitoring tool and, if used, would allow the implementation of food safety.
PubMed: 38623280
DOI: 10.4081/ijfs.2024.11635 -
International Journal of Molecular... Apr 2024Sperm cryopreservation is a procedure widely used to store gametes for later use, to preserve fertility in patients prior to gonadotoxic treatments or surgery, and for...
Sperm cryopreservation is a procedure widely used to store gametes for later use, to preserve fertility in patients prior to gonadotoxic treatments or surgery, and for sperm donation programs. The purpose of the study was to assess the impact of cryopreservation on human sperm transcriptome. Semen samples were collected from 13 normospermic men. Each sample was divided into two aliquots. The total RNA was immediately extracted from one aliquot. The second aliquot was frozen and total RNA was extracted after a week of storage in liquid nitrogen. The RNA samples were randomized in four pools, each of six donors, and analyzed by microarrays. The paired Significance Analysis of Microarray was performed. We found 219 lower abundant transcripts and 28 higher abundant transcripts in cryopreserved sperm than fresh sperm. The gene ontology analysis disclosed that cryopreservation alters transcripts of pathways important for fertility (i.e., spermatogenesis, sperm motility, mitochondria function, fertilization, calcium homeostasis, cell differentiation, and early embryo development), although the increase of some transcripts involved in immune response can compensate for the harmful effects of freezing.
Topics: Humans; Male; Semen; Transcriptome; Sperm Motility; Spermatozoa; Cryopreservation; RNA
PubMed: 38612939
DOI: 10.3390/ijms25074131 -
Plant Disease Apr 2024In July 2022, dark brown to black, angular, water-soaked lesions were observed on sesame leaves (Sesamum indicum L.) in a research plot established to assess yield...
In July 2022, dark brown to black, angular, water-soaked lesions were observed on sesame leaves (Sesamum indicum L.) in a research plot established to assess yield potential for eight varieties at the North Carolina (NC) Sandhills Research Station (Chavez 2023). Symptoms were indicative of a bacterial leaf spot (BLS). At early flowering stage, leaf spots were present on scattered plants; varieties ES108, SS3301, and ES201 exhibited up to 75% disease prevalence, with lower frequency in ES103, S39, S4302, S3251, and S3276. Symptomatic leaves from 3-4 plants were collected on four different dates from July through September. A section of symptomatic tissue was excised and macerated in sterile deionized water (SDW). A 10 µL aliquot was streaked onto SPA medium (15 g sucrose, 5.0 g proteose peptone, 0.50 g MgSO4 7H2O, 0.25 g K2HPO4, 15 g agar per liter of SDW) and incubated at 28ºC. After 72 h, numerous, smooth, white-cream colored, convex-shaped, colonies were individually isolated. Five randomly selected isolates from the different collection dates, designated as AHP108-AHP111 and AHP116, were genotyped. The 16S rRNA, gyrB, rpoD, and gapA genes were sequenced (Heuer et al. 1997; Hwang et al. 2005) and deposited to NCBI (GenBank Accessions: P213467- PP213470; OQ628040-OQ628042; PP214983-PP214994; and PP255798). These five isolates shared 100% sequence identity for gyrB and rpoD. AHP108-AHP111 shared 100% sequence identity for 16S rRNA and gapA, with 99.7% and 90.8% identity, respectively, for AHP116. A phylogenetic tree was inferred from a maximum-likelihood analysis of concatenated gyrB, rpoD, and gapA sequences of the five isolates and the top 11 hts from a blastn search of the NCBI nucleotide database. Those hits included closely related sequences from Pseudomonas syringae pv. sesami type strains ICMP 763T and ICMP 7459T. Based on this phylogenetic analysis AHP108-AHP111 and AHP116 are P. syringae pv. sesami. Recent genomic analysis suggests this pathovar is part of P. amygdali (Gomila et al. 2017), but an official name change has not been proposed. Each of the five isolates were infiltrated into leaves of sesame varieties ES108, ES103, and S327, consistently resulting in similar symptoms. Thus, strain AHP116, as a representative, was used to fulfill Koch's postulates using five, 30-day-old potted sesame plants (var. S3301). Plants were spray-inoculated with a bacterial suspension of ~108 CFU/ml until runoff; plants were incubated in moist chambers 24 h pre and post inoculation at 28ºC with 80% relative humidity and a 12 h photoperiod. At 13 days post inoculation, symptoms resembling those on plants at the Sandhills Research Stations in 2022 were evident. Reisolated bacteria were confirmed to be AHP116 through 16S rRNA and gyrB amplification and sequencing. No symptoms were observed on the five water-inoculated plants. BLS of sesame has been reported in Asia and is thought to be seedborne (Firdous et al. 2009; Prathuangwong and Yowabutra 1997). To our knowledge, this is the first report of P. syringae pv. sesami causing BLS on sesame in North Carolina. Sesame cultivation in the state increased from approximately 2,000 acres in 2022 to 13,000 acres in 2023 and there is interest in cultivating sesame as a rotational and alternative crop because it requires minimal input costs. Potential outbreaks of BLS in this warm, humid region could negatively affect sesame production, where little is known about the economic impact of the disease.
PubMed: 38587794
DOI: 10.1094/PDIS-02-24-0292-PDN