High fidelity of RecA-catalyzed recombination: a watchdog of genetic diversity

Homologous recombination plays a key role in generating genetic diversity, while maintaining protein functionality. The mechanisms by which RecA enables a single-stranded segment of DNA to recognize a homologous tract within a whole genome are poorly understood. The scale by which homology recognition takes place is of a few tens of base pairs, after which the quest for homology is over. To study the mechanism of homology recognition, RecA-promoted homologous recombination between short DNA oligomers with different degrees of heterology was studied in vitro, using fluorescence resonant energy transfer. RecA can detect single mismatches at the initial stages of recombination, and the efficiency of recombination is strongly dependent on the location and distribution of mismatches. Mismatches near the 5' end of the incoming strand have a minute effect, whereas mismatches near the 3' end hinder strand exchange dramatically. There is a characteristic DNA length above which the sensitivity to heterology decreases sharply. Experiments with competitor sequences with varying degrees of homology yield information about the process of homology search and synapse lifetime. The exquisite sensitivity to mismatches and the directionality in the exchange process support a mechanism for homology recognition that can be modeled as a kinetic proofreading cascade.Comment: http://www.weizmann.ac.il/complex/tlusty/papers/NuclAcidRes2006.pdf http://nar.oxfordjournals.org/cgi/content/short/34/18/502

A decline in avian cytokine expression with age revealed by commercially available cytokine array

Cytokines are secreted immunomodulators that are key regulators of the avian immune response. Currently, the most commonly used method to follow cytokine expression is qPCR, which measures cellular levels of mRNA, rather their extracellular circulating levels. Here we present a commercially available cytokine array designed to assay circulating expression levels of multiple cytokines and immunomodulators simultaneously. Upon minor modifications to the manufacturer protocol, background noise was reduced, leading to a significant increase in the sensitivity of the device. Our data indicate that the array is reliable and produce consistent data between biological repeats. We tested the reproducibility of the array in a biologically relevant context by assessing age-related changes in circulating cytokines. While individual features did not show a consistent pattern, our data revealed a consistent decline in the median of all cytokine values, supporting the validity of the array in studying biological processes

Characterization of community-acquired Clostridioides difficile strains in Israel, 2020–2022

BackgroundThe prevalence of community-acquired Clostridioides difficile infection (CA-CDI) has been rising, due to changes in antibiotics prescribing practices, emergence of hypervirulent strains and improved diagnostics. This study explored CA-CDI epidemiology by examining strain diversity and virulence factors of CA-CDI isolates collected across several geographical regions in Israel.MethodsStool samples of 126 CA-CDI patients were subjected to PCR and an immunoassay to identify toxin genes and proteins, respectively. Toxin loci PaLoc and PaCdt were detected by whole-genome sequencing (WGS). Biofilm production was assessed by crystal violet-based assay. Minimum inhibitory concentration was determined using the Etest technique or agar dilution. WGS and multi-locus sequence typing (MLST) were used to classify strains and investigate genetic diversity.ResultsSequence types (ST) 2 (17, 13.5%), ST42 (13, 10.3%), ST104 (10, 8%) and ST11 (9, 7.1%) were the most common. All (117, 92.8%) but ST11 belonged to Clade 1. No associations were found between ST and gender, geographic area or antibiotic susceptibility. Although all strains harbored toxins genes, 34 (27%) produced toxin A only, and 54 (42.9%) strains produced toxin B only; 38 (30.2%) produced both toxins. Most isolates were biofilm-producers (118, 93.6%), primarily weak producers (83/118, 70.3%). ST was significantly associated with both biofilm and toxin production.ConclusionC. difficile isolates in Israel community exhibit high ST diversity, with no dominant strain. Other factors may influence the clinical outcomes of CDI such as toxin production, antibiotic resistance and biofilm production. Further studies are needed to better understand the dynamics and influence of these factors on CA-CDI

Learning to look : evaluating the student experience of an interactive image appraisal activity

Introduction: Student radiographers have expressed difficulty in performing image appraisal tasks. The purpose of this study was to investigate the value of a workshop delivered to level 4 undergraduate students. All students completed an image appraisal activity, inputting their appraisal into software that displayed their response alongside an expert opinion. They were asked to identify and discuss any discrepancy. Methods: All Level 4 students participated in an image appraisal workshop and were subsequently invited to take part in a focus group immediately after the activity. Twenty-three students took part in three focus groups (n = 7; n = 8; n = 8). A thematic analysis of transcripts was performed alongside validation from observations during the image appraisal activity. Results: Findings demonstrate that despite teaching and resources being available, students had focused on learning a generic checklist for image appraisal, had not appreciated the application of projection specific criteria and felt underprepared. The use of specific criteria and repetition within the task was considered useful. They identified learning needs and misconceptions through peer discussion and via the expert opinion, highlighting the value of feedback. Students enjoyed the workshop and made suggestions for implementation into the curriculum. Conclusion: Educators must not assume that the provision of resources will result in students developing deep knowledge. Teaching and learning strategies that are task specific are recommended to avoid a surface approach to learning. Time, repetition and appropriate feedback are essential to enable learners to develop competence and confidence for complex visual tasks, such as image appraisal

An Engineering Approach to Extending Lifespan in C. elegans

We have taken an engineering approach to extending the lifespan of Caenorhabditis elegans. Aging stands out as a complex trait, because events that occur in old animals are not under strong natural selection. As a result, lifespan can be lengthened rationally using bioengineering to modulate gene expression or to add exogenous components. Here, we engineered longer lifespan by expressing genes from zebrafish encoding molecular functions not normally present in worms. Additionally, we extended lifespan by increasing the activity of four endogenous worm aging pathways. Next, we used a modular approach to extend lifespan by combining components. Finally, we used cell- and worm-based assays to analyze changes in cell physiology and as a rapid means to evaluate whether multi-component transgenic lines were likely to have extended longevity. Using engineering to add novel functions and to tune endogenous functions provides a new framework for lifespan extension that goes beyond the constraints of the worm genome

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

The addition of a developmental factor, unc-62, to already long-lived worms increases lifespan and healthspan

Aging is a complex trait that is affected by multiple genetic pathways. A relatively unexplored approach is to manipulate multiple independent aging pathways simultaneously in order to observe their cumulative effect on lifespan. Here, we report the phenotypic characterization of a strain with changes in five aging pathways: (1) mitochondrial reactive oxygen species (ROS) production, (2) innate immunity, (3) stress response, (4) metabolic control and (5) developmental regulation in old age. The quintuply modified strain has a lifespan that is 160% longer than the transgenic control strain. Additionally, the quintuply modified strain maintains several physiological markers of aging for a longer time than the transgenic control. Our results support a modular approach as a general scheme to study how multiple pathways interact to achieve extreme longevity

Arthropods as the Engine of Nutrient Cycling in Arid Ecosystems

Nutrient dynamics in most terrestrial ecosystems are regulated by moisture-dependent processes. In drylands, nutrient dynamics are often weakly associated with annual precipitation, suggesting that other factors are involved. In recent years, the majority of research on this topic focused on abiotic factors. We provide an arthropod-centric framework that aims to refocus research attention back on the fundamental role that macro-arthropods may play in regulating dryland nutrient dynamics. Macro-arthropods are prevalent in drylands and include many detritivores and burrowing taxa that remain active during long dry periods. Macro-arthropods consume and process large quantities of plant detritus and transport these nutrients to the decomposer haven within their climatically buffered and nutritionally enriched burrows. Consequently, arthropods may accelerate mineralization rates and generate a vertical nutrient recycling loop (VRL) that may assist in explaining the dryland decomposition conundrum, and how desert plants receive their nutrients when the shallow soil is dry. The burrowing activity of arthropods and the transportation of subterranean soil to the surface may alter the desert microtopography and promote desalinization, reducing resource leakage and enhancing productivity and species diversity. We conclude that these fundamental roles and the arthropods’ contribution to nutrient transportation and nitrogen fixation makes them key regulators of nutrient dynamics in drylands