Ready-to-use thermally stable mastermix pills for molecular biology applications

Rolling circle amplification (RCA), polymerase chain reaction (PCR), and loop-mediated isothermal amplification (LAMP), are powerful tools that can be used for gene manipulation, pathogen detection, and infectious disease diagnostics. However, these techniques require trained personnel, as the pipetting steps involved can lead to contamination and, consequently, erroneous results. Furthermore, many of the reagents used in molecular biology are thermally labile and must be kept within a cold-chain. In this article, we present a simple and cost-effective method that allows molecular biology reagents to be thermally stabilized into ready-to-use mastermixes via drying in pullulan and trehalose films. Our experimental results demonstrate that this method is capable of preserving the activity of RCA, PCR, LAMP, ligase, polynucleotide kinase, and Klenow fragment mastermixes for at least 3 months at ambient conditions. Thus, stabilizing reagents via drying in pullulan and trehalose film may allow for a drastic reduction in the number of pipetting steps and the elimination of the need for a cold chain. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2764, 2019.     

BMI, body composition, and physical functioning in older adults

Objective: Recent studies have emphasized the importance of muscle and fat mass in relation to age‐related decline in physical function. Our objective was to determine whether BMI, as a surrogate measurement of fat mass, may be used as a measure of risk factor for physical functioning in older adults and whether body composition measurements confer any advantage over BMI. Research Methods and Procedures: Four thousand men and women ≥65 years of age living in the community, stratified by age and sex, underwent the following measurements: body composition by DXA; grip strength; and timed 6‐m walk. Subjects were grouped into five categories of BMI using Asian criteria for health‐related risks, and between‐group differences in physical performance measures and body composition were analyzed using analysis of covariance adjusting for age, physical activity level, and presence of chronic disease. Results: Subjects in the two obese categories had a significantly greater number of instrumental activities of daily living (IADL) impairments compared with the underweight and normal‐weight groups. Those with BMI ≥30 kg/m² had the worst walking performance, and the groups with BMI in the normal and overweight range had optimal performance. Fat mass, but not appendicular muscle mass, was associated with walking speed after adjusting for BMI. Discussion: Fat mass seems to be a more important factor than appendicular muscle mass in determining walking speed in community‐living older adults, even after adjusting for BMI.     

The Current Status of Preclinical Endocrine Education in U.S. Medical Schools

ObjectiveTo characterize the current landscape of preclinical medical endocrine education in U.S. allopathic medical schools.MethodsU.S. endocrine curriculum directors were asked to voluntarily complete a 16-question email survey surveying the status of endocrine preclinical education at their medical school.ResultsSixty-nine of 155 (45%) endocrine block director respondents completed the online survey between July 2021 and September 2021. A larger incoming class, a longer duration of the endocrine curriculum, and the offering of a separate endocrine curriculum (ie apart from the teaching of other organ systems) were each independently associated with an increased number of faculty teaching the course. Schools that used a gland-/organ-based curriculum only and those that used a combination of gland-/organ-based curriculum with topic-based curriculum differed significantly in their use of large lectures, small groups, and several curriculum components, including point of care glucose testing, continuous glucose monitoring, and insulin pumps.ConclusionThis survey study reports the current landscape of preclinical endocrine education in the United States and describes opportunities to improve interest in pursuing endocrinology as a career.     

Correction to: Managing biological invasions: the cost of inaction

Biological Invasions -     

ZMYM2 restricts 53BP1 at DNA double-strand breaks to favor BRCA1 loading and homologous recombination

An inability to repair DNA double-strand breaks (DSBs) threatens genome integrity and can contribute to human diseases, including cancer. Mammalian cells repair DSBs mainly through homologous recombination (HR) and nonhomologous end-joining (NHEJ). The choice between these pathways is regulated by the interplay between 53BP1 and BRCA1, whereby BRCA1 excludes 53BP1 to promote HR and 53BP1 limits BRCA1 to facilitate NHEJ. Here, we identify the zinc-finger proteins (ZnF), ZMYM2 and ZMYM3, as antagonizers of 53BP1 recruitment that facilitate HR protein recruitment and function at DNA breaks. Mechanistically, we show that ZMYM2 recruitment to DSBs and suppression of break-associated 53BP1 requires the SUMO E3 ligase PIAS4, as well as SUMO binding by ZMYM2. Cells deficient for ZMYM2/3 display genome instability, PARP inhibitor and ionizing radiation sensitivity and reduced HR repair. Importantly, depletion of 53BP1 in ZMYM2/3-deficient cells rescues BRCA1 recruitment to and HR repair of DSBs, suggesting that ZMYM2 and ZMYM3 primarily function to restrict 53BP1 engagement at breaks to favor BRCA1 loading that functions to channel breaks to HR repair. Identification of DNA repair functions for these poorly characterized ZnF proteins may shed light on their unknown contributions to human diseases, where they have been reported to be highly dysregulated, including in several cancers.     

Measuring the repertoire of age-related behavioral changes in Drosophila melanogaster

Aging affects almost all aspects of an organism—its morphology, its physiology, its behavior. Isolating which biological mechanisms are regulating these changes, however, has proven difficult, potentially due to our inability to characterize the full repertoire of an animal’s behavior across the lifespan. Using data from fruit flies (D. melanogaster) we measure the full repertoire of behaviors as a function of age. We observe a sexually dimorphic pattern of changes in the behavioral repertoire during aging. Although the stereotypy of the behaviors and the complexity of the repertoire overall remains relatively unchanged, we find evidence that the observed alterations in behavior can be explained by changing the fly’s overall energy budget, suggesting potential connections between metabolism, aging, and behavior.     

Mating system of the rockfish, <Emphasis Type="Italic">Sebastiscus marmoratus</Emphasis> as revealed by DNA fingerprinting

The mating system of the viviparous rockfish, Sebastiscus marmoratus was studied using aquarium mating experiments and paternity testing by DNA fingerprinting. Individually specific DNA fingerprints were produced and the paternal relationships determined with the use of the restriction enzyme HinfI and the DNA probe (CAC)₅. Two types of mating experiments, multiple-male and single-male, were set up to investigate the effect of male sizes on their mating success, as determined by number of females fertilized. Male size was positively and significantly related to mating success in the multiple-male situation in which male–male interaction was present, but not in the single-male situation in which no male–male interaction was involved. Females produced one to seven batches of larvae, and all batches from individual females had identical paternity. Most females were fertilized by a single male but one case of multiple paternity was detected. The laboratory study suggested the principal mating system of S. marmoratus to be promiscuity, although multiple mating by females was uncommon.     

Role of rpoS in Escherichia coli O157:H7 Strain H32 Biofilm Development and Survival

The protein RpoS is responsible for mediating cell survival during the stationary phase by conferring cell resistance to various stressors and has been linked to biofilm formation. In this study, the role of the rpoS gene in Escherichia coli O157:H7 biofilm formation and survival in water was investigated. Confocal scanning laser microscopy of biofilms established on coverslips revealed a nutrient-dependent role of rpoS in biofilm formation, where the biofilm biomass volume of the rpoS mutant was 2.4- to 7.5-fold the size of its rpoS⁺ wild-type counterpart in minimal growth medium. The enhanced biofilm formation of the rpoS mutant did not, however, translate to increased survival in sterile double-distilled water (ddH₂O), filter-sterilized lake water, or unfiltered lake water. The rpoS mutant had an overall reduction of 3.10 and 5.30 log₁₀ in sterile ddH₂O and filter-sterilized lake water, respectively, while only minor reductions of 0.53 and 0.61 log₁₀ in viable counts were observed for the wild-type form in the two media over a 13-day period, respectively. However, the survival rates of the detached biofilm-derived rpoS⁺ and rpoS mutant cells were comparable. Under the competitive stress conditions of unfiltered lake water, the advantage conferred by the presence of rpoS was lost, and both the wild-type and knockout forms displayed similar declines in viable counts. These results suggest that rpoS does have an influence on both biofilm formation and survival of E. coli O157:H7 and that the advantage conferred by rpoS is contingent on the environmental conditions.     

Fine genetic mapping and physical delimitation of the lesion mimic gene Spl11 to a 160-kb DNA segment of the rice genome

The rice lesion mimic mutant spl11 was previously found to confer broad-spectrum disease resistance to both Magnaporthe grisea and Xanthomonas oryzae pv. oryzae. To better understand the molecular basis underlying cell death and disease resistance in rice, a map-based cloning strategy has been employed to isolate Spl11. Five Spl11-linked RAPD markers were developed and four of them were mapped to rice chromosome 12. A high-resolution genetic map was developed using a segregating population consisting of 1138 lesion mimic individuals. Recombination suppression was observed in the vicinity of Spl11. Three molecular markers tightly linked to Spl11 were identified and used to screen a BAC library. A contig spanning the Spl11 locus was constructed and physical mapping delimited Spl11 to a 160-kb DNA segment within a single BAC clone. These results provide the essential information for the final isolation of this important gene in the rice defense pathway.