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.     

The use of environmental DNA metabarcoding and quantitative PCR for molecular detection of marine invasive non-native species associated with artificial structures

Biological Invasions - Artificial coastal structures associated with coastal defences, energy generation, ports, marinas and other developments, are known to support lower levels of biodiversity...     

Proline-specific aminopeptidase P prevents replication-associated genome instability

Genotoxic stress during DNA replication constitutes a serious threat to genome integrity and causes human diseases. Defects at different steps of DNA metabolism are known to induce replication stress, but the contribution of other aspects of cellular metabolism is less understood. We show that aminopeptidase P (APP1), a metalloprotease involved in the catabolism of peptides containing proline residues near their N-terminus, prevents replication-associated genome instability. Functional analysis of C. elegans mutants lacking APP-1 demonstrates that germ cells display replication defects including reduced proliferation, cell cycle arrest, and accumulation of mitotic DSBs. Despite these defects, app-1 mutants are competent in repairing DSBs induced by gamma irradiation, as well as SPO-11-dependent DSBs that initiate meiotic recombination. Moreover, in the absence of SPO-11, spontaneous DSBs arising in app-1 mutants are repaired as inter-homologue crossover events during meiosis, confirming that APP-1 is not required for homologous recombination. Thus, APP-1 prevents replication stress without having an apparent role in DSB repair. Depletion of APP1 (XPNPEP1) also causes DSB accumulation in mitotically-proliferating human cells, suggesting that APP1’s role in genome stability is evolutionarily conserved. Our findings uncover an unexpected role for APP1 in genome stability, suggesting functional connections between aminopeptidase-mediated protein catabolism and DNA replication.     

The Distribution of Perkinsus marinus in Gulf Coast Oysters: Its Relationship with Temperature,Reproduction, and Pollutant Body Burden

Oysters from 48 Gulf of Mexico sites were examined for presence and infection intensity of the endoparasite, Perkinsus (= Dermocystidium) marinus (Mackin, Owen and Collier, 1950) as part of NOAA's Status and Trends Mussel Watch Program. Prevalence exceeded 75% at 25 sites. Infection intensity did not vary with sex or reproductive stage. Latitude, total polynuclear aromatic hydrocarbon (PAH) content and industrial and agricultural land use significantly affected the parasite's distribution. PAH and pesticide concentrations were latitudinally dependent, suggesting an impact of spawning frequency on uptake and depuration. P. marinus analysis complements the use of pollutant body burden for determining change in environmental quality because it responds differently than pollutant body burden to the biology and ecology of the oyster.     

A Cell Culture Model for Androgen Effects in Motor Neurons

Abstract: Androgens are known to alter the morphology, survival, and axonal regeneration of lower motor neurons in vivo. To understand better the molecular mechanisms of androgen action in neurons, we created a model system by stably expressing the human androgen receptor (AR) in motor neuron hybrid cells. Motor neuron hybrid cells express markers consistent with anterior horn cells and can be differentiated into a neuronal phenotype. When differentiated in the presence of androgen, AR-expressing cells, but not control cells, exhibit a dose-dependent change in morphology: androgen-treated cells develop larger cell bodies and broader neuritic processes while continuing to express neuronal markers. In addition, androgen promotes the survival of AR-expressing cells, but not control cells, under low-serum conditions. Our results demonstrate a direct trophic effect of androgens on lower motor neurons, mediated through the AR expressed in this population of neurons.