A Kinetic Analysis of Coupled (or Auxiliary) Enzyme Reactions

As a case study, we consider a coupled (or auxiliary) enzyme assay of two reactions obeying the Michaelis–Menten mechanism. The coupled reaction consists of a single-substrate, single-enzyme non-observable reaction followed by another single-substrate, single-enzyme observable reaction (indicator reaction). In this assay, the product of the non-observable reaction is the substrate of the indicator reaction. A mathematical analysis of the reaction kinetics is performed, and it is found that after an initial fast transient, the coupled reaction is described by a pair of interacting Michaelis–Menten equations. Moreover, we show that when the indicator reaction is fast, the quasi-steady-state dynamics are governed by three fast variables and one slow variable. Timescales that approximate the respective lengths of the indicator and non-observable reactions, as well as conditions for the validity of the Michaelis–Menten equations, are derived. The theory can be extended to deal with more complex sequences of enzyme-catalyzed reactions.     

Influence of Pulsed Electric Fields and Mitochondria-Cytoskeleton Interactions on Cell Respiration

Pulsed electric fields with microsecond pulse width (μsPEFs) are used clinically; namely, irreversible electroporation/Nanoknife is used for soft tissue tumor ablation. The μsPEF pulse parameters used in irreversible electroporation (0.5–1 kV/cm, 80–100 pulses, ～100 μs each, 1 Hz frequency) may cause an internal field to develop within the cell because of the disruption of the outer cell membrane and subsequent penetration of the electric field. An internal field may disrupt voltage-sensitive mitochondria, although the research literature has been relatively unclear regarding whether such disruptions occur with μsPEFs. This investigation reports the influence of clinically used μsPEF parameters on mitochondrial respiration in live cells. Using a high-throughput Agilent Seahorse machine, it was observed that μsPEF exposure comprising 80 pulses with amplitudes of 600 or 700 V/cm did not alter mitochondrial respiration in 4T1 cells measured after overnight postexposure recovery. To record alterations in mitochondrial function immediately after μsPEF exposure, high-resolution respirometry was used to measure the electron transport chain state via responses to glutamate-malate and ADP and mitochondrial membrane potential via response to carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. In addition to measuring immediate mitochondrial responses to μsPEF exposure, measurements were also made on cells permeabilized using digitonin and those with compromised cytoskeleton due to actin depolymerization via treatment with the drug latrunculin B. The former treatment was used as a control to tease out the effects of plasma membrane permeabilization, whereas the latter was used to investigate indirect effects on the mitochondria that may occur if μsPEFs impact the cytoskeleton on which the mitochondria are anchored. Based on the results, it was concluded that within the pulse parameters tested, μsPEFs alone do not hinder mitochondrial physiology but can be used to impact the mitochondria upon compromising the actin. Mitochondrial susceptibility to μsPEF after actin depolymerization provides, to our knowledge, a novel avenue for cancer therapeutics.     

A novel receptor‐like kinase involved in fungal pathogen defence in Arabidopsis thaliana

Plants are under constant attack from a variety of disease‐causing organisms. Lacking an adaptive immune system, plants repel pathogen attack via an array of pathogen recognition machinery. Receptor‐like kinases (RLKs) are involved in the recognition of pathogen‐associated molecular patterns (PAMPs) and activate resistance pathways against broad classes of pathogens. We have identified powdery mildew‐resistant kinase 1, an Arabidopsis gene encoding an RLK that is highly induced by chitin at early time points and localizes to the plasma membrane. Knockout mutants in pmrk1 are more susceptible to both Golovinomyces cichoracearum and Plectosphaerella cucumerina. Our data show that PMRK1 is essential in early stages of defence against fungi and provide evidence that PMRK1 may be unique to chitin‐induced signalling pathways. The results of this study indicate that PMRK1 is a critical component of plant innate immunity against fungal pathogens.     

Selenium unmasks protective iron armor: A possible defense against cutaneous inflammation and cancer

Background

A link between selenium deficiency and inflammatory skin diseases have been noted by many, but this link is still not well understood. We have previously studied the efficacy of ceramide analogs, based on the fire ant venom Solenopsin A, against our psoriasis animal model. Treatment of animals with solenopsin analogs resulted in significantly improved skin as well as in a coordinate downregulation of selenoproteins, namely Glutathione Peroxidase 4 (GPX4). We thus hypothesize that ferroptosis may be a physiologic process that may protect the skin from both inflammatory and neoplastic processes.

Molecular phylogeny of Plagiothecium and similar hypnalean mosses,with a revised sectional classification of Plagiothecium

We investigated the systematic relationships within the pleurocarpous moss genus Plagiothecium, based on cladistic analyses of sequence data from one nuclear (ITS) and two plastid (trnK‐psbA (matK) and rpl16 intron) DNA regions for 110 specimens of Plagiothecium and similar hypnalean mosses. Plastid and nuclear trees were mostly similar, but differed in the placement of several species of Plagiothecium, and in the relationships among other genera. The phylogenetic hypotheses based on plastid markers were well resolved; in contrast, nuclear data were insufficient to resolve some of the lowest‐level relationships within the genus. In the main Plagiothecium is natural, includes more taxa than are often recognized, and is most closely related to Isopterygiopsis and Herzogiella. Nine sections are recognized within Plagiothecium, four new: Section Pseudo‐Neckera to accommodate P. neckeroideum and its allies, Section Ortholimnobium for P. handelii and P. paleaceum, Section Struckia for P. argentatum and P. enerve, and Section Rectithecium for P. piliferum. The geographical distribution of the sections suggests that Plagiothecium originated in Asia. The derived, mainly autoicous sections Plagiothecium and Leptophyllum may have spread to the Southern Hemisphere through long distance dispersal. Three new species in section Leptophyllum (P. funale, P. pacificum and P. rhizolucidum) and two new taxa in section Pseudo‐Neckera (P. decoratum and P. neckeroideum fo. exile) are described. A limited phylogenetic hypothesis for the superficially similar hypnalean genus Taxiphyllum, which was used as outgroup, is included. A related genus (Longiella) is described, with a single species (L. plagiothecioides). The aquarium species T. barbieri is transferred to Ectropothecium.     

Variant‐specific and reciprocal Hsp40 functions in Hsp104‐mediated prion elimination

The amyloid‐based prions of Saccharomyces cerevisiae are heritable aggregates of misfolded proteins, passed to daughter cells following fragmentation by molecular chaperones including the J‐protein Sis1, Hsp70 and Hsp104. Overexpression of Hsp104 efficiently cures cell populations of the prion [PSI⁺] by an alternative Sis1‐dependent mechanism that is currently the subject of significant debate. Here, we broadly investigate the role of J‐proteins in this process by determining the impact of amyloid polymorphisms (prion variants) on the ability of well‐studied Sis1 constructs to compensate for Sis1 and ask whether any other S. cerevisiae cytosolic J‐proteins are also required for this process. Our comprehensive screen, examining all 13 members of the yeast cytosolic/nuclear J‐protein complement, uncovered significant variant‐dependent genetic evidence for a role of Apj1 (antiprion DnaJ) in this process. For strong, but not weak [PSI⁺] variants, depletion of Apj1 inhibits Hsp104‐mediated curing. Overexpression of either Apj1 or Sis1 enhances curing, while overexpression of Ydj1 completely blocks it. We also demonstrated that Sis1 was the only J‐protein necessary for the propagation of at least two weak [PSI⁺] variants and no J‐protein alteration, or even combination of alterations, affected the curing of weak [PSI⁺] variants, suggesting the possibility of biochemically distinct, variant‐specific Hsp104‐mediated curing mechanisms.     

A RAPID ASSESSMENT APPROACH FOR EVALUATING SILVER CARP GENDER

Silver carp Hypophthalmichthys molitrix were introduced into the U.S. to control water quality in aquaculture ponds. From this point of origin, silver carp escaped into nearby rivers through multiple flood events. Because of their documented negative effects on native biota, silver carp have been labeled as problematic. Therefore, evaluating the biology and ecology of these non-indigenous species is critical. Multiple parameters are needed to evaluate silver carp populations(length, weight, age, and sex). Furthermore, developing methods for rapidly acquiring these data are needed. In relation to sex determination, sexual dimorphism was observed where males exhibit distinct pectoral fin ray features. Specifically, males have pronounced ridges or a "rough patch" on the dorsal surface of pectoral fins. Therefore, to test if this was an applicable way of determining silver carp gender; silver carp were collected from Midwestern U.S. rivers(N=2015) in the fall of 2011(N=870), spring of 2012(N=645), winter of 2013—2014(N=202) and summer2015(N=323) via electrofishing. For each silver carp collected, presence(e.g., rough patch) or absence(e.g.,smooth) of pronounced ridges on the top side of the pectoral fins was recorded, and an incision was made in the body cavity to identify gender. Based on the results of our evaluation, gender was correctly identified over99% of the time(2006 out of 2015) based on the pectoral fin dimorphism. In the samples taken in the winter of 2013—2014 and summer of 2015, accuracy for silver carp shorter than 300 mm and longer than 800 mm was 53.7%(19 out of 41) while accuracy for silver carp between 300 mm and 800 mm total length was 98.9%(289 out of 292). This study provides a rapid assessment approach for evaluating silver carp gender.