A strain of Serratia marcescens pathogenic for larvae of Lymantria dispar: Infectivity and mechanisms of pathogenicity

The ED₅₀ of a strain of Serratia marcescens for microinjected instar III and IV gypsy moth larvae was 7.5 and 14.5 viable cells, respectively. Percentage and rate of mortality were found to be highly variable among replicates of the same instar and between instars in free-feeding bioassays. Mortality in second instar larvae occurred before ecdysis, whereas practically no mortality occurred in third and fourth instars until the molting period. Neither Boivin endotoxin preparations nor culture filtrates were toxic to instar III larvae when administered per os or by microinjection. Histological evidence indicated that the microorganism invaded the hemocoel of healthy or predisposed insects through the gut wall. The rapid multiplication of the bacterium in the hemocoel of infected insects, followed by death in the absence of extensive tissue damage, indicated mortality was due to a septicemia. The histological and biological evidence presented indicated that the microorganism would be less than effective if utilized as a conventional microbial insecticide.     

A protease-activatable luminescent biosensor and reporter cell line for authentic SARS-CoV-2 infection

Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.     

Effects of lipid environment on the conformational changes of an ABC importer

In order to shuttle substrates across the lipid bilayer, membrane proteins undergo a series of conformation changes that are influenced by protein structure, ligands, and the lipid environment. To test the effect of lipid on conformation change of the ABC transporter MolBC, EPR studies were conducted in lipids and detergents of variable composition. In both a detergent and lipid environment, MolBC underwent the same general conformation changes as detected by site-directed EPR spectroscopy. However, differences in activity and the details of the EPR analysis indicate conformational rigidity that is dependent on the lipid environment. From these observations, we conclude that native-like lipid mixtures provide the transporter with greater activity and conformational flexibility as well as technical advantages such as reconstitution efficiency and protein stability.     

Autoantibodies directed to novel components of the PM/Scl complex, the human exosome

The autoantigenic polymyositis/scleroderma (PM/Scl) complex was recently shown to be the human homologue of the yeast exosome, which is an RNA-processing complex. Our aim was to assess whether, in addition to targeting the known autoantigens PM/Scl-100 and PM/Scl-75, autoantibodies also target recently identified components of the PM/Scl complex. The prevalence of autoantibodies directed to six novel human exosome components (hRrp4p, hRrp40p, hRrp41p, hRrp42p, hRrp46p, hCsl4p) was determined in sera from patients with idiopathic inflammatory myopathy (n = 48), scleroderma (n = 11), or the PM/Scl overlap syndrome (n = 10). The sera were analyzed by enzyme-linked immunosorbent assays and western blotting using the affinity-purified recombinant proteins. Our results show that each human exosome component is recognized by autoantibodies. The hRrp4p and hRrp42p components were most frequently targeted. The presence of autoantibodies directed to the novel components of the human exosome was correlated with the presence of the anti-PM/Scl-100 autoantibody in the sera of patients with idiopathic inflammatory myopathy (IIM), as was previously found for the anti-PM/Scl-75 autoantibody. Other clear associations between autoantibody activities were not found. These results further support the conception that the autoimmune response may initially be directed to PM/Scl-100, whereas intermolecular epitope spreading may have caused the autoantibody response directed to the associated components.     

Convergent evolution of cysteine residues in sperm protamines of one genus of marsupials, the Planigales

A characteristic feature of the sperm P1 protamines of eutherian mammals is the constant presence of six to nine cysteine residues per molecule. During spermiogenesis these residues become oxidized to form a three-dimensional network of disulfide bridges between, and within, protamine molecules in the sperm chromatin. This covalent cross linking strongly stabilizes eutherian sperm nuclei. In contrast, protamines sequenced from teleost fish, birds, monotremes, and marsupials all lack cysteine residues and their sperm nuclei, without the stabilizing cross links, are easily decondensed in vitro. We have now found that one genus of tiny, shrewlike dasyurid marsupials, the Planigales, possess P1 protamines containing five to six cysteine residues. These residues appear to have evolved since the divergence of Planigales from other members of the family Dasyuridae, such as the marsupial mouse, Sminthopsis crassicaudata. We believe this constitutes a case of convergent evolution in a subfamily of dasyurid marsupials toward the cysteine-rich eutherian form of sperm protamine P1.      

Vectoring gypsy moth nuclear polyhedrosis virus byApanteles melanoscelus [Hym.: Braconidae]

Gypsy mothLymantria dispar L. larvae were exposed toApanteles melanoscelus (Ratzeburg) females contaminated with nuclear polyhedrosis virus. Three methods of contamination (ovipositor, total body surface, and exposure to infected hosts) and two exposure periods (2 and 24 hours) were tested. A significantly greater incidence of larval mortality caused by virus occurred among larvae exposed to contaminated than among larvae exposed to uncontaminated parasites for 2 and 24 hours. No significant differences occurred in larval mortality caused by virus for the 3 methods of contamination for the 2- and 24-hour tests or in parasite emergence from larvae parasitized by contaminated or uncontaminated parasites.     

Natural occurrence of the nucleopolyhedrosis virus of the gypsy moth,Lymantria dispar [Lep.: Lymantriidae] in wild birds and mammals

Three species of birds and 5 species of mammals were captured in the wild from 2 plots in which mortality from naturally occurring nucleopolyhedrosis virus (NPV) among gypsy moth,Lymantria dispar (L.), larvae was 15% and 70%. Bioassays of intestinal contents showed that blue jays,Cyanocitta cristata (L.), towhees,Pipilo erythrophthalmus (L.), white-footed mice,Peromyscus leucopus (Rafinesque), redback voles,Clethrionomys gapperi (Vigers), raccoons,Procyon lotor (L.), and a chipmunk,Tamias striatus (L.), contained infectious (L.), and masked shrews,Sorex cinereus (Kerr), did not. Comparisons among mice and voles indicated that those collected from the plot in which the NPV mortality was greatest (70%) contained the most virus. We concluded that birds and mammals can passively transport infectious gypsy moth NPV in the wild.

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Résumé Trois espèces d'oiseaux et 5 espèces de mammifères ont été capturées en 2 endroits où la mortalité due à la présence naturelle du virus de la nucléopolyhédrose (VNP) chez les larves deLymantria dispar (L.), était de 15% et de 70%. Le système digestif des geais bleux,Cyanocitta cristata (L.), guiraca à poitrine rose,Pipilo erythrophthalmus (L.), souris-au-pied-blanc,Peromyscus leucopus (Rafinesque), campagnols-au-dos-rouge,Clethrionomys gapperi (Vigers), ratons laveurs,Procyon lotor (L.) et d'un tamia,Tamias striatus (L.), était infecté par le virus alors que celui des rouges-gorges,Turdus migratorius (L.) et des musaraignes,Sorex cinereus (Kerr), ne l'état pas. Une comparaison entre les campagnols et les souris a indiqué que les animaux provenant de l'endroit où la mortalité par virose était la plus forte (70%) contenaient le plus de virus. Nous concluons que les oiseaux et les mammifères peuvent transporter passivement dans la nature les polyèdres nucléaires deL. dispar.

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The work reported was funded in part by a U.S. Department of Agriculture sponsored program entitled, “The Expanded Gypsy Moth Research, Development and Applications Program”.     

Study of the mitotic and meiotic chromosomes of sotol (<i>Dasylirion cedrosanum</i> Trel.)

The genus Dasylirion is a group of plants typically present in the Chihuahuan Desert, perennial, with a dioecious sexual behavior and commonly called sotoles. This genus has been little studied from the biological point of view, and the bases of its reproductive response remain unknown. In this work we studied the chromosome number and meiotic response of Dasylirion cedrosanum in the county of Saltillo, Coahuila, located at the North East of Mexico. For the preparation of mitotic chromosomes, we used a technique based on enzymatic treatment with pectolyase and cellulase, as well as staining with acetocarmin dye. For the study of meiosis, male flower buds were collected, fixed and stained for analysis with the same dye. As a result, the gametic (n = x = 19) and somatic chromosome (2n = 38) numbers of D. cedrosanum are reported for the first time, being consistent with previous findings in other Dasylirion species, which points to a constant ploidy level across the genus. Variation was observed in the morphology and size of the somatic chromosomes, with types ranging from submetacentric to subtelocentric, and sizes oscillating in a range of 4.43 µm, with an average total length of 112.38 µm for the diploid chromosome complement. This shows that the chromosome complement of D. cedrosanom would belong to a 3B classification of Stebins, with a medium variation between chromosome lengths and low chromosome asymmetry. This variation indicates the feasibility of constructing a chromosome ideotype for this species. The meiotic chromosome pairing showed a chromosome behavior consistent with a disomic inheritance characteristic of a diploid species, with prevalence of ring and chain bivalents, typically without pairing abnormalities. Bivalent configurations in all cases were symmetrical.The normal and symmetrical meiotic pairing indicates a balanced production of gametes, and suggests the absence of heteromorphic sex determination.     

An organ culture system to model early degenerative changes of the intervertebral disc

Introduction  

Back pain, a significant source of morbidity in our society, is related to the degenerative changes of the intervertebral disc. At present, the treatment of disc disease consists of therapies that are aimed at symptomatic relief. This shortcoming stems in large part from our lack of understanding of the biochemical and molecular events that drive the disease process. The goal of this study is to develop a model of early disc degeneration using an organ culture. This approach is based on our previous studies that indicate that organ culture closely models molecular events that occur in vivo in an ex vivo setting.