Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses

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Frog breeding in rain-fed wetlands after a period of severe drought: implications for predicting the impacts of climate change

Globally, rain-fed wetlands provide critical habitat for a wide range of amphibian species, however, information on the use of rain-fed wetlands by Australian frog species is extremely limited. This study examined the distribution of frog breeding in rain-fed wetlands following the first significant rain event after a period of severe drought (2002–2009) in order to predict how frog communities may be affected in the future by changed climate. Tadpole communities along with vegetation and water quality variables were described in 35 rain-fed wetlands across the South West Slopes and Riverina bioregions of inland south-eastern Australia. In addition, weekly tadpole surveys were conducted in a subset of these wetlands to describe temporal patterns of occupancy. Despite the protracted dry period prior to this study 50% of the rain-fed wetlands surveyed contained tadpoles. However, frog communities were species poor with only five species recorded. The majority of wetlands were dominated be a single species, Limnodynastes tasmaniensis which is also common within permanent waterbodies such as farm dams and irrigation infrastructure in both bioregions. Tadpoles of two burrowing species L. interioris and Neobatrachus sudelli were restricted to a small number of wetlands mostly in the South West Slopes. The composition of tadpole communities changed over time, and Crinia parinsignifera was the only species that continued to breed over winter. The dominance of generalist species within rain-fed wetlands indicates that characteristics such as dispersal capability, flexibility in breeding times and the ability to utilise created habitats may be more important than burrowing ability and longevity when predicting vulnerability to climate change.     

Characterization of the interaction between Robo1 and heparin and other glycosaminoglycans

Roundabout 1 (Robo1) is the cognate receptor for secreted axon guidance molecule, Slits, which function to direct cellular migration during neuronal development and angiogenesis. The Slit2–Robo1 signaling is modulated by heparan sulfate, a sulfated linear polysaccharide that is abundantly expressed on the cell surface and in the extracellular matrix. Biochemical studies have further shown that heparan sulfate binds to both Slit2 and Robo1 facilitating the ligand–receptor interaction. The structural requirements for heparan sulfate interaction with Robo1 remain unknown. In this report, surface plasmon resonance (SPR) spectroscopy was used to examine the interaction between Robo1 and heparin and other GAGs and determined that heparin binds to Robo1 with an affinity of ∼650 nM. SPR solution competition studies with chemically modified heparins further determined that although all sulfo groups on heparin are important for the Robo1–heparin interaction, the N-sulfo and 6-O-sulfo groups are essential for the Robo1–heparin binding. Examination of differently sized heparin oligosaccharides and different GAGs also demonstrated that Robo1 prefers to bind full-length heparin chains and that GAGs with higher sulfation levels show increased Robo1 binding affinities.     

Optimizing Human Synovial Fluid Preparation for Two-Dimensional Gel Electrophoresis

Background

Prenatal screening for Down Syndrome (DS) would benefit from an increased number of biomarkers to improve sensitivity and specificity. Improving sensitivity and specificity would decrease the need for potentially risky invasive diagnostic procedures.

Results

We have performed an in depth two-dimensional difference gel electrophoresis (2D DIGE) study to identify potential biomarkers. We have used maternal plasma samples obtained from first and second trimesters from mothers carrying DS affected fetuses compared with mothers carrying normal fetuses. Plasma samples were albumin/IgG depleted and expanded pH ranges of pH 4.5 - 5.5, pH 5.3 - 6.5 and pH 6 - 9 were used for two-dimensional gel electrophoresis (2DE). We found no differentially expressed proteins in the first trimester between the two groups. Significant up-regulation of ceruloplasmin, inter-alpha-trypsin inhibitor heavy chain H4, complement proteins C1s subcomponent, C4-A, C5, and C9 and kininogen 1 were detected in the second trimester in maternal plasma samples where a DS affected fetus was being carried. However, ceruloplasmin could not be confirmed as being consistently up-regulated in DS affected pregnancies by Western blotting.

Conclusions

Despite the in depth 2DE approach used in this study the results underline the deficiencies of gel-based proteomics for detection of plasma biomarkers. Gel-free approaches may be more productive to increase the number of plasma biomarkers for DS for non-invasive prenatal screening and diagnosis.     

Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

Ligand promotes intranuclear inclusions in a novel cell model of spinal and bulbar muscular atrophy

Spinal and bulbar muscular atrophy (SBMA, Kennedy's disease) is one of a group of progressive neurodegenerative diseases resulting from a polyglutamine repeat expansion. In SBMA the polymorphic trinucleotide CAG repeat in exon 1 of the androgen receptor (AR) gene is increased, resulting in expansion of a polyglutamine tract. Patient autopsy material reveals neuronal intranuclear inclusions (NII) in affected regions that contain only amino-terminal epitopes of the AR. Cell models have previously been unable to produce intranuclear inclusions containing only a portion of the AR. We report here the creation of an inducible cell model of SBMA that reproduces this important characteristic of disease pathology. PC12 cells expressing highly expanded AR form ubiquitinated intranuclear inclusions containing amino-terminal epitopes of the AR as well as heat shock proteins. Inclusions appear as distinct granular electron-dense structures in the nucleus by immunoelectron microscopy. Dihydrotestosterone treatment of mutant AR-expressing cells results in increased inclusion load. This model mimics the formation of ubiquitinated intranuclear inclusions containing the amino-terminal portion of AR observed in patient tissue and reveals a role for ligand in the pathogenesis of SBMA.     

Colonization of Female Watermelon Blossoms by Acidovorax avenae ssp. citrulli and the Relationship between Blossom Inoculum Dosage and Seed Infestation

The aim of this work was to investigate the ability of Acidovorax avenae ssp. citrulli, the causal agent of bacterial fruit blotch of cucurbits (BFB), to colonize female watermelon blossoms, and to explore the relationship between blossom inoculum dosage and seed infestation. Under greenhouse conditions A. avenae ssp. citrulli colonized stigmas and styles of female watermelon blossoms reaching populations of ≈10⁷ to 10⁸ colony‐forming units (CFU) per blossom for 96 h after inoculation. Acidovorax avenae ssp. citrulli growth on stigmas was slower than that of Pseudomonas syringae Cit7, a non‐pathogenic, foliar epiphyte of tomato. While pollination reduced growth of A. avenae ssp. citrulli, but P. syringae Cit7 was unaffected. Both bacteria colonized style tissues but bacterial growth in the style was significantly less than the stigma. Blossom inoculation with ≈1 × 10³A. avenae ssp. citrulli CFU/blossom led to 36–55% infested seedlots within symptomless fruits. On average 14% of the seedlings produced from these seedlots displayed BFB symptoms. There was a strong positive correlation between A. avenae ssp. citrulli inoculum concentration applied to blossoms and the percentage of infested seedlots, as determined by the seedling grow‐out assay (R² = 0.94). However, this relationship was weaker when seedlot infestation was determined by a polymerase chain reaction‐based assay (R² = 0.34). There was also a strong positive linear relationship between A. avenae ssp. citrulli blossom inoculum dose and the mean percentage of BFB‐infected seedlings (R² = 0.99) produced in seedling grow‐out assays. These data support the hypothesis that blossom colonization might be involved in seed infestation under field conditions.