Evidence of Cryptosporidium transmission between cattle and humans in northern New South Wales

Cryptosporidium is an enteric parasite of public health significance that causes diarrhoeal illness through faecal oral contamination and via water. Zoonotic transmission is difficult to determine as most species of Cryptosporidium are morphologically identical and can only be differentiated by molecular means. Transmission dynamics of Cryptosporidium in rural populations were investigated through the collection of 196 faecal samples from diarrheic (scouring) calves on 20 farms and 63 faecal samples from humans on 14 of these farms. The overall prevalence of Cryptosporidium in cattle and humans by PCR and sequence analysis of the 18S rRNA was 73.5% (144/196) and 23.8% (15/63), respectively. Three species were identified in cattle; Cryptosporidium parvum, Cryptosporidium bovis and Cryptosporidium ryanae, and from humans, C. parvum and C. bovis. This is only the second report of C. bovis in humans. Subtype analysis at the gp60 locus identified C. parvum subtype IIaA18G3R1 as the most common subtype in calves. Of the seven human C. parvum isolates successfully subtyped, five were IIaA18G3R1, one was IIdA18G2 and one isolate had a mix of IIaA18G3R1 and IIdA19G2. These findings suggest that zoonotic transmission may have occurred but more studies involving extensive sampling of both calves and farm workers are needed for a better understanding of the sources of Cryptosporidium infections in humans from rural areas of Australia.     

Separation and detection of individual Aβ aggregates by capillary electrophoresis with laser-induced fluorescence detection

The separation and detection of individual amyloid beta (Aβ) aggregates by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was demonstrated. Samples were prepared with either Aβ (1-40) or Aβ (1-42) peptides and were characterized by CE with ultraviolet (UV) absorbance detection and transmission electron microscopy (TEM). Using thioflavin T (ThT) in the electrophoresis buffer, electrophoresis of aggregate-containing samples (5.0-s injection) produced up to several hundred narrow (< 20 ms FWHM [full width at half maximum]) fluorescence peaks. Injection of Aβ (1-40) monomer samples resulted in no additional peaks compared with controls. The CE-LIF results were validated by bulk ThT fluorescence measurements for the same samples. The potential of laser-induced fluorescence anisotropy (LIFA) with CE to characterize individual Aβ aggregates also was investigated.     

Deficiency of αB crystallin augments ER stress-induced apoptosis by enhancing mitochondrial dysfunction

Endoplasmic reticulum (ER) stress is linked to several pathological conditions including age-related macular degeneration. Excessive ER stress initiates cell death cascades which are mediated, in part, through mitochondrial dysfunction. Here, we identify αB crystallin as an important regulator of ER stress-induced cell death. Retinal pigment epithelial (RPE) cells from αB crystallin (-/-) mice, and human RPE cells transfected with αB crystallin siRNA, are more vulnerable to ER stress induced by tunicamycin. ER stress-mediated cell death is associated with increased levels of reactive oxygen species, depletion of glutathione in mitochondria, decreased superoxide dismutase activity, increased release of cytochrome c, and activation of caspases 3 and 4. The ER stress signaling inhibitors, salubrinal and 4-(2-aminoethyl) benzenesulfonyl fluoride, decrease mitochondrial damage and reduce RPE apoptosis induced by ER stress. Prolonged ER stress decreases levels of αB crystallin, thus exacerbating mitochondrial dysfunction. Overexpression of αB crystallin protects RPE cells from ER stress-induced apoptosis by attenuating increases in Bax, CHOP, mitochondrial permeability transition, and cleaved caspase 3. Thus, these data collectively demonstrate that αB crystallin provides critical protection of mitochondrial function during ER stress-induced RPE apoptosis.     

Identification of a Novel Cryptosporidium Genotype in Pigs

Over a 3-year period, a total of 646 fecal samples from pigs in 22 indoor and outdoor herds from Western Australia were screened for Cryptosporidium spp. by microscopy. Results revealed that 39 of 646 samples (6.03%) were positive for Cryptosporidium. Cryptosporidium was much more common in outdoor herds (17.2%) than in indoor herds (0.5%) and was more common in animals between the ages of 5 and 8 weeks (69.2%) than in younger animals (P < 0.0001). Molecular characterization of the positive samples at the 18S ribosomal DNA locus identified two distinct genotypes of Cryptosporidium: the previously identified pig genotype I and a novel pig genotype (pig genotype II), both of which warrant species status.     

Heterocyclic aminopyrrolidine derivatives as melatoninergic agents

A series of chiral heterocyclic aminopyrrolidine derivatives was synthesized as novel melatoninergic ligands. Binding affinity assays were performed on cloned human MT₁ and MT₂ receptors, stably expressed in NIH3T3 cells. Compound 16 was identified as an orally bioavailable agonist at MT₁ and MT₂ melatonin receptors with low vasoconstrictive activity.     

Targeted disruption of TgPhIL1 in Toxoplasma gondii results in altered parasite morphology and fitness

The inner membrane complex (IMC), a series of flattened vesicles at the periphery of apicomplexan parasites, is thought to be important for parasite shape, motility and replication, but few of the IMC proteins that function in these processes have been identified. TgPhIL1, a Toxoplasma gondii protein that was previously identified through photosensitized labeling with 5-[(125)I] iodonapthaline-1-azide, associates with the IMC and/or underlying cytoskeleton and is concentrated at the apical end of the parasite. Orthologs of TgPhIL1 are found in other apicomplexans, but the function of this conserved protein family is unknown. As a first step towards determining the function of TgPhIL1 and its orthologs, we generated a T. gondii parasite line in which the single copy of TgPhIL1 was disrupted by homologous recombination. The TgPhIL1 knockout parasites have a distinctly different morphology than wild-type parasites, and normal shape is restored in the knockout background after complementation with the wild-type allele. The knockout parasites are outcompeted in culture by parasites expressing functional TgPhIL1, and they generate a reduced parasite load in the spleen and liver of infected mice. These findings demonstrate a role for TgPhIL1 in the morphology, growth and fitness of T. gondii tachyzoites.     

Seasonal freeze resistance of rainbow smelt (Osmerus mordax) is generated by differential expression of glycerol-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, and antifreeze protein genes

In winter, rainbow smelt (Osmerus mordax) accumulate glycerol and produce an antifreeze protein (AFP), which both contribute to freeze resistance. The role of differential gene expression in the seasonal pattern of these adaptations was investigated. First, cDNAs encoding smelt and Atlantic salmon (Salmo salar) phosphoenolpyruvate carboxykinase (PEPCK) and smelt glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were cloned so that all sequences required for expression analysis would be available. Using quantitative PCR, expression of beta actin in rainbow smelt liver was compared with that of GAPDH in order to determine its validity as a reference gene. Then, levels of glycerol-3-phosphate dehydrogenase (GPDH), PEPCK, and AFP relative to beta actin were measured in smelt liver over a fall-winter-spring interval. Levels of GPDH mRNA increased in the fall just before plasma glycerol accumulation, implying a driving role in glycerol synthesis. GPDH mRNA levels then declined during winter, well in advance of serum glycerol, suggesting the possibility of GPDH enzyme or glycerol conservation in smelt during the winter months. PEPCK mRNA levels rose in parallel with serum glycerol in the fall, consistent with an increasing requirement for amino acids as metabolic precursors, remained elevated for much of the winter, and then declined in advance of the decline in plasma glycerol. AFP mRNA was elevated at the onset of fall sampling in October and remained elevated until April, implying separate regulation from GPDH and PEPCK. Thus, winter freezing point depression in smelt appears to result from a seasonal cycle of GPDH gene expression, with an ensuing increase in the expression of PEPCK, and a similar but independent cycle of AFP gene expression.     

Pulmonary angiotensin-converting enzyme substrate hydrolysis during exercise.

We examined exercise-induced changes in indicator-dilution estimates of the angiotensin-converting enzyme first-order kinetic parameter, the ratio of a normalized maximal enzymatic conversion rate to the Michaelis constant (Amax/Km), which, under stable enzymatic conditions, will vary with the pulmonary vascular surface area accessible to vascular substrate, the extravascular lung water (an index of the proportion of lung tissue perfused), and the central blood volume (from pulmonary trunk to aorta). Experiments were performed in 10 mongrel dogs at rest and through two increasing levels of treadmill exercise, with the use of two vascular space tracers (labeled erythrocytes and albumin), a water space tracer ([1,8-14C]-octanediol), and a vascular endothelium surface area marker, benzoyl-Phe-Gly-Pro ([3H]BPGP), which is a pharmacologically inactive angiotensin-converting enzyme substrate. The exercise-induced increase in cardiac output was accompanied by a linear increase in central blood volume, and dilutional extravascular lung water rapidly increased to an asymptotic proportion close to 100% of postmortem vascular lung water. There was an average 55% [3H]BPGP hydrolysis, which did not vary with flow, and the computed Amax/Km increased linearly with exercise. We conclude that exercise results in complete lung tissue recruitment and increases the pulmonary vascular surface area available for BPGP hydrolysis linearly with flow, so that pulmonary vascular recruitment continues after full tissue recruitment.