The Progressive BSSG Rat Model of Parkinson's: Recapitulating Multiple Key Features of the Human Disease

The development of effective neuroprotective therapies for Parkinson''s disease (PD) has been severely hindered by the notable lack of an appropriate animal model for preclinical screening. Indeed, most models currently available are either acute in nature or fail to recapitulate all characteristic features of the disease. Here, we present a novel progressive model of PD, with behavioural and cellular features that closely approximate those observed in patients. Chronic exposure to dietary phytosterol glucosides has been found to be neurotoxic. When fed to rats, β-sitosterol β-d-glucoside (BSSG) triggers the progressive development of parkinsonism, with clinical signs and histopathology beginning to appear following cessation of exposure to the neurotoxic insult and continuing to develop over several months. Here, we characterize the progressive nature of this model, its non-motor features, the anatomical spread of synucleinopathy, and response to levodopa administration. In Sprague Dawley rats, chronic BSSG feeding for 4 months triggered the progressive development of a parkinsonian phenotype and pathological events that evolved slowly over time, with neuronal loss beginning only after toxin exposure was terminated. At approximately 3 months following initiation of BSSG exposure, animals displayed the early emergence of an olfactory deficit, in the absence of significant dopaminergic nigral cell loss or locomotor deficits. Locomotor deficits developed gradually over time, initially appearing as locomotor asymmetry and developing into akinesia/bradykinesia, which was reversed by levodopa treatment. Late-stage cognitive impairment was observed in the form of spatial working memory deficits, as assessed by the radial arm maze. In addition to the progressive loss of TH⁺ cells in the substantia nigra, the appearance of proteinase K-resistant intracellular α-synuclein aggregates was also observed to develop progressively, appearing first in the olfactory bulb, then the striatum, the substantia nigra and, finally, hippocampal and cortical regions. The slowly progressive nature of this model, together with its construct, face and predictive validity, make it ideal for the screening of potential neuroprotective therapies for the treatment of PD.     

The Discovery,Distribution, and Evolution of Viruses Associated with Drosophila melanogaster

Drosophila melanogaster is a valuable invertebrate model for viral infection and antiviral immunity, and is a focus for studies of insect-virus coevolution. Here we use a metagenomic approach to identify more than 20 previously undetected RNA viruses and a DNA virus associated with wild D. melanogaster. These viruses not only include distant relatives of known insect pathogens but also novel groups of insect-infecting viruses. By sequencing virus-derived small RNAs, we show that the viruses represent active infections of Drosophila. We find that the RNA viruses differ in the number and properties of their small RNAs, and we detect both siRNAs and a novel miRNA from the DNA virus. Analysis of small RNAs also allows us to identify putative viral sequences that lack detectable sequence similarity to known viruses. By surveying >2,000 individually collected wild adult Drosophila we show that more than 30% of D. melanogaster carry a detectable virus, and more than 6% carry multiple viruses. However, despite a high prevalence of the Wolbachia endosymbiont—which is known to be protective against virus infections in Drosophila—we were unable to detect any relationship between the presence of Wolbachia and the presence of any virus. Using publicly available RNA-seq datasets, we show that the community of viruses in Drosophila laboratories is very different from that seen in the wild, but that some of the newly discovered viruses are nevertheless widespread in laboratory lines and are ubiquitous in cell culture. By sequencing viruses from individual wild-collected flies we show that some viruses are shared between D. melanogaster and D. simulans. Our results provide an essential evolutionary and ecological context for host–virus interaction in Drosophila, and the newly reported viral sequences will help develop D. melanogaster further as a model for molecular and evolutionary virus research.     

Nonlinear response of N2O flux to incremental fertilizer addition in a continuous maize (Zea mays L.) cropping system

The relationship between nitrous oxide (N₂O) flux and N availability in agricultural ecosystems is usually assumed to be linear, with the same proportion of nitrogen lost as N₂O regardless of input level. We conducted a 3‐year, high‐resolution N fertilizer response study in southwest Michigan USA to test the hypothesis that N₂O fluxes increase mainly in response to N additions that exceed crop N needs. We added urea ammonium nitrate or granular urea at nine levels (0–292 kg N ha^?1) to four replicate plots of continuous maize. We measured N₂O fluxes and available soil N biweekly following fertilization and grain yields at the end of the growing season. From 2001 to 2003 N₂O fluxes were moderately low (ca. 20 g N₂O‐N ha^?1 day^?1) at levels of N addition to 101 kg N ha^?1, where grain yields were maximized, after which fluxes more than doubled (to >50 g N₂O‐N ha^?1 day^?1). This threshold N₂O response to N fertilization suggests that agricultural N₂O fluxes could be reduced with no or little yield penalty by reducing N fertilizer inputs to levels that just satisfy crop needs.     

ASCORBIC ACID DEFICIENCY IN CULTURED HUMAN FIBROBLASTS

Fibroblasts grown in medium containing less than 1 µg of ascorbic acid per milliliter showed evidence of ascorbic acid deficiency when compared with cells grown in medium containing 50 µg of ascorbic acid per milliliter. This was manifested morphologically by dilated endoplasmic reticulum, a decrease in number, size, and intensity of staining of the mitochondria, by defective intercellular fibril formation, and by easy disaggregation of the cells from the intercellular matrix after treatment with pronase. When 50 µg per milliliter of ascorbic acid was incorporated into the medium, the altered morphology was corrected, banded fibrils were produced which were organized into bundles, and the cells were tightly bound in a matrix which was resistant to disaggregation with a variety of proteolytic enzymes. Collagen and sulfated glycosaminoglycan synthesis were less in the control than in the ascorbic acid supplemented cells. Similar morphological and chemical changes have been reported in the connective tissue of scorbutic animals. The effects of low ascorbic acid concentration on fibroblasts in culture indicate that these cells require ascorbic acid to maintain connective tissue functions.     

Gypsophile Chemistry Unveiled: Fourier Transform Infrared (FTIR) Spectroscopy Provides New Insight into Plant Adaptations to Gypsum Soils

Gypsum soils are among the most restrictive and widespread substrates for plant life. Plants living on gypsum are classified as gypsophiles (exclusive to gypsum) and gypsovags (non-exclusive to gypsum). The former have been separated into wide and narrow gypsophiles, each with a putative different ecological strategy. Mechanisms displayed by gypsum plants to compete and survive on gypsum are still not fully understood. The aim of this study was to compare the main chemical groups in the leaves of plants with different specificity to gypsum soils and to explore the ability of Fourier transform infrared (FTIR) spectra analyzed with neural network (NN) modelling to discriminate groups of gypsum plants. Leaf samples of 14 species with different specificity to gypsum soils were analysed with FTIR spectroscopy coupled to neural network (NN) modelling. Spectral data were further related to the N, C, S, P, K, Na, Ca, Mg and ash concentrations of samples. The FTIR spectra of the three groups analyzed showed distinct features that enabled their discrimination through NN models. Wide gypsophiles stood out for the strong presence of inorganic compounds in their leaves, particularly gypsum and, in some species, also calcium oxalate crystals. The spectra of gypsovags had less inorganic chemical species, while those of narrow gypsum endemisms had low inorganics but shared with wide gypsophiles the presence of oxalate. Gypsum and calcium oxalate crystals seem to be widespread amongst gypsum specialist plants, possibly as a way to tolerate excess Ca and sulphate. However, other mechanisms such as the accumulation of sulphates in organic molecules are also compatible with plant specialization to gypsum. While gypsovags seem to be stress tolerant plants that tightly regulate the uptake of S and Ca, the ability of narrow gypsum endemisms to accumulate excess Ca as oxalate may indicate their incipient specialization to gypsum.     

A monoclonal antibody to a 48 K antigen in oestrogen-dependent human breast cancer cells

A mouse was immunised with an antigen(s) purified by oestradiol-Sepharose affinity chromatography of pooled oestrogen-receptor positive cytosols from human breast cancer tissue. One antibody secreting clone was identified which precipitated labelled antigen and which also stained MCF-7 cells. Culture supernatant and ascites fluid were used for immunofluorescence, SDS-PAGE-Western blotting, photoaffinity labelling and binding studies. The antibody staining of MCF-7 cells was inhibited by preincubation in oestrogen-receptor positive cytosol but was unaffected by oestrogen-receptor negative cytosol. MCF-7 cells stained whether cultured in the presence or absence of oestradiol. The oestrogen-receptor negative cell lines MDA-MB-231 and MDA-MB-330 did not stain. Binding studies with 16-alpha-iodooestradiol using breast cancer tissue cytosols followed by immunoprecipitation showed activity only with oestrogen-receptor positive cytosols with optimal binding activity at 4 degrees C, unaffected by molybdate, but reduced at 25 degrees C or in the presence of 0.4 M KCl. Binding studies with MCF-7, MDA-MB-231 and MDA-MB-330 cytosols and nuclear fractions only showed activity with the MCF-7 cytosol and MCF-7 particulate fractions. The antibody recognised a 48 K species in both MCF-7 cytosol and nuclear fractions but not in the cytosol and nuclear extracts of oestrogen-receptor negative cell lines. Photoaffinity labelling using 16 alpha-iodooestradiol suggests the 48 K antigen does not bind oestradiol directly. The relationship of this antigen to the classical oestrogen-receptor and receptor complex awaits further clarification.     

An inelastic multi-mechanism constitutive equation for cerebral arterial tissue

Intracranial aneurysms (ICA) are abnormal saccular dilations of cerebral arteries, commonly found at apices of arterial bifurcations and outer walls of curved arterial segments. Histological evidence suggests the stages in ICA development include the deformation of a segment of arterial wall into a “bleb” with no identifiable neck region followed by the development of an aneurysm with a clear neck. Afterwards, the aneurysm may undergo further enlargement, possibly with significant biological response including calcification and thrombosis. Past studies of the biomechanics of cerebral aneurysm tissue have been directed at modeling elastic deformations of pre-existing aneurysms. Taking this approach, the aneurysm wall is treated as a different entity than the arterial tissue from which it developed. In the current work, a nonlinear, inelastic, dual-mechanism constitutive equation for cerebral arterial tissue is developed. It is the first to model the recruitment of collagen fibers and degradation of the internal elastic lamina, two important characteristics of early stage aneurysm formation.