One species hides many: Molecular and morphological evidence for cryptic speciation in a thread snake (Leptotyphlopidae: Leptotyphlops sylvicolus Broadley & Wallach, 1997)

We investigate the phylogeographic structure of a fossorial forest‐living snake species, the forest thread snake, Leptotyphlopssylvicolus Broadley & Wallach, 1997 by sampling specimens from the Eastern Cape and KwaZulu‐Natal provinces of South Africa. Phylogenetic results, using Bayesian inferences and maximum likelihood, from the combined mitochondrial sequence data (cyt b and ND4), along with population genetic analyses suggest the presence of phylogeographic breaks broadly congruent to those exhibited by other forest‐living taxa. Divergence‐time estimates indicate that cladogenesis within the study taxon occurred during the late Miocene climatic shifts, suggesting that cladogenesis was driven by habitat fragmentation. We further investigate the species‐level divergence within L. sylvicolus by including two partial nuclear loci (PRLR and RAG1). The three species delimitation methods (ABGD, bGMYC, and STACEY), retrieved 10–12 putative species nested within the L. sylvicolus species complex. These results were corroborated by iBPP implementing molecular and morphological data in an integrative Bayesian framework. The morphological analyses exhibit large overlap among putative species but indicate differences between grassland and forest species. Due to the narrow distributions of these putative species, the results of the present study have further implications for the conservation status of the L. sylvicolus species complex and suggest that forest and grassland habitats along the east coast of South Africa may harbor significantly higher levels of diversity than currently recognized.     

Therapeutic efficacy of novel memantine nitrate MN‐08 in animal models of Alzheimer’s disease

Alzheimer''s disease (AD) is a leading cause of dementia in elderly individuals and therapeutic options for AD are very limited. Over‐activation of N‐methyl‐D‐aspartate (NMDA) receptors, amyloid β (Aβ) aggregation, a decrease in cerebral blood flow (CBF), and downstream pathological events play important roles in the disease progression of AD. In the present study, MN‐08, a novel memantine nitrate, was found to inhibit Aβ accumulation, prevent neuronal and dendritic spine loss, and consequently attenuate cognitive deficits in 2‐month‐old APP/PS1 transgenic mice (for a 6‐month preventative course) and in the 8‐month‐old triple‐transgenic (3×Tg‐AD) mice (for a 4‐month therapeutic course). In vitro, MN‐08 could bind to and antagonize NMDA receptors, inhibit the calcium influx, and reverse the dysregulations of ERK and PI3K/Akt/GSK3β pathway, subsequently preventing glutamate‐induced neuronal loss. In addition, MN‐08 had favorable pharmacokinetics, blood‐brain barrier penetration, and safety profiles in rats and beagle dogs. These findings suggest that the novel memantine nitrate MN‐08 may be a useful therapeutic agent for AD.     

A Test of Current Models for the Mechanism of Milk‐Lipid Droplet Secretion

Milk lipid is secreted by a unique process, during which triacylglycerol droplets bud from mammary cells coated with an outer bilayer of apical membrane. In all current schemes, the integral protein butyrophilin 1A1 (BTN) is postulated to serve as a transmembrane scaffold, which interacts either with itself or with the peripheral proteins, xanthine oxidoreductase (XOR) and possibly perilipin‐2 (PLIN2), to form an immobile bridging complex between the droplet and apical surface. In one such scheme, BTN on the surface of cytoplasmic lipid droplets interacts directly with BTN in the apical membrane without binding to either XOR or PLIN2. We tested these models using both biochemical and morphological approaches. BTN was concentrated in the apical membrane in all species examined and contained mature N‐linked glycans. We found no evidence for the association of unprocessed BTN with intracellular lipid droplets. BTN‐enhanced green fluorescent protein was highly mobile in areas of mouse milk‐lipid droplets that had not undergone post‐secretion changes, and endogenous mouse BTN comprised only 0.5–0.7% (w/w) of the total protein, i.e. over 50‐fold less than in the milk‐lipid droplets of cow and other species. These data are incompatible with models of milk‐lipid secretion in which BTN is the major component of an immobile global adhesive complex and suggest that interactions between BTN and other proteins at the time of secretion are more transient than previously predicted. The high mobility of BTN in lipid droplets marks it as a potential mobile signaling molecule in milk .     

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.     

Oridonin ameliorates neuropathological changes and behavioural deficits in a mouse model of cerebral amyloidosis

Alzheimer's disease (AD) is the most common form of neurodegeneration and the major cause of dementia. This multifactorial disorder is clinically defined by progressive behavioural and cognitive deficits, and neuropathologically characterized by β‐amyloid aggregation, hyperphosphorylated tau and neuroinflammation. Oridonin, a diterpenoid isolated from Chinese herb Rabdosia rubescens, has multiple biological properties, especially anti‐inflammatory and neuroregulatory activities. Potential therapeutic effects of Oridonin were investigated in an animal model of cerebral amyloidosis for AD, transgenic APP/PS1 mice. Oridonin was suspended in carboxymethylcellulose or loaded with a nanostructured emulsion, and was orally administrated or injected. Before, during and following the experimental treatments, behavioural tests were performed with these transgenic mice and their naive littermates. Following relatively short‐term treatments of 10 days, brain tissue of mice were removed for immunohistochemical assays. The results indicate that both oral treatment and injection of Oridonin significantly attenuated β‐amyloid deposition, plaque‐associated APP expression and microglial activation in brain of transgenic mice. Furthermore, injection of Oridonin‐nanoemulsion ameliorated deficits in nesting, an important affiliative behaviour, and in social interaction. Additional in vitro studies indicated that Oridonin effectively attenuated inflammatory reaction of macrophage and microglial cell lines. Our results suggest that Oridonin might be considered a promising therapeutic option for human AD or other neurodegenerative diseases.     

Combinatorial materials research applied to the development of new surface coatings IV. A high-throughput bacterial biofilm retention and retraction assay for screening fouling-release performance of coatings

Abstract

A high-throughput bacterial biofilm retention screening method has been augmented to facilitate the rapid analysis and down-selection of fouling-release coatings for identification of promising candidates. Coatings were cast in modified 24-well tissue culture plates and inoculated with the marine bacterium Cytophaga lytica for attachment and biofilm growth. Biofilms retained after rinsing with deionised water were dried at ambient laboratory conditions. During the drying process, retained biofilms retracted through a surface de-wetting phenomenon on the hydrophobic silicone surfaces. The retracted biofilms were stained with crystal violet, imaged, and analysed for percentage coverage. Two sets of experimental fouling-release coatings were analysed with the high-throughput biofilm retention and retraction assay (HTBRRA). The first set consisted of a series of model polysiloxane coatings that were systematically varied with respect to ratios of low and high MW silanol-terminated PDMS, level of cross-linker, and amount of silicone oil. The second set consisted of cross-linked PDMS-polyurethane coatings varied with respect to the MW of the PDMS and end group functionality. For the model polysiloxane coatings, HTBRRA results were compared to data obtained from field immersion testing at the Indian River Lagoon at the Florida Institute of Technology. The percentage coverage calculations of retracted biofilms correlated well to barnacle adhesion strength in the field (R² = 0.82) and accurately identified the best and poorest performing coating compositions. For the cross-linked PDMS-polyurethane coatings, the HTBRRA results were compared to combinatorial pseudobarnacle pull-off adhesion data and good agreement in performance was observed. Details of the developed assay and its implications in the rapid discovery of new fouling-release coatings are discussed.