Discrete nuclear structures in actively growing neuroblastoma cells are revealed by antibodies raised against phosphorylated neurofilament proteins

BACKGROUND: Nuclear objects that have in common the property of being recognized by monoclonal antibodies specific for phosphoprotein epitopes and cytoplasmic intermediate filaments (in particular, SMI-31 and RT-97) have been reported in glial and neuronal cells, in situ and in vitro. Since neurofilament and glial filaments are generally considered to be restricted to the cytoplasm, we were interested in exploring the identity of the structures labeled in the nucleus as well as the conditions under which they could be found there. RESULTS: Using confocal microscopy and western analysis techniques, we determined 1) the immunolabeled structures are truly within the nucleus; 2) the phosphoepitope labeled by SMI-31 and RT-97 is not specific to neurofilaments (NFs) and it can be identified on other intermediate filament proteins (IFs) in other cell types; and 3) there is a close relationship between DNA synthesis and the amount of nuclear staining by these antibodies thought to be specific for cytoplasmic proteins. Searches of protein data bases for putative phosphorylation motifs revealed that lamins, NF-H, and GFAP each contain a single tyrosine phosphorylation motif with nearly identical amino acid sequence. CONCLUSION: We therefore suggest that this sequence may be the epitope recognized by SMI-31 and RT-97 mABs, and that the nuclear structures previously reported and shown here are likely phosphorylated lamin intermediate filaments, while the cytoplasmic labeling revealed by the same mABs indicates phosphorylated NFs in neurons or GFAP in glia

Supporting marine spatial planning with an ecosystem model of Algoa Bay, South Africa

The Ecopath with Ecosim (EwE) modelling framework was used to develop a model of Algoa Bay and test the ecosystem impacts of the  implementation of the Addo Elephant National Park Marine Protected Area (MPA). The Ecopath model included 37 functional groups ranging from  phytoplankton to top predators and was fitted to 12 and 14 time-series of biomass and landings, respectively, from 2010 to 2019 (calibration  period), using Ecosim. Two scenarios representing different degrees of fisheries closures in the MPA were explored through a 30% and a 100%  reduction in fishing effort. Temporal simulations were run until 2059. The fitting procedure identified the best-fit model as the one including the  effects of fishing, the six most-sensitive predator–prey interactions, and environmental forcing (primary production anomaly on small  phytoplankton). Overall, the predicted biomass and catch time-series reasonably reproduced the observed time-series for 2010–2019, with the  biomass of sardine Sardinops sagax, round herring Etrumeus whiteheadi, and African penguins Spheniscus demersus showing the best fits to data.  Both MPA scenarios resulted in higher total biomass compared with the baseline by the end of the simulation and decreased catches due to less  fishing effort. The most profound biomass increases under the MPA scenarios were observed in apex and pelagic elasmobranchs, yellowtail Seriola  lalandi and African penguins. Future research is needed to improve the more-uncertain model parameters and include other key sectors in Algoa  Bay, such as shipping. However, this model provides a good foundation for future work including the application of spatially explicit modelling of the  bay using Ecospace

Optical systems for point-of-care diagnostic instrumentation: analysis of imaging performance and cost

One of the key elements in point-of-care (POC) diagnostic test instrumentation is the optical system required for signal detection and / or imaging. Many tests which use fluorescence, absorbance, or colorimetric optical signals are under development for management of infectious diseases in resource limited settings, where the overall size and cost of the device is of critical importance. At present, high-performance lenses are expensive to fabricate and difficult to obtain commercially, presenting barriers for developers of in vitro POC tests or microscopic image-based diagnostics. We recently described a compact “hybrid” objective lens incorporating both glass and plastic optical elements, with a numerical aperture of 1.0 and field-of-view of 250 μm. This design concept may potentially enable mass-production of high-performance, low-cost optical systems which can be easily incorporated in the readout path of existing and emerging POC diagnostic assays. In this paper, we evaluate the biological imaging performance of these lens systems in three broad POC diagnostic application areas; (1) bright field microscopy of histopathology slides, (2) cytologic examination of blood smears, and (3) immunofluorescence imaging. We also break down the fabrication costs and draw comparisons with other miniature optical systems. The hybrid lenses provided images with quality comparable to conventional microscopy, enabling examination of neoplastic pathology and infectious parasites including malaria and cryptosporidium. We describe how these components can be produced at below $10 per unit in full-scale production quantities, making these systems well suited for use within POC diagnostic instrumentation

Oligonucleotide-Gold Nanoparticle Networks for Detection of Cryptosporidium parvum Heat Shock Protein 70 mRNA ▿

We report on a novel strategy for the detection of mRNA targets derived from Cryptosporidium parvum oocysts by the use of oligonucleotide-gold nanoparticles. Gold nanoparticles are functionalized with oligonucleotides which are complementary to unique sequences present on the heat shock protein 70 (HSP70) DNA/RNA target. The results indicate that the presence of HPS70 targets of increasing complexity causes the formation of oligonucleotide-gold nanoparticle networks which can be visually monitored via a simple colorimetric readout measured by a total internal reflection imaging setup. Furthermore, the induced expression of HSP70 mRNA in Cryptosporidium parvum oocysts via a simple heat shock process provides nonenzymatic amplification such that the HSP70 mRNA derived from as few as 5 × 103 purified C. parvum oocysts was successfully detected. Taken together, these results support the use of oligonucleotide-gold nanoparticles for the molecular diagnosis of cryptosporidiosis, offering new opportunities for the further development of point-of-care diagnostic assays with low-cost, robust reagents and simple colorimetric detection