Prospective large-scale field study generates predictive model identifying major contributors to colony losses.

Over the last decade, unusually high losses of colonies have been reported by beekeepers across the USA. Multiple factors such as Varroa destructor, bee viruses, Nosema ceranae, weather, beekeeping practices, nutrition, and pesticides have been shown to contribute to colony losses. Here we describe a large-scale controlled trial, in which different bee pathogens, bee population, and weather conditions across winter were monitored at three locations across the USA. In order to minimize influence of various known contributing factors and their interaction, the hives in the study were not treated with antibiotics or miticides. Additionally, the hives were kept at one location and were not exposed to potential stress factors associated with migration. Our results show that a linear association between load of viruses (DWV or IAPV) in Varroa and bees is present at high Varroa infestation levels (>3 mites per 100 bees). The collection of comprehensive data allowed us to draw a predictive model of colony losses and to show that Varroa destructor, along with bee viruses, mainly DWV replication, contributes to approximately 70% of colony losses. This correlation further supports the claim that insufficient control of the virus-vectoring Varroa mite would result in increased hive loss. The predictive model also indicates that a single factor may not be sufficient to trigger colony losses, whereas a combination of stressors appears to impact hive health.

Protection of pancreatic beta-cells from various stress conditions is mediated by DJ-1.

Pancreatic beta-cells are vulnerable to multiple stresses, leading to dysfunction and apoptotic death. Deterioration in beta-cells function and mass is associated with type 2 diabetes. Comparative two-dimensional gel electrophoresis from pancreatic MIN6 cells that were maintained at varying glucose concentrations was carried out. An induced expression of a protein spot, detected in MIN6 cells experiencing high glucose concentration, was identified by mass spectrometry as the oxidized form of DJ-1. DJ-1 (park7) is a multifunctional protein implicated in familial Parkinsonism and neuroprotection in response to oxidative damage. The DJ-1 protein and its oxidized form were also induced following exposure to oxidative and endoplasmic reticulum stress in MIN6 and betaTC-6 cells and also in mouse pancreatic islets. Suppression of DJ-1 levels by small interfering RNA led to an accelerated cell death, whereas an increase in DJ-1 levels by adenovirus-based infection attenuated cell death induced by H(2)O(2) and thapsigargin in beta-cell lines and mouse pancreatic islets. Furthermore, DJ-1 improved regulated insulin secretion under basal as well as oxidative and endoplasmic reticulum stress conditions in a dose-dependent manner. We identified TFII-I (Gtf2i) as DJ-1 partner in the cytosol, whereas the binding of TFII-I to DJ-1 prevented TFII-I translocation to the nucleus. The outcome was attenuation of the stress response. Our results suggest that DJ-1 together with TFII-I operate in concert to cope with various insults and to sustain pancreatic beta-cell function.

Cellular processes underlying maturation of P19 neurons: Changes in protein folding regimen and cytoskeleton organization.

Embryonal carcinoma P19 cells provide an ideal model to study molecular programs along differentiation. Upon induction by retinoic acid (RA), the cells undergo a program of differentiation that generates functioning neurons within 60 h. RA induced cells that were plated as sparse (1000 cells/mm(2)) or dense (4000 cells/mm(2)) cultures showed a marked difference in the culture morphology with the dense cultures exhibiting rapid maturation and accelerated neurite outgrowth. The protein expression levels of the sparse and dense cultures were compared 48 h following RA. Cell extracts were separated by 1-DE and 2-DE and differential expression (>four-fold) proteins were identified by MS. Here, we focus on 20 proteins associated with cytoskeletal regulation and stress-dependent protein refolding. The first group includes drebrin, cofilin, alpha-internexin, vimentin, and nestin. Among the proteins in the second group are subunits of the TCP-1, and several chaperones of the Hsp70 and Hsp90 families. We show that coordinated remodeling of the cytoskeleton and modulations in chaperone activity underlie the change in neurite extension rate. Furthermore, a proteomics-based analysis applied on P19 neurons demonstrated pathways underlying neuronal outgrowth, suggesting that a malfunction of such pathways leads to neuropathological conditions.

Evolutional insights on uncharacterized SARS coronavirus genes.

The complete genome of the severe acute respiratory syndrome coronavirus (SARS-CoV) and many of its variants has been determined by several laboratories. The genome contains fourteen predicted open reading frames (ORFs). However, a function had been clearly assigned for only six of these ORFs, in the viral replication, transcription and structural constituents. The others are herein referred to as uncharacterized ORFs (UC-ORFs). Here, we try to provide a relational insight on those UC-ORFs, suggesting that a number of them are remotely related to structural proteins of coronaviruses and other viruses infecting mammalian hosts. Surprisingly, several of the UC-ORFs exhibit considerable similarity with other SARS-CoV ORFs. These observations may provide clues on the evolution and genome dynamics of the SARS-CoV.

PROCEED: A proteomic method for analysing plasma membrane proteins in living mammalian cells.

Elucidating the profile of extracellular integral membrane proteins on live cells is vital for uncovering diagnostic disease biomarkers, therapeutic agents and drug receptor candidates. Exploring the realm of these proteins has proved to be an intricate task, mainly due to their hydrophobic nature and low abundance. Furthermore, the level of purity achieved by classical methods of purification and cell fractionation is insufficient. These restrictions pose major limitations for gel electrophoresis or chromatography-based separation techniques as the preferred methodologies for high-throughput analysis. Mass spectrometry has alleviated most of the difficulties in the identification of proteins in general; however, the Achilles' heel is still the isolation and separation of membrane proteins. In order to circumvent these limitations, a high-throughput platform has been devised, whereby proteases are applied to whole intact living cells. The resulting peptide fragments are then analysed by liquid chromatology followed by tandem MS (LC-MS/MS) technology to provide a detailed profile of proteins exposed on the surface of the plasma membrane. This kind of protein trimming offers the advantages that no prior manipulation or fractionation of the cell is required, contaminating proteins are remarkably reduced and the procedure is adequate for high-throughput purposes. This method, referred to as PROCEED (PROteome of Cell Exposed Extracellular Domains) is compatible with isotope labelling techniques which facilitate comparative protein expression studies. The methodology is extendable to all cell types including yeast and bacteria. Finally, the advantages and the limitations of PROCEED are discussed in view of other current technologies.