Automated 100-position specimen loader and image acquisition system for transmission electron microscopy

We report the development of a novel, multi-specimen imaging system for high-throughput transmission electron microscopy. Our cartridge-based loading system, called the "Gatling", permits the sequential examination of as many as 100 specimens in the microscope for room temperature electron microscopy using mechanisms for rapid and automated specimen exchange. The software for the operation of the Gatling and automated data acquisition has been implemented in an updated version of our in-house program AutoEM. In the current implementation of the system, the time required to deliver 95 specimens into the microscope and collect overview images from each is about 13 h. Regions of interest are identified from a low magnification atlas generation from each specimen and an unlimited number of higher magnifications images can be subsequently acquired from these regions using fully automated data acquisition procedures that can be controlled from a remote interface. We anticipate that the availability of the Gatling will greatly accelerate the speed of data acquisition for a variety of applications in biology, materials science, and nanotechnology that require rapid screening and image analysis of multiple specimens.     

Co-expressed Cyclin D variants cooperate to regulate proliferation of germline nuclei in a syncytium

The role of the G1-phase Cyclin D-CDK 4/6 regulatory module in linking germline stem cell (GSC) proliferation to nutrition is evolutionarily variable. In invertebrate Drosophila and C. elegans GSC models, G1 is nearly absent and Cyclin E is expressed throughout the cell cycle, whereas vertebrate spermatogonial stem cells have a distinct G1 and Cyclin D1 plays an important role in GSC renewal. In the invertebrate, chordate, Oikopleura, where germline nuclei proliferate asynchronously in a syncytium, we show a distinct G1-phase in which 2 Cyclin D variants are co-expressed. Cyclin Dd, present in both somatic endocycling cells and the germline, localized to germline nuclei during G1 before declining at G1/S. Cyclin Db, restricted to the germline, remained cytoplasmic, co-localizing in foci with the Cyclin-dependent Kinase Inhibitor, CKIa. These foci showed a preferential spatial distribution adjacent to syncytial germline nuclei at G1/S. During nutrient-restricted growth arrest, upregulated CKIa accumulated in arrested somatic endoreduplicative nuclei but did not do so in germline nuclei. In the latter context, Cyclin Dd levels gradually decreased. In contrast, the Cyclin Dbβ splice variant, lacking the Rb-interaction domain and phosphodegron, was specifically upregulated and the number of cytoplasmic foci containing this variant increased. This upregulation was dependent on stress response MAPK p38 signaling. We conclude that under favorable conditions, Cyclin Dbβ-CDK6 sequesters CKIa in the cytoplasm to cooperate with Cyclin Dd-CDK6 in promoting germline nuclear proliferation. Under nutrient-restriction, this sequestration function is enhanced to permit continued, though reduced, cycling of the germline during somatic growth arrest.     

Dose-dependent proteomic analysis of glioblastoma cancer stem cells upon treatment with γ-secretase inhibitor

Notch signaling has been demonstrated to have a central role in glioblastoma (GBM) cancer stem cells (CSCs) and we have demonstrated recently that Notch pathway blockade by γ-secretase inhibitor (GSI) depletes GBM CSCs and prevents tumor propagation both in vitro and in vivo. In order to understand the proteome alterations involved in this transformation, a dose-dependent quantitative mass spectrometry (MS)-based proteomic study has been performed based on the global proteome profiling and a target verification phase where both Immunoassay and a multiple reaction monitoring (MRM) assay are employed. The selection of putative protein candidates for confirmation poses a challenge due to the large number of identifications from the discovery phase. A multilevel filtering strategy together with literature mining is adopted to transmit the most confident candidates along the pipeline. Our results indicate that treating GBM CSCs with GSI induces a phenotype transformation towards non-tumorigenic cells with decreased proliferation and increased differentiation, as well as elevated apoptosis. Suppressed glucose metabolism and attenuated NFR2-mediated oxidative stress response are also suggested from our data, possibly due to their crosstalk with Notch Signaling. Overall, this quantitative proteomic-based dose-dependent work complements our current understanding of the altered signaling events occurring upon the treatment of GSI in GBM CSCs.     

Hypochlorous acid-induced heme degradation from lactoperoxidase as a novel mechanism of free iron release and tissue injury in inflammatory diseases

Lactoperoxidase (LPO) is the major consumer of hydrogen peroxide (H(2)O(2)) in the airways through its ability to oxidize thiocyanate (SCN(-)) to produce hypothiocyanous acid, an antimicrobial agent. In nasal inflammatory diseases, such as cystic fibrosis, both LPO and myeloperoxidase (MPO), another mammalian peroxidase secreted by neutrophils, are known to co-localize. The aim of this study was to assess the interaction of LPO and hypochlorous acid (HOCl), the final product of MPO. Our rapid kinetic measurements revealed that HOCl binds rapidly and reversibly to LPO-Fe(III) to form the LPO-Fe(III)-OCl complex, which in turn decayed irreversibly to LPO Compound II through the formation of Compound I. The decay rate constant of Compound II decreased with increasing HOCl concentration with an inflection point at 100 μM HOCl, after which the decay rate increased. This point of inflection is the critical concentration of HOCl beyond which HOCl switches its role, from mediating destabilization of LPO Compound II to LPO heme destruction. Lactoperoxidase heme destruction was associated with protein aggregation, free iron release, and formation of a number of fluorescent heme degradation products. Similar results were obtained when LPO-Fe(II)-O(2), Compound III, was exposed to HOCl. Heme destruction can be partially or completely prevented in the presence of SCN(-). On the basis of the present results we concluded that a complex bi-directional relationship exists between LPO activity and HOCl levels at sites of inflammation; LPO serve as a catalytic sink for HOCl, while HOCl serves to modulate LPO catalytic activity, bioavailability, and function.     

pTARGET [corrected] a new method for predicting protein subcellular localization in eukaryotes

MOTIVATION: There is a scarcity of efficient computational methods for predicting protein subcellular localization in eukaryotes. Currently available methods are inadequate for genome-scale predictions with several limitations. Here, we present a new prediction method, pTARGET that can predict proteins targeted to nine different subcellular locations in the eukaryotic animal species. RESULTS: The nine subcellular locations predicted by pTARGET include cytoplasm, endoplasmic reticulum, extracellular/secretory, golgi, lysosomes, mitochondria, nucleus, plasma membrane and peroxisomes. Predictions are based on the location-specific protein functional domains and the amino acid compositional differences across different subcellular locations. Overall, this method can predict 68-87% of the true positives at accuracy rates of 96-99%. Comparison of the prediction performance against PSORT showed that pTARGET prediction rates are higher by 11-60% in 6 of the 8 locations tested. Besides, the pTARGET method is robust enough for genome-scale prediction of protein subcellular localizations since, it does not rely on the presence of signal or target peptides. AVAILABILITY: A public web server based on the pTARGET method is accessible at the URL http://bioinformatics.albany.edu/~ptarget. Datasets used for developing pTARGET can be downloaded from this web server. Source code will be available on request from the corresponding author.     

Common binding site for disialyllactose and tri-peptide in C-fragment of tetanus neurotoxin

Clostridial neurotoxins are comprised of botulinum (BoNT) and tetanus (TeNT), which share significant structural and functional similarity. Crystal structures of the binding domain of TeNT complexed with disialyllactose (DiSia) and a tri-peptide Tyr-Glu-Trp (YEW) have been determined to 2.3 and 2.2 A, respectively. Both DiSia and YEW bind in a shallow cleft region on the surface of the molecule in the beta-trefoil domain, interacting with a set of common residues, Asp1147, Asp1214, Asn1216, and Arg1226. DiSia and YEW binding at the same site in tetanus toxin provides a putative site that could be occupied either by a ganglioside moiety or a peptide. Soaking experiments with a mixture of YEW and DiSia show that YEW competes with DiSia, suggesting that YEW can be used to block ganglioside binding. A comparison with the TeNT binding domain in complex with small molecules, BoNT/A and /B, provides insight into the different modes of ganglioside binding.     

Effects of vitamin restriction and supplementation on rat intestinal epithelial cell apoptosis

Diet influences intestinal growth and function and vitamins modulate intestinal cell turnover. We have assessed the effects of chronic, moderate (50% of control) vitamin restriction and supplementation on intestinal epithelial cell (IEC) apoptosis and the relevance of this to alterations in tissue oxidative stress and antioxidant status. Feeding a vitamin-restricted diet to male, weanling WNIN rats for 20 weeks significantly increased IEC apoptosis, but only in the villi region, as evident from increased annexin V staining, M30 positivity, histological observations, DNA ladder formation, and reduced expression of Bcl-2. This was associated with elevated levels of lipid peroxides and protein carbonyls in the intestinal mucosa despite the increased activities of superoxide dismutase, catalase, and glutathione peroxidase. Consistent with the increased oxidative stress and apoptosis, structural and functional integrity of the villi were compromised as evident from the lowered ratio of villus height:crypt depth and the decreased activities of the membrane marker enzymes alkaline phosphatase and Lys-Ala dipeptidyl aminopeptidase. These changes were reversed by supplementation with a vitamin mixture or vitamin E alone, whereas riboflavin or folic acid supplementation reduced the apoptotic rates, but only partially. Further, oxidative stress was the least in vitamin E- or vitamin mixture-supplemented rats and correlated well with their IEC apoptotic rates. Increased tissue oxidative stress seems to mediate the vitamin-restriction-induced apoptosis of the IECs in rats.     

The IFNAR1 subunit of the type I IFN receptor complex contains a functional nuclear localization sequence

A nuclear localization sequence (NLS) in the type II interferon (IFN) IFN gamma, which is responsible for the nuclear translocation of both the ligand and the alpha-subunit (IFNGR1) of the receptor complex, has previously been characterized and its role in signaling examined in detail. We have now identified an NLS in the type I IFN receptor (IFNAR) common subunit IFNAR1 from humans and show that the human IFNAR1 subunit can translocate to the nucleus following human IFN beta stimulation. An NLS in human IFNAR1 is located in the extracellular domain of IFNAR1 within the sequence (382)RKIIEKKT (numbered for the precursor form). Nuclear import by the NLS functions in a conventional fashion requiring cytosolic import factors, is energy-dependent and inhibited by the prototypical NLS of the SV40 large T-antigen. These studies provide a mechanism for nuclear import of IFNAR1, as well as for type I IFN ligands, and a starting point for studying an alternate role for IFNAR1 in nuclear signaling within the type I IFN system.