The Online Bioinformatics Resources Collection at the University of Pittsburgh Health Sciences Library System—a one-stop gateway to online bioinformatics databases and software tools

To bridge the gap between the rising information needs of biological and medical researchers and the rapidly growing number of online bioinformatics resources, we have created the Online Bioinformatics Resources Collection (OBRC) at the Health Sciences Library System (HSLS) at the University of Pittsburgh. The OBRC, containing 1542 major online bioinformatics databases and software tools, was constructed using the HSLS content management system built on the Zope(®) Web application server. To enhance the output of search results, we further implemented the Vivísimo Clustering Engine(®), which automatically organizes the search results into categories created dynamically based on the textual information of the retrieved records. As the largest online collection of its kind and the only one with advanced search results clustering, OBRC is aimed at becoming a one-stop guided information gateway to the major bioinformatics databases and software tools on the Web. OBRC is available at the University of Pittsburgh's HSLS Web site ()

Identifying Molecular Signatures of Distinct Modes of Collective Migration in Response to the Microenvironment Using Three-Dimensional Breast Cancer Models

Collective cell migration is a key feature of transition of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) among many other cancers, yet the microenvironmental factors and underlying mechanisms that trigger collective migration remain poorly understood. Here, we investigated two microenvironmental factors, tumor-intrinsic hypoxia and tumor-secreted factors (secretome), as triggers of collective migration using three-dimensional (3D) discrete-sized microtumor models that recapitulate hallmarks of DCIS-IDC transition. Interestingly, the two factors induced two distinct modes of collective migration: directional and radial migration in the 3D microtumors generated from the same breast cancer cell line model, T47D. Without external stimulus, large (600 µm) T47D microtumors exhibited tumor-intrinsic hypoxia and directional migration, while small (150 µm), non-hypoxic microtumors exhibited radial migration only when exposed to the secretome of large microtumors. To investigate the mechanisms underlying hypoxia- and secretome-induced directional vs. radial migration modes, we performed differential gene expression analysis of hypoxia- and secretome-induced migratory microtumors compared with non-hypoxic, non-migratory small microtumors as controls. We propose unique gene signature sets related to tumor-intrinsic hypoxia, hypoxia-induced epithelial-mesenchymal transition (EMT), as well as hypoxia-induced directional migration and secretome-induced radial migration. Gene Set Enrichment Analysis (GSEA) and protein-protein interaction (PPI) network analysis revealed enrichment and potential interaction between hypoxia, EMT, and migration gene signatures for the hypoxia-induced directional migration. In contrast, hypoxia and EMT were not enriched in the secretome-induced radial migration, suggesting that complete EMT may not be required for radial migration. Survival analysis identified unique genes associated with low survival rate and poor prognosis in TCGA-breast invasive carcinoma dataset from our tumor-intrinsic hypoxia gene signature (CXCR4, FOXO3, LDH, NDRG1), hypoxia-induced EMT gene signature (EFEMP2, MGP), and directional migration gene signature (MAP3K3, PI3K3R3). NOS3 was common between hypoxia and migration gene signature. Survival analysis from secretome-induced radial migration identified ATM, KCNMA1 (hypoxia gene signature), and KLF4, IFITM1, EFNA1, TGFBR1 (migration gene signature) to be associated with poor survival rate. In conclusion, our unique 3D cultures with controlled microenvironments respond to different microenvironmental factors, tumor-intrinsic hypoxia, and secretome by adopting distinct collective migration modes and their gene expression analysis highlights the phenotypic heterogeneity and plasticity of epithelial cancer cells

Temporal transcriptional response to latency reversing agents identifies specific factors regulating HIV-1 viral transcriptional switch

Background: Latent HIV-1 reservoirs are identified as one of the major challenges to achieve HIV-1 cure. Currently available strategies are associated with wide variability in outcomes both in patients and CD4+ T cell models. This underlines the critical need to develop innovative strategies to predict and recognize ways that could result in better reactivation and eventual elimination of latent HIV-1 reservoirs. Results and discussion: In this study, we combined genome wide transcriptome datasets post activation with Systems Biology approach (Signaling and Dynamic Regulatory Events Miner, SDREM analyses) to reconstruct a dynamic signaling and regulatory network involved in reactivation mediated by specific activators using a latent cell line. This approach identified several critical regulators for each treatment, which were confirmed in follow-up validation studies using small molecule inhibitors. Results indicate that signaling pathways involving JNK and related factors as predicted by SDREM are essential for virus reactivation by suberoylanilide hydroxamic acid. ERK1/2 and NF-κB pathways have the foremost role in reactivation with prostratin and TNF-aα, respectively. JAK-STAT pathway has a central role in HIV-1 transcription. Additional evaluation, using other latent J-Lat cell clones and primary T cell model, also confirmed that many of the cellular factors associated with latency reversing agents are similar, though minor differences are identified. JAK-STAT and NF-κB related pathways are critical for reversal of HIV-1 latency in primary resting T cells. Conclusion: These results validate our combinatorial approach to predict the regulatory cellular factors and pathways responsible for HIV-1 reactivation in latent HIV-1 harboring cell line models. JAK-STAT have a role in reversal of latency in all the HIV-1 latency models tested, including primary CD4+ T cells, with additional cellular pathways such as NF-κB, JNK and ERK 1/2 that may have complementary role in reversal of HIV-1 latency

A study of the interaction of a 67kDa glycoprotein (p67) with eukaryotic initiation factor 2 (eIF-2) and other cellular proteins

The eukaryotic initiation factor 2 (eIF-2) is a hetero-trimer, $\alpha\beta\gamma.$ The eIF-2 kinases (HRI and PKR) phosphorylate specifically the $\alpha$ subunit of eIF-2. This inactivates eIF-2 activity and inhibits protein synthesis. A 67 kDa glycoprotein (p\sp{67}) protects eIF-2 $\alpha$ subunit from inhibitory phosphorylation and thus promotes protein synthesis in the presence of active eIF-2 kinase(s). We have now studied the interactions between the eIF-2 subunits and p67 using the yeast two-hybrid system. We expressed the cDNAs for each subunits of eIF-2 and p\sp{67} in yeast as a fusion with the DNA binding or the transcription activation domain of the yeast GAL4 transcription activator. We report that (i) eIF-2 $\gamma$ interacted with all three polypeptides eIF-2 $\alpha,\ \beta$ and p\sp{67}, (ii) No interaction was observed between eIF-2 $\alpha,$ eIF-2 $\beta$ and p\sp{67}, (iii) Only p\sp{67} formed an oligomer. eIF-2 $\alpha,$ eIF-2 $\beta$ and eIF-2 $\gamma$ did not form oligomers. Molecular weight determination using Sephacryl S-300 gel filtration chromatography revealed that p\sp{67} exits as a dimer. The two hybrid system was also used to screen for binding proteins of the eIF-2 associated 67 kDa glycoprotein, p\sp{67}. The plasmid pGBT9-p\sp{67}, having a full length rat p\sp{67} fused to the DNA binding domain of GAL4 transcription factor, was prepared. The two hybrid mouse liver cDNA library containing cDNAs fused to the GAL4 trans-activation domain was used. Yeast cells (HF7c) were transformed with the plasmids and screened for clones, whose translational products interact with p\sp{67}. Positive clones were obtained and their DNA sequences were partially determined. Two clones (clone 1 and 4) showed strong sequence identity with rat p\sp{67}. Sequence analysis revealed that the clone 1 shares 98% sequence identity with the amino acid sequence 39-110 of rat p\sp{67} and the clone 4 shares 97% sequence identity with the amino acid sequence 87-151 of rat p\sp{67}. One clone (clone 3) showed 93% sequence identity to the heat shock cognate protein HSc 70 (319-406 aa). The significance of p\sp{67}: HSc 70 interaction was further studied using in vitro experiments. The results show: (i) p\sp{67} bound to HSc 70 and was co-immunoprecipitated with the HSc 70 monoclonal antibodies. (ii) A p\sp{67} deglycosylase activated in heme-deficient reticulocyte lysate, rapidly deglycosylated exogenously added p\sp{67}. Addition of HSc 70 significantly protected p\sp{67} from this deglycosylation reaction. We suggest that HSc 70 binds to p\sp{67} inside the cells and protects p\sp{67} against deglycosylation and subsequent degradation