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Objectives: Reliable biomarkers for renal fibrosis are needed for clinical care and for research. Existing non-invasive biomarkers are imprecise, which has limited their utility. Methods: We developed a method to quantify fibrosis by subject size-adjusted CT Hounsfield units. This was accomplished using CT measurements of renal cortex in previously irradiated non-human primates. Results: Renal cortex mean CT Hounsfield units that were adjusted for body size had a very good direct correlation with renal parenchymal fibrosis, with an area under the curve of 0.93. Conclusions: This metric is a promising and simple non-invasive biomarker for renal fibrosis. [ABSTRACT FROM AUTHOR]

We investigated the cytogenetic and gene expression responses of peripheral blood cells of non-human primates (NHP, Macaca mulatta) that were whole-thorax irradiated with a single dose of 10 Gy. In this model, partial irradiation of NHPs in the thoracic region (Whole Thorax Lung Irradiation, WTLI) allows the study of late radiation-induced lung injury, while avoiding acute radiation syndromes related to hematopoietic and gastrointestinal injury. A transient drop in circulating lymphocytes and platelets was seen by 9 days, followed by elevations in respiratory rate, circulating neutrophils, lymphocytes, and monocytes at 60–100 days, corresponding to computed tomography (CT) and histologic evidence of pneumonitis, and elective euthanasia of four animals. To evaluate long-term DNA damage in NHP peripheral blood lymphocytes after 10 Gy WTLI, we used the cytokinesis-block micronucleus (CBMN) assay to measure chromosomal aberrations as post-mitotic micronuclei in blood samples collected up to 8 months after irradiation. Regression analysis showed significant induction of micronuclei in NHP blood cells that persisted with a gradual decline over the 8-month study period, suggesting long-term DNA damage in blood lymphocytes after WTLI. We also report transcriptomic changes in blood up to 30 days after WTLI. We isolated total RNA from peripheral blood at 3 days before and then at 2, 5 and 30 days after irradiation. We identified 1187 transcripts that were significantly changed across the 30-day time course. From changes in gene expression, we identified biological processes related to immune responses, which persisted across the 30-day study. Response to oxygen-containing compounds and bacteria were implicated by gene-expression changes at the earliest day 2 and latest, day 30 time-points. Gene expression changes suggest a persistent altered state of the immune system, specifically response to infection, for at least a month after WTLI. [ABSTRACT FROM AUTHOR]

Genome packaging for viruses with segmented genomes is often a complex problem. This is particularly true for influenza viruses and other orthomyxoviruses, whose genome consists of multiple negative-sense RNAs encapsidated as ribonucleoprotein (RNP) complexes. To better understand the structural features of orthomyxovirus RNPs that allow them to be packaged, we determined the crystal structure of the nucleoprotein (NP) of a fish orthomyxovirus, the infectious salmon anemia virus (ISAV) (genus Isavirus). As the major protein component of the RNPs, ISAV-NP possesses a bi-lobular structure similar to the influenza virus NP. Because both RNA-free and RNA-bound ISAV NP forms stable dimers in solution, we were able to measure the NP RNA binding affinity as well as the stoichiometry using recombinant proteins and synthetic oligos. Our RNA binding analysis revealed that each ISAV-NP binds ∼12 nts of RNA, shorter than the 24–28 nts originally estimated for the influenza A virus NP based on population average. The 12-nt stoichiometry was further confirmed by results from electron microscopy and dynamic light scattering. Considering that RNPs of ISAV and the influenza viruses have similar morphologies and dimensions, our findings suggest that NP-free RNA may exist on orthomyxovirus RNPs, and selective RNP packaging may be accomplished through direct RNA-RNA interactions. [ABSTRACT FROM AUTHOR]

The blood-brain-barrier (BBB) presents a significant obstacle to the delivery of systemically administered chemotherapeutics for the treatment of brain cancer. Irreversible electroporation (IRE) is an emerging technology that uses pulsed electric fields for the non-thermal ablation of tumors. We hypothesized that there is a minimal electric field at which BBB disruption occurs surrounding an IRE-induced zone of ablation and that this transient response can be measured using gadolinium (Gd) uptake as a surrogate marker for BBB disruption. The study was performed in a Good Laboratory Practices (GLP) compliant facility and had Institutional Animal Care and Use Committee (IACUC) approval. IRE ablations were performed in vivo in normal rat brain (n = 21) with 1-mm electrodes (0.45 mm diameter) separated by an edge-to-edge distance of 4 mm. We used an ECM830 pulse generator to deliver ninety 50-µs pulse treatments (0, 200, 400, 600, 800, and 1000 V/cm) at 1 Hz. The effects of applied electric fields and timing of Gd administration (-5, +5, +15, and +30 min) was assessed by systematically characterizing IRE-induced regions of cell death and BBB disruption with 7.0-T magnetic resonance imaging (MRI) and histopathologic evaluations. Statistical analysis on the effect of applied electric field and Gd timing was conducted via Fit of Least Squares with α = 0.05 and linear regression analysis. The focal nature of IRE treatment was confirmed with 3D MRI reconstructions with linear correlations between volume of ablation and electric field. Our results also demonstrated that IRE is an ablation technique that kills brain tissue in a focal manner depicted by MRI (n = 16) and transiently disrupts the BBB adjacent to the ablated area in a voltage-dependent manner as seen with Evan's Blue (n = 5) and Gd administration. [ABSTRACT FROM AUTHOR]

To replicate in mammalian hosts, bacterial pathogens must acquire iron. The majority of iron is coordinated to the protoporphyrin ring of heme, which is further bound to hemoglobin. Pathogenic bacteria utilize secreted hemophores to acquire heme from heme sources such as hemoglobin. Bacillus anthracis, the causative agent of anthrax disease, secretes two hemophores, IsdX1 and IsdX2, to acquire heme from host hemoglobin and enhance bacterial replication in iron-starved environments. Both proteins contain NEAr-iron Transporter (NEAT) domains, a conserved protein module that functions in heme acquisition in Gram-positive pathogens. Here, we report the structure of IsdX1, the first of a Gram-positive hemophore, with and without bound heme. Overall, IsdX1 forms an immunoglobin-like fold that contains, similar to other NEAT proteins, a 310-helix near the heme-binding site. Because the mechanistic function of this helix in NEAT proteins is not yet defined, we focused on the contribution of this region to hemophore and NEAT protein activity, both biochemically and biologically in cultured cells. Site-directed mutagenesis of amino acids in and adjacent to the helix identified residues important for heme and hemoglobin association, with some mutations affecting both properties and other mutations affecting only heme stabilization. IsdX1 with mutations that reduced the ability to associate with hemoglobin and bind heme failed to restore the growth of a hemophore-deficient strain of B. anthracis on hemoglobin as the sole iron source. These data indicate that not only is the 310-helix important for NEAT protein biology, but also that the processes of hemoglobin and heme binding can be both separate as well as coupled, the latter function being necessary for maximal heme-scavenging activity. These studies enhance our understanding of NEAT domain and hemophore function and set the stage for structure-based inhibitor design to block NEAT domain interaction with upstream ligands. [ABSTRACT FROM AUTHOR]

The article presents a study which evaluates the teaching strategy named research deconstruction in providing evidence-based science instruction to 1st and 2nd year college students. The research deconstruction course with an hour-long seminar was demonstrated for 10 days, which allowed the students identify hypothesis, experimental approaches, and analyze data. The authors conclude that strategy is efficient in offering evidence-based science education to a big number of early-stage students.

Background: A major challenge in oncology is the selection of the most effective chemotherapeutic agents for individual patients, while the administration of ineffective chemotherapy increases mortality and decreases quality of life in cancer patients. This emphasizes the need to evaluate every patient's probability of responding to each chemotherapeutic agent and limiting the agents used to those most likely to be effective. Methods and Results: Using gene expression data on the NCI-60 and corresponding drug sensitivity, mRNA and microRNA profiles were developed representing sensitivity to individual chemotherapeutic agents. The mRNA signatures were tested in an independent cohort of 133 breast cancer patients treated with the TFAC (paclitaxel, 5-fluorouracil, adriamycin, and cyclophosphamide) chemotherapy regimen. To further dissect the biology of resistance, we applied signatures of oncogenic pathway activation and performed hierarchical clustering. We then used mRNA signatures of chemotherapy sensitivity to identify alternative therapeutics for patients resistant to TFAC. Profiles from mRNA and microRNA expression data represent distinct biologic mechanisms of resistance to common cytotoxic agents. The individual mRNA signatures were validated in an independent dataset of breast tumors (P = 0.002, NPV = 82%). When the accuracy of the signatures was analyzed based on molecular variables, the predictive ability was found to be greater in basal-like than non basal-like patients (P = 0.03 and P = 0.06). Samples from patients with co-activated Myc and E2F represented the cohort with the lowest percentage (8%) of responders. Using mRNA signatures of sensitivity to other cytotoxic agents, we predict that TFAC non-responders are more likely to be sensitive to docetaxel (P = 0.04), representing a viable alternative therapy. Conclusions: Our results suggest that the optimal strategy for chemotherapy sensitivity prediction integrates molecular variables such as ER and HER2 status with corresponding microRNA and mRNA expression profiles. Importantly, we also present evidence to support the concept that analysis of molecular variables can present a rational strategy to identifying alternative therapeutic opportunities. [ABSTRACT FROM AUTHOR]

The chemotherapeutic doxorubicin (DOX) induces DNA double-strand break (DSB) damage. In order to identify conserved genes that mediate DOX resistance, we screened the Saccharomyces cerevisiae diploid deletion collection and identified 376 deletion strains in which exposure to DOX was lethal or severely reduced growth fitness. This diploid screen identified 5-fold more DOX resistance genes than a comparable screen using the isogenic haploid derivative. Since DSB damage is repaired primarily by homologous recombination in yeast, and haploid cells lack an available DNA homolog in G1 and early S phase, this suggests that our diploid screen may have detected the loss of repair functions in G1 or early S phase prior to complete DNA replication. To test this, we compared the relative DOX sensitivity of 30 diploid deletion mutants identified under our screening conditions to their isogenic haploid counterpart, most of which (n = 26) were not detected in the haploid screen. For six mutants (bem1Delta, ctf4Delta, ctk1Delta, hfi1Delta,nup133Delta, tho2Delta) DOX-induced lethality was absent or greatly reduced in the haploid as compared to the isogenic diploid derivative. Moreover, unlike WT, all six diploid mutants displayed severe G1/S phase cell cycle progression defects when exposed to DOX and some were significantly enhanced (ctk1Delta and hfi1Delta) or deficient (tho2Delta) for recombination. Using these and other "THO2-like" hypo-recombinogenic, diploid-specific DOX sensitive mutants (mft1Delta, thp1Delta, thp2Delta) we utilized known genetic/proteomic interactions to construct an interactive functional genomic network which predicted additional DOX resistance genes not detected in the primary screen. Most (76%) of the DOX resistance genes detected in this diploid yeast screen are evolutionarily conserved suggesting the human orthologs are candidates for mediating DOX resistance by impacting on checkpoint and recombination functions in G1 and/or early S phases.

BRCA1 has been implicated in numerous DNA repair pathways that maintain genome integrity, however the function responsible for its tumor suppressor activity in breast cancer remains obscure. To identify the most highly conserved of the many BRCA1 functions, we screened the evolutionarily distant eukaryote Saccharomyces cerevisiae for mutants that suppressed the G1 checkpoint arrest and lethality induced following heterologous BRCA1 expression. A genome-wide screen in the diploid deletion collection combined with a screen of ionizing radiation sensitive gene deletions identified mutants that permit growth in the presence of BRCA1. These genes delineate a metabolic mRNA pathway that temporally links transcription elongation (SPT4, SPT5, CTK1, DEF1) to nucleopore-mediated mRNA export (ASM4, MLP1, MLP2, NUP2, NUP53, NUP120, NUP133, NUP170, NUP188, POM34) and cytoplasmic mRNA decay at P-bodies (CCR4, DHH1). Strikingly, BRCA1 interacted with the phosphorylated RNA polymerase II (RNAPII) carboxy terminal domain (P-CTD), phosphorylated in the pattern specified by the CTDK-I kinase, to induce DEF1-dependent cleavage and accumulation of a RNAPII fragment containing the P-CTD. Significantly, breast cancer associated BRCT domain defects in BRCA1 that suppressed P-CTD cleavage and lethality in yeast also suppressed the physical interaction of BRCA1 with human SPT5 in breast epithelial cells, thus confirming SPT5 as a relevant target of BRCA1 interaction. Furthermore, enhanced P-CTD cleavage was observed in both yeast and human breast cells following UV-irradiation indicating a conserved eukaryotic damage response. Moreover, P-CTD cleavage in breast epithelial cells was BRCA1-dependent since damage-induced P-CTD cleavage was only observed in the mutant BRCA1 cell line HCC1937 following ectopic expression of wild type BRCA1. Finally, BRCA1, SPT5 and hyperphosphorylated RPB1 form a complex that was rapidly degraded following MMS treatment in wild type but not BRCA1 mutant breast cells. These results extend the mechanistic links between BRCA1 and transcriptional consequences in response to DNA damage and suggest an important role for RNAPII P-CTD cleavage in BRCA1-mediated cancer suppression.