Cone beam computed tomography of plastinated hearts for instruction of radiological anatomy.

Radiological anatomy education is an important aspect of the medical curriculum. The purpose of this study was to establish and demonstrate the use of plastinated anatomical specimens, specifically human hearts, for use in radiological anatomy education. Four human hearts were processed with routine plastination procedures at room temperature. Specimens were subjected to cone beam computed tomography and a graphics program (ER3D) was applied to generate 3D cardiac models. A comparison was conducted between plastinated hearts and their corresponding computer models based on a list of morphological cardiac features commonly studied in the gross anatomy laboratory. Results showed significant correspondence between plastinations and CBCT-generated 3D models (98 %; p < .01) for external structures and 100 % for internal cardiac features, while 85 % correspondence was achieved between plastinations and 2D CBCT slices. Complete correspondence (100 %) was achieved between key observations on the plastinations and internal radiological findings typically required of medical student. All pathologic features seen on the plastinated hearts were also visualized internally with the CBCT-generated models and 2D slices. These results suggest that CBCT-derived slices and models can be successfully generated from plastinated material and provide accurate representations for radiological anatomy education.

Quantitative assessment of automated purification and concentration of E. coli bacteria.

Automated methods for rapidly purifying and concentrating bacteria from environmental interferents are needed in next-generation applications for anything from water purification to biological weapons detection. Though previous work has been performed by other researchers in this area, there is still a need to create an automated system that can both purify and concentrate target pathogens in a timely manner with readily available and replaceable components that could be easily integrated with a detection mechanism. Thus, the objective of this work was to design, build, and demonstrate the effectiveness of an automated system, the Automated Dual-filter method for Applied Recovery, or aDARE. aDARE uses a custom LABVIEW program that guides the flow of bacterial samples through a pair of size-based separation membranes to capture and elute the target bacteria. Using aDARE, we eliminated 95% of the interfering beads of a 5 mL-sample volume containing 107 CFU/mL of E. coli contaminated with 2 µm and 10 µm polystyrene beads at 106 beads/mL concentration., The target bacteria were concentrated to more than twice the initial concentration in 900 µL of eluent, resulting in an enrichment ratio for the target bacteria of 42 ± 13 in 5.5 min. These results show the feasibility and effectiveness of using size-based filtration membranes to purify and concentrate a target bacterium, in this case E. coli, in an automated system.

ESPRIT: estimating species richness using large collections of 16S rRNA pyrosequences.

Recent metagenomics studies of environmental samples suggested that microbial communities are much more diverse than previously reported, and deep sequencing will significantly increase the estimate of total species diversity. Massively parallel pyrosequencing technology enables ultra-deep sequencing of complex microbial populations rapidly and inexpensively. However, computational methods for analyzing large collections of 16S ribosomal sequences are limited. We proposed a new algorithm, referred to as ESPRIT, which addresses several computational issues with prior methods. We developed two versions of ESPRIT, one for personal computers (PCs) and one for computer clusters (CCs). The PC version is used for small- and medium-scale data sets and can process several tens of thousands of sequences within a few minutes, while the CC version is for large-scale problems and is able to analyze several hundreds of thousands of reads within one day. Large-scale experiments are presented that clearly demonstrate the effectiveness of the newly proposed algorithm. The source code and user guide are freely available at http://www.biotech.ufl.edu/people/sun/esprit.html.

Alternatively initiated gene 50/RTA transcripts expressed during murine and human gammaherpesvirus reactivation from latency.

In the process of characterizing the requirements for expression of the essential immediate-early transcriptional activator (RTA) encoded by gene 50 of murine gammaherpesvirus 68 (MHV68), a recombinant virus was generated in which the known gene 50 promoter was deleted (G50pKO). Surprisingly, the G50pKO mutant retained the ability to replicate in permissive murine fibroblasts, albeit with slower kinetics than wild-type MHV68. 5'-rapid amplification of cDNA ends analyses of RNA prepared from G50pKO-infected fibroblasts revealed a novel upstream transcription initiation site, which was also utilized during wild-type MHV68 infection of permissive cells. Furthermore, the region upstream of the distal gene 50/RTA transcription initiation site exhibited promoter activity in both permissive NIH 3T12 fibroblasts as well as in the murine macrophage cell line RAW 264.7. In addition, in RAW 264.7 cells the activity of the distal gene 50/RTA promoter was strongly upregulated (>20-fold) by treatment of the cells with lipopolysaccharide. Reverse transcriptase PCR analyses of RNA prepared from Kaposi's sarcoma-associated herpesvirus- and Epstein-Barr virus-infected B-cell lines, following induction of virus reactivation, also revealed the presence of gene 50/RTA transcripts initiating upstream of the known transcription initiation site. The latter argues that alternative initiation of gene 50/RTA transcription is a strategy conserved among murine and human gammaherpesviruses. Infection of mice with the MHV68 G50pKO demonstrated the ability of this mutant virus to establish latency in the spleen and peritoneal exudate cells (PECs). However, the G50pKO mutant was unable to reactivate from latently infected splenocytes and also exhibited a significant reactivation defect from latently infected PECs, arguing in favor of a model where the proximal gene 50/RTA promoter plays a critical role in virus reactivation from latency, particularly from B cells. Finally, analyses of viral genome methylation in the regions upstream of the proximal and distal gene 50/RTA transcription initiation sites revealed that the distal promoter is partially methylated in vivo and heavily methylated in MHV68 latently infected B-cell lines, suggesting that DNA methylation may serve to silence the activity of this promoter during virus latency.

Estrogen-related receptor alpha1 transcriptional activities are regulated in part via the ErbB2/HER2 signaling pathway.

We previously showed that (a) estrogen-related receptor alpha1 (ERRalpha1) down-modulates estrogen receptor (ER)-stimulated transcription in low ErbB2-expressing MCF-7 mammary carcinoma cells, and (b) ERRalpha and ErbB2 mRNA levels positively correlate in clinical breast tumors. We show here that ERRalpha1 represses ERalpha-mediated activation in MCF-7 cells because it failed to recruit the coactivator glucocorticoid receptor interacting protein 1 (GRIP1) when bound to an estrogen response element. In contrast, ERRalpha1 activated estrogen response element- and ERR response element-mediated transcription in ERalpha-positive, high ErbB2-expressing BT-474 mammary carcinoma cells, activation that was enhanced by overexpression of GRIP1. Likewise, regulation of the endogenous genes pS2, progesterone receptor, and ErbB2 by ERRalpha1 reflected the cell type-specific differences observed with our reporter plasmids. Importantly, overexpression of activated ErbB2 in MCF-7 cells led to transcriptional activation, rather than repression, by ERRalpha1. Two-dimensional PAGE of radiophosphate-labeled ERRalpha1 indicated that it was hyperphosphorylated in BT-474 relative to MCF-7 cells; incubation of these cells with anti-ErbB2 antibody led to reduction in the extent of ERRalpha1 phosphorylation. Additionally, mitogen-activated protein kinases (MAPK) and Akts, components of the ErbB2 pathway, phosphorylated ERRalpha1 in vitro. ERRalpha1-activated transcription in BT-474 cells was inhibited by disruption of ErbB2/epidermal growth factor receptor signaling with trastuzumab or gefitinib or inactivation of downstream components of this signaling, MAPK kinase/MAPK, and phosphatidylinositol-3-OH kinase/Akt, with U0126 or LY294002, respectively. Thus, ERRalpha1 activities are regulated, in part, via ErbB2 signaling, with ERRalpha1 likely positively feedback-regulating ErbB2 expression. Taken together, we conclude that ERRalpha1 phosphorylation status shows potential as a biomarker of clinical course and antihormonal- and ErbB2-based treatment options, with ERRalpha1 serving as a novel target for drug development.

Collaboration with Regulators to Support Quality and Accountability Following Medical Errors: The Communication and Resolution Program Certification Pilot.

OBJECTIVE: Communication and resolution programs (CRPs) involve institutions responding to adverse events using transparency with patients, event analysis, recurrence prevention, and compensation. Collaboration with regulators around CRPs could enhance health care quality. SETTING AND PARTICIPANTS: Health care institutions, liability insurers, and the Medical Quality Assurance Commission (MQAC, board of medicine) in Washington State. STUDY DESIGN: MQAC has collaborated with the Foundation for Health Care Quality (FHCQ) on the CRP Certification pilot. A panel of physicians, risk managers, and patient advocates at FHCQ will review cases for use of the CRP key elements. Cases meeting this standard will be "CRP Certified." If MQAC determines that the CRP enhanced patient safety comparable or better than board action, the Commission may close the case. PRINCIPAL FINDINGS: Developing this process identified the following issues: (1) protecting information submitted for CRP Certification; (2) determining what information the Commission needs to assess whether additional investigation is warranted; (3) preserving the Commission's responsibility to protect the public while working with health care organizations; and (4) addressing concerns that CRP Certification not shield incompetent providers. CONCLUSIONS: The CRP Certification program is a promising example of collaboration among institutions, insurers, and regulators to promote patient-centered accountability and learning following adverse events.

Tessellation analysis of glomerular spatial arrangement in mice with heritable renal hypoplasia.

Renal hypoplasia results from an insufficient kidney volume caused, in part, by a deficient number of glomeruli. The purpose of this study was to apply tessellation analysis to determine whether glomerular point patterns differed between adult normal (WT) and mutant (Br) mice with heritable renal hypoplasia and to delineate a spatial distribution accounting for the observed patterns. Kidneys from adult WT and Br mice were collected, processed with routine light histology and representative transverse sections were photographed. Cortical area and perimeter were calculated from traced tissue contours and glomeruli were identified and digitized. Voronoi tessellations were constructed and average parameters for Voronoi polygon number, area, perimeter and edge counts as well as spatial metrics comprising nearest neighbor and centroidal distances were calculated and compared. Point distributions were simulated by randomizing glomerular coordinates from each section and plotting the new points utilizing uniform random, Gaussian random, or isotropic functions. Average nearest neighbor distances were generated for each specimen and ranked with respect to corresponding values generated from 1,000 iterations for each simulated set. Results showed that WT and Br were significantly different for each parameter suggesting that WT kidneys possessed more glomeruli, but these were less clustered compared to Br. Simulations suggested that WT and Br demonstrated similar, but not identical, underlying glomerular spatial distributions. Defective gene expression in Br is important for determining glomerular number and the defective pattern likely results from a heterochronic disturbance consisting of a truncated growth trajectory during embryonic kidney development.

A gammaherpesvirus 68 gene 50 null mutant establishes long-term latency in the lung but fails to vaccinate against a wild-type virus challenge.

The gammaherpesvirus immediate-early genes are critical regulators of virus replication and reactivation from latency. Rta, encoded by gene 50, serves as the major transactivator of the lytic program and is highly conserved among all the gammaherpesviruses, including Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus, and murine gammaherpesvirus 68 (gammaHV68). Introduction of a translation stop codon in gammaHV68 gene 50 (gene 50.stop gammaHV68) demonstrated that Rta is essential for virus replication in vitro. To investigate the role that virus replication plays in the establishment and maintenance of latency, we infected mice with gene 50.stop gammaHV68. Notably, the gene 50.stop virus established a long-term infection in lung B cells following intranasal infection of mice but was unable to establish latency in the spleen. This complete block in the establishment of latency in the spleen was also seen when lytic virus production was inhibited by treating mice infected with wild-type virus with the antiviral drug cidofovir, implicating virus replication and not an independent function of Rta in the establishment of splenic latency. Furthermore, we showed that gene 50.stop gammaHV68 was unable to prime the immune system and was unable to protect against a challenge with wild-type gammaHV68, despite its ability to chronically infect lung B cells. These data indicate gammaherpesviruses that are unable to undergo lytic replication in vivo may not be viable vaccine candidates despite the detection of cells harboring viral genome at late times postinfection.

Cell type-specific replication of simian virus 40 conferred by hormone response elements in the late promoter.

The late genes of SV40 are not expressed at significant levels until after the onset of viral DNA replication. We previously identified two hormone response elements (HREs) in the late promoter that contribute to this delay. Mutants defective in these HREs overexpress late RNA at early, but not late, times after transfection of CV-1PD cells. Overexpression of nuclear receptors (NRs) that recognize these HREs leads to repression of the late promoter in a sequence-specific and titratable manner, resulting in a delay in late gene expression. These observations led to a model in which the late promoter is repressed at early times after infection by NRs, with this repression being relieved by titration of these repressors through simian virus 40 (SV40) genome replication to high copy number. Here, we tested this model in the context of the viral life cycle. SV40 genomes containing mutations in either or both HREs that significantly reduce NR binding without altering the coding of any proteins were constructed. Competition for replication between mutant and wild-type viruses in low-multiplicity coinfections indicated that the +1 HRE offered a significant selective advantage to the virus within a few cycles of infection in African green monkey kidney cell lines CV-1, CV-1P, TC-7, MA-134, and Vero but not in CV-1PD' cells. Interestingly, the +55 HRE offered a selective disadvantage in MA-134 cells but had no effect in CV-1, CV-1P, TC-7, Vero, and CV-1PD' cells. Thus, we conclude that these HREs are biologically important to the virus, but in a cell type-specific manner.

Hormone response element in SV40 late promoter directly affects synthesis of early as well as late viral RNAs.

We previously demonstrated that the presence of a hormone response element surrounding the transcription initiation site of the SV40 major late promoter (+1 HRE) confers a replication advantage to the virus in a cell-type-specific manner. We determine here the mechanism by which the +1 HRE confers this advantage by analyzing in detail the various stages of the viral life cycle of wild-type versus a +1 HRE mutant in MA-134 cells. We show that the mutant overexpresses late genes at the expense of early genes at early times after infection. This initial underproduction of early RNA leads, subsequently, to an underproduction of large T-antigen, viral DNA, and infectious virions. We conclude that the +1 HRE is necessary for the proper initial regulation of transcription from the early as well as late promoter so the cascade of subsequent events can be executed for the optimal production of virions.

Estrogen-related receptor alpha 1 actively antagonizes estrogen receptor-regulated transcription in MCF-7 mammary cells.

The estrogen-related receptor alpha (ERRalpha) is an orphan member of the nuclear receptor superfamily. We show that the major isoform of the human ERRalpha gene, ERRalpha1, can sequence-specifically bind a consensus palindromic estrogen response element (ERE) and directly compete with estrogen receptor alpha (ERalpha) for binding. ERRalpha1 activates or represses ERE-regulated transcription in a cell type-dependent manner, repressing in ER-positive MCF-7 cells while activating in ER-negative HeLa cells. Thus, ERRalpha1 can function both as a modulator of estrogen responsiveness and as an estrogen-independent activator. Repression likely occurs in the absence of exogenous ligand since charcoal treatment of the serum had no effect on silencing activity. Mutational analysis revealed that repression is not simply the result of competition between ERalpha and ERRalpha1 for binding to the DNA. Rather, it also requires the presence of sequences within the carboxyl-terminal E/F domain of ERRalpha1. Thus, ERRalpha1 can function as either an active repressor or a constitutive activator of ERE-dependent transcription. We hypothesize that ERRalpha1 can play a critical role in the etiology of some breast cancers, thereby providing a novel therapeutic target in their treatment.