Generation, annotation, evolutionary analysis, and database integration of 20,000 unique sea urchin EST clusters

Together with the hemichordates, sea urchins represent basal groups of nonchordate invertebrate deuterostomes that occupy a key position in bilaterian evolution. Because sea urchin embryos are also amenable to functional studies, the sea urchin system has emerged as one of the leading models for the analysis of the function of genomic regulatory networks that control development. We have analyzed a total of 107,283 cDNA clones of libraries that span the development of the sea urchin Strongylocentrotus purpuratus. Normalization by oligonucleotide fingerprinting, EST sequencing and sequence clustering resulted in an EST catalog comprised of 20,000 unique genes or gene fragments. Around 7000 of the unique EST consensus sequences were associated with molecular and developmental functions. Phylogenetic comparison of the identified genes to the genome of the urochordate Ciona intestinalis indicate that at least one quarter of the genes thought to be chordate specific were already present at the base of deuterostome evolution. Comparison of the number of gene copies in sea urchins to those in chordates and vertebrates indicates that the sea urchin genome has not undergone extensive gene or complete genome duplications. The established unique gene set represents an essential tool for the annotation and assembly of the forthcoming sea urchin genome sequence. All cDNA clones and filters of all analyzed libraries are available from the resource center of the German genome project at http://www.rzpd.de.     

Values and ethics: a collection of curricular reforms for a new generation of physicians.

In recent years, medical educators have expressed concern that the reductionist-positivist mode of medical education fails to equip physicians with the skills and attitudes to meet the full range of patients' physical and emotional needs. Indeed, the authors suggest that neither patients nor physicians are satisfied. Among the factors responsible are a pervasive industrialization of clinical practice, a progressive segmentation of patient care, and a deepening shortage of both primary care and specialty physicians. But underlying these system issues is a lack of adequate schooling in the values, ethics, and culture of caring. Today's physicians must simultaneously be analytical, perceptive, and self-reflective. They must have the capacity to see their patients as individuals with differing psychological, social, and historical natures. And they must have insight into their own values and behaviors. All of this contributes to making a competent and humane physician. To aid medical students in achieving these characteristics, the authors contend that medical education must be radically restructured so that knowledge and skills are taught within the context of values and ethics. This commentary explores such reform through the lens of three articles published in the current issue of Academic Medicine, by Litzelman and Cottingham, Kanter and colleagues, and Dobie. These articles are the product of a national call that resulted in more than thirty abstracts, testimony to the fertile thinking already being applied to this problem. It is the authors' hope that this series of papers will stimulate still more thinking and lead to the curricular reform that future generations of physicians deserve.     

Proximal tubular epithelial cell integrins respond to high glucose by altered cell-matrix interactions and differentially regulate matrixin expression

Thickening of the tubular basement membrane (TBM) occurs in diabetic nephropathy, but the effects of high glucose on the functional aspects of proximal tubular epithelial cells are not clearly understood. In the present study, we examined the effects of elevated glucose concentrations on (a) integrin expression by human proximal tubular epithelial cells (HK-2) and integrin-mediated interactions with type IV collagen (colIV) and laminin, major components of TBM; (b) the expression of matrixins/matrix metalloproteinases (MMPs), which is regulated by integrins; and (c) the expression of tissue inhibitors of metalloproteinases (TIMPs). HK-2 cells cultured in 25 mM glucose underwent a reduction of the expression of alpha3, beta1, alpha(v)beta3, and alpha5 integrin subunits, with a concomitant increase of the alpha2 subunit, compared with cells grown in 5 mM glucose. Adhesion experiments demonstrated that high glucose led to increased cell adhesion on either colIV or laminin. Experiments of competition of adhesion using anti-integrin antibodies indicated that HK-2 cells in 5 mM glucose used mainly alpha(v)beta3 and alpha5beta1 integrins to adhere to colIV, whereas in 25 mM glucose they additionally used alpha2beta1. In the case of laminin, a beta1-mediated adhesion was observed when HK-2 cells were in 5 mM glucose, whereas in 25 mM glucose, alpha2beta1 and alpha(v)beta3 were also involved. Elevated glucose concentrations resulted in decreased expression of MMP-9 and MMP-2, whereas an increase in TIMP-1 and a decrease in TIMP-2 expression were observed. We also examined which integrins mediated the expression and secretion of matrixins MMP-2 and MMP-9. Ligation of alpha3beta1 with mAbs resulted in induction of MMP-2 expression and secretion, whereas antibody ligation of alpha(v)beta3 led to down-regulation of MMP-9. The above data implicate integrins of proximal tubular epithelial cells in the regulation of MMPs and in the development of TBM thickening in diabetic nephropathy.     

High rates of clustering of strains causing tuberculosis in Harare, Zimbabwe: a molecular epidemiological study

We examined the pattern of tuberculosis (TB) transmission (i.e., reactivation versus recent transmission) and the impact of human immunodeficiency virus (HIV) infection in Harare, Zimbabwe. Consecutive adult smear-positive pulmonary TB patients presenting to an urban hospital in Harare were enrolled. A detailed epidemiological questionnaire was completed, and tests for HIV type 1 and CD4 cell counts were performed for each patient. Molecular fingerprinting of the genomic DNA recovered from cultures of sputum was performed by two molecular typing methods: spacer oligonucleotide typing (spoligotyping) and analysis of variable number of tandem DNA repeats (VNTRs). A cluster was defined as isolates from two or more patients that shared the same spoligotype pattern or the same VNTR pattern, or both. DNA suitable for typing was recovered from 224 patients. The prevalence of HIV infection was 79%. Of 187 patient isolates (78.6%) typed by both spoligotyping and analysis of VNTRs, 147 were identified as part of a cluster by both methods. By spoligotyping alone, 84.1% of patient isolates were grouped into 20 clusters. The cluster size was generally <8 patient isolates, although three large clusters comprised 68, 25, and 23 patient isolates. A total of 89.4% of the patient isolates grouped into 12 clusters defined by analysis of VNTRs, with 2 large clusters consisting of 127 and 13 patient isolates, respectively. Thirty-six percent of patient isolates with a shared spoligotype and 17% with a shared VNTR pattern were geographically linked within Harare, but they were not linked on the basis of the patient's home district. In a multivariate analysis, there were no independent predictors of clustering, including HIV infection status. Comparison with the International Spoligotype database (Pasteur Institute, Pointe a Pitre, Guadeloupe) demonstrated that our three largest spoligotype clusters are well recognized and ubiquitous in Africa. In this epidemiologically well characterized urban population with a high prevalence of HIV infection, we identified a very high level of strain clustering, indicating substantial ongoing recent TB transmission. Geographic linkage could be detected in a proportion of these clusters. A small group of actively circulating strains accounted for most of the cases of TB transmission.     

Pre-clinical Study of <Superscript>213</Superscript>Bi Labeled PAI2 for the Control of Micrometastatic Pancreatic Cancer

Purpose: The urokinase plasminogen activator (uPA) and its receptor (uPAR) are expressed by pancreatic cancer cells and can be targeted by the plasminogen activator inhibitor type 2 (PAI2). We have labeled PAI2 with ²¹³Bi to form the alpha conjugate (AC), and have studied its in vitro cytotoxicity and in vivo efficacy. Methods and Materials: The expression of uPA/uPAR on pancreatic cell lines, human pancreatic cancer tissues, lymph node metastases, and mouse xenografts were detected by immunohistochemistry, confocal microscopy, and flow cytometry. Cytotoxicity was assessed by the MTS and TUNEL assay. At 2 days post-cancer cell subcutaneous inoculation, mice were injected with AC by local or systemic injection. Results: uPA/uPAR is strongly expressed on pancreatic cancer cell lines and cancer tissues. The AC can target and kill cancer cells in vitro in a concentration-dependent fashion. Some 90% of TUNEL positive cells were found after incubation with 1.2 MBq/ml of AC. A single local injection of ~222 MBq/kg 2 days post-cell inoculation can completely inhibit tumor growth over 12 weeks, and an intraperitoneal injection of 111 MBq/kg causes significant tumor growth delay. Conclusions: ²¹³Bi-PAI2 can specifically target pancreatic cancer cells in vitro and inhibit tumor growth in vivo. ²¹³Bi-PAI2 may be a useful agent for the treatment of post-surgical pancreatic cancer patients with minimum residual disease.     

Silencing of Fas, but not caspase-8, in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis

Apoptosis and inflammation play an important role in the pathogenesis of direct/pulmonary acute lung injury (ALI). However, the role of the Fas receptor-driven apoptotic pathway in indirect/nonpulmonary ALI is virtually unstudied. We hypothesized that if Fas or caspase-8 plays a role in the induction of indirect ALI, their local silencing using small interfering RNA (siRNA) should be protective in hemorrhage-induced septic ALI. Initially, as a proof of principle, green fluorescent protein-siRNA was administered intratracheally into transgenic mice overexpressing green fluorescent protein. Twenty-four hours after siRNA delivery, lung sections revealed a significant decrease in green fluorescence. Intratracheally administered Cy-5-labeled Fas-siRNA localized primarily in pulmonary epithelial cells. Intratracheal instillation of siRNA did not induce lung inflammation via toll-like receptor or protein kinase PKR pathways as assessed by lung tissue interferon-alpha, tumor necrosis factor-alpha, and interleukin (IL)-6 levels. Mice subjected to hemorrhagic shock and sepsis received either Fas-, caspase-8-, or control-siRNA intratracheally 4 hours after hemorrhage. Fas- or caspase-8-siRNA significantly reduced lung tissue Fas or caspase-8 mRNA, respectively. Only Fas-siRNA markedly diminished lung tissue tumor necrosis factor-alpha, IL-6, IL-10, interferon-gamma, IL-12, and caspase-3 activity. Fas-siRNA also preserved alveolar architecture and reduced lung neutrophil infiltration and pulmonary epithelial apoptosis. These data indicate the pathophysiological significance of Fas activation in nonpulmonary/shock-induced ALI and the feasibility of intrapulmonary administration of anti-apoptotic siRNA in vivo.     

The time course of glycogen depletion in single fibers of chronically stimulated rabbit fast-twitch muscle

A time course study was conducted to investigate the possibility of a relationship between fiber degeneration and glycogen depletion in chronically nerve-stimulated extensor digitorum longus muscle of the rabbit. Muscles were stimulated 12 h daily at 10 Hz using alternating one-hour periods of stimulation and rest. When measured for the first time after 3 h (1 h stimulation, 1 h rest, 1 h stimulation), microphotometry revealed complete glycogen depletion of all fiber types (fast glycolytic, FG; fast oxidative glycolytic, FOG; slow oxidative, SO). Different responses were noted beginning at day 4. At this time point, all FOG and SO fibers recovered their glycogen stores with some of the FOG population attaining levels higher than the FOG fibers in the unstimulated, contralateral muscle. Approximately 28% of the FG fibers recovered to normal glycogen values, whereas 58% remained depleted and 14% displayed overshoting glycogen levels. Fifteen percent of all fibers were glycogen-depleted after 12 days of stimulation. At this time, classic fiber types could no longer be distinguished. Fiber degeneration, which was recognized by the invasion of nonmuscle cells, began after 6 days and was restricted to the glycogen-depleted fibers. By this time, there was also a significant increase in DNA content. Exhaustions of glycogen, the main fuel of the FG fibers, is believed to cause a collapse of energy-supply and ATP-driven ionic pumps. The latter could be the initial step of fiber deterioration.     

Immediate breast reconstruction-impact on radiation management

Breast reconstruction is an option for women undergoing modified radical mastectomy due to a diagnosis of breast cancer. In certain patients, breast reconstruction is performed by insertion of a temporary tissue expander prior to the placement of permanent breast implants. Some of these patients, following mastectomy, may require chest wall irradiation to prevent loco regional relapse. The compatibility of radiation and tissue expanders placed in the chest wall is of major concern to the radiation oncologist. Clinically undetectable changes can occur in the tissue expander during the course of radiation therapy. This can lead to radiation treatment set-up changes, variation in tissue expansion resulting in unwanted cosmesis, and deviation from the prescribed radiation dose leading to over and/or under dosing of tumor burden. At Howard University hospital, a CT scan was utilized to evaluate the status of the temporary tissue expander during radiation treatment to enable us to prevent radiation treatment related complications resulting from dosimetric discrepancies. CT images of the tissue expander were obtained through the course of treatment. To avoid a 'geographic miss' the amount of fluid injected into the tissue expander was kept constant following patient's satisfaction with the size of the breast mound. The CT scans allowed better visualization of the prosthesis and its relation to the surrounding tumor bed. This technique ensured that anatomical changes occurring during radiation treatment, if any, were minimized. Repeated dosimetry evaluations showed no changes to the prescribed dose distribution. A CT of the reconstructed breast provides an important quality control. Further studies with greater number of patients are required for confirming this impact on radiation treatment.     

Procedure for Fluorescent-Antibody Staining of Virus-Infected Cell Cultures in Plastic Plates

Acetone fixation and fluorescent-antibody staining of virus-infected cell cultures were performed in plastic plates. Proper addition of acetone as a fixative did not alter the plastic.