Faculty Bibliography

A recent study presents a technique allowing one to image transcription from a single gene copy in live cells, and highlights the dynamic nature of transcriptional regulation.

Membrane proteins are critical to cell physiology, playing roles in signaling, trafficking, transport, adhesion, and recognition. Despite their relative abundance in the proteome and their prevalence as targets of therapeutic drugs, structural information about membrane proteins is in short supply. This chapter describes the use of electron crystallography as a tool for determining membrane protein structures. Electron crystallography offers distinct advantages relative to the alternatives of X-ray crystallography and NMR spectroscopy. Namely, membrane proteins are placed in their native membranous environment, which is likely to favor a native conformation and allow changes in conformation in response to physiological ligands. Nevertheless, there are significant logistical challenges in finding appropriate conditions for inducing membrane proteins to form two-dimensional arrays within the membrane and in using electron cryo-microscopy to collect the data required for structure determination. A number of developments are described for high-throughput screening of crystallization trials and for automated imaging of crystals with the electron microscope. These tools are critical for exploring the necessary range of factors governing the crystallization process. There have also been recent software developments to facilitate the process of structure determination. However, further innovations in the algorithms used for processing images and electron diffraction are necessary to improve throughput and to make electron crystallography truly viable as a method for determining atomic structures of membrane proteins.

We provide a brief description of a Wikibook open-content textbook that was set up with a goal of providing a comprehensive and continually updated list of all of the software packages of interest to the cryo-EM community. While the content of the Wikibook will change over time, here we provide a snapshot of the current state of software tools available, and encourage the members of this community to view the pages, add content, correct errors, and make any other contributions that might be useful

BACKGROUND: A very early step in splice site recognition is exon definition, a process that is as yet poorly understood. Communication between the two ends of an exon is thought to be required for this step. We report genome-wide evidence for exons being defined through the combinatorial activity of motifs located in flanking intronic regions. RESULTS: Strongly co-occurring motifs were found to specifically reside in four intronic regions surrounding a large number of human exons. These paired motifs occur around constitutive and alternative exons but not pseudo exons. Most co-occurring motifs are limited to intronic regions within 100 nucleotides of the exon. They are preferentially associated with weaker exons. Their pairing is conserved in evolution and they exhibit a lower frequency of single nucleotide polymorphism when paired. Paired motifs display specificity with respect to distance from the exon borders and in constitutive versus alternative splicing. Many resemble binding sites for heterogeneous nuclear ribonucleoproteins. Specific pairs are associated with tissue-specific genes, the higher expression of which coincides with that of the pertinent RNA binding proteins. Tested pairs acted synergistically to enhance exon inclusion, and this enhancement was found to be exon-specific. CONCLUSIONS: The exon-flanking sequence pairs identified here by genomic analysis promote exon inclusion and may play a role in the exon definition step in pre-mRNA splicing. We propose a model in which multiple concerted interactions are required between exonic sequences and flanking intronic sequences to effect exon definition.

beta-Catenin plays important roles in mammary development and tumorigenesis through its functions in cell adhesion, signal transduction and regulation of cell-context-specific gene expression. Studies in mice have highlighted the critical role of beta-catenin signaling for stem cell biology at multiple stages of mammary development. Deregulated beta-catenin signaling disturbs stem and progenitor cell dynamics and induces mammary tumors in mice. Recent data showing deregulated beta-catenin signaling in metaplastic and basal-type tumors suggest a similar link to reactivated developmental pathways and human breast cancer. The present review will discuss beta-catenin as a central transducer of numerous signaling pathways and its role in mammary development and breast cancer

Patients from the Indian subcontinent have a distinct cardiovascular risk profile with profound health consequences. South Asians tend to develop more severe coronary artery disease at a younger age, and may also suffer from earlier myocardial infarction and heart failure. The genesis of this risk is multi-factorial. One important culprit is increased insulin resistance, possibly due to recently identified genetic polymorphisms. Another possible explanation is subclinical inflammation and a prothrombotic environment, as evidenced by increased levels of homocysteine, plasminogen activator inhibitor-1, and fibrinogen. The lipid profile of South Asians may play a role, as this population is known to have elevated levels of lipoprotein (a), as well as lower levels of HDL. In addition, this HDL may be dysfunctional, as this population may have a higher prevalence of low levels of HDL2b, as well as an increased preponderance of smaller HDL. Current guidelines for primary and secondary prevention have not reflected our growing insight into the unique characteristics of the South Asian population, and may need to evolve to reflect our knowledge

Mass spectrometry is a method of choice for quantifying low-abundance proteins and peptides in many biological studies. Here, we describe a range of computational aspects of protein and peptide quantitation, including methods for finding and integrating mass spectrometric peptide peaks, and detecting interference to obtain a robust measure of the amount of proteins present in samples

Purpose: Survival of bone implants depends on biological fixation, and prosthesis loosening can be catastrophic leading to replacement of prostheses. Inflammation and osteoclast-mediated bone resorption in response to wear particles near prostheses contribute to loosening. Because we have demonstrated that adenosine A_2Areceptor activation is anti-inflammatory and prevents osteoclast formation and function we hypothesized that adenosine A_2Areceptor agonists might prevent osteoclast-mediated bone resorption at the site of prosthesis wear in a calvarial model of wear particle-induced bone resorption. Methods: Eighteen C57Bl/6 mice age 6-8 weeks were anesthetized by intraperitoneal injection of ketamine and xylazine and a 1cm midline sagittal incision was made over the calvarium anterior to the line connecting both ears. Six animals received no particles (control), and 12 received 15 ml of polyethylene particle suspension. Of the 12 mice receiving particulate, 6 were injected subcutaneously at the surgical site with 20 ml of 10 muM CGS21680 (A_2Areceptor agonist), and 6 mice were injected with saline 0.9%, beginning immediately after incision closure and continuing every other day until sacrifice. Animals were sacrificed after 14 days and the calvaria were removed, fixed, and prepared for microCT and histological staining with TRAP. Results: Histologic examination of calvaria demonstrated lymphocytic infiltration in both particle-exposed groups. TRAP staining revealed a reduction in osteoclast differentiation after treatment with CGS21680. mCT showed pitting and increased porosity in both particle-exposed groups compared to controls, although in CGS21680-treated mice the reduction in cortical bone was significantly less than in the untreated particle-exposed mice (p<0.01). Control bone volume/trabecular volume was significantly greater (p<0.005) than in either particulate group, however, calvarial bone from CGS21680-treated mice had significantly greater mean bone volume than did the untreated group (p<0.0005). Trabecular thickness was significantly reduced in both CGS21680-treated and untreated particle-exposed groups as compared to control mice (p<0.05). Finally, digital morphometric analysis of microCTs reveals that CGS21680 significantly reduced the area of bone pitting compared to control particle-treated mice (p<0.05). (Table presented) Conclusions: Adenosine A_2Areceptor activation reduces inflammation and bone destruction due to prosthetic wear particles. This observation suggests that delivery of an adenosine A_2Aagonist in the cement may enhance orthopedic implant survival, delaying or eliminating the need for revision arthroplasty surgery

Purpose: Organ injury induced by antibodies characteristic of Sjogren Syndrome and Systemic Lupus Erythematosus, while varied in the adult and fetus, may share in common a link between apoptosis and ultimate fibrosis. In congenital heart block (CHB), binding of maternal anti-Ro/La antibodies to apoptotic cardiocytes impairs their removal by healthy cardiocytes and increases uPA/uPAR-dependent plasmin activation. Immunological staining of CHB hearts reveals AV node TGFb staining and TGFb activation promotes fibroblast trans-differentiation, a scarring phenotype. Since the uPA/uPAR system plays a role in TGFb activation, this study evaluated whether anti-Ro/La binding to apoptotic cardiocytes via plasmin activation stimulates TGFb and promotes a profibrosing phenotype. Methods and Results: Initial analysis showed increased TGFb in supernatants from co-cultures of healthy cards and apoptotic cards incubated with IgG fractions from mothers whose sera contain anti-Ro/La antibodies and who had a child with CHB (apo-CHB-IgG) compared to co-cultures of healthy cards and apoptotic cards incubated with control IgG (apo-nl-IgG). Using a luciferase bioassay of active TGFb, supernatants from co-cultures of healthy cards and apo-CHB-IgG cards exhibited increased levels of active TGFb compared to those cocultured with apo-nl-IgG cards (511pg/ml CHB-IgG RLU vs 217 nl-IgG RLU; p=0.007; n=7). Abrogation of RLU via anti-TGFb antibody or TGFb inhibitor (SB431542) confirmed TGFb activation was solely due to TGFb. Significantly increased uPA levels (903 pg/ml vs 508 pg/ml; p =0.01; n=5) and uPA activity (0.8 units vs 0.04 units; p=0.005; n=3) were demonstrated in supernatants generated from coculture of healthy cards and apo CHB-IgG cards compared to healthy cards and apo nl-IgG, respectively. To determine whether uPA activity was responsible for TGFb activation, coculture experiments were conducted in which the apo-CHB-IgG cards were treated with anti-uPAR or anti-uPA antibodies or the plasmin inhibitor aprotinin prior to coculturing with healthy cards. In all instances treatments attenuated TGFb activation and uPA activity. To evaluate the profibrotic role of the observed TGFb activation, supernatants from either apo-CHB-IgG or apo-nl-IgG cocultures with healthy cards were applied to serum deprived cardiac fibroblasts. Only supernatants derived from cocultures of healthy cards and apo-CHB-IgG cardiocytes promoted transdifferentiation as evidenced by increased SMAc staining, an effect that was decreased when fibroblasts were treated with supernatants where cocultures were pretreated with uPAR antibodies. Supporting the hypothesis that increased uPA activity is causally related to the pathogenesis of CHB, cord blood samples from 12 of 18 children with CHB exhibited increased uPA activity and uPAR cleavage compared to 4 of 14 non CHB children exposed to maternal anti-Ro antibodies. Conclusions: These data suggest that binding of anti-Ro/La antibodies to apoptotic cardiocytes by virtue of increased uPAR-dependent uPA activity trigger TGFb activation thus initiating and amplifying a cascade of events that promote myofibroblast transdifferentiation and scar