6041 Cadillac Ave Internal Medicine Module 2D
Los Angeles CA 90034
Medical School: Yale University School Of Medicine - 1996
Accepts Medicare: Yes
Participates In eRX: No
Participates In PQRS: No
Participates In EHR: No
License #: A065639
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Novel components of the Toxoplasma inner membrane complex revealed by BioID. - mBio
The inner membrane complex (IMC) of Toxoplasma gondii is a peripheral membrane system that is composed of flattened alveolar sacs that underlie the plasma membrane, coupled to a supporting cytoskeletal network. The IMC plays important roles in parasite replication, motility, and host cell invasion. Despite these central roles in the biology of the parasite, the proteins that constitute the IMC are largely unknown. In this study, we have adapted a technique named proximity-dependent biotin identification (BioID) for use in T.Â gondii to identify novel components of the IMC. Using IMC proteins in both the alveoli and the cytoskeletal network as bait, we have uncovered a total of 19 new IMC proteins in both of these suborganellar compartments, two of which we functionally evaluate by gene knockout. Importantly, labeling of IMC proteins using this approach has revealed a group of proteins that localize to the sutures of the alveolar sacs that have been seen in their entirety in Toxoplasma species only by freeze fracture electron microscopy. Collectively, our study greatly expands the repertoire of known proteins in the IMC and experimentally validates BioID as a strategy for discovering novel constituents of specific cellular compartments of T.Â gondii.The identification of binding partners is critical for determining protein function within cellular compartments. However, discovery of protein-protein interactions within membrane or cytoskeletal compartments is challenging, particularly for transient or unstable interactions that are often disrupted by experimental manipulation of these compartments. To circumvent these problems, we adapted an in vivo biotinylation technique called BioID for Toxoplasma species to identify binding partners and proximal proteins within native cellular environments. We used BioID to identify 19 novel proteins in the parasite IMC, an organelle consisting of fused membrane sacs and an underlying cytoskeleton, whose protein composition is largely unknown. We also demonstrate the power of BioID for targeted discovery of proteins within specific compartments, such as the IMC cytoskeleton. In addition, we uncovered a new group of proteins localizing to the alveolar sutures of the IMC. BioID promises to reveal new insights on protein constituents and interactions within cellular compartments of Toxoplasma.Copyright Â© 2015 Chen et al.
Induction and inhibition of CPAF activity during analysis of Chlamydia-infected cells. - Pathogens and disease
Studies of the chlamydial protease CPAF have been complicated by difficulties in distinguishing bona fide intracellular proteolysis from in vitro proteolysis. This confounding issue has been attributed to CPAF activity in lysates from Chlamydia-infected cells. We compared three methods that have been used to inhibit in vitro CPAF-mediated proteolysis: (1) pre-treatment of infected cells with the inhibitor clasto-lactacystin, (2) direct cell lysis in 8 M urea and (3) direct lysis in hot 1% SDS buffer. We identified a number of experimental conditions that reduce the effectiveness of each method in preventing CPAF activity during lysate preparation. The amount of in vitro proteolysis in a lysate was variable and depended on factors such as the specific substrate and the time in the intracellular infection. Additionally, we demonstrated for the first time that artifactual CPAF activity is induced before cell lysis by standard cell detachment methods, including trypsinization. Protein analysis of Chlamydia-infected cells therefore requires precautions to inhibit CPAF activity during both cell detachment and lysate preparation, followed by verification that the cell lysates do not contain residual CPAF activity. These concerns about artifactual proteolysis extend beyond studies of CPAF function because they have the potential to affect the analyses of host and chlamydial proteins from Chlamydia-infected cells.Â© The Author 2015. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: firstname.lastname@example.org.
RON5 is critical for organization and function of the Toxoplasma moving junction complex. - PLoS pathogens
Apicomplexans facilitate host cell invasion through formation of a tight-junction interface between parasite and host plasma membranes called the moving junction (MJ). A complex of the rhoptry neck proteins RONs 2/4/5/8 localize to the MJ during invasion where they are believed to provide a stable anchoring point for host penetration. During the initiation of invasion, the preformed MJ RON complex is injected into the host cell where RON2 spans the host plasma membrane while RONs 4/5/8 localize to its cytosolic face. While much attention has been directed toward an AMA1-RON2 interaction supposed to occur outside the cell, little is known about the functions of the MJ RONs positioned inside the host cell. Here we provide a detailed analysis of RON5 to resolve outstanding questions about MJ complex organization, assembly and function during invasion. Using a conditional knockdown approach, we show loss of RON5 results in complete degradation of RON2 and mistargeting of RON4 within the parasite secretory pathway, demonstrating that RON5 plays a key role in organization of the MJ RON complex. While RON8 is unaffected by knockdown of RON5, these parasites are unable to invade new host cells, providing the first genetic demonstration that RON5 plays a critical role in host cell penetration. Although invasion is not required for injection of rhoptry effectors into the host cytosol, parasites lacking RON5 also fail to form evacuoles suggesting an intact MJ complex is a prerequisite for secretion of rhoptry bulb contents. Additionally, while the MJ has been suggested to function in egress, disruption of the MJ complex by RON5 depletion does not impact this process. Finally, functional complementation of our conditional RON5 mutant reveals that while proteolytic separation of RON5 N- and C-terminal fragments is dispensable, a portion of the C-terminal domain is critical for RON2 stability and function in invasion.
DNA topoisomerase I drugs and radiotherapy for lung cancer. - Journal of thoracic disease
Lung cancer represents the most common cause of cancer-related mortality in the United States and around the world. DNA topoisomerase I (TOP1) drugs such as irinotecan and topotecan represent a unique class of chemotherapeutic agents that exhibit not only potent cytotoxic effect, but also tumor-selective radiation-sensitizing effect. The mechanism of cytotoxicity and radiation sensitization by TOP1 drugs has been intensely investigated. Modern radiotherapy, aided by improved imaging and treatment delivery technology, is capable of targeting tumors more precisely, while sparing surrounding critical structures. Clinical trials with camptothecin derivatives and radiotherapy have been conducted in lung cancers. Combined modality therapy with TOP1 drugs and radiotherapy offers a new frontier for lung cancer therapy. We review the present state of TOP1-targeted chemotherapy and modern radiotherapy for lung cancer.
CPAF: a Chlamydial protease in search of an authentic substrate. - PLoS pathogens
Bacteria in the genus Chlamydia are major human pathogens that cause an intracellular infection. A chlamydial protease, CPAF, has been proposed as an important virulence factor that cleaves or degrades at least 16 host proteins, thereby altering multiple cellular processes. We examined 11 published CPAF substrates and found that there was no detectable proteolysis when CPAF activity was inhibited during cell processing. We show that the reported proteolysis of these putative CPAF substrates was due to enzymatic activity in cell lysates rather than in intact cells. Nevertheless, Chlamydia-infected cells displayed Chlamydia-host interactions, such as Golgi reorganization, apoptosis resistance, and host cytoskeletal remodeling, that have been attributed to CPAF-dependent proteolysis of host proteins. Our findings suggest that other mechanisms may be responsible for these Chlamydia-host interactions, and raise concerns about all published CPAF substrates and the proposed roles of CPAF in chlamydial pathogenesis.
A Chlamydia-specific C-terminal region of the stress response regulator HrcA modulates its repressor activity. - Journal of bacteriology
Chlamydial heat shock proteins have important roles in Chlamydia infection and immunopathogenesis. Transcription of chlamydial heat shock genes is controlled by the stress response regulator HrcA, which binds to its cognate operator CIRCE, causing repression by steric hindrance of RNA polymerase. All Chlamydia spp. encode an HrcA protein that is larger than other bacterial orthologs because of an additional, well-conserved C-terminal region. We found that this unique C-terminal tail decreased HrcA binding to CIRCE in vitro as well as HrcA-mediated transcriptional repression in vitro and in vivo. When we isolated HrcA from chlamydiae, we only detected the full-length protein, but we found that endogenous HrcA had a higher binding affinity for CIRCE than recombinant HrcA. To examine this difference further, we tested the effect of the heat shock protein GroEL on the function of HrcA since endogenous chlamydial HrcA has been previously shown to associate with GroEL as a complex. GroEL enhanced the ability of HrcA to bind CIRCE and to repress transcription in vitro, but this stimulatory effect was greater on full-length HrcA than HrcA lacking the C-terminal tail. These findings demonstrate that the novel C-terminal tail of chlamydial HrcA is an inhibitory region and provide evidence that its negative effect on repressor function can be counteracted by GroEL. These results support a model in which GroEL functions as a corepressor that interacts with HrcA to regulate chlamydial heat shock genes.
Induction of amyloid fibrils by the C-terminal fragments of TDP-43 in amyotrophic lateral sclerosis. - Journal of the American Chemical Society
TAR DNA-binding protein 43 (TDP-43) has been identified as the major ubiquitinated aggregates in the inclusion bodies in the patients of amyotrophic lateral sclerosis (ALS) since 2006 and become a crucial culprit for ALS and related motor neuron diseases. Recent literature has further indicated that the major components of these aggregates are hyper-phosphorylated TDP-43 C-terminus. In an effort to clarify the conformational and physical properties of its disordered C-terminal domain, we have synthesized several peptide fragments and shown that only D1 within D1-4 can form twisted fibrils with a cross section of approximately 11 nm in width under the incubation of phosphate buffer. In contrast, the D2-4 peptides all formed amorphous aggregates, showing different aggregation propensities. In addition to D1, two pathological mutant peptides, A315T and G294A, can also form fibrils that share similar shape and morphology with neuronal cytoplasmic inclusions. We propose that the residues with this region (287-322), which contains myriads of glycine repeats, may contribute significantly to the fiber formation as well as aggregation propensity. Moreover, from the conformational characterizations of D1, A315T, and G294A with EM, CD, fluorescence, and Raman spectroscopy, we found that all three peptides formed an amyloid structure, providing insights into the nature of its aggregation vis a vis the other fragments in the C-terminus of TDP-43.
The option of Linac-based radiosurgery in a Gamma Knife radiosurgery center. - Clinical neurology and neurosurgery
Due to the fundamental differences in treatment delivery, linear-accelerator-based radiosurgery can be complementary to Gamma Knife (GK) for intracranial lesions. We reviewed the effect of adding GK to an existing linear accelerator (Linac)-based radiosurgery practice and analyzed case selections for the two modalities.UC Davis Medical Center installed a Leksell Gamma Knife Model C in October 2003 to supplement an established Linac-based radiosurgery program. Radiosurgery indications for the 15 months before and after installation were compared.Radiosurgery cases expanded by twofold from 68 patients before GK installation to 139 after, with 106 treated by GK and 33 by Linac. Besides a major increase for trigeminal neuralgia and a general growth for acoustic neuroma, meningioma and brain metastases, case numbers for glioma and arteriovenous malformation (AVM) remained stable. Considering case selections for Linac, glioma decreased from 28 to 18%, while meningioma and metastases increased from 9 to 21% and 38-46%, respectively. The Linac patients receiving fractionated treatment also increased from 37 to 61%.While the majority of patients were treated with GK, a significant proportion was judged to be suited for Linac treatment. This latter group included particularly patients who benefit from fractionated therapy.
Chemotherapy for brain metastases in small-cell lung cancer. - Clinical lung cancer
Brain metastasis occurs commonly in patients with small-cell lung cancer (SCLC). Herein, we report the efficacy of irinotecan and carboplatin in the treatment of brain metastases from SCLC. In addition, we review the existing data on chemotherapy for brain metastases in SCLC.Eighty patients with metastatic or relapsed SCLC were enrolled in a phase II trial of irinotecan and carboplatin. Patients naive to chemotherapy were treated with irinotecan 200 mg/m2 and carboplatin AUC of 5, and patients previously treated with chemotherapy received irinotecan 150 mg/m2 and carboplatin AUC of 5, every 21 days for 6 cycles.Among the 80 patients, 15 (19%) presented with brain metastases. An analysis of 14 assessable patients with brain metastases revealed an overall response rate of 65% after 2 cycles of chemotherapy and a median survival of 6 months (range, 1-24 months). Upon review of the literature, 8 studies were identified as having > 10 patients who received chemotherapy for brain metastases from SCLC. Based on these studies, the response rate of brain metastases from SCLC to a variety of chemotherapy and median survival of patients ranged from 22% to 85% and 3 months to 9 months, respectively.Chemotherapy, including the regimen of irinotecan and carboplatin, is an effective treatment for SCLC brain metastases.
The impact of concurrent granulocyte macrophage-colony stimulating factor on radiation-induced mucositis in head and neck cancer patients: a double-blind placebo-controlled prospective phase III study by Radiation Therapy Oncology Group 9901. - International journal of radiation oncology, biology, physics
Based on early clinical evidence of potential mucosal protection by granulocyte-macrophage colony stimulating factor (GM-CSF), the Radiation Therapy Oncology Group conducted a double-blind, placebo-controlled, randomized study to test the efficacy and safety of GM-CSF in reducing the severity and duration of mucosal injury and pain (mucositis) associated with curative radiotherapy (RT) in head-and-neck cancer patients.Eligible patients included those with head-and-neck cancer with radiation ports encompassing >50% of oral cavity and/or oropharynx. Standard RT ports were used to cover the primary tumor and regional lymphatics at risk in standard fractionation to 60-70 Gy. Concurrent cisplatin chemotherapy was allowed. Patients were randomized to receive subcutaneous injection of GM-CSF 250 microg/m2 or placebo 3 times a week. Mucosal reaction was assessed during the course of RT using the National Cancer Institute Common Toxicity Criteria and the protocol-specific scoring system.Between October 2000 and September 2002, 130 patients from 36 institutions were accrued. Nine patients (7%) were excluded from the analysis, 3 as a result of drug unavailability. More than 80% of the patients participated in the quality-of-life endpoint of this study. The GM-CSF did not cause any increase in toxicity compared with placebo. There was no statistically significant difference in the average mean mucositis score in the GM-CSF and placebo arms by a t test (p = 0.4006).This placebo-controlled, randomized study demonstrated no significant effect of GM-CSF given concurrently compared with placebo in reducing the severity or duration of RT-induced mucositis in patients undergoing definitive RT for head-and-neck cancer.
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