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Hypoxia that develops in solid tumors stabilizes the hypoxiainducible factor-1A (HIF-1A) subunit of the HIF-1 transcription factor, leading to up-regulation of dozens of hypoxia-regulated genes that increase glycolysis and oxygen delivery. HIF-1A and its downstream target gene CA9 have both been used as surrogate hypoxia markers, and, in general, high expression predicts for a poor response to treatment. Combinations of hypoxia markers offer the opportunity to measure changes in tumordoi:10.1158/0008-5472.can-04-4480 pmid:16103077 fatcat:g6r7qati65hkpcnqdaqgnb7fqi
more »... n that may be relevant to tumor response to treatment. We compared the degree of colocalization of two endogenous markers for hypoxia, HIF-1A and carbonic anhydrase IX (CAIX), with a chemical marker for hypoxia, pimonidazole. Unexpectedly, expression of HIF-1A was reduced in the most hypoxic regions that border necrosis in xenograft tumors composed of SiHa cervical carcinoma, WiDr colon carcinoma, or M006 astrocytoma cells. Similar results were obtained for samples from three cervical cancer biopsies. However, CAIX was present in these perinecrotic cells that were also capable of metabolizing and binding a chemical marker for hypoxia, pimonidazole. In vitro experiments using tumor cells and tumor cubes incubated under anoxic conditions indicated that nutrient deprivation seems to be largely responsible for the lack of HIF-1A expression in perinecrotic regions. The half-life of CAIX was sufficiently long that, once formed, it remained for days in the absence of continued HIF-1A expression. These results have implications for the use of HIF-1A as an indicator of tumor hypoxia and aggressiveness as well as development of hypoxia-directed antitumor therapies based on the expression of HIF-1A. (Cancer Res 2005; 65(16): 7259-66)
From humble beginnings of a contaminated petri dish, b-lactam antibiotics have distinguished themselves among some of the most powerful drugs in human history. The devastating effects of antibiotic resistance have nevertheless led to an "arms race" with disquieting prospects. The emergence of multidrug resistant bacteria threatens an ever-dwindling antibiotic arsenal, calling for new discovery, rediscovery, and innovation in b-lactam research. Here the current state of b-lactam antibiotics from a structural perspective was reviewed.doi:10.1002/pro.2889 pmid:26813250 pmcid:PMC4941212 fatcat:wiybq3cyprhzzdxxyor2asi3w4
In recent years, there has been a growing interest in teichoic acids as targets for antibiotic drug design against major clinical pathogens such as Staphylococcus aureus, reflecting the disquieting increase in antibiotic resistance and the historical success of bacterial cell wall components as drug targets. It is now becoming clear that β-O-GlcNAcylation of S. aureus wall teichoic acids plays a major role in both pathogenicity and antibiotic resistance. Here we present the first structure ofdoi:10.1371/journal.ppat.1006067 pmid:27973583 pmcid:PMC5156392 fatcat:wvnqfcw72fbvjogize4h6hzv3i
more »... aureus TarS, the enzyme responsible for polyribitol phosphate β-O-GlcNAcylation. Using a divide and conquer strategy, we obtained crystal structures of various TarS constructs, mapping high resolution overlapping N-terminal and C-terminal structures onto a lower resolution full-length structure that resulted in a high resolution view of the entire enzyme. Using the N-terminal structure that encapsulates the catalytic domain, we furthermore captured several snapshots of TarS, including the native structure, the UDP-GlcNAc donor complex, and the UDP product complex. These structures along with structure-guided mutants allowed us to elucidate various catalytic features and identify key active site residues and catalytic loop rearrangements that provide a valuable platform for anti-MRSA drug design. We furthermore observed for the first time the presence of a trimerization domain composed of stacked carbohydrate binding modules, commonly observed in starch active enzymes, but adapted here for a poly sugar-phosphate glycosyltransferase. Author Summary Historically, β-lactam class antibiotics such as methicillin have been very successful in the treatment of bacterial infections, effectively destroying bacteria by rupturing their cell PLOS Pathogens |
Motivation: Structural characterization of protein interactions is necessary for understanding and modulating biological processes. On one hand, X-ray crystallography or NMR spectroscopy provide atomic resolution structures but the data collection process is typically long and the success rate is low. On the other hand, computational methods for modeling assembly structures from individual components frequently suffer from high false-positive rate, rarely resulting in a unique solution.doi:10.1093/bioinformatics/bts628 pmid:23093611 pmcid:PMC3519461 fatcat:ck4vkfpaezcgtef4nfhbnwcfgu
more »... Here, we present a combined approach that computationally integrates data from a variety of fast and accessible experimental techniques for rapid and accurate structure determination of protein-protein complexes. The integrative method uses atomistic models of two interacting proteins and one or more datasets from five accessible experimental techniques: a small-angle X-ray scattering (SAXS) profile, 2D class average images from negative-stain electron microscopy micrographs (EM), a 3D density map from single-particle negative-stain EM, residue type content of the protein-protein interface from NMR spectroscopy and chemical cross-linking detected by mass spectrometry. The method is tested on a docking benchmark consisting of 176 known complex structures and simulated experimental data. The near-native model is the top scoring one for up to 61% of benchmark cases depending on the included experimental datasets; in comparison to 10% for standard computational docking. We also collected SAXS, 2D class average images and 3D density map from negative-stain EM to model the PCSK9 antigen-J16 Fab antibody complex, followed by validation of the model by a subsequently available X-ray crystallographic structure. Availability:
Unique to Gram-positive bacteria, wall teichoic acids are anionic glycopolymers cross-stitched to a thick layer of peptidoglycan. The polyol phosphate subunits of these glycopolymers are decorated with GlcNAc sugars that are involved in phage binding, genetic exchange, host antibody response, resistance, and virulence. The search for the enzymes responsible for GlcNAcylation in Staphylococcus aureus has recently identified TarM and TarS with respective αand β-(1-4) glycosyltransferasedoi:10.1073/pnas.1418084112 pmid:25624472 pmcid:PMC4330757 fatcat:ylkx4cvz2vccdhvm4owvkrc6nq
more »... . The stereochemistry of the GlcNAc attachment is important in balancing biological processes, such that the interplay of TarM and TarS is likely important for bacterial pathogenicity and survival. Here we present the crystal structure of TarM in an unusual ternary-like complex consisting of a polymeric acceptor substrate analog, UDP from a hydrolyzed donor, and an α-glyceryl-GlcNAc product formed in situ. These structures support an internal nucleophilic substitution-like mechanism, lend new mechanistic insight into the glycosylation of glycopolymers, and reveal a trimerization domain with a likely role in acceptor substrate scaffolding.
Hypoxic cells in solid tumours are known to resist radiotherapy as well as forms of chemotherapy. Hypoxia is also believed to promote tumour aggressiveness and metastasis. It is therefore important to develop a practical method to identify hypoxic tumour cells in order to assess which patients could benefit from hypoxic cell-targeted therapies. The goal of this study was to develop an immunohistochemical approach to the detection of hypoxia in xenograft models that may be applied to clinicaldoi:10.14288/1.0091847 fatcat:75yvl3xjmjhj3p3jivx6oifbk4
more »... ples. The hypotheses to be tested were that areas of chronic or transient hypoxia could be distinguished based on specific patterns of hypoxia markers, and that HIF-1α could be comparable as a hypoxia marker to pimonidazole. The objectives were: 1) to characterise the kinetics of development under anoxia and loss upon reoxygenation of the exogenous hypoxia marker pimonidazole and the endogenous hypoxia marker HIF-1α, 2) to use quantitative fluorescence image analysis to compare patterns of pimonidazole binding and HIF-1α expression in WiDr, SiHa and M006 human tumour xenografts, and to measure marker response under various oxygen- breathing conditions, and 3) to compare hypoxia marker patterns in xenograft tumours with patterns observed in cervical cancer biopsies. HIF-1α and pimonidazole colocalised in regions distant from blood vessels; perinecrotic regions however showed pimonidazole binding but no HIF-la expression. This lack was not a result of anoxia but likely a result of glucose and serum starvation. With time after pimonidazole administration, the extent of colocalisation with HIF-1α was reduced from 60% at 90min to 7% at 48hr, consistent with the movement of pimonidazole-labelled cells into necrosis. Patterns of HIF-1α and pimonidazole binding in clinical samples confirmed the dissociation between the markers when pimonidazole was administered 24hr before tumour excision. Overall our xenograft results indicated HIF-1α to be a reliable indicator of tumour hypoxia; however, it over-estimate [...]
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics which may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555 (also known as bamlanivimab), adoi:10.1126/scitranslmed.abf1906 pmid:33820835 fatcat:h54ddozr7fgkldidjmzhianjjm
more »... potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days, and clearance of 0.22 mL/hr/kg, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study Day 6 following viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.
SARS-CoV-2 poses a public health threat for which therapeutic agents are urgently needed. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555, a potent anti-spike neutralizing antibody from a convalescent COVID-19 patient. Biochemical, structural, and functional characterization revealed high-affinity binding to the receptor-binding domain, ACE2 binding inhibition, and potent neutralizing activity. In a rhesus macaquedoi:10.1101/2020.09.30.318972 pmid:33024963 pmcid:PMC7536866 fatcat:lbksshvgfrdexi5c4rvj3mligi
more »... hallenge model, prophylaxis doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract. These data demonstrate that high-throughput screening can lead to the identification of a potent antiviral antibody that protects against SARS-CoV-2 infection.
Structure and mechanism of Staphylococcus aureus TarM, the wall teichoic acid α-glycosyltransferase Solmaz Sobhanifar, Liam James Worrall, Robert J. Gruninger, Gregory A. ...doi:10.1073/pnas.ss11206 fatcat:pdw5535dfbau5hqikgx3dfru74