Structural basis of broad-spectrum β-lactam resistance in Staphylococcus aureus.


Journal

Nature
ISSN: 1476-4687
Titre abrégé: Nature
Pays: England
ID NLM: 0410462

Informations de publication

Date de publication:
01 2023
Historique:
received: 06 06 2022
accepted: 18 11 2022
pubmed: 5 1 2023
medline: 14 1 2023
entrez: 4 1 2023
Statut: ppublish

Résumé

Broad-spectrum β-lactam antibiotic resistance in Staphylococcus aureus is a global healthcare burden

Identifiants

pubmed: 36599987
doi: 10.1038/s41586-022-05583-3
pii: 10.1038/s41586-022-05583-3
pmc: PMC9834060
doi:

Substances chimiques

Anti-Bacterial Agents 0
Bacterial Proteins 0
beta-Lactams 0
BlaR1 protein, Staphylococcus aureus EC 3.4.24.-

Types de publication

Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Langues

eng

Sous-ensembles de citation

IM

Pagination

375-382

Subventions

Organisme : NIGMS NIH HHS
ID : U24 GM129547
Pays : United States
Organisme : NIAID NIH HHS
ID : R01 AI100291
Pays : United States
Organisme : NIAID NIH HHS
ID : R21 AI142501
Pays : United States

Informations de copyright

© 2023. The Author(s).

Références

Turner, N. A. et al. Methicillin-resistant Staphylococcus aureus: an overview of basic and clinical research. Nat. Rev. Microbiol. 17, 203–218 (2019).
doi: 10.1038/s41579-018-0147-4
Murray, C. J. et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet 399, 629–655 (2022).
doi: 10.1016/S0140-6736(21)02724-0
Hackbarth, C. J. & Chambers, H. F. blaI and blaR1 regulate β-lactamase and PBP 2a production in methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 37, 1144–1149 (1993).
doi: 10.1128/AAC.37.5.1144
Zhang, H. Z., Hackbarth, C. J., Chansky, K. M. & Chambers, H. F. A proteolytic transmembrane signaling pathway and resistance to β-lactams in staphylococci. Science 291, 1962–1965 (2001).
doi: 10.1126/science.1055144
Herzberg, O. & Moult, J. Bacterial resistance to β-lactam antibiotics: crystal structure of β-lactamase from Staphylococcus aureus PC1 at 2.5 Å resolution. Science 236, 694–701 (1987).
doi: 10.1126/science.3107125
Lim, D. & Strynadka, N. C. J. Structural basis for the β-lactam resistance of PBP2a from methicillin-resistant Staphylococcus aureus. Nat. Struct. Biol. 9, 870–876 (2002).
Blázquez, B. et al. Regulation of the expression of the β-lactam antibiotic-resistance determinants in methicillin-resistant Staphylococcus aureus (MRSA). Biochemistry 53, 1548–1550 (2014).
doi: 10.1021/bi500074w
Amoroso, A. et al. A peptidoglycan fragment triggers β-lactam resistance in Bacillus licheniformis. PLoS Pathog. 8, e1002571 (2012).
doi: 10.1371/journal.ppat.1002571
Rodvold, K. A. & Mcconeghy, K. W. Methicillin-resistant Staphylococcus aureus therapy: past, present, and future. Clin. Infect. Dis. 58, S20–S27 (2014).
doi: 10.1093/cid/cit614
Wu, Q. et al. Systematic review and meta-analysis of the epidemiology of vancomycin-resistance Staphylococcus aureus isolates. Antimicrob. Resist. Infect. Control 10, 101 (2021).
doi: 10.1186/s13756-021-00967-y
Rossi, F. et al. Transferable vancomycin resistance in a community-associated MRSA lineage. N. Engl. J. Med. 370, 1524–1531 (2014).
doi: 10.1056/NEJMoa1303359
Otero, L. H. et al. How allosteric control of Staphylococcus aureus penicillin binding protein 2a enables methicillin resistance and physiological function. Proc. Natl Acad. Sci. USA 110, 16808–16813 (2013).
doi: 10.1073/pnas.1300118110
Katayama, Y., Ito, T. & Hiramatsu, K. A new class of genetic element, Staphylococcus cassette chromosome mec, encodes methicillin resistance in Staphylococcus aureus. Antimicrob. Agents Chemother. 44, 1549–1555 (2000).
doi: 10.1128/AAC.44.6.1549-1555.2000
Arêde, P., Ministro, J. & Oliveira, D. C. Redefining the role of the β-lactamase locus in methicillin-resistant Staphylococcus aureus: β-lactamase regulators disrupt the MecI-mediated strong repression on mecA and optimize the phenotypic expression of resistance in strains with constitutive mecA. Antimicrob. Agents Chemother. 57, 3037–3045 (2013).
doi: 10.1128/AAC.02621-12
Alexander, J. A. N. et al. Structural analysis of avibactam-mediated activation of the bla and mec divergons in methicillin-resistant Staphylococcus aureus. J. Biol. Chem. 295, 10870–10884 (2020).
doi: 10.1074/jbc.RA120.013029
McKinney, T. K., Sharma, V. K., Craig, W. A. & Archer, G. L. Transcription of the gene mediating methicillin resistance in Staphylococcus aureus (mecA) is corepressed but not coinduced by cognate mecA and β-lactamase regulators transcription of the gene mediating methicillin resistance in Staphylococcus aureus. J. Bacteriol. 183, 6862–6868 (2001).
doi: 10.1128/JB.183.23.6862-6868.2001
Lakhundi, S. & Zhang, K. Methicillin-resistant Staphylococcus aureus: molecular characterization, evolution, and epidemiology. Clin Microbiol. Rev. 31, e00020-18 (2018).
doi: 10.1128/CMR.00020-18
Liu, C. et al. A population-based study of the incidence and molecular epidemiology of methicillin-resistant Staphylococcus aureus disease in San Francisco, 2004–2005. Clin. Infect. Dis. 46, 1637–1646 (2008).
doi: 10.1086/587893
Hanique, S. et al. Evidence of an intramolecular interaction between the two domains of the BlaR1 penicillin receptor during the signal transduction. J. Biol. Chem. 279, 14264–14272 (2004).
doi: 10.1074/jbc.M313488200
Frederick, T. E., Wilson, B. D., Cha, J., Mobashery, S. & Peng, J. W. Revealing cell-surface intramolecular interactions in the BlaR1 protein of methicillin-resistant Staphylococcus aureus by NMR spectroscopy. Biochemistry 53, 10–12 (2014).
doi: 10.1021/bi401552j
Belluzo, B. S. et al. An experiment-informed signal transduction model for the role of the Staphylococcus aureus MecR1 protein in β-lactam resistance. Sci. Rep. 9, 19558 (2019).
doi: 10.1038/s41598-019-55923-z
Sala, C. et al. Genome-wide regulon and crystal structure of BlaI (Rv1846c) from Mycobacterium tuberculosis. Mol. Microbiol. 71, 1102–1116 (2009).
doi: 10.1111/j.1365-2958.2008.06583.x
Sandhu, B. K., Edwards, A. N., Anderson, S. E., Woods, E. C. & McBride, S. M. Regulation and anaerobic function of the Clostridioides difficile β-lactamase. Antimicrob. Agents Chemother. 64, e01496-19 (2019).
doi: 10.1128/AAC.01496-19
Sobhanifar, S., Prehna, G. & Strynadka, N. in Handbook of Proteolytic Enzymes (eds Barrett, A. et al.) 1237–1243 (Academic, 2012).
Mierau, I. & Kleerebezem, M. 10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis. Appl. Microbiol. Biotechnol. 68, 705–717 (2005).
doi: 10.1007/s00253-005-0107-6
Llarrull, L. I. & Mobashery, S. Dissection of events in the resistance to β-lactam antibiotics mediated by the protein BlaR1 from Staphylococcus aureus. Biochemistry 51, 4642–4649 (2012).
doi: 10.1021/bi300429p
Zhao, G., Meier, T. I., Kahl, S. D., Gee, K. R. & Blaszczak, L. C. BOCILLIN FL, a sensitive and commercially available reagent for detection of penicillin-binding proteins. Antimicrob. Agents Chemother. 43, 1124–1128 (1999).
doi: 10.1128/AAC.43.5.1124
Llarrull, L. I., Toth, M., Champion, M. M. & Mobashery, S. Activation of BlaR1 protein of methicillin-resistant Staphylococcus aureus, its proteolytic processing, and recovery from induction of resistance. J. Biol. Chem. 286, 38148–38158 (2011).
doi: 10.1074/jbc.M111.288985
Viklund, H., Granseth, E. & Elofsson, A. Structural classification and prediction of reentrant regions in α-helical transmembrane proteins: application to complete genomes. J. Mol. Biol. 361, 591–603 (2006).
doi: 10.1016/j.jmb.2006.06.037
Jongeneel, C. V., Bouvier, J. & Bairoch, A. A unique signature identifies a family of zinc-dependent metallopeptidases. FEBS Lett. 242, 211–214 (1989).
doi: 10.1016/0014-5793(89)80471-5
Granseth, E., Daley, D. O., Rapp, M., Melén, K. & von Heijne, G. Experimentally constrained topology models for 51,208 bacterial inner membrane proteins. J. Mol. Biol. 352, 489–494 (2005).
doi: 10.1016/j.jmb.2005.07.053
Daley, D. O. et al. Global topology analysis of the Escherichia coli inner membrane proteome. Science 308, 1321–1323 (2005).
doi: 10.1126/science.1109730
White, D. C. & Frerman, F. E. Extraction, characterization, and cellular localization of the lipids of Staphylococcus aureus. J. Bacteriol. 94, 1854–1867 (1967).
doi: 10.1128/jb.94.6.1854-1867.1967
Young, S. A., Desbois, A. P. & Coote, P. J. Characterisation of Staphylococcus aureus lipids by nanoelectrospray ionisation tandem mass spectrometry (nESI-MS/MS). Preprint at bioRxiv https://doi.org/10.1101/593483 (2019).
Schleifer, K. H. et al. Transfer of Streptococcus lactis and related Streptococci to the genus Lactococcus gen. nov. Syst. Appl. Microbiol. 6, 183–195 (1985).
doi: 10.1016/S0723-2020(85)80052-7
Driessen, A. J. M., Zheng, T., Veld, G. I. T., Op Den Kamp, J. A. F. & Konings, W. N. Lipid requirement of the branched-chain amino acid transport system of Streptococcus cremoris. Biochemistry 27, 865–872 (1988).
doi: 10.1021/bi00403a005
Wilke, M. S., Hills, T. L., Zhang, H.-Z., Chambers, H. F. & Strynadka, N. C. J. Crystal structures of the Apo and penicillin-acylated forms of the BlaR1 β-lactam sensor of Staphylococcus aureus. J. Biol. Chem. 279, 47278–47287 (2004).
doi: 10.1074/jbc.M407054200
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
doi: 10.1038/s41586-021-03819-2
Dimaio, F. et al. Atomic-accuracy models from 4.5-Å cryo-electron microscopy data with density-guided iterative local refinement. Nat. Methods 12, 361–365 (2015).
doi: 10.1038/nmeth.3286
Staude, M. W. et al. Investigation of signal transduction routes within the sensor/transducer protein BlaR1 of Staphylococcus aureus. Biochemistry 54, 1600–1610 (2015).
doi: 10.1021/bi501463k
Hooper, N. M. Families of zinc metalloproteases. FEBS Lett. 354, 1–6 (1994).
doi: 10.1016/0014-5793(94)01079-X
Marie-Claire, C. et al. Evidence by site-directed mutagenesis that arginine 203 of thermolysin and arginine 717 of neprilysin (neutral endopeptidase) play equivalent critical roles in substrate hydrolysis and inhibitor binding. Biochemistry 36, 13938–13945 (1997).
doi: 10.1021/bi9712495
Tyndall, J. D. A., Nall, T. & Fairlie, D. P. Proteases universally recognize beta strands in their active sites. Chem. Rev. 105, 973–999 (2005).
doi: 10.1021/cr040669e
Gomis-Rüth, F. X., Botelho, T. O. & Bode, W. A standard orientation for metallopeptidases. Biochim. Biophys. Acta 1824, 157–163 (2012).
doi: 10.1016/j.bbapap.2011.04.014
Punjani, A. & Fleet, D. J. 3D variability analysis: resolving continuous flexibility and discrete heterogeneity from single particle cryo-EM. J. Struct. Biol. 213, 107702 (2021).
doi: 10.1016/j.jsb.2021.107702
Kobayashi, T., Zhu, Y. F., Nicholls, N. J. & Lampen, J. O. A second regulatory gene, blaR1, encoding a potential penicillin-binding protein required for induction of β-lactamase in Bacillus licheniformis. J. Bacteriol. 169, 3873–3878 (1987).
doi: 10.1128/jb.169.9.3873-3878.1987
Quigley, A. et al. The structural basis of ZMPSTE24-dependent laminopathies. Science 339, 1604–1607 (2013).
doi: 10.1126/science.1231513
Pryor, E. E. et al. Structure of the integral membrane protein CAAX protease Ste24p. Science 339, 1600–1604 (2013).
doi: 10.1126/science.1232048
Berzigotti, S., Benlafya, K., Sépulchre, J., Amoroso, A. & Joris, B. Bacillus licheniformis BlaR1 L3 loop is a zinc metalloprotease activated by self-proteolysis. PLoS ONE 7, e36400 (2012).
doi: 10.1371/journal.pone.0036400
Beaumont, A. et al. The role of histidine 231 in thermolysin-like enzymes: a site-directed mutagenesis study. J. Biol. Chem. 270, 16803–16808 (1995).
doi: 10.1074/jbc.270.28.16803
Boudreau, M. A., Fishovitz, J., Llarrull, L. I., Xiao, Q. & Mobashery, S. Phosphorylation of BlaR1 in manifestation of antibiotic resistance in methicillin-resistant Staphylococcus aureus and its abrogation by small molecules. ACS Infect. Dis. 1, 454–459 (2015).
doi: 10.1021/acsinfecdis.5b00086
Powers, M. E. et al. Type I signal peptidase and protein secretion in Staphylococcus epidermidis. J. Bacteriol. 193, 2677–2686 (2011).
Kuipers, O. P., de Ruyter, P. G. G. A., Kleerebezem, M. & de Vos, W. M. Quorum sensing-controlled gene expression in lactic acid bacteria. J. Biotechnol. 64, 15–21 (1998).
doi: 10.1016/S0168-1656(98)00100-X
van den Ent, F. & Löwe, J. RF cloning: a restriction-free method for inserting target genes into plasmids. J. Biochem. Biophys. Methods 67, 67–74 (2006).
doi: 10.1016/j.jbbm.2005.12.008
Geertsma, E. R. & Poolman, B. High-throughput cloning and expression in recalcitrant bacteria. Nat. Methods 4, 705–707 (2007).
doi: 10.1038/nmeth1073
Terzaghi, B. & Sandine, W. Improved medium for lactic streptococci and their bacteriophages. Appl. Microbiol. 29, 807–813 (1975).
doi: 10.1128/am.29.6.807-813.1975
Gasteiger, E. et al. in The Proteomics Protocols Handbook (ed. Walker, J. M.) 571–607 (Springer, 2005); https://doi.org/10.1385/1-59259-890-0:571
Hauer, F. et al. GraDeR: membrane protein complex preparation for single-particle cryo-EM. Structure 23, 1769–1775 (2015).
doi: 10.1016/j.str.2015.06.029
Coombs, D. H. & Watts, N. R. M. Generating sucrose gradients in three minutes by tilted tube rotation. Anal. Biochem. 148, 254–259 (1985).
doi: 10.1016/0003-2697(85)90654-2
Grossman, M. J. & Lampen, J. O. Purification and DNA binding properties of the blal gene product, repressor for the (3-lactamase gene, blaP, of Bacillus licheniformis. Nucleic Acids Res. 15, 6049–6062 (1987).
Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).
doi: 10.1016/j.jsb.2005.07.007
Zheng, S. Q. et al. MotionCor2—anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
doi: 10.1038/nmeth.4193
Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).
doi: 10.1016/j.jsb.2015.08.008
Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).
doi: 10.1016/j.jsb.2012.09.006
Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. CryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).
doi: 10.1038/nmeth.4169
Asarnow, D., Palovcak, E. & Cheng, Y. UCSF pyem v0.5. Zenodo https://doi.org/10.5281/zenodo.3576630 (2019).
Zivanov, J., Nakane, T. & Scheres, S. H. W. A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis. IUCrJ 6, 5–17 (2019).
doi: 10.1107/S205225251801463X
Zivanov, J., Nakane, T. & Scheres, S. H. W. Estimation of high-order aberrations and anisotropic magnification from cryo-EM data sets in RELION-3.1. IUCrJ 7, 253–267 (2020).
doi: 10.1107/S2052252520000081
Chen, S. et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013).
doi: 10.1016/j.ultramic.2013.06.004
Zimmermann, L. et al. A completely reimplemented MPI bioinformatics toolkit with a new HHpred server at its core. J. Mol. Biol. 430, 2237–2243 (2018).
doi: 10.1016/j.jmb.2017.12.007
Webb, B. & Sali, A. Comparative protein structure modeling using MODELLER. Curr. Protoc. Bioinform. 54, 5.6.1–5.6.37 (2016).
doi: 10.1002/cpbi.3
Casañal, A., Lohkamp, B. & Emsley, P. Current developments in Coot for macromolecular model building of electron cryo-microscopy and crystallographic data. Protein Sci. 29, 1069–1078 (2020).
doi: 10.1002/pro.3791
Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D 74, 531–544 (2018).
doi: 10.1107/S2059798318006551
Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D 75, 861–877 (2019).
doi: 10.1107/S2059798319011471
Williams, C. J. et al. MolProbity: more and better reference data for improved all‐atom structure validation. Protein Sci. 27, 293 (2018).
doi: 10.1002/pro.3330
Afonine, P. V. et al. New tools for the analysis and validation of cryo-EM maps and atomic models. Acta Crystallogr. D 74, 814–840 (2018).
doi: 10.1107/S2059798318009324
Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
doi: 10.1002/jcc.20084
Pettersen, E. et al. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).
doi: 10.1002/pro.3943
Jo, S., Kim, T., Iyer, V. G. & Im, W. CHARMM-GUI: a web-based graphical user interface for CHARMM. J. Comput. Chem. 29, 1859–1865 (2008).
doi: 10.1002/jcc.20945
Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).
doi: 10.1016/j.jmb.2007.05.022
Ashkenazy, H. et al. ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Res. 44, W344–W350 (2016).
doi: 10.1093/nar/gkw408
Chan, L. C. et al. PBP 4 mediates high-level resistance to new-generation cephalosporins in Staphylococcus aureus. Antimicrob. Agents Chemother. 60, 3934–3941 (2016).
doi: 10.1128/AAC.00358-16
Chan, L. C. et al. Ceftobiprole- and Ceftaroline-resistant methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 59, 2960–2963 (2015).
doi: 10.1128/AAC.05004-14
Lowy, F. D. Antimicrobial resistance: the example of Staphylococcus aureus. J. Clin. Invest. 111, 1265–1273 (2003).
doi: 10.1172/JCI18535

Auteurs

J Andrew N Alexander (JAN)

Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia, Canada.
Centre for Blood Research, The University of British Columbia, Vancouver, British Columbia, Canada.

Liam J Worrall (LJ)

Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia, Canada.
Centre for Blood Research, The University of British Columbia, Vancouver, British Columbia, Canada.
HRMEM Facility, The University of British Columbia, Vancouver, British Columbia, Canada.

Jinhong Hu (J)

Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia, Canada.
Centre for Blood Research, The University of British Columbia, Vancouver, British Columbia, Canada.

Marija Vuckovic (M)

Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia, Canada.
Centre for Blood Research, The University of British Columbia, Vancouver, British Columbia, Canada.

Nidhi Satishkumar (N)

Department of Microbial Pathogenesis, School of Dentistry, University of Maryland, Baltimore, MD, USA.
Institute of Marine and Environmental Technology, Baltimore, MD, USA.

Raymond Poon (R)

Department of Microbial Pathogenesis, School of Dentistry, University of Maryland, Baltimore, MD, USA.
Institute of Marine and Environmental Technology, Baltimore, MD, USA.

Solmaz Sobhanifar (S)

Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia, Canada.
Centre for Blood Research, The University of British Columbia, Vancouver, British Columbia, Canada.

Federico I Rosell (FI)

Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia, Canada.
Centre for Blood Research, The University of British Columbia, Vancouver, British Columbia, Canada.

Joshua Jenkins (J)

Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia, Canada.
Centre for Blood Research, The University of British Columbia, Vancouver, British Columbia, Canada.

Daniel Chiang (D)

Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.

Wesley A Mosimann (WA)

Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia, Canada.
Centre for Blood Research, The University of British Columbia, Vancouver, British Columbia, Canada.

Henry F Chambers (HF)

Division of Infectious Diseases, School of Medicine, University of California, San Francisco, San Francisco, CA, USA.

Mark Paetzel (M)

Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.

Som S Chatterjee (SS)

Department of Microbial Pathogenesis, School of Dentistry, University of Maryland, Baltimore, MD, USA.
Institute of Marine and Environmental Technology, Baltimore, MD, USA.

Natalie C J Strynadka (NCJ)

Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia, Canada. ncjs@mail.ubc.ca.
Centre for Blood Research, The University of British Columbia, Vancouver, British Columbia, Canada. ncjs@mail.ubc.ca.
HRMEM Facility, The University of British Columbia, Vancouver, British Columbia, Canada. ncjs@mail.ubc.ca.

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