Analytic philosophy for biomedical research: the imperative of applying yesterday's timeless messages to today's impasses

The mantra that "the best way to predict the future is to invent it" (attributed to the computer scientist Alan Kay) exemplifies some of the expectations from the technical and innovative sides of biomedical research at present. However, for technical advancements to make real impacts both on patient health and genuine scientific understanding, quite a number of lingering challenges facing the entire spectrum from protein biology all the way to randomized controlled trials should start to be overcome. The proposal in this chapter is that philosophy is essential in this process. By reviewing select examples from the history of science and philosophy, disciplines which were indistinguishable until the mid-nineteenth century, I argue that progress toward the many impasses in biomedicine can be achieved by emphasizing theoretical work (in the true sense of the word 'theory') as a vital foundation for experimental biology. Furthermore, a philosophical biology program that could provide a framework for theoretical investigations is outlined

Membrane anchoring stabilizes and favors secretion of New Delhi metallo-β-lactamase

Carbapenems, 'last-resort' β-lactam antibiotics, are inactivated by zinc-dependent metallo-β-lactamases (MBLs). The host innate immune response withholds nutrient metal ions from microbial pathogens by releasing metal-chelating proteins such as calprotectin. We show that metal sequestration is detrimental for the accumulation of MBLs in the bacterial periplasm, because those enzymes are readily degraded in their nonmetallated form. However, the New Delhi metallo-β-lactamase (NDM-1) can persist under conditions of metal depletion. NDM-1 is a lipidated protein that anchors to the outer membrane of Gram-negative bacteria. Membrane anchoring contributes to the unusual stability of NDM-1 and favors secretion of this enzyme in outer-membrane vesicles (OMVs). OMVs containing NDM-1 can protect nearby populations of bacteria from otherwise lethal antibiotic levels, and OMVs from clinical pathogens expressing NDM-1 can carry this MBL and the bla[subscript NDM] gene. We show that protein export into OMVs can be targeted, providing possibilities of new antibacterial therapeutic strategies.Kinship Foundation. Searle Scholars ProgramMassachusetts Institute of Technology. Department of Chemistr

Biochemical and Spectroscopic Observation of Mn(II) Sequestration from Bacterial Mn(II) Transport Machinery by Calprotectin

Human calprotectin (CP, S100A8/S100A9 oligomer) is a metal-sequestering host-defense protein that prevents bacterial acquisition of Mn(II). In this work, we investigate Mn(II) competition between CP and two solute-binding proteins that Staphylococcus aureus and Streptococcus pneumoniae, Gram-positive bacterial pathogens of significant clinical concern, use to obtain Mn(II) when infecting a host. Biochemical and electron paramagnetic resonance (EPR) spectroscopic analyses demonstrate that CP outcompetes staphylococcal MntC and streptococcal PsaA for Mn(II). This behavior requires the presence of excess Ca(II) ions, which enhance the Mn(II) affinity of CP. This report presents new spectroscopic evaluation of two Mn(II) proteins important for bacterial pathogenesis, direct observation of Mn(II) sequestration from bacterial Mn(II) acquisition proteins by CP, and molecular insight into the extracellular battle for metal nutrients that occurs during infection

The Hexahistidine Motif of Host-Defense Protein Human Calprotectin Contributes to Zinc Withholding and Its Functional Versatility

Human calprotectin (CP, S100A8/S100A9 oligomer, MRP-8/MRP-14 oligomer) is an abundant host-defense protein that is involved in the metal-withholding innate immune response. CP coordinates a variety of divalent first-row transition metal ions, which is implicated in its antimicrobial function, and its ability to sequester nutrient Zn­(II) ions from microbial pathogens has been recognized for over two decades. CP has two distinct transition-metal-binding sites formed at the S100A8/S100A9 dimer interface, including a histidine-rich site composed of S100A8 residues His17 and His27 and S100A9 residues His91 and His95. In this study, we report that CP binds Zn­(II) at this site using a hexahistidine motif, completed by His103 and His105 of the S100A9 C-terminal tail and previously identified as the high-affinity Mn­(II) and Fe­(II) coordination site. Zn­(II) binding at this unique site shields the S100A9 C-terminal tail from proteolytic degradation by proteinase K. X-ray absorption spectroscopy and Zn­(II) competition titrations support the formation of a Zn­(II)-His₆ motif. Microbial growth studies indicate that the hexahistidine motif is important for preventing microbial Zn­(II) acquisition from CP by the probiotic <i>Lactobacillus plantarum</i> and the opportunistic human pathogen <i>Candida albicans</i>. The Zn­(II)-His₆ site of CP expands the known biological coordination chemistry of Zn­(II) and provides new insight into how the human innate immune system starves microbes of essential metal nutrients

Synthesis of a Tris(phosphaalkene)phosphine Ligand and Fundamental Organometallic Reactions on Its Sterically Shielded Metal Complexes

A new tris­(phosphaalkene)­phosphine ligand (<b>1</b>) was synthesized via phospha-Wittig methodology. Metalation of <b>1</b> with [RhCl­(C₂H₄)₂]₂ and [IrCl­(COE)₂]₂ (COE = cyclooctene) produced trigonal bipyramidal metal chlorides <b>2a</b> (M = Rh) and <b>2b</b> (M = Ir) in which the ligand coordinates in a tetradentate fashion. X-ray crystallographic studies on <b>1</b>·1.5THF, <b>2a</b>·5CHCl₃, and <b>2b</b>·2.5CHCl₃ combined with DFT calculations revealed a pronounced change in hybridization of the phosphaalkene phosphorus atoms upon coordination to the Rh/Ir centers, resulting in highly sterically congested metal complexes. Nucleophilic substitution on <b>2a</b> with NaN₃ afforded Rh–N₃ complex <b>3</b>; computational analysis, IR spectroscopy, and ¹⁵N­{¹H} NMR spectroscopy on isotopologue ^<b>15</b><b>N-3</b> provided additional structural insights. Halide abstraction of the chloride in <b>2b</b> with AgOTf in the presence of acetonitrile afforded cationic Ir–NCMe complex <b>4</b>. Evidence of the bound acetonitrile unit was obtained by 2D NMR spectroscopy and deuterium labeling studies