A Binding-Site Barrier Affects Imaging Efficiency of High Affinity Amyloid-Reactive Peptide Radiotracers In Vivo

Amyloid is a complex pathology associated with a growing number of diseases including Alzheimer’s disease, type 2 diabetes, rheumatoid arthritis, and myeloma. The distribution and extent of amyloid deposition in body organs establishes the prognosis and can define treatment options; therefore, determining the amyloid load by using non-invasive molecular imaging is clinically important. We have identified a heparin-binding peptide designated p5 that, when radioiodinated, was capable of selectively imaging systemic visceral AA amyloidosis in a murine model of the disease. The p5 peptide was posited to bind effectively to amyloid deposits, relative to similarly charged polybasic heparin-reactive peptides, because it adopted a polar α helix secondary structure. We have now synthesized a variant, p5R, in which the 8 lysine amino acids of p5 have been replaced with arginine residues predisposing the peptide toward the α helical conformation in an effort to enhance the reactivity of the peptide with the amyloid substrate. The p5R peptide had higher affinity for amyloid and visualized AA amyloid in mice by using SPECT/CT imaging; however, the microdistribution, as evidenced in micro-autoradiographs, was dramatically altered relative to the p5 peptide due to its increased affinity and a resultant “binding site barrier” effect. These data suggest that radioiodinated peptide p5R may be optimal for the in vivo detection of discreet, perivascular amyloid, as found in the brain and pancreatic vasculature, by using molecular imaging techniques; however, peptide p5, due to its increased penetration, may yield more quantitative imaging of expansive tissue amyloid deposits.     

The preservation of ultrastructure in saturated phosphatidyl cholines by tannic acid in model systems and type II pneumocytes

The preservation for electron microscopy of saturated phospholipids in general, and phosphatidyl choline (PC)in particular, remains and unsolved problem since OsO(4) and glutaraldehyde are incapable of interacting with PC directly. However, by introducing tannic acid preceding osmication, we were able to demonstrate highly ordered, preserved lamellar structures in model experiments with saturated PC, and in vivo experiments type II pneumocytes of lung tissue. The secretory bodies of the latter are known to contain a high proportion of these saturated phospholipids. In both cases, the repeating periodicity approximated 45 A. It was determined that tannic acid interacts with the choline component of PC to form a "complex," which then could be stabilized by treatment with OsO(4). In the absence of osmication, the PC-tannic acid complex acid did not survive conventional dehydration techniques, but osmication permitted conventional Epon embedment. Sphingomyelin (SPH), which contains choline, behaved similarly in model experiments. But there was no evidence of a comparable reaction with tannic acid using phosphatidyl ethanolamine (PEA), phosphatidyl serine (PS), or phosphstidy inositol (PI). Chemical studies indicted a high pH dependency for the formation of the PC- tannic acid complex. Also, experiments demonstrated its dissociation in various organic solvents. Sharp delineation and great contrast of the polar zones in the ordered lamellar structures was achieved by additional staining with lead citrate thus leading to the conclusion that tannic acid serves as a multivalent agent, capable of simultaneous interaction with saturated PC, OsO(4), and lead citrate stains.     

LplA1-dependent utilization of host lipoyl peptides enables Listeria cytosolic growth and virulence

The bacterial pathogen Listeria monocytogenes replicates within the cytosol of mammalian cells. Mechanisms by which the bacterium exploits the host cytosolic environment for essential nutrients are poorly defined. L. monocytogenes is a lipoate auxotroph and must scavenge this critical cofactor, using lipoate ligases to facilitate attachment of the lipoyl moiety to metabolic enzyme complexes. Although the L. monocytogenes genome encodes two putative lipoate ligases, LplA1 and LplA2, intracellular replication and virulence require only LplA1. Here we show that LplA1 enables utilization of host-derived lipoyl peptides by L. monocytogenes. LplA1 is dispensable for growth in the presence of free lipoate, but necessary for growth on low concentrations of mammalian lipoyl peptides. Furthermore, we demonstrate that the intracellular growth defect of the DeltalplA1 mutant is rescued by addition of exogenous lipoic acid to host cells, suggesting that L. monocytogenes dependence on LplA1 is dictated by limiting concentrations of available host lipoyl substrates. Thus, the ability of L. monocytogenes and other intracellular pathogens to efficiently use host lipoyl peptides as a source of lipoate may be a requisite adaptation for life within the mammalian cell.     

Structural Basis for the Recognition of Peptide RJPXD33 by Acyltransferases in Lipid A Biosynthesis

UDP-N-acetylglucosamine acyltransferase (LpxA) and UDP-3-O-(acyl)-glucosamine acyltransferase (LpxD) constitute the essential, early acyltransferases of lipid A biosynthesis. Recently, an antimicrobial peptide inhibitor, RJPXD33, was identified with dual affinity for LpxA and LpxD. To gain a fundamental understanding of the molecular basis of inhibitor binding, we determined the crystal structure of LpxA from Escherichia coli in complex with RJPXD33 at 1.9 Å resolutions. Our results suggest that the peptide binds in a unique modality that mimics (R)-β-hydroxyacyl pantetheine binding to LpxA and displays how the peptide binds exclusive of the native substrate, acyl-acyl carrier protein. Acyltransferase binding studies with photo-labile RJPXD33 probes and truncations of RJPXD33 validated the structure and provided fundamental insights for future design of small molecule inhibitors. Overlay of the LpxA-RJPXD33 structure with E. coli LpxD identified a complementary peptide binding pocket within LpxD and serves as a model for further biochemical characterization of RJPXD33 binding to LpxD.     

Bone marrow-derived stromal cells home to and remain in the infarcted rat heart but fail to improve function: an in vivo cine-MRI study

Basic and clinical studies have shown that bone marrow cell therapy can improve cardiac function following infarction. In experimental animals, reported stem cell-mediated changes range from no measurable improvement to the complete restoration of function. In the clinic, however, the average improvement in left ventricular ejection fraction is around 2% to 3%. A possible explanation for the discrepancy between basic and clinical results is that few basic studies have used the magnetic resonance (MR) imaging (MRI) methods that were used in clinical trials for measuring cardiac function. Consequently, we employed cine-MR to determine the effect of bone marrow stromal cells (BMSCs) on cardiac function in rats. Cultured rat BMSCs were characterized using flow cytometry and labeled with iron oxide particles and a fluorescent marker to allow in vivo cell tracking and ex vivo cell identification, respectively. Neither label affected in vitro cell proliferation or differentiation. Rat hearts were infarcted, and BMSCs or control media were injected into the infarct periphery (n = 34) or infused systemically (n = 30). MRI was used to measure cardiac morphology and function and to determine cell distribution for 10 wk after infarction and cell therapy. In vivo MRI, histology, and cell reisolation confirmed successful BMSC delivery and retention within the myocardium throughout the experiment. However, no significant improvement in any measure of cardiac function was observed at any time. We conclude that cultured BMSCs are not the optimal cell population to treat the infarcted heart.     

Structural search and retrieval using a tableau representation of protein folding patterns

Comparison and classification of folding patterns from a database of protein structures is crucial to understand the principles of protein architecture, evolution and function. Current search methods for proteins with similar folding patterns are slow and computationally intensive. The sharp growth in the number of known protein structures poses severe challenges for methods of structural comparison. There is a need for methods that can search the database of structures accurately and rapidly. We provide several methods to search for similar folding patterns using a concise tableau representation of proteins that encodes the relative geometry of secondary structural elements. Our first approach allows the extraction of identical and very closely-related protein folding patterns in constant-time (per hit). Next, we address the hard computational problem of extraction of maximally-similar subtableaux, when comparing two tableaux. We solve the problem using Quadratic and Linear integer programming formulations and demonstrate their power to identify subtle structural similarities, especially when protein structures significantly diverge. Finally, we describe a rapid and accurate method for comparing a query structure against a database of protein domains, TableauSearch. TableauSearch is rapid enough to search the entire structural database in seconds on a standard desktop computer. Our analysis of TableauSearch on many queries shows that the method is very accurate in identifying similarities of folding patterns, even between distantly related proteins. AVAILABILITY: A web server implementing the TableauSearch is available from http://hollywood.bx.psu.edu/TabSearch.     

Computer modeling of a potential agent against SARS-Cov-2 (COVID-19) protease

We have modeled modifications of a known ligand to the SARS-CoV-2 (COVID-19) protease, that can form a covalent adduct, plus additional ligand-protein hydrogen bonds.     

Seasonally Dependent Relationships between Indicators of Malaria Transmission and Disease Provided by Mathematical Model Simulations

Evaluating the effectiveness of malaria control interventions on the basis of their impact on transmission as well as impact on morbidity and mortality is becoming increasingly important as countries consider pre-elimination and elimination as well as disease control. Data on prevalence and transmission are traditionally obtained through resource-intensive epidemiological and entomological surveys that become difficult as transmission decreases. This work employs mathematical modeling to examine the relationships between malaria indicators allowing more easily measured data, such as routine health systems data on case incidence, to be translated into measures of transmission and other malaria indicators. Simulations of scenarios with different levels of malaria transmission, patterns of seasonality and access to treatment were run with an ensemble of models of malaria epidemiology and within-host dynamics, as part of the OpenMalaria modeling platform. For a given seasonality profile, regression analysis mapped simulation results of malaria indicators, such as annual average entomological inoculation rate, prevalence, incidence of uncomplicated and severe episodes, and mortality, to an expected range of values of any of the other indicators. Results were validated by comparing simulated relationships between indicators with previously published data on these same indicators as observed in malaria endemic areas. These results allow for direct comparisons of malaria transmission intensity estimates made using data collected with different methods on different indicators. They also address key concerns with traditional methods of quantifying transmission in areas of differing transmission intensity and sparse data. Although seasonality of transmission is often ignored in data compilations, the models suggest it can be critically important in determining the relationship between transmission and disease. Application of these models could help public health officials detect changes of disease dynamics in a population and plan and assess the impact of malaria control interventions.