Antimicrobial Peptide-Poly(ethylene glycol) Conjugates: Connecting Molecular Architecture, Solution Properties, and Functional Performance.

Antimicrobial peptides (AMPs) are promising alternatives to conventional antibiotics for treating infections caused by drug-resistant bacteria; yet, many peptides are limited by toxicity to eukaryotic cells and instability in biological environments. Conjugation to linear polymers that reduce cytotoxicity and improve stability, however, often decreases antimicrobial activity. In this work, we combine the biocompatibility advantages of poly(ethylene glycol) (PEG) with the efficacy merits of nonlinear polymer architectures that accommodate multiple AMPs per molecule. By conjugating a chemokine-derived AMP, stapled Ac-P9, to linear and star-shaped PEG with various arm numbers and lengths, we investigated the role of molecular architecture in solution properties (i.e., ζ-potential, size, and morphology) and performance (i.e., antimicrobial activity, hemolysis, and protease resistance). Linear, 4-arm, and 8-arm conjugates with 2-2.5 kDa PEG arms were found to form nanoscale structures in solution with lower ζ-potentials relative to the unconjugated AMP, suggesting that the polymer partially shields the cationic AMP. Reducing the length of the PEG arms of the 8-arm conjugate to 1.25 kDa appeared to better reveal the peptide, seen by the increased ζ-potential, and promote assembly into particles with a larger size and defined spherical morphology. The antimicrobial effects exerted by the short 8-arm conjugate rivaled that of the unconjugated peptide, and the AMP constituents of the short 8-arm conjugate were protected from proteolytic degradation. All other conjugates examined also imparted a degree of protease resistance, but exhibited some reduced level of antimicrobial activity as compared to the AMP alone. None of the conjugates caused significant cytotoxic effects, which bodes well for their future potential to treat infections. While enhancing proteolytic stability often comes with the cost of lower antimicrobial activity, we have found that presenting AMPs at high density on a neutral nonlinear polymer strikes a favorable balance, exhibiting both enhanced stability and high antimicrobial activity.

Enterobacterales draft genome sequences: 15 historical (1998-2004) and 30 contemporary (2015-2016) clinical isolates from Pakistan.

The continued emergence and spread of antimicrobial resistance among pathogenic bacteria are ever-growing threats to health and economy. Here, we report the draft genomes for 45 Enterobacterales clinical isolates, including historical and contemporary drug-resistant organisms, obtained in Pakistan between 1998 and 2016: 5 Serratia, 3 Salmonella, 3 Enterobacter, and 34 Klebsiella.

Investigation of multidrug-resistant plasmids from carbapenemase-producing Klebsiella pneumoniae clinical isolates from Pakistan.

Objectives: The study aim was to investigate multidrug-resistant (MDR) plasmids from a collection of 10 carbapenemase-producing Klebsiella pneumoniae clinical isolates identified within the same healthcare institution in Pakistan. Full characterization of the MDR plasmids including structure, typing characteristics, and AMR content as well as determination of their plasmid-based antimicrobial susceptibility profiles were carried out. Methods: Plasmids were isolated from 10 clinical isolates of Klebsiella pneumoniae, and from a corresponding set of Escherichia coli transconjugants, then sequenced using Nanopore/Illumina technology to generate plasmid hybrid assemblies. Full characterization of MDR plasmids, including determination of next generation sequencing (NGS)-based AMR profiles, plasmid incompatibility groups, and types, was carried out. The structure of MDR plasmids was analyzed using the Galileo AMR platform. For E. coli transconjugants, the NGS-based AMR profiles were compared to NGS-predicted AMR phenotypes and conventional broth microdilution (BMD) antimicrobial susceptibility testing (AST) results. Results: All carbapenemase-producing K. pneumoniae isolates (carrying either blaNDM-1, or/and blaOXA-48) carried multiple AMR plasmids encoding 34 antimicrobial resistance genes (ARGs) conferring resistance to antimicrobials from 6 different classes. The plasmid incompatibility groups and types identified were: IncC (types 1 and 3), IncFIA (type 26) IncFIB, IncFII (types K1, K2, K7, and K9), IncHI1B, and IncL. None of the blaNDM-1 and blaESBL-plasmids identified in this study were previously described. Most blaNDM-1-plasmids shared identical AMR regions suggesting potential genetic material/plasmid exchange between K. pneumoniae isolates of this collection. The majority of NGS-based AMR profiles from the E. coli transconjugants correlated well with both NGS-based predicted and conventional AST results. Conclusion: This study highlights the complexity and diversity of the plasmid-based genetic background of carbapenemase-producing clinical isolates from Pakistan. This study emphasizes the need for characterization of MDR plasmids to determine their complete molecular background and monitor AMR through plasmid transmission between multi-resistant bacterial pathogens.

Disparate Regions of the Human Chemokine CXCL10 Exhibit Broad-Spectrum Antimicrobial Activity against Biodefense and Antibiotic-Resistant Bacterial Pathogens.

CXCL10 is a pro-inflammatory chemokine produced by the host in response to microbial infection. In addition to canonical, receptor-dependent actions affecting immune-cell migration and activation, CXCL10 has also been found to directly kill a broad range of pathogenic bacteria. Prior investigations suggest that the bactericidal effects of CXCL10 occur through two distinct pathways that compromise the cell envelope. These observations raise the intriguing notion that CXCL10 features a separable pair of antimicrobial domains. Herein, we affirm this possibility through peptide-based mapping and structure/function analyses, which demonstrate that discrete peptides derived from the N- and C-terminal regions of CXCL10 mediate bacterial killing. The N-terminal derivative, peptide P1, exhibited marked antimicrobial activity against Bacillus anthracis vegetative bacilli and spores, as well as antibiotic-resistant clinical isolates of Klebsiella pneumoniae, Acinetobacter baumannii, Enterococcus faecium, and Staphylococcus aureus, among others. At bactericidal concentrations, peptide P1 had a minimal degree of chemotactic activity, but did not cause red blood cell hemolysis or cytotoxic effects against primary human cells. The C-terminal derivative, peptide P9, exhibited antimicrobial effects, but only against Gram-negative bacteria in low-salt medium─conditions under which the peptide can adopt an α-helical conformation. The introduction of a hydrocarbon staple induced and stabilized α-helicity; accordingly, stapled peptide P9 displayed significantly improved bactericidal effects against both Gram-positive and Gram-negative bacteria in media containing physiologic levels of salt. Together, our findings identify and characterize the antimicrobial regions of CXCL10 and functionalize these novel determinants as discrete peptides with potential therapeutic utility against difficult-to-treat pathogens.

The Pro-Inflammatory Chemokines CXCL9, CXCL10 and CXCL11 Are Upregulated Following SARS-CoV-2 Infection in an AKT-Dependent Manner.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible RNA virus that is the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Patients with severe COVID-19 may develop acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) and require mechanical ventilation. Key features of SARS-CoV-2 induced pulmonary complications include an overexpression of pro-inflammatory chemokines and cytokines that contribute to a 'cytokine storm.' In the current study an inflammatory state in Calu-3 human lung epithelial cells was characterized in which significantly elevated transcripts of the immunostimulatory chemokines CXCL9, CXCL10, and CXCL11 were present. Additionally, an increase in gene expression of the cytokines IL-6, TNFα, and IFN-γ was observed. The transcription of CXCL9, CXCL10, IL-6, and IFN-γ was also induced in the lungs of human transgenic angiotensin converting enzyme 2 (ACE2) mice infected with SARS-CoV-2. To elucidate cell signaling pathways responsible for chemokine upregulation in SARS-CoV-2 infected cells, small molecule inhibitors targeting key signaling kinases were used. The induction of CXCL9, CXCL10, and CXCL11 gene expression in response to SARS-CoV-2 infection was markedly reduced by treatment with the AKT inhibitor GSK690693. Samples from COVID-19 positive individuals also displayed marked increases in CXCL9, CXCL10, and CXCL11 transcripts as well as transcripts in the AKT pathway. The current study elucidates potential pathway specific targets for reducing the induction of chemokines that may be contributing to SARS-CoV-2 pathogenesis via hyperinflammation.

Molecular engineering of antimicrobial peptide (AMP)-polymer conjugates.

As antimicrobial resistance becomes an increasing threat, bringing significant economic and health burdens, innovative antimicrobial treatments are urgently needed. While antimicrobial peptides (AMPs) are promising therapeutics, exhibiting high activity against resistant bacterial strains, limited stability and toxicity to mammalian cells has hindered clinical development. Attaching AMPs to polymers provides opportunities to present AMPs in a way that maximizes bacterial killing while enhancing compatibility with mammalian cells, stability, and solubility. Conjugation of an AMP to a linear hydrophilic polymer yields the desired improvements in stability, mammalian cell compatibility, and solubility, yet often markedly reduces bactericidal effects. Non-linear polymer architectures and supramolecular assemblies that accommodate multiple AMPs per polymer chain afford AMP-polymer conjugates that strike a superior balance of antimicrobial activity, mammalian cell compatibility, stability, and solubility. Therefore, we review the design criteria, building blocks, and synthetic strategies for engineering AMP-polymer conjugates, emphasizing the connection between molecular architecture and antimicrobial performance to inspire and enable further innovation to advance this emerging class of biomaterials.

Draft Genome Sequences of Two Extensively Drug-Resistant Strains of Acinetobacter baumannii Isolated from Clinical Samples in Pakistan.

Infections in immunocompromised patients that are caused by extensively drug-resistant (XDR) Acinetobacter baumannii strains have been increasingly reported worldwide. In particular, carbapenem-resistant A. baumannii strains are a prominent cause of health care-associated infections. Here, we report draft genome assemblies for two clinical XDR A. baumannii isolates obtained from hospitalized patients in Pakistan.

Draft Genome Sequences of Antimicrobial-Resistant Shigella Clinical Isolates from Pakistan.

Shigella spp. are the most common cause of dysentery in developing countries and the second leading cause of diarrheal deaths worldwide. Multidrug-resistant (MDR) Shigella spp. are a serious threat to global health. Herein, we report draft genome sequences for three MDR Shigella isolates from Pakistan, two Shigella flexneri isolates and one Shigella sonnei isolate.

Mechanistic insights and therapeutic opportunities of antimicrobial chemokines.

Chemokines are a family of small proteins best known for their ability to orchestrate immune cell trafficking and recruitment to sites of infection. Their role in promoting host defense is multiplied by a number of additional receptor-dependent biological activities, and most, but not all, chemokines have been found to mediate direct antimicrobial effects against a broad range of microorganisms. The molecular mechanism(s) by which antimicrobial chemokines kill bacteria remains unknown; however, recent observations have expanded our fundamental understanding of chemokine-mediated bactericidal activity to reveal increasingly diverse and complex actions. In the current review, we present and consider mechanistic insights of chemokine-mediated antimicrobial activity against bacteria. We also discuss how contemporary advances are reshaping traditional paradigms and opening up new and innovative avenues of research with translational implications. Towards this end, we highlight a developing framework for leveraging chemokine-mediated bactericidal and immunomodulatory effects to advance pioneering therapeutic approaches for treating bacterial infections, including those caused by multidrug-resistant pathogens.

DksA-DnaJ redox interactions provide a signal for the activation of bacterial RNA polymerase.

RNA polymerase is the only known protein partner of the transcriptional regulator DksA. Herein, we demonstrate that the chaperone DnaJ establishes direct, redox-based interactions with oxidized DksA. Cysteine residues in the zinc finger of DksA become oxidized in Salmonella exposed to low concentrations of hydrogen peroxide (H2O2). The resulting disulfide bonds unfold the globular domain of DksA, signaling high-affinity interaction of the C-terminal α-helix to DnaJ. Oxidoreductase and chaperone activities of DnaJ reduce the disulfide bonds of its client and promote productive interactions between DksA and RNA polymerase. Simultaneously, guanosine tetraphosphate (ppGpp), which is synthesized by RelA in response to low concentrations of H2O2, binds at site 2 formed at the interface of DksA and RNA polymerase and synergizes with the DksA/DnaJ redox couple, thus activating the transcription of genes involved in amino acid biosynthesis and transport. However, the high concentrations of ppGpp produced by Salmonella experiencing oxidative stress oppose DksA/DnaJ-dependent transcription. Cumulatively, the interplay of DksA, DnaJ, and ppGpp on RNA polymerase protects Salmonella from the antimicrobial activity of the NADPH phagocyte oxidase. Our research has identified redox-based signaling that activates the transcriptional activity of the RNA polymerase regulator DksA.

Guanosine tetraphosphate relieves the negative regulation of Salmonella pathogenicity island-2 gene transcription exerted by the AT-rich ssrA discriminator region.

The repressive activity of ancestral histone-like proteins helps integrate transcription of foreign genes with discrepant AT content into existing regulatory networks. Our investigations indicate that the AT-rich discriminator region located between the -10 promoter element and the transcription start site of the regulatory gene ssrA plays a distinct role in the balanced expression of the Salmonella pathogenicity island-2 (SPI2) type III secretion system. The RNA polymerase-binding protein DksA activates the ssrAB regulon post-transcriptionally, whereas the alarmone guanosine tetraphosphate (ppGpp) relieves the negative regulation imposed by the AT-rich ssrA discriminator region. An increase in the GC-content of the ssrA discriminator region enhances ssrAB transcription and SsrB translation, thus activating the expression of downstream SPI2 genes. A Salmonella strain expressing a GC-rich ssrA discriminator region is attenuated in mice and grows poorly intracellularly. The combined actions of ppGpp and DksA on SPI2 expression enable Salmonella to grow intracellularly, and cause disease in a murine model of infection. Collectively, these findings indicate that (p)ppGpp relieves the negative regulation associated with the AT-rich discriminator region in the promoter of the horizontally-acquired ssrA gene, whereas DksA activates ssrB gene expression post-transcriptionally. The combined effects of (p)ppGpp and DksA on the ssrAB locus facilitate a balanced SPI2 virulence gene transcription that is essential for Salmonella pathogenesis.

Resistome of carbapenem- and colistin-resistant Klebsiella pneumoniae clinical isolates.

The emergence and dissemination of carbapenemases, bacterial enzymes able to inactivate most ß-lactam antibiotics, in Enterobacteriaceae is of increasing concern. The concurrent spread of resistance against colistin, an antibiotic of last resort, further compounds this challenge further. Whole-genome sequencing (WGS) can play a significant role in the rapid and accurate detection/characterization of existing and emergent resistance determinants, an essential aspect of public health surveillance and response activities to combat the spread of antimicrobial resistant bacteria. In the current study, WGS data was used to characterize the genomic content of antimicrobial resistance genes, including those encoding carbapenemases, in 10 multidrug-resistant Klebsiella pneumoniae isolates from Pakistan. These clinical isolates represented five sequence types: ST11 (n = 3 isolates), ST14 (n = 3), ST15 (n = 1), ST101 (n = 2), and ST307 (n = 1). Resistance profiles against 25 clinically-relevant antimicrobials were determined by broth microdilution; resistant phenotypes were observed for at least 15 of the 25 antibiotics tested in all isolates except one. Specifically, 8/10 isolates were carbapenem-resistant and 7/10 isolates were colistin-resistant. The blaNDM-1 and blaOXA-48 carbapenemase genes were present in 7/10 and 5/10 isolates, respectively; including 2 isolates carrying both genes. No plasmid-mediated determinants for colistin resistance (e.g. mcr) were detected, but disruptions and mutations in chromosomal loci (i.e. mgrB and pmrB) previously reported to confer colistin resistance were observed. A blaOXA-48-carrying IncL/M-type plasmid was found in all blaOXA-48-positive isolates. The application of WGS to molecular epidemiology and surveillance studies, as exemplified here, will provide both a more complete understanding of the global distribution of MDR isolates and a robust surveillance tool useful for detecting emerging threats to public health.

CXC Chemokines Exhibit Bactericidal Activity against Multidrug-Resistant Gram-Negative Pathogens.

The continued rise and spread of antimicrobial resistance among bacterial pathogens pose a serious challenge to global health. Countering antimicrobial-resistant pathogens requires a multifaceted effort that includes the discovery of novel therapeutic approaches. Here, we establish the capacity of the human CXC chemokines CXCL9 and CXCL10 to kill multidrug-resistant Gram-negative bacteria, including New Delhi metallo-beta-lactamase-1-producing Klebsiella pneumoniae and colistin-resistant members of the family Enterobacteriaceae that harbor the mobile colistin resistance protein MCR-1 and thus possess phosphoethanolamine-modified lipid A. Colistin-resistant K. pneumoniae isolates affected by genetic mutation of the PmrA/PmrB two-component system, a chromosomally encoded regulator of lipopolysaccharide modification, and containing 4-amino-4-deoxy-l-arabinose-modified lipid A were also found to be susceptible to chemokine-mediated antimicrobial activity. However, loss of PhoP/PhoQ autoregulatory control, caused by disruption of the gene encoding the negative regulator MgrB, limited the bactericidal effects of CXCL9 and CXCL10 in a variable, strain-specific manner. Cumulatively, these findings provide mechanistic insight into chemokine-mediated antimicrobial activity, highlight disparities amongst determinants of colistin resistance, and suggest that chemokine-mediated bactericidal effects merit additional investigation as a therapeutic avenue for treating infections caused by multidrug-resistant pathogens.IMPORTANCE As bacterial pathogens become resistant to multiple antibiotics, the infections they cause become increasingly difficult to treat. Carbapenem antibiotics provide an essential clinical barrier against multidrug-resistant bacteria; however, the dissemination of bacterial enzymes capable of inactivating carbapenems threatens the utility of these important antibiotics. Compounding this concern is the global spread of bacteria invulnerable to colistin, a polymyxin antibiotic considered to be a last line of defense against carbapenem-resistant pathogens. As the effectiveness of existing antibiotics erodes, it is critical to develop innovative antimicrobial therapies. To this end, we demonstrate that the chemokines CXCL9 and CXCL10 kill the most concerning carbapenem- and colistin-resistant pathogens. Our findings provide a unique and timely foundation for therapeutic strategies capable of countering antibiotic-resistant "superbugs."

Genome Sequences of Multidrug-Resistant, Colistin-Susceptible and -Resistant Klebsiella pneumoniae Clinical Isolates from Pakistan.

The emergence and spread of colistin resistance among multidrug-resistant (MDR) Klebsiella pneumoniae represent a critical threat to global health. Here, we report the complete genome sequences of 10 MDR, colistin-susceptible and -resistant K. pneumoniae clinical isolates obtained in Pakistan between 2010 and 2013.

DksA-Dependent Transcriptional Regulation in Salmonella Experiencing Nitrosative Stress.

Redox-based signaling is fundamental to the capacity of bacteria to sense, and respond to, nitrosative and oxidative stress encountered in natural and host environments. The conserved RNA polymerase regulatory protein DksA is a thiol-based sensor of reactive nitrogen and oxygen species. DksA-dependent transcriptional control promotes antinitrosative and antioxidative defenses that contribute to Salmonella pathogenesis. The specific adaptive changes mediated by DksA in response to reactive species, however, have not been elucidated. Herein, we characterize DksA-dependent changes in gene expression in Salmonella enterica experiencing nitrosative stress. Genome-wide expression analysis of wild-type and ΔdksA Salmonella exposed to the nitric oxide ((•)NO) donor DETA NONOate demonstrated (•)NO- and DksA-dependent regulatory control of 427 target genes. Transcriptional changes centered primarily on genes encoding aspects of cellular metabolism. Several antioxidants and oxidoreductases important in redox buffering, (•)NO detoxification, and damage repair were also observed to be up-regulated in an (•)NO- and DksA-dependent manner. Compared to wild-type bacteria, (•)NO-treated ΔdksA Salmonella exhibited a de-repression of genes encoding components of iron homeostasis and failed to activate sulfur assimilation and cysteine biosynthetic operons. As cysteine is integral to efficient antinitrosative and antioxidative defense and repair programs, we further examined the redox-responsive transcriptional control of cysteine biosynthesis by DksA. These investigations revealed that the activation of genes comprising cysteine biosynthesis also occurs in response to hydrogen peroxide, is dependent upon the redox-sensing zinc finger motif of DksA, and requires the transcriptional regulator CysB. Our observations demonstrate that DksA mediates global adaptation to nitrosative stress in Salmonella and provide unique insight into a novel regulatory mechanism by which cysteine biosynthesis is controlled in response to reactive oxygen and nitrogen species.

Redox-Active Sensing by Bacterial DksA Transcription Factors Is Determined by Cysteine and Zinc Content.

UNLABELLED: The four-cysteine zinc finger motif of the bacterial RNA polymerase regulator DksA is essential for protein structure, canonical control of the stringent response to nutritional limitation, and thiol-based sensing of oxidative and nitrosative stress. This interdependent relationship has limited our understanding of DksA-mediated functions in bacterial pathogenesis. Here, we have addressed this challenge by complementing ΔdksA Salmonella with Pseudomonas aeruginosa dksA paralogues that encode proteins differing in cysteine and zinc content. We find that four-cysteine, zinc-bound (C4) and two-cysteine, zinc-free (C2) DksA proteins are able to mediate appropriate stringent control in Salmonella and that thiol-based sensing of reactive species is conserved among C2 and C4 orthologues. However, variations in cysteine and zinc content determine the threshold at which individual DksA proteins sense and respond to reactive species. In particular, zinc acts as an antioxidant, dampening cysteine reactivity and raising the threshold of posttranslational thiol modification with reactive species. Consequently, C2 DksA triggers transcriptional responses in Salmonella at levels of oxidative or nitrosative stress normally tolerated by Salmonella expressing C4 orthologues. Inappropriate transcriptional regulation by C2 DksA increases the susceptibility of Salmonella to the antimicrobial effects of hydrogen peroxide and nitric oxide, and attenuates virulence in macrophages and mice. Our findings suggest that the redox-active sensory function of DksA proteins is finely tuned to optimize bacterial fitness according to the levels of oxidative and nitrosative stress encountered by bacterial species in their natural and host environments. IMPORTANCE: In order to cause disease, pathogenic bacteria must rapidly sense and respond to antimicrobial pressures encountered within the host. Prominent among these stresses, and of particular relevance to intracellular pathogens such as Salmonella, are nutritional restriction and the enzymatic generation of reactive oxygen and nitrogen species. The conserved transcriptional regulator DksA controls adaptive responses to nutritional limitation, as well as to oxidative and nitrosative stress. Here, we demonstrate that each of these functions contributes to bacterial pathogenesis. Our observations highlight the importance of metabolic adaptation in bacterial pathogenesis and show the mechanism by which DksA orthologues are optimized to sense the levels of oxidative and nitrosative stress encountered in their natural habitats. An improved understanding of the conserved processes used by bacteria to sense, respond to, and limit host defense will inform the development of novel strategies to treat infections caused by pathogenic, potentially multidrug-resistant bacteria.

The 4-cysteine zinc-finger motif of the RNA polymerase regulator DksA serves as a thiol switch for sensing oxidative and nitrosative stress.

We show that thiols in the 4-cysteine zinc-finger motif of DksA, an RNA polymerase accessory protein known to regulate the stringent response, sense oxidative and nitrosative stress. Hydrogen peroxide- or nitric oxide (NO)-mediated modifications of thiols in the DksA 4-cysteine zinc-finger motif release the metal cofactor and drive reversible changes in the α-helicity of the protein. Wild-type and relA spoT mutant Salmonella, but not isogenic dksA-deficient bacteria, experience the downregulation of r-protein and amino acid transport expression after NO treatment, suggesting that DksA can regulate gene expression in response to NO congeners independently of the ppGpp alarmone. Oxidative stress enhances the DksA-dependent repression of rpsM, while preventing the activation of livJ and hisG gene transcription that is supported by reduced, zinc-bound DksA. The inhibitory effects of oxidized DksA on transcription are reversible with dithiothreitol. Our investigations indicate that sensing of reactive species by DksA redox active thiols fine-tunes the expression of translational machinery and amino acid assimilation and biosynthesis in accord with the metabolic stress imposed by oxidative and nitrosative stress. Given the conservation of Cys(114) , and neighbouring hydrophobic and charged amino acids in DksA orthologues, phylogenetically diverse microorganisms may use the DksA thiol switch to regulate transcriptional responses to oxidative and nitrosative stress.

Individualized patient-centered lifestyle recommendations: an expert system for communicating patient specific cardiovascular risk information and prioritizing lifestyle options.

We propose a proof-of-concept machine-learning expert system that learned knowledge of lifestyle and the associated 10-year cardiovascular disease (CVD) risks from individual-level data (i.e., Atherosclerosis Risk in Communities Study, ARIC). The expert system prioritizes lifestyle options and identifies the one that maximally reduce an individual's 10-year CVD risk by (1) using the knowledge learned from the ARIC data and (2) communicating for patient-specific cardiovascular risk information and personal limitations and preferences (as defined by variables used in this study). As a result, the optimal lifestyle is not only prioritized based on an individual's characteristics but is also relevant to personal circumstances. We also explored probable uses and tested the system in several examples using real-world scenarios and patient preferences. For example, the system identifies the most effective lifestyle activities as the starting point for an individual's behavior change, shows different levels of BMI changes and the associated CVD risk reductions to encourage weight loss, identifies whether weight loss or smoking cessation is the most urgent change for a diabetes patient, etc. Answers to the questions noted above vary based on an individual's characteristics. Our validation results from clinical trial simulations, which compared original with the optimal lifestyle using an independent dataset, show that the optimal individualized patient-centered lifestyle consistently reduced 10-year CVD risks.

A simulation platform to examine heterogeneity influence on treatment.

Although a protocol aims to guide treatment management and optimize overall outcomes, the benefits and harms for each individual vary due to heterogeneity. Some protocols integrate clinical and genetic variation to provide treatment recommendation; it is not clear whether such integration is sufficient. If not, treatment outcomes may be sub-optimal for certain patient sub-populations. Unfortunately, running a clinical trial to examine such outcome responses is cost prohibitive and requires a significant amount of time to conduct the study. We propose a simulation approach to discover this knowledge from electronic medical records; a rapid method to reach this goal. We use the well-known drug warfarin as an example to examine whether patient characteristics, including race and the genes CYP2C9 and VKORC1, have been fully integrated into dosing protocols. The two genes mentioned above have been shown to be important in patient response to warfarin.

Identification of the bacterial protein FtsX as a unique target of chemokine-mediated antimicrobial activity against Bacillus anthracis.

Chemokines are a family of chemotactic cytokines that function in host defense by orchestrating cellular movement during infection. In addition to this function, many chemokines have also been found to mediate the direct killing of a range of pathogenic microorganisms through an as-yet-undefined mechanism. As an understanding of the molecular mechanism and microbial targets of chemokine-mediated antimicrobial activity is likely to lead to the identification of unique, broad-spectrum therapeutic targets for effectively treating infection, we sought to investigate the mechanism by which the chemokine CXCL10 mediates bactericidal activity against the Gram-positive bacterium Bacillus anthracis, the causative agent of anthrax. Here, we report that disruption of the gene ftsX, which encodes the transmembrane domain of a putative ATP-binding cassette transporter, affords resistance to CXCL10-mediated antimicrobial effects against vegetative B. anthracis bacilli. Furthermore, we demonstrate that in the absence of FtsX, CXCL10 is unable to localize to its presumed site of action at the bacterial cell membrane, suggesting that chemokines interact with specific, identifiable bacterial components to mediate direct microbial killing. These findings provide unique insight into the mechanism of CXCL10-mediated bactericidal activity and establish, to our knowledge, the first description of a bacterial component critically involved in the ability of host chemokines to target and kill a bacterial pathogen. These observations also support the notion of chemokine-mediated antimicrobial activity as an important foundation for the development of innovative therapeutic strategies for treating infections caused by pathogenic, potentially multidrug-resistant microorganisms.