Computer-aided drug design to generate a unique antibiotic family.

The World Health Organization has identified antibiotic resistance as one of the three greatest threats to human health. The need for antibiotics is a pressing matter that requires immediate attention. Here, computer-aided drug design is used to develop a structurally unique antibiotic family targeting holo-acyl carrier protein synthase (AcpS). AcpS is a highly conserved enzyme essential for bacterial survival that catalyzes the first step in lipid synthesis. To the best of our knowledge, there are no current antibiotics targeting AcpS making this drug development program of high interest. We synthesize a library of > 700 novel compounds targeting AcpS, from which 33 inhibit bacterial growth in vitro at ≤ 2 µg/mL. We demonstrate that compounds from this class have stand-alone activity against a broad spectrum of Gram-positive organisms and synergize with colistin to enable coverage of Gram-negative species. We demonstrate efficacy against clinically relevant multi-drug resistant strains in vitro and in animal models of infection in vivo including a difficult-to-treat ischemic infection exemplified by diabetic foot ulcer infections in humans. This antibiotic family could form the basis for several multi-drug-resistant antimicrobial programs.

Clinical activism in community-based practice: The case of LGBT affirmative care at the Eromin Center, Philadelphia, 1973-1984.

The Eromin Center was founded in Philadelphia in 1973, aiming to provide lesbian, gay, bisexual, and transgender (LGBT) affirmative mental health treatment 6 months before the American Psychiatric Association voted to remove homosexuality from the Diagnostic and Statistical Manual of Mental Disorders (DSM-II; American Psychiatric Association, 1968). This study of archival records and oral histories with Eromin Center staff and volunteers reveals an iterative and improvisational approach to community-based affirmative care. Rather than waiting for national leadership or institutional change, they aimed to respond directly to otherwise unrecognized needs of LGBT people through psychotherapy and social services-what we are calling clinical activism. Without training or guidance from research, they tended to base their work on their own experiences, an approach with inherent limitations in particular because most of the staff were White, cisgender, and identified as gay and lesbian. They attempted to address these limitations until Eromin's closing in 1984. Largely overshadowed by the broader policy changes in mental health care, Eromin's work provides a crucial case study in community-based clinical activism and affirmative practice with continuing salience today. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Synchronization of energy consumption by human societies throughout the Holocene.

We conduct a global comparison of the consumption of energy by human populations throughout the Holocene and statistically quantify coincident changes in the consumption of energy over space and time-an ecological phenomenon known as synchrony. When populations synchronize, adverse changes in ecosystems and social systems may cascade from society to society. Thus, to develop policies that favor the sustained use of resources, we must understand the processes that cause the synchrony of human populations. To date, it is not clear whether human societies display long-term synchrony or, if they do, the potential causes. Our analysis begins to fill this knowledge gap by quantifying the long-term synchrony of human societies, and we hypothesize that the synchrony of human populations results from (i) the creation of social ties that couple populations over smaller scales and (ii) much larger scale, globally convergent trajectories of cultural evolution toward more energy-consuming political economies with higher carrying capacities. Our results suggest that the process of globalization is a natural consequence of evolutionary trajectories that increase the carrying capacities of human societies.

Deliberative curriculum inquiry for integration in an MD curriculum: Dalhousie University's curriculum renewal process.

BACKGROUND: Dalhousie University's MD Programme faced a one-year timeline for renewal of its undergraduate curriculum. AIM: Key goals were renewed faculty engagement for ongoing quality improvement and increased collaboration across disciplines for an integrated curriculum, with the goal of preparing physicians for practice in the twenty-first century. METHODS: We engaged approximately 600 faculty members, students, staff and stakeholders external to the faculty of medicine in a process described by Harris (1993) as 'deliberative curriculum inquiry'. Temporally overlapping and networked intraprofessional and interprofessional teams developed programme outcomes, completed environment scans of emerging content and best practices, and designed curricular units. RESULTS: The resulting curriculum is the product of new collaborations among faculty and exemplifies distinct forms of integration. Innovations include content and cases shared by concurrent units, foundations courses at the beginning of each year and integrative experiences at the end, and an interprofessional community health mentors programme. CONCLUSION: The use of deliberative inquiry for pre-med curriculum renewal on a one-year time frame is feasible, in part through the use of technology. Ongoing structures for integration remain challenging. Although faculty collaboration fosters integration, a learner-centred lens must guide its design.

Species-specific responses of Late Quaternary megafauna to climate and humans.

Despite decades of research, the roles of climate and humans in driving the dramatic extinctions of large-bodied mammals during the Late Quaternary period remain contentious. Here we use ancient DNA, species distribution models and the human fossil record to elucidate how climate and humans shaped the demographic history of woolly rhinoceros, woolly mammoth, wild horse, reindeer, bison and musk ox. We show that climate has been a major driver of population change over the past 50,000 years. However, each species responds differently to the effects of climatic shifts, habitat redistribution and human encroachment. Although climate change alone can explain the extinction of some species, such as Eurasian musk ox and woolly rhinoceros, a combination of climatic and anthropogenic effects appears to be responsible for the extinction of others, including Eurasian steppe bison and wild horse. We find no genetic signature or any distinctive range dynamics distinguishing extinct from surviving species, emphasizing the challenges associated with predicting future responses of extant mammals to climate and human-mediated habitat change.

NMR solution structure and biophysical characterization of Vibrio harveyi acyl carrier protein A75H: effects of divalent metal ions.

Bacterial acyl carrier protein (ACP) is a highly anionic, 9 kDa protein that functions as a cofactor protein in fatty acid biosynthesis. Escherichia coli ACP is folded at neutral pH and in the absence of divalent cations, while Vibrio harveyi ACP, which is very similar at 86% sequence identity, is unfolded under the same conditions. V. harveyi ACP adopts a folded conformation upon the addition of divalent cations such as Ca(2+) and Mg(2+) and a mutant, A75H, was previously identified that restores the folded conformation at pH 7 in the absence of divalent cations. In this study we sought to understand the unique folding behavior of V. harveyi ACP using NMR spectroscopy and biophysical methods. The NMR solution structure of V. harveyi ACP A75H displays the canonical ACP structure with four helices surrounding a hydrophobic core, with a narrow pocket closed off from the solvent to house the acyl chain. His-75, which is charged at neutral pH, participates in a stacking interaction with Tyr-71 in the far C-terminal end of helix IV. pH titrations and the electrostatic profile of ACP suggest that V. harveyi ACP is destabilized by anionic charge repulsion around helix II that can be partially neutralized by His-75 and is further reduced by divalent cation binding. This is supported by differential scanning calorimetry data which indicate that calcium binding further increases the melting temperature of V. harveyi ACP A75H by ∼20 °C. Divalent cation binding does not alter ACP dynamics on the ps-ns timescale as determined by (15)N NMR relaxation experiments, however, it clearly stabilizes the protein fold as observed by hydrogen-deuterium exchange studies. Finally, we demonstrate that the E. coli ACP H75A mutant is similarly unfolded as wild-type V. harveyi ACP, further stressing the importance of this particular residue for proper protein folding.

Intein-mediated cyclization of bacterial acyl carrier protein stabilizes its folded conformation but does not abolish function.

Bacterial acyl carrier protein (ACP) is essential for the synthesis of fatty acids and serves as the major acyl donor for the formation of phospholipids and other lipid products. Acyl-ACP encloses attached fatty acyl groups in a hydrophobic pocket within a four-helix bundle, but must at least partially unfold to present the acyl chain to the active sites of its multiple enzyme partners. To further examine the constraints of ACP structure and function, we have constructed a cyclic version of Vibrio harveyi ACP, using split-intein technology to covalently join its closely apposed N and C termini. Cyclization stabilized ACP in a folded helical conformation as indicated by gel electrophoresis, circular dichroism, fluorescence, and mass spectrometry. Molecular dynamics simulations also indicated overall decreased polypeptide chain mobility in cyclic ACP, although no major conformational rearrangements over a 10-ns period were noted. In vivo complementation assays revealed that cyclic ACP can functionally replace the linear wild-type protein and support growth of an Escherichia coli ACP-null mutant strain. Cyclization of a folding-deficient ACP mutant (F50A) both restored its ability to adopt a folded conformation and enhanced complementation of growth. Our results thus suggest that ACP must be able to adopt a folded conformation for biological activity, and that its function does not require complete unfolding of the protein.

A case of Salmonella enterica serotype typhi in a patient without a history of international travel.

Typhoid fever, endemic in the developing world, is associated with international travel in developed nations. We present a case of Salmonella enterica infection in a patient without a history of international travel acquired from his traveling ex-wife. History of overseas travel in family members should be investigated when evaluating suspected cases.

Tryptophan fluorescence reveals induced folding of Vibrio harveyi acyl carrier protein upon interaction with partner enzymes.

We have introduced tryptophan as a local fluorescent probe to monitor the conformation of Vibrio harveyi acyl carrier protein (ACP), a small flexible protein that is unfolded at neutral pH but must undergo reversible conformational change during the synthesis and delivery of bacterial fatty acids. Consistent with known 3D structures of ACP, steady-state fluorescence and quenching experiments indicated that Trp at positions 46, 50, and 72 are buried in the hydrophobic core upon Mg(2+)-induced ACP folding, whereas residues 25 and 45 remain in a hydrophilic environment on the protein surface. Attachment of fatty acids to the phosphopantetheine prosthetic group progressively stabilized the folded conformation of all Trp-substituted ACPs, but longer chains (14:0) were less effective than medium chains (8:0) in shielding Trp from acrylamide quenching in the L46W protein. Interaction with ACP-dependent enzymes LpxA and holo-ACP synthase also caused folding of L46W; fluorescence quenching indicated proximity of Trp-45 in helix II of ACP in LpxA binding. Our results suggest that divalent cations and fatty acylation produce differing environments in the ACP core and also reveal enzyme partner-induced folding of ACP, a key feature of "natively unfolded" proteins.

Unusual case of Pneumocystis jiroveci pneumonia during primary HIV infection.

Symptomatic primary HIV infection occurs in an estimated 50% to 90% of patients. A constellation of symptoms that most closely resembles those of acute infectious mononucleosis characterizes the syndrome. On rare occasions, opportunistic infections present simultaneously with primary HIV infection. We describe a patient who presented with an episode of severe Pneumocystis jiroveci pneumonia during what appeared to be a prolonged primary HIV infection. Serological testing demonstrated the progressive development of reactive bands on serial Western blot determinations. This case highlights how primary HIV infection can produce profound immunosuppression through CD4 lymphocytopenia predisposing patients to opportunistic infection.

Acyl carrier protein: structure-function relationships in a conserved multifunctional protein family.

Acyl carrier protein (ACP) is a universal and highly conserved carrier of acyl intermediates during fatty acid synthesis. In yeast and mammals, ACP exists as a separate domain within a large multifunctional fatty acid synthase polyprotein (type I FAS), whereas it is a small monomeric protein in bacteria and plastids (type II FAS). Bacterial ACPs are also acyl donors for synthesis of a variety of products, including endotoxin and acylated homoserine lactones involved in quorum sensing; the distinct and essential nature of these processes in growth and pathogenesis make ACP-dependent enzymes attractive antimicrobial drug targets. Additionally, ACP homologues are key components in the production of secondary metabolites such as polyketides and nonribosomal peptides. Many ACPs exhibit characteristic structural features of natively unfolded proteins in vitro, with a dynamic and flexible conformation dominated by 3 parallel alpha helices that enclose the thioester-linked acyl group attached to a phosphopantetheine prosthetic group. ACP conformation may also be influenced by divalent cations and interaction with partner enzymes through its "recognition" helix II, properties that are key to its ability to alternately sequester acyl groups and deliver them to the active sites of ACP-dependent enzymes. This review highlights recent progress in defining how the structural features of ACP are related to its multiple carrier roles in fatty acid metabolism.

Human newborn polymorphonuclear neutrophils exhibit decreased levels of MyD88 and attenuated p38 phosphorylation in response to lipopolysaccharide.

PURPOSE: Human newborn infants have increased susceptibility to gram-negative bacterial infection. Since lipopolysaccharide (LPS) primes polymorphonuclear neutrophils (PMN) to enhance host defense functions, we investigated its effect on adult and newborn PMN in vitro. METHODS: PMN were isolated from blood of healthy adults and umbilical cords of full term newborns using dextran and Ficoll-Paque gradient sedimentation. Gel electrophoresis and Western blotting of membranes were used to probe for Mitogen-Activated Protein (MAP) kinase p38 phosphorylation, Toll-like Receptor-4 (TLR-4) and Myeloid Differentiation Factor 88 (MyD88) on isolated PMN membranes using specific antibodies. LPS induced degranulation was assessed using CD66 expression on PMN measured by flow cytometry. RESULTS: We show that p38 phosphorylation in newborn PMN is attenuated in response to LPS stimulation even though adult and newborn PMN have similar amounts of p38 protein. The degree of attenuation in newborn PMN is dependent on the osmolarity of the medium. In addition, LPS-induced degranulation, a process that is p38 dependent, was also absent in newborn PMN. Although the LPS receptor TLR-4 is present at similar levels on newborn and adult PMN, its downstream adaptor protein MyD88 was significantly diminished in newborn PMN compared to adult cells. CONCLUSIONS: Although the mechanism of PMN priming by LPS is not fully understood, our results suggest that MyD88 and p38 phosphorylation are important pathways in the process and contribute to attenuated response of newborn PMN to LPS in vitro.

Electrospray ionization mass spectra of acyl carrier protein are insensitive to its solution phase conformation.

Electrospray ionization mass spectrometry (ESI-MS) can be used to monitor conformational changes of proteins in solution based on the charge state distribution (CSD) of the corresponding gas-phase ions, although relatively few studies of acidic proteins have been reported. Here, we have compared the CSD and solution structure of recombinant Vibrio harveyi acyl carrier protein (rACP), a small acidic protein whose secondary and tertiary structure can be manipulated by pH, fatty acylation, and site-directed mutagenesis. Circular dichroism and intrinsic fluorescence demonstrated that apo-rACP adopts a folded helical conformation in aqueous solution below pH 6 or in 50% acetonitrile/0.1% formic acid, but is unfolded at neutral and basic pH values. A rACP mutant, in which seven conserved acidic residues were replaced with their corresponding neutral amides, was folded over the entire pH range of 5 to 9. However, under the same solvent conditions, both wild type and mutant ACPs exhibited similar CSDs (6(+)-9(+) species) at all pH values. Covalent attachment of myristic acid to the phosphopantetheine prosthetic group of rACP, which is known to stabilize a folded conformation in solution, also had little influence on its CSD in either positive or negative ion modes. Overall, our results are consistent with ACP as a "natively unfolded" protein in a dynamic conformational equilibrium, which allows access to (de)protonation events during the electrospray process.

Rickettsia parkeri infection after tick bite, Virginia.

We describe a man with a febrile illness and an eschar that developed at the site of a tick bite. Rickettsia parkeri was detected and isolated from the eschar. This report represents the second documented case of R. parkeri rickettsiosis in a US serviceman in eastern Virginia.

Qualitative and quantitative evaluation of protein extraction protocols for apple and strawberry fruit suitable for two-dimensional electrophoresis and mass spectrometry analysis.

A modified phenol-based protocol and a phenol-free protocol that involves hot SDS extraction followed by TCA precipitation in acetone were qualitatively and quantitatively compared and evaluated on apple peel and strawberry fruit. The phenol protocol resulted in significantly higher protein yields of 2.35 +/- 0.1 and 0.46 +/- 0.06 mg/g of FW from apple and strawberry fruit, respectively, compared to the SDS protocol, which produced 0.74 +/- 0.1 and 0.27 +/- 0.02 mg/g of FW, respectively. 2-DE analysis of apple protein extracts revealed 1422 protein spots associated with the phenol protocol and 849 spots associated with the SDS protocol. Of these, 761 were present only in phenol gels, whereas 23 were exclusive to SDS samples. For strawberry, SDS extraction produced poor-quality spots with a high degree of streaking, indicating possible contamination. The application of a cleanup procedure resulted in a purified protein extract with high-quality spots. 2-DE analysis of strawberry protein extracts revealed 1368 spots for the phenol protocol and 956 spots for the SDS protocol accompanied by the cleanup procedure. Of these, 599 spots were present only in phenol gels, whereas 109 were present only in SDS samples. Spots from each fruit tissue and extraction procedure were selected, and a total of 26 were identified by LC-MS/MS. Overall, this study demonstrates the complexity of protein extraction of fruit tissues and suggests that a phenol-based protein extraction protocol should be used as a standard procedure for recalcitrant fruit tissues, whereas a SDS protocol with or without a cleanup procedure may be used as an alternative protocol.

Neutralization of acidic residues in helix II stabilizes the folded conformation of acyl carrier protein and variably alters its function with different enzymes.

Acyl carrier protein (ACP), a small protein essential for bacterial growth and pathogenesis, interacts with diverse enzymes during the biosynthesis of fatty acids, phospholipids, and other specialized products such as lipid A. NMR and hydrodynamic studies have previously shown that divalent cations stabilize native helical ACP conformation by binding to conserved acidic residues at two sites (A and B) at either end of the "recognition" helix II. To examine the roles of these amino acids in ACP structure and function, site-directed mutagenesis was used to replace individual site A (Asp-30, Asp-35, Asp-38) and site B (Glu-47, Glu-53, Asp-56) residues in recombinant Vibrio harveyi ACP with the corresponding amides, along with combined mutations at each site (SA, SB) or both sites (SA/SB). Like native V. harveyi ACP, all individual mutants were unfolded at neutral pH but adopted a helical conformation in the presence of millimolar Mg(2+) or upon fatty acylation. Mg(2+) binding to sites A or B independently stabilized native ACP conformation, whereas mutant SA/SB was folded in the absence of Mg(2+), suggesting that charge neutralization is largely responsible for ACP stabilization by divalent cations. Asp-35 in site A was critical for holo-ACP synthase activity, while acyl-ACP synthetase and UDP-N-acetylglucosamine acyltransferase (LpxA) activities were more affected by mutations in site B. Both sites were required for fatty acid synthase activity. Overall, our results indicate that divalent cation binding site mutations have predicted effects on ACP conformation but unpredicted and variable consequences on ACP function with different enzymes.

Expression of MARCKS effector domain mutants alters phospholipase D activity and cytoskeletal morphology of SK-N-MC neuroblastoma cells.

Stable overexpression of myristoylated alanine-rich C-kinase substrate (MARCKS) is known to enhance phorbol ester stimulation of phospholipase D (PLD) activity and protein kinase Calpha (PKCalpha) levels in SK-N-MC neuroblastoma cells. In contrast, expression of MARCKS mutants (S152A or S156A) lacking key PKC phosphorylation sites within the central basic effector domain (ED) had no significant effect on PLD activity or PKCalpha levels relative to vector control cells. Like control cells, those expressing wild type MARCKS were elongated and possessed longitudinally oriented stress fibers, although these cells were more prone to detach from the substratum and undergo cell death upon phorbol ester treatment. However, cells expressing MARCKS ED mutants were irregularly shaped and stress fibers were either shorter or less abundant, and cell adhesion and viability were not affected. These results suggest that intact phosphorylation sites within the MARCKS ED are required for PLD activation and influence both membrane-cytoskeletal organization and cell viability.

Role of protein tyrosine kinase p53/56lyn in diminished lipopolysaccharide priming of formylmethionylleucyl- phenylalanine-induced superoxide production in human newborn neutrophils.

Human newborns are more susceptible than adults to bacterial infection. With gram-negative bacteria, this may be due to a diminished response of newborn leukocytes to lipopolysaccharide (LPS). Since protein tyrosine kinase inhibition abolishes LPS priming in adult cells, we hypothesized that protein tyrosine kinases may have a critical role in LPS priming of polymorphonuclear neutrophils (PMNs) and that newborn PMNs may have altered protein tyrosine kinase activities. In the present study, we investigated the role of src family protein tyrosine kinases in the LPS response of newborn PMNs compared to adult cells. In a respiratory assay, the LPS-primed increase in formylmethionylleucylphenylalanine (fMLP)-triggered O2- release by adult PMNs was greatly decreased by PP1 [4-amino-5-(4-methyphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine], a src kinase inhibitor, to the level of untreated newborn PMNs, in which LPS failed to prime. LPS activated the src-like kinases p59hck (HCK) and p58fgr (FGR) in both adult and newborn PMNs but increased the activation of p53/56lyn (LYN) only in adult cells. In newborn PMNs, LYN was highly phosphorylated independent of LPS. We evaluated subcellular fractions of PMNs and found that the phosphorylated form of LYN was mainly in the Triton-extractable, cytosolic fraction in adult PMNs, while in newborn cells it was located mainly in Triton-insoluble, granule- and membrane-associated fractions. In contrast, the phosphorylated mitogen-activated protein kinases ERK1/2 and p38 were mainly detected in the cytosol in both adult and newborn PMNs. These data indicate a role for LYN in the regulation of LPS priming. The trapping of phosphorylated LYN in the membrane-granule fraction in newborn PMNs may contribute to the deficiency of newborn cells in responding to LPS stimulation.