Heidar Ali Panahi, Azam Bolhassani, Gholamreza Javadi, Zahra Noormohammadi

by Heidar Ali Panahi, Azam Bolhassani, Gholamreza Javadi, Zahra Noormohammadi Human papillomaviruses (HPVs) are a group of circular double-stranded DNA viruses, showing severe tropism to mucosal tissues. A subset of HPVs, especially HPV16 and 18, are the primary etiological cause for several epithelial cell malignancies, causing about 5.2% of all cancers worldwide. Due to the high prevalence and mortality, HPV-associated cancers have remained as a significant health problem in human society, making an urgent need to develop an effective therapeutic vaccine against them. Achieving this goal is primarily dependent on the identification of efficient tumor-associated epitopes, inducing a robust cell-mediated immune response. Previous information has shown that E5, E6, and E7 early proteins are responsible for the induction and maintenance of HPV-associated cancers. Therefore, the prediction of major histocompatibility complex (MHC) class I T cell epitopes of HPV16, 18, 31 and 45 oncoproteins was targeted in this study. For this purpose, a two-step plan was designed to identify the most probable CD8+ T cell epitopes. In the first step, MHC-I and II binding, MHC-I processing, MHC-I population coverage and MHC-I immunogenicity prediction analyses, and in the second step, MHC-I and II protein-peptide docking, epitope conservation, and cross-reactivity with host antigens’ analyses were carried out successively by different tools. Finally, we introduced five probable CD8+ T cell epitopes for each oncoprotein of the HPV genotypes (60 epitopes in total), which obtained better scores by an integrated approach. These predicted epitopes are valuable candidates for in vitro or in vivo therapeutic vaccine studies against the HPV-associated cancers. Additionally, this two-step plan that each step includes several analyses to find appropriate epitopes provides a rational basis for DNA- or peptide-based vaccine development.

Hsu C, Chang I, Hsieh P, et al.

AbstractKlebsiella pneumoniae is an important human pathogen causing hospital-acquired and community-acquired infections. Systemic K. pneumoniae infections may be preceded by gastrointestinal colonization, but the basis of this bacterium’s interaction with the intestinal epithelium remains unclear. Here, we report that the K. pneumoniae Sap (sensitivity to antimicrobial peptides) transporter contributes to bacterial–host cell interactions and in vivo virulence. Gene deletion showed that sapA is required for the adherence of a K. pneumoniae blood isolate to intestinal epithelial, lung epithelial, urinary bladder epithelial, and liver cells. The ΔsapA mutant was deficient for translocation across intestinal epithelial monolayers, macrophage interactions, and induction of proinflammatory cytokines. In a mouse gastrointestinal infection model, ΔsapA yielded significantly decreased bacterial loads in liver, spleen and intestine, reduced liver abscess generation, and decreased mortality. These findings offer new insights into the pathogenic interaction of K. pneumoniae with the host gastrointestinal tract to cause systemic infection.

Dubé M, Chan E, Lake J, et al.

AbstractBackgroundDipeptidyl peptidase-4 (DPP-4) inhibitors have pleotropic anti-inflammatory and immune regulatory effects in addition to their glucoregulatory actions. We evaluated inflammation and immune markers in suppressed HIV infection during treatment with the DPP-4 inhibitor sitagliptin.MethodsVirologically suppressed adults with HIV without diabetes mellitus on stable ART with ≥100/mm 3 CD4 cells were randomized to 16 weeks of sitagliptin 100 mg/d vs. placebo in a multicenter trial. The primary endpoint was the change in plasma soluble CD14 (sCD14) from baseline to week 15/16. Additional soluble biomarkers, and lymphocyte and monocyte activation were assessed.ResultsNinety participants were randomized, and 42 from each arm were included in per-protocol analyses. Evaluable participants were 45% non-Hispanic white, 38% non-Hispanic black, 15% Hispanic, median age of 51 years, 83% male, with median CD4 count 602 cells/mm 3. At week 15/16, there was no difference in sCD14 change between the two arms (p=0.69). Relative to placebo, the sitagliptin arm had 47% greater decline in CXCL10 (95% CI:-57,-35) at week 15 (p

Abstract The ongoing transmission of Mycobacterium (M.) leprae reflected in a very slow decline in leprosy incidence, forces us to be innovative and conduct cutting-edge research. Single dose rifampicin (SDR) as post-exposure prophylaxis (PEP) for contacts of leprosy patients, reduces their risk to develop leprosy by 60%. This is a promising new preventive measure that can be integrated into routine leprosy control programmes, as is being demonstrated in the Leprosy Post-Exposure Programme that is currently ongoing in eight countries. The limited (60%) effectiveness of SDR is likely due to the fact that some contacts have a preclinical infection beyond the early stages for which SDR is not sufficient to prevent the development of clinical signs and symptoms of leprosy. An enhanced regimen, more potent against a higher load of leprosy bacteria, would increase the effectiveness of this preventive measure significantly. The Netherlands Leprosy Relief (NLR) is developing a multi-country study aiming to show that breaking the chain of transmission of M. leprae is possible, evidenced by a dramatic reduction in incidence. In this study the assessment of the effectiveness of an enhanced prophylactic regimen for leprosy is an important component. To define the so called PEP++ regimen for this intervention study, NLR convened an Expert Meeting that was attended by clinical leprologists, public health experts, pharmacologists, dermatologists and microbiologists. The Expert Meeting advised on combinations of available drugs, with known efficacy against leprosy, as well as on the duration of the intake, aiming at a risk reduction of 80–90%. To come to a conclusion the Expert Meeting considered the bactericidal, sterilising and bacteriostatic activity of the potential drugs. The criteria used to determine an optimal enhanced regimen were: effectiveness, safety, acceptability, availability, affordability, feasibility and not inducing drug resistance. The Expert Meeting concluded that the enhanced regimen for the PEP++ study should comprise three standard doses of rifampicin 600 mg (weight adjusted when given to children) plus moxifloxacin 400 mg given at four-weekly intervals. For children and for adults with contraindications for moxifloxacin, moxifloxacin should be replaced by clarithromycin 300 mg (weight adjusted).…

Andreoni, F., Toyofuku, M., Menzi, C., Kalawong, R., Mairpady Shambat, S., Francois, P., Zinkernagel, A. S., Eberl, L.

Bacterial membrane vesicles research has so far mainly focussed on Gram-negative bacteria. Only recently Gram-positive bacteria have been demonstrated to produce and release extracellular membrane vesicles (MVs) that contribute to bacterial virulence. Although treatment of bacteria with antibiotics is a well-established trigger of bacterial MVs formation, the underlying mechanisms are poorly understood. In this study we show that antibiotics can induce MVs through different routes in the important human pathogen Staphylococcus aureus. DNA damaging agents and antibiotics inducing the SOS response triggered vesicle formation in lysogenic strains of S. aureus but not in their phage-devoid counterparts. β-lactam antibiotics flucloxacillin and ceftaroline increased vesicle formation in a prophage-independent manner by weakening the peptidoglycan layer. We present evidence that the amount of DNA associated with MVs formed by phage lysis is higher than that of MVs formed by β-lactam antibiotics-induced blebbing. The purified MVs derived from S. aureus protected the bacteria from challenge with daptomycin, a membrane-targeting antibiotic, both in vitro and ex vivo in whole blood. In addition, the MVs protected S. aureus from killing in whole blood, indicating that antibiotic-induced MVs function as a decoy and thereby contribute to the survival of the bacterium.…

Carlos Iniesta, Débora Álvarez-del Arco, Luis Miguel García-Sousa, Belén Alejos, Asunción Díaz, Nieves Sanz, Jorge Garrido, Michael Meulbroek, Ferran Pujol, Santiago Moreno, María José Fuster-Ruiz de Apocada, Pep Coll, Antonio Antela, Jorge del Romero, Oskar Ayerdi, Melchor Riera, Juanse Hernández, Julia del Amo

by Carlos Iniesta, Débora Álvarez-del Arco, Luis Miguel García-Sousa, Belén Alejos, Asunción Díaz, Nieves Sanz, Jorge Garrido, Michael Meulbroek, Ferran Pujol, Santiago Moreno, María José Fuster-Ruiz de Apocada, Pep Coll, Antonio Antela, Jorge del Romero, Oskar Ayerdi, Melchor Riera, Juanse Hernández, Julia del Amo Objective To assess the awareness, knowledge, use, and willingness to use and need of PrEP among men who have sex with men (MSM) and transgender women (TW) who attended World Gay Pride (WGP) 2017 in Madrid. Design and methods Online survey. Participants were recruited through gay-oriented dating apps and HIV Non-Governmental Organizations´ social media. Inclusion criteria included being MSM or TW, age 18 years old or above, and having attended WGP in Madrid. Information regarding the participant’s awareness and knowledge, use or willingness to use, and need for PrEP was collected, as well as sociodemographic characteristics. Participants were considered to be in need of PrEP if they met one of the following indication criteria: having practiced unprotected anal intercourse with more than 2 partners, having practiced chemsex, or having engaged in commercial sex—all in the preceding 6 months. Descriptive and multivariable analyses with logistic regression were conducted. Results 472 participants met the inclusion criteria and completed the questionnaire. The mean age was 38, 97.7% were MSM, 77% had a university education, and 85% were living in Spain, mostly in big cities. Overall, 64% of participants were aware of PrEP, but only 33% knew correctly what PrEP was. 67% of HIV-negative participants were willing to take PrEP, although only 5% were taking it during WGP, mostly due to lack of access. 43% of HIV-negative respondents met at least one PrEP indication criteria. For HIV-negative men living in Spain, university education and living in big cities was associated with PrEP awareness. Lower education level and meeting PrEP criteria was associated with willingness to use PrEP. Conclusions Our study shows that among MSM attending WGP 2017 in Madrid, there was limited PrEP awareness, low accuracy of PrEP knowledge, and a high need and willingness to use PrEP. Health authorities should strengthen existing preventive strategies and implement PrEP.…

Patrick Ebner, Friedrich Götz

The excretion of cytoplasmic and signal-peptide-less proteins (ECP) by microorganisms and eukaryotes remains a fascinating topic. In principle, it appears to be a waste of energy. However, it turns out that – extracellularly – some cytoplasmic proteins (CPs) exert a completely different function such as contributing to pathogenicity or evasion of the immune system. Such CPs have been referred to as ‘moonlighting’ proteins. ECP is boosted by many endogenous or external factors that impair the membrane or cell wall structure.

Helene Botella, Julien Vaubourgeix

Fluorescent amino acid analogs have proven to be useful tools for studying the dynamics of peptidoglycan metabolism. García-Heredia and colleagues showed that their route of incorporation differs depending on the adjunct fluorophore and applied this property to investigate mycobacterial peptidoglycan synthesis and remodeling with heightened granularity.

Derek B. Laskar, Michael Rose, Raavi Gupta, Herbert B. Tanowitz and M. A. Haseeb

Abstract. A 63-year-old woman who migrated from Nigeria to the United States was found to have an elevated total serum protein, anemia, and eosinophilia. Serum protein electrophoresis (SPEP) and serum protein immunofixation electrophoresis (SPIFE) demonstrated monoclonal immunoglobulin G (IgG) κ restricted bands (IgG 3,820 mg/dL; κ/λ ratio 4.47), indicative of monoclonal gammopathy of unknown significance (MGUS). A rapid diagnostic test (RDT) for malaria was positive for Plasmodium falciparum (BinaxNOW®; Alere Scarborough Inc., Scarborough, ME). Giemsa-stained blood smears were negative for malarial parasites, however, Loa loa microfilariae were identified. Reverse transcription polymerase chain reaction for P. falciparum, Plasmodium ovale, Plasmodium malariae, and Plasmodium vivax yielded a negative result. She was treated for loiasis with diethylcarbamazine and received no malaria medication. Treatment resulted in a resolution of the microfilaremia and eosinophilia, a negative RDT for malaria, and marked reduction in the monoclonal gammopathy. This is the first reported human case of MGUS associated with loiasis and its resolution after antiparasitic treatment.…

Wuerth, K., Lee, A. H. Y., Falsafi, R., Gill, E. E., Hancock, R. E. W.

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial pneumonia and infects patients with cystic fibrosis. P. aeruginosa lung infections are difficult to treat due to bacterial resistance to antibiotics, and strains with multi-drug resistance are becoming more prevalent. Here we examined the use of a small host defense peptide, innate defense regulator 1002 (IDR-1002), in an acute P. aeruginosa lung infection in vivo. IDR-1002 significantly reduced the bacterial burden in the bronchoalveolar lavage fluid (BALF) as well as MCP-1 in the BALF and serum, KC in the serum, and IL-6 in the BALF. RNA-Seq was conducted on lungs and whole blood and the effects of P. aeruginosa, IDR-1002, or the combination of P. aeruginosa and IDR-1002 were evaluated. Differential gene expression analysis showed that P. aeruginosa increased multiple inflammatory and innate immune pathways as well as affected hemostasis, matrix metalloproteinases, collagen biosynthesis, and various metabolism pathways in the lungs and/or blood. Infected mice treated with IDR-1002 had significant changes in gene expression compared to untreated infected mice, with fewer differentially expressed genes associated with the inflammatory and innate immune responses to microbial infection, and treatment also affected morphogenesis, certain metabolic pathways, and lymphocyte activation. Overall, these results show that IDR-1002 was effective in treating P. aeruginosa acute lung infections and associated inflammation.…

Habib Hasan Farooqui, Sakthivel Selvaraj, Aashna Mehta, David L. Heymann

by Habib Hasan Farooqui, Sakthivel Selvaraj, Aashna Mehta, David L. Heymann India was the largest consumer of antibiotics in 2010 in the world. Evidence suggests that countries with high per-capita antibiotic consumption have higher rates of antibiotic resistance. To control antibiotic resistance, not only reduction in antibiotic consumption is required, socio-economic factors like access to clean water and sanitation, regulation of private healthcare sector and better governance are equally important. The key objective of this research was to investigate the five year trends in consumption of major antibiotic classes in India and compare them with European Surveillance of Antimicrobial Consumption Network (ESAC-Net) countries. We used Intercontinental Marketing Statistics (IMS) Health (now IQVIA) medicine sales audit data of antibiotic sales in the retail private sector (excluding the hospitals sector) in India. We then standardized dosage trends and assigned defined daily dose (DDD) to all formulations based on the ATC/DDD index. We expressed our data in standardized matrices of DDD per 1000 inhabitants’ per day (DID) to compare antibiotic use in India with ESAC-Net countries. The antibiotic use was plotted and reported by year and antibiotic class. Our main findings are—per capita antibiotic consumption in the retail sector in India has increased from 13.1 DID in 2008 to 16.0 DID in 2012—an increase of ~22%; use of newer class of antibiotics like carbapenems (J01DH), lincosamides (J01FF), glycopeptides (J01XA), 3rd generation cephalosporins (J01DD) and penicillin’s with beta-lactamase inhibitors has risen; and antibiotic consumption rates in India are still low as compared to ESAC-Net countries (16.0 DID vs. 21.54 DID). To conclude our study has provided the first reliable estimates of antibiotic use in the retail sector in India vis-à-vis ESAC-Net countries. In addition, our study could provide a reference point to measure the impact of interventions directed towards reducing antibiotic use.

To the Editor: In their study of the value of copeptin for diagnosing diabetes insipidus, Fenske et al. (Aug. 2 issue) overlook two possible methodologic flaws. First, some of their patients appear to have had an element of a solute diuresis. For example, among patients with primary polydipsia, the……

Hiroshi Wada, Atsushi Shimizu, Toshihiro Osada, Yuki Tanaka, Satoshi Fukaya, Eiji Sasaki

by Hiroshi Wada, Atsushi Shimizu, Toshihiro Osada, Yuki Tanaka, Satoshi Fukaya, Eiji Sasaki…

Seydlova, G., Sokol, A., Liskova, P., Konopasek, I., Fiser, R.

Daptomycin is a calcium-dependent lipodepsipeptide antibiotic clinically used to treat serious infections caused by Gram-positive pathogens. Its precise mode of action is somewhat controversial; the biggest issue is daptomycin pore formation, which we directly investigated here. We first performed a screening experiment using propidium iodide (PI) entry to Bacillus subtilis cells and chose the optimum and therapeutically relevant conditions (10 µg/mL daptomycin and 1.25 mM CaCl2) for the subsequent analyses. Using conductance measurements on planar lipid bilayers, we show that daptomycin forms non-uniform oligomeric pores with conductance ranging from 120 pS up to 14 nS. The smallest conductance unit is probably a dimer, however tetramers and pentamers occur in the membrane most frequently. Moreover, daptomycin pore-forming activity is exponentially dependent on the applied membrane voltage. We further analyzed the membrane permeabilizing activity in B. subtilis cells using fluorescence methods (PI, DiSC3(5)). Daptomycin most rapidly permeabilizes cells with high initial membrane potential and dissipates it within a few minutes. Low initial membrane potential hinders daptomycin pore formation.…

Barros, E. M., Martin, M. J., Selleck, E. M., Lebreton, F., Sampaio, J. L. M., Gilmore, M. S.

Lipopeptide daptomycin is a last line cell membrane-targeting antibiotic to treat multidrug-resistant Staphylococcus aureus. Alarmingly, daptomycin-resistant S. aureus isolates have emerged. The mechanisms underlying daptomycin resistance are diverse, share similarities with resistance to cationic antimicrobial peptides and other lipopeptides, but remain to be fully elucidated. We selected mutants with increased resistance to daptomycin from a library of transposon insertions in ST8 S. aureus HG003. Insertions in conferring increased daptomycin resistance were localized to two genes, one coding for a hypothetical lipoprotein (SAOUHSC_00362, Dsp1), and the other for an alkaline shock protein (SAOUHSC_02441, Asp23). Markerless loss of function mutants were then generated for comparison. All transposon mutants and knockout strains exhibited increased daptomycin resistance compared to wild type and complemented strains. Null and transposon insertion mutants also exhibited increased resistance to cationic antimicrobial peptides. Interestingly, dsp1 also showed increased resistance to vancomycin, a cell wall targeting drug with a different mode of action. Null mutations in both dsp1 and asp23 displayed increased tolerance as reflected by reduced killing to both daptomycin and vancomycin, as well as an increased tolerance to surfactant (Triton X-100). Neither mutant exhibited increased resistance to lysostaphin, a cell wall targeting endopeptidase. These findings identified two genes core to the S. aureus species, that make previously uncharacterized contributions to antimicrobial resistance and tolerance in S. aureus.…

Phillips, Tamsin K; Wilson, Ira B; Brittain, Kirsty; Zerbe, Allison; Mellins, Claude A; Remien, Robert H; Orrell, Catherine; Abrams, Elaine J; Myer, Landon

Introduction: Routine HIV viral load (VL) monitoring is recommended for patients on antiretroviral therapy (ART) but frequent VL testing, required in pregnant and postpartum women, is often not feasible. Self-reported adherence can be valuable, but little is known about its longitudinal characteristics. Methods: We followed women living with HIV from ART initiation in pregnancy through 18 months postpartum in Cape Town, South Africa, with repeated measurement of VL and self-reported adherence using a three-item scale. We used generalized estimating equations (with results presented as odds ratios [OR] with 95% confidence intervals [CI]) to investigate the association between viremia and change in adherence over pairs of consecutive visits. Results: Among 2085 visit pairs from 433 women, a decrease in self-reported adherence relative to the previous visit on any of three self-report items, or the combined scale, was associated with VL >50 and >1000 copies/mL. The best performing thresholds to predict VL >50 copies/mL were a single level decrease on the Likert response item “how good a job did you do at taking your HIV medicines in the way that you were supposed to?” (OR 2.08 95% CI 1.48-2.91), and a decrease equivalent to ≥5 missed doses or a one level decrease in score on either of two Likert items (OR 1.34 95% CI 1.06-1.69). Conclusion: Longitudinal changes in self-reported adherence can help identify patients with viremia. This approach warrants consideration in settings where frequent viral load monitoring or other objective adherence measures are not possible. Corresponding author: Tamsin K Phillips, Level 5 Falmouth Building, School of Public Health & Family Medicine, University of Cape Town, Cape Town, South Africa, Tel: +27 21 650 1646, Email: tk.phillips@uct.ac.za Conflicts of interest and sources of funding: This research was supported by the President’s Emergency Plan for AIDS Relief (PEPFAR) through the National Institute of Child Health and Human Development (NICHD), grant number 1R01HD074558. Additional funding comes from the Elizabeth Glaser Pediatric AIDS Foundation. Dr. Wilson is partially supported by the Providence/Boston Center for AIDS Research (P30AI042853) and by Institutional Development Award Number U54GM115677 from the National Institute of General Medical Sciences of the National Institutes of Health, which funds Advance Clinical and Translational Research (Advance-CTR) from the Rhode Island IDeA-CTR award (U54GM115677). Dr Orrell is partially supported through DAIDS grants (1R01AI122300 – 01, 1R34MH108393-01 and 2UM1AI0695-08). Drs. Mellins and Remien are partially supported by NIMH Center Grant (P30-MH43520). For the remaining authors none were declared. Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved.

Kayana Suryadi, Nancy Shine

by Kayana Suryadi, Nancy Shine Bacillus anthracis is a major biological warfare threat. The inhalation form of infection can kill quickly. While antibiotic treatment is effective, if diagnosis is delayed, the rapidly produced toxin may already be present in lethal amounts. This report describes a fast, sensitive, specific and accurate method for detection of active infection by Bacillus anthracis in plasma. One of the virulence factors, anthrax lethal factor, is an endopeptidase present in blood early in the infection. However, the use of peptidic substrates to detect endopetidases is problematic in plasma due to the presence of other proteases and the likelihood of nonspecific cleavage of the substrate. The fluorescently labeled peptide substrate MAPKKide Plus designed in this study is not cleaved by plasma proteases and thus is specific for lethal factor. Three detection strategies are described. Two include enrichment by capture from plasma using lethal factor antibody-coated microtiter plates or similarly coated immuno-tubes. The captured lethal factor is exposed to the MAPKKide Plus, and the amount of cleavage is determined either by HPLC or microplate reader. Concentration of lethal factor using the antibody-coated plates aplnd HPLC allows for detection of less than 5 pg lethal factor/ml of neat plasma after 2 hours of incubation. Using antibody-coated immuno-tubes, 20 pg lethal factor/ml plasma can be detected in 5 hours by a simple end point read of fluorescence in a microplate reader. For a third strategy, the substrate is added directly to diluted plasma, and cleavage is monitored by the increase in fluorescence as a function of time. The limit of detection by this simple method is 25 ng lethal factor/ml of plasma in 15 minutes, 5 ng/ml after 45 minutes, and…

Q. Wu, J. Liu, Y. Wang, Q. Zhou, X. Wang, Z. Xuan, L. Zhang, Y. Gao, B. Chen, Y. Hu

Although administration of a second dose of varicella vaccine (2nd-dose VarV) to individuals who have previously received one-dose VarV has been recommended as a post-exposure prophylaxis (PEP) strategy for outbreak control, the effectiveness of this strategy remains unclear. We evaluated the vaccination effectiveness (VE) of 2nd-dose VarV as PEP among students involved in 129 varicella outbreaks in Shanghai, China from 2013 to 2016.

Kavanagh, A., Ramu, S., Gong, Y., Cooper, M. A., Blaskovich, M. A. T.

The determination of antibiotic potency against bacterial strains by assessment of their minimum inhibitory concentration normally uses a standardized broth microdilution assay procedure developed more than 50 years ago. However, certain antibiotics require modified assay conditions in order to observe optimal activity. For example, daptomycin requires media supplemented with Ca2+ and the lipoglycopeptides dalbavancin and oritavancin require Tween-80 to be added to the growth media to prevent depletion of free drug via adsorption to the plastic microplate. In this report we examine systematically the effects of several different plate types on microdilution broth MIC values for a set of antibiotics against Gram-positive and Gram-negative bacteria, both in media alone and in media supplemented with commonly used additives Tween-80, lysed horse blood, and 50% human serum. We observe very significant differences in measured MICs (up to 100-fold) for some lipophilic antibiotics, such as the Gram-positive lipoglycopeptide dalbavancin and the Gram-negative lipopeptide polymyxins, and find that non-specific binding plates can replace the need for surfactant additives. Microtitre plate types and any additives should be specified when reporting broth dilution MIC values as results can vary dramatically for some classes of antibiotics.…

Anne-Laure Flamar, Henri Bonnabau, Sandra Zurawski, Christine Lacabaratz, Monica Montes, Laura Richert, Aurelie Wiedemann, Lindsey Galmin, Deborah Weiss, Anthony Cristillo, Lauren Hudacik, Andres Salazar, Cécile Peltekian, Rodolphe Thiebaut, Gerard Zurawski, Yves Levy

by Anne-Laure Flamar, Henri Bonnabau, Sandra Zurawski, Christine Lacabaratz, Monica Montes, Laura Richert, Aurelie Wiedemann, Lindsey Galmin, Deborah Weiss, Anthony Cristillo, Lauren Hudacik, Andres Salazar, Cécile Peltekian, Rodolphe Thiebaut, Gerard Zurawski, Yves Levy HIV-1 infection can be controlled by anti-retroviral drug therapy, but this is a lifetime treatment and the virus remains latent and rapidly rebounds if therapy is stopped. HIV-1-infected individuals under this drug regimen have increased rates of cancers, cardiovascular diseases, and autoimmunity due to compromised immunity. A therapeutic vaccine boosting cellular immunity against HIV-1 is therefore desirable and, possibly combined with other immune modulating agents, could obviate the need for long-term drug therapies. An approach to elicit strong T cell-based immunity is to direct virus protein antigens specifically to dendritic cells (DCs), which are the key cell type for controlling immune responses. For eliciting therapeutic cellular immunity in HIV-1-infected individuals, we developed vaccines comprised of five T cell epitope-rich regions of HIV-1 Gag, Nef, and Pol (HIV5pep) fused to monoclonal antibodies that bind either, the antigen presenting cell activating receptor CD40, or the endocytic dendritic cell immunoreceptor DCIR. The study aimed to demonstrate vaccine safety, establish efficacy for broad T cell responses in both primed and naïve settings, and identify one candidate vaccine for human therapeutic development. The vaccines were administered to Rhesus macaques by intradermal injection with poly-ICLC adjuvant. The animals were either i) naïve or, ii) previously primed with modified vaccinia Ankara vector (MVA) encoding HIV-1 Gag, Pol, and Nef (MVA GagPolNef). In the MVA-primed groups, both DC-targeting vaccinations boosted HIV5pep-specific blood CD4+ T cells producing multiple cytokines, but did not affect the MVA-elicited CD8+ T cell responses. In the naive groups, both DC-targeting vaccines elicited antigen-specific polyfunctional CD4+ and CD8+ T cell responses to multiple epitopes and these responses were unchanged by a subsequent MVA GagPolNef boost. In both settings, the T cell responses elicited via the CD40-targeting vaccine were more robust and were detectable in all the animals, favoring further development of the CD40-targeting vaccine for therapeutic vaccination of HIV-1-infected individuals.

Bennett M, Bombardi R, Kose N, et al.

AbstractThe nutrient metal iron plays a key role in the survival of microorganisms. The iron-regulated surface determinant (Isd) system scavenges heme-iron from the human host, enabling acquisition of iron in iron-deplete conditions in Staphylococcus aureus during infection. The cell surface receptors IsdB and IsdH bind hemoproteins and transfer heme to IsdA, the final surface protein before heme-iron is transported through the peptidoglycan. To define the human B-cell response to IsdA, we isolated human monoclonal antibodies (mAbs) specific to the surface Isd proteins and determined their mechanism of action. We describe the first isolation of fully human IsdA and IsdH mAbs, as well as cross-reactive Isd mAbs. Two of the identified IsdA mAbs worked in a murine septic model of infection to reduce bacterial burden during staphylococcal infection. Their protection was a result of both heme-blocking and Fc-mediated effector functions, underscoring the importance of targeting S. aureus using diverse mechanisms.

Jeroen Corver, Jeff Sen, Bastian V.H. Hornung, Bart J. Mertens, Eric K.L. Berssenbrugge, Céline Harmanus, Ingrid M.J.G. Sanders, Nitin Kumar, Trevor D. Lawley, Ed J. Kuijper, Paul J. Hensbergen, Simone Nicolardi

Clostridioides difficile infection (CDI) has become the main cause of nosocomial infective diarrhoea. To survey and control the spreading of different C. difficile strains, there is a need for suitable rapid tests. The aim of this study was to identify peptide/protein markers for the rapid recognition of C. difficile strains by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).

Abstract Background For the success of the malaria control and eradication programme it is essential to reduce parasite transmission by mosquito vectors. In the midguts of mosquitoes fed with parasite-infected blood, sexual-stage parasites fertilize to develop into motile ookinetes that traverse midgut epithelial cells and reside adjacent the basal lamina. Therefore, the ookinete is a promising target of transmission-blocking vaccines to break the parasite lifecycle in mosquito vectors. However, the molecular mechanisms of ookinete formation and invasion of epithelial cells have not been fully elucidated. A unique structure called the crystalloid body has been identified in the ookinete cytoplasm by electron microscopy, but its biological functions remain unclear. Methods A recombinant protein of a novel molecule, designated as crystalloid body specific PH domain-containing protein of Plasmodium yoelii (PyCryPH), was synthesized using a wheat germ cell-free system. Specific rabbit antibodies against PyCryPH were obtained to characterize the expression and localization of PyCryPH during sexual-stage parasite development. In addition, PyCryPH knockout parasites were generated by targeted gene disruption to examine PyCryPH function in mosquito-stage parasite development. Results Western blot and immunofluorescence assays using specific antibodies showed that PyCryPH is specifically expressed in zygotes and ookinetes. By immunoelectron microscopy it was demonstrated that PyCryPH is localized within crystalloid bodies. Parasites with a disrupted PyCryPH gene developed normally into ookinetes and formed oocysts on the basal lamina of midguts. In addition, the number of sporozoites residing in salivary glands was comparable to that of wild-type parasites. Conclusions CryPH, containing a signal peptide and PH domain, is predominantly expressed in zygotes and ookinetes and is localized to crystalloid bodies in P. yoelii. CryPH accumulates in vesicle-like structures prior to the appearance of typical crystalloid bodies. Unlike other known crystalloid body localized proteins, CryPH does not appear to have a multiple domain architecture characteristic of the LAP/CCp family proteins. Although CryPH is highly conserved among Plasmodium, Babesia, Theileria, and Cryptosporidium, PyCryPH is dispensable for the development of invasive ookinetes and sporozoites in mosquito bodies.…

Abstract Background Plasmodium falciparum haemozoin, a detoxification product of digested haemoglobin from infected erythrocytes, is released into the bloodstream upon schizont rupture and accumulates in leukocytes. High levels of haemozoin correlate with disease severity. Some studies have shown that concentrations of the substrate of inducible nitric oxide synthase (iNOS), l-arginine, as well as nitric oxide are low in patients infected with P. falciparum malaria. The present study investigates, in vitro, the role of P. falciparum haemozoin on nitric oxide production, iNOS expression in macrophages, and the possible interaction between l-arginine and haemozoin. Methods Plasmodium falciparum haemozoin was obtained from in vitro cultures through magnetic isolation. Phagocytosis of haemozoin by immortalized bone marrow derived macrophages was detected by confocal reflection combined with fluorescence microscopy. Nitrite concentrations in the supernatants was evaluated by Griess assay as a standard indication of nitric oxide production, while iNOS expression was detected on cell extracts by western blotting. Detection of l-arginine in haemozoin-treated or untreated media was achieved by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Results Haemozoin synergizes in vitro with interferon-gamma to produce nitric oxide. However, when mouse macrophages were stimulated with haemozoin, a proportional increase of nitric oxide was observed up to 25 μM of haemozoin, followed by a decrease with doses up to 100 μM, when nitric oxide release was completely abrogated. This was not due to reactive oxygen species production, nor to an effect on iNOS activity. Interestingly, when at 24 h, haemozoin-treated macrophages were washed and incubated in fresh medium for further 24 h, the nitric oxide production was restored in a dose–response manner. Similar results were seen when l-arginine-enriched media was used in the stimulation. Moreover, muramyldipeptide, a strong nitric oxide inducer, was unable to activate macrophages to release nitric oxide in the presence of haemozoin-treated medium. By LC–MS/MS a complete depletion of l-arginine was observed in this haemozoin-treated, conditioned medium. Conclusions It is proposed that haemozoin interacts with l-arginine reducing its availability for iNOS, and thus decreasing nitric oxide production. The clinical (or pathological) implications of these results are discussed.…

Igor Iatsenko, Jean-Philippe Boquete, Bruno Lemaitre

Iatsenko et al. find that flies bearing a mutation in a peptidoglycan recognition protein exhibit dysbiosis and shortened lifespan. In these flies, the unrestricted growth of a commensal bacterium generates excessive lactate, which triggers ROS production in intestinal cells and subsequent dysplasia. This ROS-producing pathway is conserved, suggesting relevance of such host-microbe metabolic crosstalk in age-related pathologies in humans.

Adriana Gielbert, Jemma K. Thorne, Jane M. Plater, Leigh Thorne, Peter C. Griffiths, Marion M. Simmons, Claire A. Cassar

by Adriana Gielbert, Jemma K. Thorne, Jane M. Plater, Leigh Thorne, Peter C. Griffiths, Marion M. Simmons, Claire A. Cassar The prion hypothesis proposes a causal relationship between the misfolded prion protein (PrPSc) molecular entity and the disease transmissible spongiform encephalopathy (TSE). Variations in the conformation of PrPSc are associated with different forms of TSE and different risks to animal and human health. Since the discovery of atypical forms of bovine spongiform encephalopathy (BSE) in 2003, scientists have progressed the molecular characterisation of the associated PrPSc in order to better understand these risks, both in cattle as the natural host and following experimental transmission to other species. Here we report the development of a mass spectrometry based assay for molecular characterisation of bovine proteinase K (PK) treated PrPSc (PrPres) by quantitative identification of its N-terminal amino acid profiles (N-TAAPs) and tryptic peptides. We have applied the assay to classical, H-type and L-type BSE prions purified from cattle, transgenic (Tg) mice expressing the bovine (Tg110 and Tg1896) or ovine (TgEM16) prion protein gene, and sheep brain. We determined that, for classical BSE in cattle, the G96 N-terminal cleavage site dominated, while the range of cleavage sites was wider following transmission to Tg mice and sheep. For L-BSE in cattle and Tg bovinised mice, a C-terminal shift was identified in the N-TAAP distribution compared to classical BSE, consistent with observations by Western blot (WB). For L-BSE transmitted to sheep, both N-TAAP and tryptic peptide profiles were found to be changed compared to cattle, but less so following transmission to Tg ovinised mice. Relative abundances of aglycosyl peptides were found to be significantly different between the atypical BSE forms in cattle as well as in other hosts. The enhanced resolution provided by molecular analysis of PrPres using mass spectrometry has improved insight into the molecular changes following transmission of atypical BSE to other species.

Schaub, R. E., Perez-Medina, K. M., Hackett, K. T., Garcia, D. L., Dillard, J. P.

Neisseria gonorrhoeae releases peptidoglycan fragments during growth, and these molecules induce an inflammatory response in the human host. The proinflammatory molecules include peptidoglycan monomers, peptidoglycan dimers, and free peptides. These molecules can be released by the actions of lytic transglycosylases or an amidase. However, over 40% of the gonococcal cell wall is crosslinked, where the peptide stem on one peptidoglycan strand is linked to the peptide stem on a neighboring strand, suggesting that endopeptidases may be required for release of many peptidoglycan fragments. Therefore, we characterized mutants with individual or combined mutations in genes for the low molecular mass penicillin-binding proteins PBP3 and PBP4. Mutations in either dacB encoding PBP3 or pbpG encoding PBP4 did not significantly reduce peptidoglycan monomer or free peptide release. A mutation in dacB caused the appearance of a larger-sized peptidoglycan monomer, the pentapeptide monomer, and an increased release of peptidoglycan dimers, suggesting the involvement of this enzyme in both the removal of C-terminal d-Ala residues from stem peptides and the cleavage of crosslinked peptidoglycan. Mutation of both dacB and pbpG eliminated release of tripeptide-containing peptidoglycan fragments concomitant with the appearance of penta-peptide and di-peptide peptidoglycan fragments and higher molecular weight peptidoglycan dimers. Consistent with the loss of tripeptide peptidoglycan fragments, human NOD1 activation by the dacB pbpG mutants was significantly reduced compared to wild type. We conclude that PBP3 and PBP4 overlap in function for crosslink cleavage and that these endopeptidases act in the normal release of peptidoglycan fragments during growth.…

Edna O. Viegas, Arne Kroidl, Patricia J. Munseri, Marco Missanga, Charlotta Nilsson, Nelson Tembe, Asli Bauer, Agricola Joachim, Sarah Joseph, Philipp Mann, Christof Geldmacher, Sue Fleck, Wolfgang Stöhr, Gabriella Scarlatti, Said Aboud, Muhammad Bakari, Leonard Maboko, Michael Hoelscher, Britta Wahren, Merlin L. Robb, Jonathan Weber, Sheena McCormack, Gunnel Biberfeld, Ilesh V. Jani, Eric Sandström, Eligius Lyamuya, TaMoVac study group

by Edna O. Viegas, Arne Kroidl, Patricia J. Munseri, Marco Missanga, Charlotta Nilsson, Nelson Tembe, Asli Bauer, Agricola Joachim, Sarah Joseph, Philipp Mann, Christof Geldmacher, Sue Fleck, Wolfgang Stöhr, Gabriella Scarlatti, Said Aboud, Muhammad Bakari, Leonard Maboko, Michael Hoelscher, Britta Wahren, Merlin L. Robb, Jonathan Weber, Sheena McCormack, Gunnel Biberfeld, Ilesh V. Jani, Eric Sandström, Eligius Lyamuya, TaMoVac study group Background We evaluated the safety and immunogenicity of (i) an intradermal HIV-DNA regimen given with/without intradermal electroporation (EP) as prime and (ii) the impact of boosting with modified vaccinia virus Ankara (HIV-MVA) administered with or without subtype C CN54rgp140 envelope protein adjuvanted with Glucopyranosyl Lipid A (GLA-AF) in volunteers from Tanzania and Mozambique. Methods Healthy HIV-uninfected adults (N = 191) were randomized twice; first to one of three HIV-DNA intradermal priming regimens by needle-free ZetaJet device at weeks 0, 4 and 12 (Group I: 2x0.1mL [3mg/mL], Group II: 2x0.1mL [3mg/mL] plus EP, Group III: 1x0.1mL [6mg/mL] plus EP). Second the same volunteers received 108 pfu HIV-MVA twice, alone or combined with CN54rgp140/GLA-AF, intramuscularly by syringe, 16 weeks apart. Additionally, 20 volunteers received saline placebo. Results Vaccinations and electroporation did not raise safety concerns. After the last vaccination, the overall IFN-γ ELISpot response rate to either Gag or Env was 97%. Intradermal electroporation significantly increased ELISpot response rates to HIV-DNA-specific Gag (66% group I vs. 86% group II, p = 0.026), but not to the HIV-MVA vaccine-specific Gag or Env peptide pools nor the magnitude of responses. Co-administration of rgp140/GLA-AF with HIV-MVA did not impact the frequency of binding antibody responses against subtype B gp160, C gp140 or E gp120 antigens (95%, 99%, 79%, respectively), but significantly enhanced the magnitude against subtype B gp160 (2700 versus 300, p

Skerry, C., Goldman, W. E., Carbonetti, N. H.

Incidence of whooping cough (pertussis), a bacterial infection of the respiratory tract caused by the bacterium Bordetella pertussis, has reached levels not seen since the 1950s. Antibiotics fail to improve the course of disease unless administered early in infection. Therefore, there is an urgent need for the development of anti-pertussis therapeutics. Sphingosine-1-phosphate receptor (S1PR) agonists have been shown to reduce pulmonary inflammation during Bordetella pertussis infection in mouse models. However, the mechanisms by which S1PR agonists attenuate pertussis disease are unknown. We report the results of an RNAseq study examining pulmonary transcriptional responses in B. pertussis-infected mice treated with S1PR agonist AAL-R or vehicle control. This study identified peptidoglycan recognition protein 4 (PGLYRP4) as one of the most highly upregulated genes in the lungs of infected mice following S1PR agonism. PGLYRP4, a secreted, innate mediator of host defenses was found to limit early inflammatory pathology in knock-out mouse studies. Further, S1PR agonist AAL-R failed to attenuate pertussis disease in PGLYRP4 KO mice. B. pertussis virulence factor tracheal cytotoxin (TCT), a secreted peptidoglycan breakdown product, induces host tissue damage. TCT over-secreting strains were found to drive an early inflammatory response similar to that observed in PGLYRP4 KO mice. Further, TCT over-secreting strains induced significantly greater pathology in PGLYRP4 deficient animals than their wild-type counterparts. Together these data indicate that S1PR agonist mediated protection against pertussis disease is mediated via PGLYRP4. Our data suggest PGLYRP4 functions, in part, by preventing TCT induced airway damage.…

Govindasamy, K., Khan, R., Snyder, M., Lou, H. J., Du, P., Kudyba, H. M., Muralidharan, V., Turk, B. E., Bhanot, P.

Malaria is caused by the protozoan parasite, Plasmodium, which undergoes a complex life-cycle in a human host and a mosquito vector. The parasite’s cGMP dependent protein kinase (PKG) is essential at multiple steps of the life-cycle. Phosphoproteomic studies in P. falciparum eyrthocytic stages and P. berghei ookinetes have identified proteolysis as a major biological pathway dependent on PKG activity. To further understand PKG’s mechanism of action, we screened a yeast two-hybrid library for P. falciparum proteins that interact with P. falciparum PKG (PfPKG), and tested peptide libraries to identify its phosphorylation site preferences. Our data suggest that PfPKG has a distinct phosphorylation site and that PfPKG directly phosphorylates parasite RPT1, one of six AAA+ ATPases present in the 19S regulatory particle of the proteasome. PfPKG and RPT1 interact in vitro, the interacting fragment of RPT1 carries a PfPKG consensus phosphorylation site, a peptide carrying this consensus site competes with the RPT1 fragment for binding to PfPKG and is efficiently phosphorylated by PfPKG. These data suggest that PfPKG’s phosphorylation of RPT1 could contribute to its regulation of parasite proteolysis. We demonstrate that proteolysis plays an important role in a biological process known to require Plasmodium PKG - invasion by sporozoites of hepatocytes. A small molecule inhibitor of proteasomal activity blocks sporozoite invasion in an additive manner when combined with a Plasmodium PKG-specific inhibitor. Mining the previously described parasite PKG-dependent phosphoproteomes using the consensus phosphorylation motif identified additional proteins that are likely to be direct substrates of the enzyme.…

Rasch, J., Ünal, C. M., Klages, A., Karsli, U., Heinsohn, N., Brouwer, R. M. H. J., Richter, M., Dellmann, A., Steinert, M.

The gamma-proteobacterium Legionella pneumophila is the causative agent of Legionnaires’ disease, an atypical pneumonia that manifests itself with severe lung damage. L. pneumophila, a common inhabitant of fresh water environments, replicates in free-living amoebae, and persists in biofilms in natural and man-made water systems. Its environmental versatility is reflected in its ability to survive and grow within a broad temperature range as well as its capability to colonize and infect a wide range of hosts including protozoa and humans. Peptidyl-prolyl-cis/trans-isomerases (PPIases) are multifunctional proteins that mainly are involved in protein folding and secretion in bacteria. In L. pneumophila the surface associated PPIase Mip was shown to facilitate the establishment of the intracellular infection cycle in its early stages. The cytoplasmic PpiB was shown to promote cold tolerance. Here, we set out to analyze the interrelationship of these two relevant PPIases in the context of environmental fitness and infection. We demonstrate that the PPIases Mip and PpiB are important for surfactant dependent sliding motility, and adaptation to suboptimal temperatures, features that contribute to the environmental fitness of L. pneumophila. Furthermore, they contribute to the infection of the natural host Acanthamoeba castellanii as well as human macrophages and human explanted lung tissue. These effects were additive in case of sliding motility, or synergistic in case of temperature tolerance and infection as assessed by the behavior of the double mutant. Accordingly, we propose that Mip and PpiB are virulence modulators of L. pneumophila with compensatory action and pleiotropic effects.…

Lucina Kimaro, Juma Adinan, Damian J. Damian, Bernard Njau

by Lucina Kimaro, Juma Adinan, Damian J. Damian, Bernard Njau Background Infection with Human Immunodeficiency Virus is a serious public health problem that threatens the lives of many people including health care workers. Health care workers are frequently exposed to occupational hazards throughout their careers. Health care workers are at risk of being infected by the virus when caring for patients in health care facilities. Utilization of HIV Post-exposure Prophylaxis (HIV PEP) is very vital once an individual is exposed. Aim The aim of this study is to determine the prevalence of occupational exposure, knowledge of, availability and utilization of post exposure prophylaxis among health care workers in Singida District Council, Tanzania. Methods A descriptive cross sectional study was conducted from April to May 2013. Health care workers actively treating patients were enrolled from 18 heath facilities in Singida District Council. Data were collected using a self-administered questionnaire, and analysed using Stata version 12. Results Out of 239 participants, slightly more than half, 124 (52%) had inadequate overall knowledge of HIV PEP. Of the 239, 121(50.6%) participants experienced occupational exposure. Two leading types of exposure were blood splash 57(47.1%) and needle stick injuries 45 (37.2%),respectively. Among the 121 exposed participants, 83(68.6%) reported the exposure incident, 91(75.2%) had an HIV test, 32 (26.4%), started HIV PEP after testing, 28 (23.1%), completed HIV PEP, and 65 (53.7%) had a follow-up HIV test. About two thirds (159/239), of participants reported that HIV PEP services were available at the time the study was conducted, and 49 (20.5%), reported daily access to HIV PEP service. Conclusion The prevalence of occupational exposure among health care workers is high with low utilization of HIV PEP. The majority of healthcare workers had inadequate knowledge of HIV PEP. The findings highlight the need to improve the level of knowledge of HIV PEP and utilization of PEP among this at-high-risk-group in Singida.…

Kang, H. K., Seo, C. H., Luchian, T., Park, Y.

Pseudin-2, isolated from the frog Pseudis paradoxa, exhibits potent antibacterial activity but also cytotoxicity. In an effort to develop clinically applicable AMPs, we designed pseudin-2 analogs with Lys substitutions, resulting in elevated amphipathic α-helical structure and cationicity. In addition, truncated analogs of pseudin-2 and Lys-substituted peptides were synthesized to produce linear 18-residue amphipathic α-helices, which were further investigated for their mechanism and functions. These truncated analogs exhibited higher antimicrobial activity and lower cytotoxicity than pseudin-2. In particular, Pse-T2 showed marked pore formation, permeabilization of the outer/inner bacterial membranes, and DNA binding. Fluorescence spectroscopy and scanning electron microscopy showed that Pse-T2 kills bacterial cells by disrupting membrane integrity. In vivo, wounds infected with MDR Pseudomonas aeruginosa healed significantly faster when treated Pse-T2 than did untreated wounds or wounds treated with ciprofloxacin. Moreover, Pse-T2 facilitated infected wound closure by reducing inflammation through suppression of IL-1β, IL-6, and TNF-α. These data suggest the small antimicrobial peptide Pse-T2 could be useful for future development of therapeutic agents effective against MDR bacterial strains.…

Tomoyoshi Inoue, Yoshiya Ito, Nobuyuki Nishizawa, Koji Eshima, Ken Kojo, Fumisato Otaka, Tomohiro Betto, Sakiko Yamane, Kazutake Tsujikawa, Wasaburo Koizumi, Masataka Majima

by Tomoyoshi Inoue, Yoshiya Ito, Nobuyuki Nishizawa, Koji Eshima, Ken Kojo, Fumisato Otaka, Tomohiro Betto, Sakiko Yamane, Kazutake Tsujikawa, Wasaburo Koizumi, Masataka Majima The significance of the relationship between the nervous and immune systems with respect to disease course is increasingly apparent. Immune cells in the liver and spleen are responsible for the development of acute liver injury, yet the regulatory mechanisms of the interactions remain elusive. Calcitonin gene-related peptide (CGRP), which is released from the sensory nervous system, regulates innate immune activation via receptor activity-modifying protein 1 (RAMP1), a subunit of the CGRP receptor. Here, we show that RAMP1 in Kupffer cells (KCs) plays a critical role in the etiology of immune-mediated hepatitis. RAMP1-deficient mice with concanavalin A (ConA)-mediated hepatitis, characterized by severe liver injury accompanied by infiltration of immune cells and increased secretion of pro-inflammatory cytokines by KCs and splenic T cells, showed poor survival. Removing KCs ameliorated liver damage, while depleting T cells or splenectomy led to partial amelioration. Adoptive transfer of splenic T cells from RAMP1-deficient mice led to a modest increase in liver injury. Co-culture of KCs with splenic T cells led to increased cytokine expression by both cells in a RAMP1-dependent manner. Thus, immune-mediated hepatitis develops via crosstalk between immune cells. RAMP1 in KCs is a key regulator of immune responses.

Freeman, R., Han, M., Alvarez, Z., Lewis, J. A., Wester, J. R., Stephanopoulos, N., McClendon, M. T., Lynsky, C., Godbe, J. M., Sangji, H., Luijten, E., Stupp, S. I.

Soft structures in nature such as protein assemblies can organize reversibly into functional and often hierarchical architectures through noncovalent interactions. Molecularly encoding this dynamic capability in synthetic materials has remained an elusive goal. We report on hydrogels of peptide-DNA conjugates and peptides that organize into superstructures of intertwined filaments that disassemble upon the addition of molecules or changes in charge density. Experiments and simulations demonstrate that this response requires large scale spatial redistribution of molecules directed by strong noncovalent interactions among them. Simulations also suggest that the chemically reversible structures can only occur within a limited range of supramolecular cohesive energies. Storage moduli of the hydrogels change reversibly as superstructures form and disappear, as does the phenotype of neural cells in contact with these materials.…

Antoine Couture-Cossette, Alex Carignan, Adam Mercier, Claudine Desruisseaux, Louis Valiquette, Jacques Pépin

by Antoine Couture-Cossette, Alex Carignan, Adam Mercier, Claudine Desruisseaux, Louis Valiquette, Jacques Pépin Objectives To examine secular changes in the incidence of invasive beta-hemolytic streptococcal infections, and to assess the efficacy of immunoglobulins and clindamycin as adjunctive therapies in the management of Streptococcus pyogenes infections. Methods Retrospective cohort study of all cases of invasive group A (GAS), B (GBS), C or G (GCGS) streptococcal infections managed in a Canadian tertiary center from 1996–2016. Population incidence was measured for diabetics and non-diabetics. Adjusted odds ratios (AOR) and their 95% confidence intervals (CI) were calculated by logistic regression. Results 741 cases were identified (GAS: 249; GBS: 304; GCGS: 188). While the incidence of invasive GAS infections fluctuated with no clear trend, incidence of invasive GBS and GCGS increased over time and were 8.4 and 6.3 times higher in diabetics. Mortality of invasive GAS infections decreased from 16% (6/37) in 1996–2001 to 4% (4/97) in 2011–15. Among patients with GAS infections, clindamycin administered concomitantly with a beta-lactam within 24 hours of admission decreased mortality (AOR: 0.04, 95%CI: 0.003–0.55, P = 0.02. Immunoglobulins had no such effect (AOR: 1.66, 95%CI: 0.16–17.36, P = 0.67). The protective effect of clindamycin was similar in patients with pneumonia/empyema compared to all others. Conclusion Incidence of GBS and GCGS infections increased due to an expansion of the high-risk population (elderly diabetics), but also rose in non-diabetics. No such secular change was seen for invasive GAS infections. The decrease in mortality in patients with invasive GAS infections presumably reflects better case-management. Adjunctive clindamycin reduced mortality in invasive GAS infections; immunoglobulins did not, but power was limited. The highest mortality was seen in patients with GAS pneumonia/empyema, for whom clindamycin was protective but underused.…

Yuan Liao, Jiao Gong, Wenying Zhou, Huimin Dong, Jiayin Liang, Minqi Luo, Bo Hu

Chronic liver inflammation caused by chronic hepatitis B virus (CHB) infection leads to liver cirrhosis and hepatocellular carcinoma. Recently, the role of alanine aminotransferase (ALT) as a predictor of liver inflammation has been questioned. The aim of this study was to investigate the utility of noninvasive fibrosis markers including hyaluronic acid (HA), collagen type IV (CIV), N‐terminal propeptide of type III procollagen (PIIINP), and laminin (LN) in identifying significant liver inflammation in patients with CHB, especially in patients with normal or near‐normal ALT. A total of 242 CHB patients who underwent liver biopsy were enrolled. The serum levels of ALT, aspartate aminotransferase, HA, CIV, PIIINP, and LN were quantified and the relationship between histological staging and serum markers was systematically analyzed. Serum CIV, PIIINP, HA, and LN levels increased significantly along with the increasing severity of liver inflammation. Multivariate analysis showed that CIV and LN were independently associated with significant inflammation. CIV, PIIINP, HA, and LN levels were found to have high diagnostic values for predicting significant inflammation in patients with CHB (area under the curve, AUC = 0.807, 0.795, 0.767, and 0.703, respectively). The combined index for the identification of significant inflammation, including CIV, PIIINP, HA, and LN levels, significantly improved diagnostic performance (AUC = 0.851). Moreover, the combined index also achieved excellent diagnostic accuracy (AUC = 0.861) in patients with CHB with normal or near‐normal ALT. In conclusion, the combined index may be a strong indicator for discriminating significant liver inflammation, especially in patients with CHB with normal or near‐normal ALT.

Allart, R., Bourrier, V., Lovis, C., Ehrenreich, D., Spake, J. J., Wyttenbach, A., Pino, L., Pepe, F., Sing, D. K., Lecavelier des Etangs, A.

Stellar heating causes atmospheres of close-in exoplanets to expand and escape. These extended atmospheres are difficult to observe because their main spectral signature—neutral hydrogen at ultraviolet wavelengths—is strongly absorbed by interstellar medium. We report the detection of the near-infrared triplet of neutral helium in the transiting warm Neptune-mass exoplanet HAT-P-11b using ground-based, high-resolution observations. The helium feature is repeatable over two independent transits, with an average absorption depth of 1.08 ± 0.05%. Interpreting absorption spectra with 3D simulations of the planet’s upper atmosphere suggests it extends beyond 5 planetary radii, with a large scale height and a helium mass loss rate 3x105 g·s–1. A net blue-shift of the absorption might be explained by high-altitude winds flowing at 3 km·s–1 from day to night-side.…

van Haren, Frank; Pham, Tài; Brochard, Laurent; Bellani, Giacomo; Laffey, John; Dres, Martin; Fan, Eddy; Goligher, Ewan C.; Heunks, Leo; Lynch, Joan; Wrigge, Hermann; McAuley, Danny; and the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) and the European Society of Intensiv

Objectives: To describe the characteristics and outcomes of patients with acute respiratory distress syndrome with or without spontaneous breathing and to investigate whether the effects of spontaneous breathing on outcome depend on acute respiratory distress syndrome severity. Design: Planned secondary analysis of a prospective, observational, multicentre cohort study. Setting: International sample of 459 ICUs from 50 countries. Patients: Patients with acute respiratory distress syndrome and at least 2 days of invasive mechanical ventilation and available data for the mode of mechanical ventilation and respiratory rate for the 2 first days. Interventions: Analysis of patients with and without spontaneous breathing, defined by the mode of mechanical ventilation and by actual respiratory rate compared with set respiratory rate during the first 48 hours of mechanical ventilation. Measurements and Main Results: Spontaneous breathing was present in 67% of patients with mild acute respiratory distress syndrome, 58% of patients with moderate acute respiratory distress syndrome, and 46% of patients with severe acute respiratory distress syndrome. Patients with spontaneous breathing were older and had lower acute respiratory distress syndrome severity, Sequential Organ Failure Assessment scores, ICU and hospital mortality, and were less likely to be diagnosed with acute respiratory distress syndrome by clinicians. In adjusted analysis, spontaneous breathing during the first 2 days was not associated with an effect on ICU or hospital mortality (33% vs 37%; odds ratio, 1.18 [0.92–1.51]; p = 0.19 and 37% vs 41%; odds ratio, 1.18 [0.93–1.50]; p = 0.196, respectively ). Spontaneous breathing was associated with increased ventilator-free days (13 [0–22] vs 8 [0–20]; p = 0.014) and shorter duration of ICU stay (11 [6–20] vs 12 [7–22]; p = 0.04). Conclusions: Spontaneous breathing is common in patients with acute respiratory distress syndrome during the first 48 hours of mechanical ventilation. Spontaneous breathing is not associated with worse outcomes and may hasten liberation from the ventilator and from ICU. Although these results support the use of spontaneous breathing in patients with acute respiratory distress syndrome independent of acute respiratory distress syndrome severity, the use of controlled ventilation indicates a bias toward use in patients with higher disease severity. In addition, because the lack of reliable data on inspiratory effort in our study, prospective studies incorporating the magnitude of inspiratory effort and adjusting for all potential severity confounders are required. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. European Society of Intensive Care Medicine, St Michael’s Hospital and University of Milan-Bicocca had no role in the design and conduct of the study; management, analysis, and interpretation of the data; preparation, review, or approval of the article; or decision to submit the article for publication. Drs. van Haren and Pham are co-first authors. Drs. van Haren, Pham, Brochard, Bellani, Laffey, Wrigge, McAuley conceptualized and designed the article. All authors analyzed and interpreted the article; drafted and revised the article for important intellectual content; and approved the final article. Members of the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) steering committee, national coordinators and site investigators are listed in the supplemental data (Supplemental Digital Content 1, http://links.lww.com/CCM/E146). Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (http://journals.lww.com/ccmjournal). Supported, in part, by the European Society of Intensive Care Medicine (ESICM), Brussels, Belgium, by St Michael’s Hospital, Toronto, Canada, and by the University of Milan-Bicocca, Monza, Italy. The ESICM provided support in data collection and study coordination. Dr. Brochard received funding from Medtronic Covodien, Air Liquide, and Fisher Paykel, and he received equipment from Philips. Dr. Heunks’ institution received funding from European Respiratory Society and Ventfree, and he received funding from Maquet (speaker’s fee). Dr. Wrigge received funding from Dräger Medical, Merck Sharp & Dohme, and InfectoPharm. Dr. McAuley’s institution received funding from National Institute for Health Research, Wellcome Trust and other grant funders; and he received funding from Bayer/GlaxoSmithKline, and Boehringer Ingelheim/Peptinnovate. The remaining authors have disclosed that they do not have any potential conflicts of interest. For information regarding this article, E-mail: fvanharen@me.com Copyright © by 2018 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.

Itskanov, S., Park, E.

The Sec61 protein-conducting channel mediates transport of many proteins, such as secretory proteins, across the endoplasmic reticulum (ER) membrane during or after translation. Posttranslational transport is enabled by two additional membrane proteins associated with the channel, Sec63 and Sec62, but its mechanism is poorly understood. We determined a structure of the Sec complex (Sec61-Sec63-Sec71-Sec72) from Saccharomyces cerevisiae by cryo-electron microscopy (cryo-EM). The structure shows that Sec63 tightly associates with Sec61 though interactions in cytosolic, transmembrane, and ER-luminal domains, prying open Sec61’s lateral gate and translocation pore and thus activating the channel for substrate engagement. Furthermore, Sec63 optimally positions binding sites for cytosolic and luminal chaperones in the complex to enable efficient polypeptide translocation. Our study provides mechanistic insights into eukaryotic posttranslational protein translocation.…

Janet Saul, Gretchen Bachman, Shannon Allen, Nora F. Toiv, Caroline Cooney, Ta’Adhmeeka Beamon

by Janet Saul, Gretchen Bachman, Shannon Allen, Nora F. Toiv, Caroline Cooney, Ta’Adhmeeka Beamon In sub-Saharan Africa, adolescent girls and young women (AGYW) are 5 to 14 times more likely to be infected with HIV than their male peers. Every day, more than 750 AGYW are infected with HIV. Many factors make girls and young women particularly vulnerable to HIV, including gender-based violence, exclusion from economic opportunities, and a lack of access to secondary school. The President’s Emergency Plan for AIDS Relief (PEPFAR) is dedicating significant resources through the Determined, Resilient, Empowered, AIDS-free, Mentored, and Safe (DREAMS) partnership to impact the lives of women and girls based on PEPFAR’s mission to help countries achieve epidemic control of HIV/AIDS. The data show that new HIV infections must be reduced in AGYW, or the global community risks losing the extensive progress made towards reaching epidemic control. With support from PEPFAR and private sector partners—the Bill & Melinda Gates Foundation, Gilead Sciences, Girl Effect, Johnson & Johnson and ViiV Healthcare, DREAMS works together with partner governments to deliver a core package of interventions that combines evidence-based approaches that go beyond the health sector, addressing the structural drivers that directly and indirectly increase girls’ HIV risk. Not only is DREAMS an effort to reduce new HIV infections, but it aims to reduce other critical vulnerabilities such as gender-based violence. When girls and young women thrive, the effects are felt throughout their families, communities and countries.

Lewis, Lara; Maughan-Brown, Brendan; Grobler, Anneke; Cawood, Cherie; Khanyile, David; Glenshaw, Mary; BM Kharsany, Ayesha

Background: In several sub-groups of South Africa, the percentage of HIV-positive individuals aware of their status falls well below the UNAIDS 90% target. This study examined the impact that home-based HIV testing services (HBHTS) had on knowledge of status in a hyperendemic area of South Africa. Methods: We analysed data from the second cross-sectional HIV Incidence Provincial Surveillance System survey (2015/2016), a representative sample (n=10236) of individuals aged 15-49 years. Participants completed a questionnaire, provided blood samples for laboratory testing (used to estimate HIV prevalence) and were offered HBHTS. The proportion of people living with HIV (n=3870) made aware of their status through HBHTS was measured and factors associated with HBHTS uptake were identified. Results: Knowledge of HIV-positive status at the time of the survey was 62.9% among men and 73.4% among women. Through HBHTS, the percentage of HIV-positive men and women who knew their status rose to 74.2% and 80.5% respectively. The largest impact was observed among youth (15-24 years). Knowledge of status increased from 36.6% to 59.3% and from 50.8% to 64.8% among young men and women respectively. Additionally, 51.4% of those who had previously never tested received their first test. Key reasons for declining HBHTS among undiagnosed HIV-positive individuals included fear and self-report of a HIV-negative status. Conclusions: HBHTS was effective in increasing awareness of HIV-positive status, particularly among youth, men and those who had never tested. HBHTS could have a marked impact on progress towards the UNAIDS 90-90-90 targets within these sub-groups. Corresponding author: Brendan Maughan-Brown; Southern Africa Labour and Development Research Unit, University of Cape Town, Private Bag, Rondebosch, 7701, Cape Town, South Africa; brendan.maughanbrown@gmail.com; Telephone: +27-21-650-5695; Fax: +27-21-650-5697 Conflicts of interest and source of funding: We have no conflicts of interest to declare. The HIV Incidence Provincial Surveillance System (HIPSS) is funded by the US President’s Emergency Plan for AIDS Relief (PEPFAR) through the Centers for Disease Control and Prevention (CDC) under the terms of cooperative agreement 3U2GGH000372-02 W1. Support was provided to BMB by the National Research Foundation, South Africa, through the Research Career Advancement Fellowship. ABMK is supported by a joint South Africa–U.S. Program for Collaborative Biomedical Research, National Institutes of Health grant (R01HD083343). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official position of the funding agencies. * These authors have contributed equally to the work. Authors’ contributions: ABMK is the principal investigator of HIPSS. ABMK, CC, and DK, were responsible for HIPSS study operations and quality assurance. LL analysed the data. LL, BMB, AG and ABMK contributed to analysis and the interpretation of the data of the first draft. All authors contributed to subsequent drafts, and approved the final version of the report. Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved.

Walsh, S. I., Peters, D. S., Smith, P. A., Craney, A., Dix, M. M., Cravatt, B. F., Romesberg, F. E.

At sufficient concentrations, antibiotics effectively eradicate many bacterial infections. However, during therapy bacteria are unavoidably exposed to lower antibiotic concentrations, and sub-minimum inhibitory concentration (MIC) exposure can result in a wide variety of other effects, including the induction of virulence, which can complicate therapy, or horizontal gene transfer (HGT), which can accelerate the spread of resistance genes. Bacterial type I signal peptidase (SPase) is an essential protein that acts at the final step of the general secretory pathway. This pathway is required for the secretion of many proteins, including many required for virulence, and the arylomycins are a class of natural product antibiotics that target SPase. Here, we investigated the consequences of exposing Escherichia coli to sub-MIC levels of an arylomycin. Using multidimensional protein identification technology mass spectrometry, we find that arylomycin treatment inhibits the proper extracytoplasmic localization of many proteins, both those that appear to be SPase substrates as well as several that do not. The identified proteins are involved in a broad range of extracytoplasmic processes and include a number of virulence factors. The effects of arylomycin on several processes required for virulence were then individually examined, and we found that even at sub-MIC levels, the arylomycins potently inhibit flagellation, motility, biofilm formation, and the dissemination of antibiotic resistance via HGT. Thus, we conclude that the arylomycins represent promising novel therapeutics with the potential to eradicate infections while simultaneously reducing virulence and the dissemination of resistance.…