Exploring the free-energy landscape of carbohydrate-protein complexes: development and validation of scoring functions considering the binding-site topology

Carbohydrates play a key role in a variety of physiological and pathological processes and, hence, represent a rich source for the development of novel therapeutic agents. Being able to predict binding mode and binding affinity is an essential, yet lacking, aspect of the structure-based design of carbohydrate-based ligands. We assembled a diverse data set comprising 273 carbohydrate-protein crystal structures with known binding affinity and evaluated the prediction accuracy of a large collection of well-established scoring and free-energy functions, as well as combinations thereof. Unfortunately, the tested functions were not capable of reproducing binding affinities in the studied complexes. To simplify the complex free-energy surface of carbohydrate-protein systems, we classified the studied proteins according to the topology and solvent exposure of the carbohydrate-binding site into five distinct categories. A free-energy model based on the proposed classification scheme reproduced binding affinities in the carbohydrate data set with an r 2 of 0.71 and root-mean-squared-error of 1.25kcal/mol (N=236). The improvement in model performance underlines the significance of the differences in the local micro-environments of carbohydrate-binding sites and demonstrates the usefulness of calibrating free-energy functions individually according to binding-site topology and solvent exposure. Graphical Abstract

Mutation of Tyr137 of the universal Escherichia coli fimbrial adhesin FimH relaxes the tyrosine gate prior to mannose binding

The most prevalent diseases manifested by Escherichia coli are acute and recurrent bladder infections and chronic inflammatory bowel diseases such as Crohn's disease. E. coli clinical isolates express the FimH adhesin, which consists of a mannose-specific lectin domain connected via a pilin domain to the tip of type 1 pili. Although the isolated FimH lectin domain has affinities in the nanomolar range for all high-mannosidic glycans, differentiation between these glycans is based on their capacity to form predominantly hydrophobic interactions within the tyrosine gate at the entrance to the binding pocket. In this study, novel crystal structures of tyrosine-gate mutants of FimH, ligand-free or in complex with heptyl α - D - O -mannopyranoside or 4-biphenyl α - D - O- mannopyranoside, are combined with quantum-mechanical calculations and molecular-dynamics simulations. In the Y48A FimH crystal structure, a large increase in the dynamics of the alkyl chain of heptyl α - D - O -mannopyranoside attempts to compensate for the absence of the aromatic ring; however, the highly energetic and stringent mannose-binding pocket of wild-type FimH is largely maintained. The Y137A mutation, on the other hand, is the most detrimental to FimH affinity and specificity: (i) in the absence of ligand the FimH C-terminal residue Thr158 intrudes into the mannose-binding pocket and (ii) ethylenediaminetetraacetic acid interacts strongly with Glu50, Thr53 and Asn136, in spite of multiple dialysis and purification steps. Upon mutation, pre-ligand-binding relaxation of the backbone dihedral angles at position 137 in the tyrosine gate and their coupling to Tyr48 via the interiorly located Ile52 form the basis of the loss of affinity of the FimH adhesin in the Y137A mutant

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

Surgical site infection after gastrointestinal surgery in high-income, middle-income, and low-income countries: a prospective, international, multicentre cohort study

Background: Surgical site infection (SSI) is one of the most common infections associated with health care, but its importance as a global health priority is not fully understood. We quantified the burden of SSI after gastrointestinal surgery in countries in all parts of the world. Methods: This international, prospective, multicentre cohort study included consecutive patients undergoing elective or emergency gastrointestinal resection within 2-week time periods at any health-care facility in any country. Countries with participating centres were stratified into high-income, middle-income, and low-income groups according to the UN's Human Development Index (HDI). Data variables from the GlobalSurg 1 study and other studies that have been found to affect the likelihood of SSI were entered into risk adjustment models. The primary outcome measure was the 30-day SSI incidence (defined by US Centers for Disease Control and Prevention criteria for superficial and deep incisional SSI). Relationships with explanatory variables were examined using Bayesian multilevel logistic regression models. This trial is registered with ClinicalTrials.gov, number NCT02662231. Findings: Between Jan 4, 2016, and July 31, 2016, 13 265 records were submitted for analysis. 12 539 patients from 343 hospitals in 66 countries were included. 7339 (58·5%) patient were from high-HDI countries (193 hospitals in 30 countries), 3918 (31·2%) patients were from middle-HDI countries (82 hospitals in 18 countries), and 1282 (10·2%) patients were from low-HDI countries (68 hospitals in 18 countries). In total, 1538 (12·3%) patients had SSI within 30 days of surgery. The incidence of SSI varied between countries with high (691 [9·4%] of 7339 patients), middle (549 [14·0%] of 3918 patients), and low (298 [23·2%] of 1282) HDI (p < 0·001). The highest SSI incidence in each HDI group was after dirty surgery (102 [17·8%] of 574 patients in high-HDI countries; 74 [31·4%] of 236 patients in middle-HDI countries; 72 [39·8%] of 181 patients in low-HDI countries). Following risk factor adjustment, patients in low-HDI countries were at greatest risk of SSI (adjusted odds ratio 1·60, 95% credible interval 1·05–2·37; p=0·030). 132 (21·6%) of 610 patients with an SSI and a microbiology culture result had an infection that was resistant to the prophylactic antibiotic used. Resistant infections were detected in 49 (16·6%) of 295 patients in high-HDI countries, in 37 (19·8%) of 187 patients in middle-HDI countries, and in 46 (35·9%) of 128 patients in low-HDI countries (p < 0·001). Interpretation: Countries with a low HDI carry a disproportionately greater burden of SSI than countries with a middle or high HDI and might have higher rates of antibiotic resistance. In view of WHO recommendations on SSI prevention that highlight the absence of high-quality interventional research, urgent, pragmatic, randomised trials based in LMICs are needed to assess measures aiming to reduce this preventable complication

Molecular simulations of carbohydrate-protein complexes

I. Generation and validation of a free-energy model for carbohydrate binding. Carbohy-drates play a key role in a variety of physiological and pathological processes and, hence, represent a rich source for the development of novel therapeutic agents. Being able to predict binding mode and binding affinity is an essential, yet lacking, aspect of the stru-cture-based design of carbohydrate-based ligands. To this end, we assembled a diverse data set of 316 carbohydrate–protein crystal structures with known binding affinity. We eval-uated the prediction accuracy of a large collection of well-established scoring and free-energy functions, as well as empirical combinations thereof. Unfortunately, the tested func-tions were not capable of reproducing carbohydrates binding affinities in our complexes. To simplify the complex free-energy surface of carbohydrate–protein systems, we classified the studied complexes according to the topology and solvent exposure of the carbohydrate-binding site into five distinct categories. A free-energy model based on the proposed classi-fication scheme reproduced binding affinities in the carbohydrate data set with an r2 of 0.69 and root-mean-squared-error of 1.36 kcal/mol. The improvement in model performance underlines the significance of the differences in the local micro-environments of carbo-hydrate-binding sites and demonstrates the usefulness of calibrating free-energy functions individually according to binding-site topology and surface exposure. II. Simulating the binding of Lewis-type ligands to DC-SIGN. Dendritic cells (DCs) have the function of presenting antigens to other processing cells of the immune system, particularly T-cells. DC-SIGN (DC-specific intercellular adhesion molecule-3-grabbing non-integrin) is one of the major receptors on DCs involved in the uptake of pathogens and has gained increasing interest over the last decade as it is crucially involved in infections caused by HIV-1, Ebola virus, Mycobacterium tuberculosis, and various other pathogens. High-mannosylated N-glycans or L-Fuc-containing trisaccharide motifs such as the Lewis (Le) blood group antigens Lea and Lex, which are surface components of these microorganisms, mediate binding to DC-SIGN. Crystallographic data for DC-SIGN in complex with a Lex-containing pentasaccharide suggest that the terminal sugar residues, L-Fuc and D-Gal, are predominantly involved in binding. We elucidated the interaction of DC-SIGN with Lea and Lex bearing two different aglycones. Binding assays together with STD NMR analysis, molecular modeling and mutagenesis studies revealed distinct binding modes dependent on the nature of the aglycone. Introduction of phenyl aglycones at the Le trisaccharides offers the establishment of an additional hydrophobic contact with Phe313 in the binding site of DC-SIGN, which entails a switch of the binding mode. Based on this information a new series of DC-SIGN antagonists can be designed. III. Developing a molecular modeling toolbox for medicinal chemists. In the current era of high-throughput drug discovery and development, molecular modeling has become an indispensable tool for identifying, optimizing and prioritizing small-molecule drug candidates. The required background in computational chemistry and the knowledge of how to handle the complex underlying protocols, however, might keep medicinal chemists from routinely using in silico technologies. Our objective is to encourage those researchers to exploit existing modeling technologies more frequently through easy-to-use graphical user interfaces. In this account, we present two innovative tools (which we are prepared to share with academic institutions) facilitating computational tasks commonly utilized in drug discovery and development: (1) the VirtualDesignLab estimates the binding affinity of small molecules by simulating and quantifying their binding to the three-dimensional structure of a target protein; and (2) the MD Client launches molecular dynamics simulations aimed at exploring the time-dependent stability of ligand–protein complexes and provides residue-based interaction energies. This allows medicinal chemists to identify sites of potential improvement in their candidate molecule. As a case study, we present the application of our tools towards the design of novel antagonists for the FimH adhesin

CYTOTOXICITY AND ELECTRON MICROSCOPIC

Interleukin-2 (IL-2) - activated lymphocytes, known as lymphokine activated laller/ natural laller ce11s (LAKlNK) were generated by incubation of human mononuclear blood cells in medium containing different doses (100, 250, 500 and 1000 mimI) of IL-2 for 3-5 days. These cells were tested for their cytotoxic activity against K562 target cells in short term (4 h) and long term (18 h) SlCr-release assay at different effector/target (E/T) ratios (100/1, SOil, 25/1, 12.5/1 and 6.25/1) Non-IL-2 treated mononuclear cells were able to lysis the target cells up to 9.67 % and 27.6 % in short term and long term assays, respectively at EIT (100:1). Incubation of effector (LA KINK) cells with different doses of IL-2 showed increase in the cytotoxicity percentage at all EIT ratios. The maximum cytotoxic activity of LAKINK cells was detected at 50/1 (EIT) ratio in presence of 1000 IU/ml of IL-2 either at 4 h (70 %) or at 18 h (81.2 %) by using 51Cr-release assay. There were direct relationship. between the percentages of cytotoxicity induced by LAKINK cells and the concentrations of ll_-2 as well as increased Err ratios. IL-2 is a potent inducer of LAK activity against K562 tumor celJ line. The ultrastructural results of this investigation allowed us to study the most important moments of interaction between LAKINK cells and K562 target cells in vitro. Effector (LAKlNK) cells undergo several ultrastructural changes, they started to develop border irregularity and protrusions that came in direct contact with target cells and established various degrees of adh..esion up to the development of desmosomes

Exploring the free-energy landscape of carbohydrate-protein complexes : development and validation of scoring functions considering the binding-site topology

Carbohydrates play a key role in a variety of physiological and pathological processes and, hence, represent a rich source for the development of novel therapeutic agents. Being able to predict binding mode and binding affinity is an essential, yet lacking, aspect of the structure-based design of carbohydrate-based ligands. We assembled a diverse data set comprising 273 carbohydrate-protein crystal structures with known binding affinity and evaluated the prediction accuracy of a large collection of well-established scoring and free-energy functions, as well as combinations thereof. Unfortunately, the tested functions were not capable of reproducing binding affinities in the studied complexes. To simplify the complex free-energy surface of carbohydrate-protein systems, we classified the studied proteins according to the topology and solvent exposure of the carbohydrate-binding site into five distinct categories. A free-energy model based on the proposed classification scheme reproduced binding affinities in the carbohydrate data set with an r (2) of 0.71 and root-mean-squared-error of 1.25 kcal/mol (N = 236). The improvement in model performance underlines the significance of the differences in the local micro-environments of carbohydrate-binding sites and demonstrates the usefulness of calibrating free-energy functions individually according to binding-site topology and solvent exposure

Design, synthesis, antimicrobial evaluation and molecular docking studies of some new 2,3-dihydrothiazoles and 4-thiazolidinones containing sulfisoxazole

<div><p></p><p>Microbial resistance to the available drugs poses a serious threat in modern medicine. We report the design, synthesis and <i>in vitro</i> antimicrobial evaluation of new functionalized 2,3-dihydrothiazoles and 4-thiazolidinones tagged with sulfisoxazole moiety. Compound <b>8d</b> was most active against <i>Bacillis subtilis</i> (MIC, 0.007 µg/mL). Moreover, compounds <b>7c</b>–<b>d</b> and <b>8c</b> displayed significant activities against <i>B. subtilis</i> and <i>Streptococcus pneumoniae</i> (MIC, 0.03–0.06 µg/mL and 0.06–0.12 µg/mL versus ampicillin 0.24 µg/mL and 0.12 µg/mL; respectively). Compounds <b>7a</b> and <b>7c</b>–<b>d</b> were highly potent against <i>Escherichia coli</i> (MIC, 0.49–0.98 µg/mL versus gentamycin 1.95 µg/mL). On the other hand, compounds <b>7e</b> and <b>9c</b> were fourfolds more active than amphotericin B against <i>Syncephalastrum racemosum</i>. Molecular docking studies showed that the synthesized compounds could act as inhibitors for the dihydropteroate synthase enzyme (DHPS). This study is a platform for the future design of more potent antimicrobial agents.</p></div

Iatrogenic bile duct injuries after laparoscopic cholecystectomy: evaluation by MRCP before management

Abstract Background Iatrogenic bile duct injuries are unusual but possibly associated with fatal complications with increased incidence since the introduction of laparoscopic cholecystectomy. Appropriate estimation of these injuries is essential for proper management. Imaging is vital for the initial diagnosis, extent assessment and consequently, treatment guidance of bile duct injury with an ideal outcome. In this study, MRCP was carried out in 37 cases (28 females and 9 males, age range from 19 to 58 years) with suspected BDI following laparoscopic cholecystectomy. MRCP images were assessed for bile duct transection injury, strictures, biliary leakage, and intrahepatic biliary radicles (IHBR) dilatation. In positive cases, Strasberg classification system was used with the definitive diagnosis was done regarding the surgical findings and/or findings on endoscopic retrograde cholangiopancreatography (ERCP) or percutaneous transhepatic cholangiography (PTC). Results Our study includes 37 cases with biliary injuries. On MRCP our cases were stratified regarding the Strasberg-Bismuth classification into five types (A to E). Most BDIs were type E2 (29.7%), followed by type E1 (18.9%), type A (16.2%), type E3 (10.8%), type E4 (8.2%), type C (5.4%), and type D and finally type E5 and B injuries with each one representing 2.7%. Twenty cases presented with biliary leakage and seventeen with bile duct obstruction, whether duct ligation or stricture. Conclusion MRCP is an essential imaging modality for assessment of iatrogenic BDIs enabling the radiologists to classify these injuries and helps to govern the management