Identification and analysis of conserved pockets on protein surfaces

BACKGROUND: The interaction between proteins and ligands occurs at pockets that are often lined by conserved amino acids. These pockets can represent the targets for low molecular weight drugs. In order to make the research for new medicines as productive as possible, it is necessary to exploit "in silico" techniques, high throughput and fragment-based screenings that require the identification of druggable pockets on the surface of proteins, which may or may not correspond to active sites. RESULTS: We developed a tool to evaluate the conservation of each pocket detected on the protein surface by CastP. This tool was named DrosteP because it recursively searches for optimal input sequences to be used to calculate conservation. DrosteP uses a descriptor of statistical significance, Poisson p-value, as a target to optimize the choice of input sequences. To benchmark DrosteP we used monomeric or homodimer human proteins with known 3D-structure whose active site had been annotated in UniProt. DrosteP is able to detect the active site with high accuracy because in 81% of the cases it coincides with the most conserved pocket. Comparing DrosteP with analogous programs is difficult because the outputs are different. Nonetheless we could assess the efficacy of the recursive algorithm in the identification of active site pockets by calculating conservation with the same input sequences used by other programs. We analyzed the amino-acid composition of conserved pockets identified by DrosteP and we found that it differs significantly from the amino-acid composition of non conserved pockets. CONCLUSIONS: Several methods for predicting ligand binding sites on protein surfaces, that combine 3D-structure and evolutionary sequence conservation, have been proposed. Any method relying on conservation mainly depends on the choice of the input sequences. DrosteP chooses how deeply distant homologs must be collected to evaluate conservation and thus optimizes the identification of active site pockets. Moreover it recognizes conserved pockets other than those coinciding with the sites annotated in UniProt that might represent useful druggable sites. The distinctive amino-acid composition of conserved pockets provides useful hints on the fundamental principles underlying protein-ligand interaction. AVAILABILITY: http://www.icb.cnr.it/project/drosteppy

A thermodynamic assay to test pharmacological chaperones for Fabry disease

AbstractBackgroundThe majority of the disease-causing mutations affect protein stability, but not functional sites and are amenable, in principle, to be treated with pharmacological chaperones. These drugs enhance the thermodynamic stability of their targets. Fabry disease, a disorder caused by mutations in the gene encoding lysosomal alpha-galactosidase, represents an excellent model system to develop experimental protocols to test the efficiency of such drugs.MethodsThe stability of lysosomal alpha-galactosidase under different conditions was studied by urea-induced unfolding followed by limited proteolysis and Western blotting.ResultsWe measured the concentration of urea needed to obtain half-maximal unfolding because this parameter represents an objective indicator of protein stability.ConclusionsUrea-induced unfolding is a versatile technique that can be adapted to cell extracts containing tiny amounts of wild-type or mutant proteins. It allows testing of protein stability as a function of pH, in the presence or in the absence of drugs. Results are not influenced by the method used to express the protein in transfected cells.General significanceScarce and dispersed populations pose a problem for the clinical trial of drugs for rare diseases. This is particularly true for pharmacological chaperones that must be tested on each mutation associated with a given disease. Diverse in vitro tests are needed. We used a method based on chemically induced unfolding as a tool to assess whether a particular Fabry mutation is responsive to pharmacological chaperones, but, by no means is our protocol limited to this disease

Prediction of the responsiveness to pharmacological chaperones: lysosomal human alpha-galactosidase, a case of study

<p>Abstract</p> <p>Background</p> <p>The pharmacological chaperones therapy is a promising approach to cure genetic diseases. It relies on substrate competitors used at sub-inhibitory concentration which can be administered orally, reach difficult tissues and have low cost. Clinical trials are currently carried out for Fabry disease, a lysosomal storage disorder caused by inherited genetic mutations of alpha-galactosidase. Regrettably, not all genotypes respond to these drugs.</p> <p>Results</p> <p>We collected the experimental data available in literature on the enzymatic activity of ninety-six missense mutants of lysosomal alpha-galactosidase measured in the presence of pharmacological chaperones. We associated with each mutation seven features derived from the analysis of 3D-structure of the enzyme, two features associated with their thermo-dynamic stability and four features derived from sequence alone. Structural and thermodynamic analysis explains why some mutants of human lysosomal alpha-galactosidase cannot be rescued by pharmacological chaperones: approximately forty per cent of the non responsive cases examined can be correctly associated with a negative prognostic feature. They include mutations occurring in the active site pocket, mutations preventing disulphide bridge formation and severely destabilising mutations. Despite this finding, prediction of mutations responsive to pharmacological chaperones cannot be achieved with high accuracy relying on combinations of structure- and thermodynamic-derived features even with the aid of classical and state of the art statistical learning methods.</p> <p>We developed a procedure to predict responsive mutations with an accuracy as high as 87%: the method scores the mutations by using a suitable position-specific substitution matrix. Our approach is of general applicability since it does not require the knowledge of 3D-structure but relies only on the sequence.</p> <p>Conclusions</p> <p>Responsiveness to pharmacological chaperones depends on the structural/functional features of the disease-associated protein, whose complex interplay is best reflected on sequence conservation by evolutionary pressure. We propose a predictive method which can be applied to screen novel mutations of alpha galactosidase. The same approach can be extended on a genomic scale to find candidates for therapy with pharmacological chaperones among proteins with unknown tertiary structures.</p

Axillary Ectopic Carcinoma of the Breast. Report of Two Cases with Different Clinical Presentation and Review of the Literature.

Aims: Primary ectopic breast cancer (PEBC) is a rare and often misdiagnosed condition. Through the discussion of two clinical cases, we want to focus on clinical presentation, outcomes and treatment of PEBC, to lead clinicians to awareness and optimal management. Methods: We present the case of a 47-year-old patient, with a 30 mm axillary mass, that was diagnosed as a PEBC (infiltrating lobular carcinoma, triple negative). The patient underwent systemic staging: diffuse metastatic bone lesions and leptomeningeal metastasis were found. The second patient is a 73-year-old woman with personal history of right breast tumor. She came to our attention for a 9 mm left axillary mass, suspicious for a metastatic lymph node. A fine-needle cytology revealed the absence of lymphoid cells but the presence of atypical epithelial cells, as in a primary breast carcinoma. She was treated with local excision and sentinel node biopsy. Results: The first patient presented with metastatic disease at the time of diagnosis and she deceased after three months from the diagnosis, despite systemic chemotherapy. The diagnosis was performed at an early stage in the second patient. She underwent surgery, complementary endocrine therapy and radiotherapy. She has no evident disease after two years from surgery. Conclusion: Primary ectopic breast cancer is a rare clinical entity, often misdiagnosed or diagnosed with a long delay. The treatment of PEBC is analogous to that of orthotopic breast cancer, but we strongly recommend to approach the patient with a multidisciplinary team to provide the best staging workout and therapie

Biological and chemo-diverse characterization of Amazonian (Ecuador) Citrus petitgrains

Six Amazonian petitgrains samples from C. nobilis Lour., C. aurantium L., C. limon L. and mixture of Citrus spp.(Rutaceae), named CN, CA, CL1, CL2, C1 and C2, were chemically characterized by GC-MS and 13C NMR and evaluated for antioxidant acitivity (DPPH and b-carotene bleaching tests), for antimicrobial properties (disk diffusion method) and for antifungal capacity (agar vapour assay). CN, C1, C2 samples evidenced the most interesting results: CN (g-terpinene/linalool chemotype: 14.3%/41.6%, with a considerable amount of thymol: 9.0%), and C1 (linalool, 18.3%; sabinene, 11.6%; thymol, 5.5%), showed relevant antioxidant activity with both DPPH (IC50=3.52 and 5.48 mg/ml, respectively) and b-carotene (IC50=0.387and 0.491 mg/ml, respectively). Antibacterial properties of CN and C1 against P. mirabilis (MIC=0.61 mg/ml for both)and B. subtilis (MIC=0.61 and 0.44 mg/ml, respectively) were most probably due to thymol.C2 (geranial: 34.7%, neral: 33.1%) evidenced a valuable bioactivity against Candida albicans (MIC=0.44 mg/ml).The 50% growth inhibition (IC50) of the dermatophytes T. mentagrophytes and N. cajetani was reached with amounts ofC1, C2 and CN less than 4 ml/plate. Bioactivity of Amazonian Citrus spp. CN, C1 and C2 essential oils suggests their potential use as food preservatives or additives in cosmeceuticals as preventive against dermatophytic fungal infections

Il progetto Lab2Go per la diffusione della pratica laboratoriale nelle Scuole Secondarie di II grado

Even if laboratory practice is essential for all scientific branches of knowledge, it is often neglected at High School, due to lack of time and/or resources. To establish a closer contact between school and experimental sciences, Sapienza Università di Roma and the Istituto Nazionale di Fisica Nucleare (INFN) launched the Lab2Go project, with the goal of spreading laboratory practice among students and teachers in high schools

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

Infected pancreatic necrosis: outcomes and clinical predictors of mortality. A post hoc analysis of the MANCTRA-1 international study

: The identification of high-risk patients in the early stages of infected pancreatic necrosis (IPN) is critical, because it could help the clinicians to adopt more effective management strategies. We conducted a post hoc analysis of the MANCTRA-1 international study to assess the association between clinical risk factors and mortality among adult patients with IPN. Univariable and multivariable logistic regression models were used to identify prognostic factors of mortality. We identified 247 consecutive patients with IPN hospitalised between January 2019 and December 2020. History of uncontrolled arterial hypertension (p = 0.032; 95% CI 1.135-15.882; aOR 4.245), qSOFA (p = 0.005; 95% CI 1.359-5.879; aOR 2.828), renal failure (p = 0.022; 95% CI 1.138-5.442; aOR 2.489), and haemodynamic failure (p = 0.018; 95% CI 1.184-5.978; aOR 2.661), were identified as independent predictors of mortality in IPN patients. Cholangitis (p = 0.003; 95% CI 1.598-9.930; aOR 3.983), abdominal compartment syndrome (p = 0.032; 95% CI 1.090-6.967; aOR 2.735), and gastrointestinal/intra-abdominal bleeding (p = 0.009; 95% CI 1.286-5.712; aOR 2.710) were independently associated with the risk of mortality. Upfront open surgical necrosectomy was strongly associated with the risk of mortality (p &lt; 0.001; 95% CI 1.912-7.442; aOR 3.772), whereas endoscopic drainage of pancreatic necrosis (p = 0.018; 95% CI 0.138-0.834; aOR 0.339) and enteral nutrition (p = 0.003; 95% CI 0.143-0.716; aOR 0.320) were found as protective factors. Organ failure, acute cholangitis, and upfront open surgical necrosectomy were the most significant predictors of mortality. Our study confirmed that, even in a subgroup of particularly ill patients such as those with IPN, upfront open surgery should be avoided as much as possible. Study protocol registered in ClinicalTrials.Gov (I.D. Number NCT04747990)

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries