Probing the druggability of the Notch1 ankyrin domain using a fragment-based approach

Notch signalling is a highly conserved pathway that is important in the developmental processes that control cell differentiation and cell fates. This canonical pathway involves binding of a transmembrane ligand in one cell to the extraceullular domain of a transmembrane Notch receptor in an adjacent cell. Ligand binding triggers two sequential proteolytic cleavages that shed a Notch intracellular domain (NICD). This is followed by translocation of NICD to the nucleus where it interacts with a transcription factor CSL and forms an activated Notch transcription complex, which induces the transcription of Notch target genes. Abnormal expression or mutations in the different components of the pathway are associated with a number of diseases and cancers. An enhanced activity of Notch signalling resulting from a mutation in the extracellular domain is implicated in the progression of T-acute lymphoblastic leukaemia (T-ALL). Several therapeutic agents have been developed to target the Notch signalling pathway such as, γ-secretase inhibitors, antibodies targeting different regions of the Notch receptor and recently a synthetic stapled peptide, which was found to inhibit the formation of the transcription complex. The current inhibitors have their own disadvantages including lack of selectivity, cost of goods and delivery to the target. Thus, a more selective approach to target downstream proteinprotein interactions by small molecules would provide an attractive approach to the design of new therapeutic agents that target this pathway. Here I report a fragment-based approach to target the ankyrin domain, a historically known but challenging, often-considered “undruggable” target. In this dissertation I describe the application of various biophysical and computational approaches to find, characterise and design compounds. The initial screening of a commercial fragment-library exploited a fluorescent-based thermal shift assay that identified 36 fragment hits. Some of the fragments were kinetically characterised by Surface Plasmon Resonance (SPR) and their affinities were found to be in the millimolar range. Several attempts at soaking vii and co-crystallising the fragments in the ankyrin domain crystal resulted in only two successful crystal structures that clearly define the positions of the fragments and their interactions with the ankyrin domain. One fragment binds to a pre-defined hotspot residue at the interface between the ankyrin domain and CSL. The other fragment is located at the interface between the ankyrin domain and Mastermind (MAML). The structural and kinetic data assisted the design of larger compounds with more extensive interactions using drug design software such as SPROUT and a docking program (GOLD). However, the optimised fragments did not show much improvement in affinity underlying the difficulty of flat protein-protein interface. The results reported here show the first structures of small molecules binding to the ankyrin domain of Notch1 receptor

Effects of omega-3 fatty acids and pioglitazone combination on insulin resistance through fibroblast growth factor 21 in type 2 diabetes mellitus

AbstractFibroblast growth factor 21 (FGF21) is an effective regulator of glucose and lipid metabolism. It is mainly regulated by peroxisome proliferator activated receptors, and is widely associated with cases of insulin resistance as obesity and type 2 diabetes (T2D). Our study aimed to investigate the potential effects of omega-3 fatty acids, pioglitazone, and their combination on serum and liver FGF21 concentrations, and its hepatic gene expression in a rat model of T2D. We also studied the modulating effects of these treatments on blood glucose, lipid profile, and insulin resistance. T2D was induced in male Sprague–Dawley rats by combination of high fat diet and low dose streptozotocin (35 mg/kg). Diabetic rats were treated with omega-3 fatty acids (10%W/W diet), pioglitazone (20 mg/kg), and their combination for a period of 4 weeks. Serum FGF21 concentration was significantly increased in diabetic rats. In contrast, hepatic FGF21 concentration, and gene expression were significantly decreased. Omega-3 fatty acids, pioglitazone, and their combination significantly decreased serum FGF21. Omega-3 fatty acids and combination therapy significantly decreased liver FGF21 concentration, with non-significant changes in gene expression. On the other hand, pioglitazone significantly increased hepatic FGF21 concentration and gene expression. Omega-3 fatty acids, pioglitazone and their combination significantly improved lipid profile. Pioglitazone and combination significantly decreased blood glucose levels and improved insulin resistance. In conclusion, this study introduces the first evidence regarding the antidiabetic effects of omega-3 fatty acids and pioglitazone combination, such effects are mediated through FGF21

Antimicrobial susceptibility pattern of genital Mycoplasmas among a group of pregnant women

AbstractMycoplasma hominis (MH) and Ureaplasma urealyticum (UU) are important members of genital Mycoplasmas. They are implicated in urogenital infections and complicated pregnancy (chorioamnionitis, preterm delivery, abortion, and preterm birth) as well as bacterial vaginosis and cervicitis. The administration of antimicrobial agents to pregnant women with preterm rupture of the membranes (PROM) may extend the gestation period and decrease the risks of associated complications and neonatal infections. Despite empirical therapy is the rule in cases suspected to have genital infection in Egypt, the surveillance of the susceptibilities of used antibiotics is mandatory to ensure treatment efficacy and good prevention of any possible complications. This study aimed to assess the infection rate of genital Mycoplasmas (MH and UU) among pregnant females and their antimicrobial susceptibility pattern to provide a provisional idea about the effectiveness of antibiotics used empirically to treat cases of genital infections in pregnant women. High vaginal swabs of 50 pregnant females were examined using Mycoplasma IES kit, for identification of UU and MH. The kit also provides the antimicrobial susceptibility results for 12 antimicrobials of five different classes. UU and MH were detected in 26/50 (52%), and 7/50 (14%) of cases respectively, of which 5 cases showed mixed infection with both organisms. UU was most sensitive to quinolones (90–95%), followed by tetracyclines (80–85%). The least sensitivity was detected with chloramphenicol and clindamycin (40% and 30% respectively). The two MH isolates (100%) were sensitive to the three tested quinolones in addition to clindamycin and thiamphenicol. MH showed 100% sensitivity to clindamycin and 75% of UU isolates were sensitive to azithromycin. Further studies are needed to detect any future changes in the susceptibility pattern for these drugs or other antibiotics

COMPARATIVE STUDY BETWEEN PERIOSTIN AND OSTEOCALCIN AS BIOMARKERS FOR OSTEOPOROSIS AND FRACTURE RISK IN EGYPTIAN POSTMENOPAUSAL WOMEN

Objective: This study aimed to compare between periostin and osteocalcin as biomarkers in Egyptian postmenopausal women with osteoporosis and to explore their possible relationship with fracture risk. Methods: This study included 90 postmenopausal females recruited from Al-Hussein University Hospital, Cairo, Egypt; divided into three groups; 35 postmenopausal osteoporotic females with low fracture risk (group I), 35 postmenopausal osteoporotic females with high fracture risk (group II), and 20 apparently healthy controls. Serum periostin, osteocalcin, and estrogen were measured by Enzyme Linked Immunosorbent Assay (ELISA). Fracture risk assessment was calculated. Alkaline phosphatase (ALP), total and ionized calcium, Aspartate transaminase (AST), and Alanine transaminase (ALT) were measured spectrophotometrically. Results: The diagnostic performance of periostin for discriminating high fracture risk from low fracture risk groups showed the specificity of (68.6 %) and sensitivity of (100 %), while for osteocalcin the specificity was (51.4 %) and the sensitivity was (68.6 %) respectively. Moreover, the multi Receiver Operating Characteristics (multi-ROC) curve for periostin and osteocalcin together revealed improved specificity and sensitivity of (100 %) each. Conclusion: Periostin was superior to osteocalcin in discriminating high fracture risk from low fracture risk postmenopausal osteoporotic groups. Moreover, dual use of both markers gave the highest discriminative power between low and high fracture risk groups with 100 % specificity and sensitivity

Hydrophobin Film Structure for HFBI and HFBII and Mechanism for Accelerated Film Formation

Peer reviewe

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

Global burden of 288 causes of death and life expectancy decomposition in 204 countries and territories and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BACKGROUND Regular, detailed reporting on population health by underlying cause of death is fundamental for public health decision making. Cause-specific estimates of mortality and the subsequent effects on life expectancy worldwide are valuable metrics to gauge progress in reducing mortality rates. These estimates are particularly important following large-scale mortality spikes, such as the COVID-19 pandemic. When systematically analysed, mortality rates and life expectancy allow comparisons of the consequences of causes of death globally and over time, providing a nuanced understanding of the effect of these causes on global populations. METHODS The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 cause-of-death analysis estimated mortality and years of life lost (YLLs) from 288 causes of death by age-sex-location-year in 204 countries and territories and 811 subnational locations for each year from 1990 until 2021. The analysis used 56 604 data sources, including data from vital registration and verbal autopsy as well as surveys, censuses, surveillance systems, and cancer registries, among others. As with previous GBD rounds, cause-specific death rates for most causes were estimated using the Cause of Death Ensemble model-a modelling tool developed for GBD to assess the out-of-sample predictive validity of different statistical models and covariate permutations and combine those results to produce cause-specific mortality estimates-with alternative strategies adapted to model causes with insufficient data, substantial changes in reporting over the study period, or unusual epidemiology. YLLs were computed as the product of the number of deaths for each cause-age-sex-location-year and the standard life expectancy at each age. As part of the modelling process, uncertainty intervals (UIs) were generated using the 2·5th and 97·5th percentiles from a 1000-draw distribution for each metric. We decomposed life expectancy by cause of death, location, and year to show cause-specific effects on life expectancy from 1990 to 2021. We also used the coefficient of variation and the fraction of population affected by 90% of deaths to highlight concentrations of mortality. Findings are reported in counts and age-standardised rates. Methodological improvements for cause-of-death estimates in GBD 2021 include the expansion of under-5-years age group to include four new age groups, enhanced methods to account for stochastic variation of sparse data, and the inclusion of COVID-19 and other pandemic-related mortality-which includes excess mortality associated with the pandemic, excluding COVID-19, lower respiratory infections, measles, malaria, and pertussis. For this analysis, 199 new country-years of vital registration cause-of-death data, 5 country-years of surveillance data, 21 country-years of verbal autopsy data, and 94 country-years of other data types were added to those used in previous GBD rounds. FINDINGS The leading causes of age-standardised deaths globally were the same in 2019 as they were in 1990; in descending order, these were, ischaemic heart disease, stroke, chronic obstructive pulmonary disease, and lower respiratory infections. In 2021, however, COVID-19 replaced stroke as the second-leading age-standardised cause of death, with 94·0 deaths (95% UI 89·2-100·0) per 100 000 population. The COVID-19 pandemic shifted the rankings of the leading five causes, lowering stroke to the third-leading and chronic obstructive pulmonary disease to the fourth-leading position. In 2021, the highest age-standardised death rates from COVID-19 occurred in sub-Saharan Africa (271·0 deaths [250·1-290·7] per 100 000 population) and Latin America and the Caribbean (195·4 deaths [182·1-211·4] per 100 000 population). The lowest age-standardised death rates from COVID-19 were in the high-income super-region (48·1 deaths [47·4-48·8] per 100 000 population) and southeast Asia, east Asia, and Oceania (23·2 deaths [16·3-37·2] per 100 000 population). Globally, life expectancy steadily improved between 1990 and 2019 for 18 of the 22 investigated causes. Decomposition of global and regional life expectancy showed the positive effect that reductions in deaths from enteric infections, lower respiratory infections, stroke, and neonatal deaths, among others have contributed to improved survival over the study period. However, a net reduction of 1·6 years occurred in global life expectancy between 2019 and 2021, primarily due to increased death rates from COVID-19 and other pandemic-related mortality. Life expectancy was highly variable between super-regions over the study period, with southeast Asia, east Asia, and Oceania gaining 8·3 years (6·7-9·9) overall, while having the smallest reduction in life expectancy due to COVID-19 (0·4 years). The largest reduction in life expectancy due to COVID-19 occurred in Latin America and the Caribbean (3·6 years). Additionally, 53 of the 288 causes of death were highly concentrated in locations with less than 50% of the global population as of 2021, and these causes of death became progressively more concentrated since 1990, when only 44 causes showed this pattern. The concentration phenomenon is discussed heuristically with respect to enteric and lower respiratory infections, malaria, HIV/AIDS, neonatal disorders, tuberculosis, and measles. INTERPRETATION Long-standing gains in life expectancy and reductions in many of the leading causes of death have been disrupted by the COVID-19 pandemic, the adverse effects of which were spread unevenly among populations. Despite the pandemic, there has been continued progress in combatting several notable causes of death, leading to improved global life expectancy over the study period. Each of the seven GBD super-regions showed an overall improvement from 1990 and 2021, obscuring the negative effect in the years of the pandemic. Additionally, our findings regarding regional variation in causes of death driving increases in life expectancy hold clear policy utility. Analyses of shifting mortality trends reveal that several causes, once widespread globally, are now increasingly concentrated geographically. These changes in mortality concentration, alongside further investigation of changing risks, interventions, and relevant policy, present an important opportunity to deepen our understanding of mortality-reduction strategies. Examining patterns in mortality concentration might reveal areas where successful public health interventions have been implemented. Translating these successes to locations where certain causes of death remain entrenched can inform policies that work to improve life expectancy for people everywhere. FUNDING Bill & Melinda Gates Foundation

Brainstem Adenosine A1 Receptor Signaling Masks Phosphorylated Extracellular Signal-Regulated Kinase 1/2-Dependent Hypotensive Action of Clonidine in Conscious Normotensive Rats

Central adenosine A1 and A2A receptors mediate pressor and depressor responses, respectively. The adenosine subtype A2A receptor (A2AR)-evoked enhancement of phosphorylated extracellular signal-regulated kinase (pERK) 1/2 production in the rostral ventrolateral medulla (RVLM), a major neuroanatomical target for clonidine, contributes to clonidine-evoked hypotension, which is evident in conscious aortic barodenervated (ABD) but not in conscious sham-operated (SO) normotensive rats. We conducted pharmacological and cellular studies to test the hypothesis that the adenosine A2AR-mediated (pERK1/2-dependent) hypotensive action of clonidine is not expressed in SO rats because it is counterbalanced by fully functional central adenosine subtype A1 receptor (A1R) signaling. We first demonstrated an inverse relationship between A1R expression in RVLM and clonidine-evoked hypotension in ABD and SO rats. The functional (pharmacological) relevance of the reduced expression of RVLM A1R in ABD rats was verified by the smaller dose-dependent pressor responses elicited by the selective A1R agonist N6-cyclopentyladenosine in ABD versus SO rats. It is important that after selective blockade of central A1R with 8-cyclopentyl-1,3-dipropylxanthine in conscious SO rats, clonidine lowered blood pressure and significantly increased neuronal pERK1/2 in the RVLM. In contrast, central A1R blockade had no influence on the hypotensive response or the increase in RVLM pERK1/2 elicited by clonidine in ABD rats. These findings support the hypothesis that central adenosine A1R signaling opposes the adenosine A2AR-mediated (pERK1/2-dependent) hypotensive response and yield insight into a cellular mechanism that explains the absence of clonidine-evoked hypotension in conscious normotensive rats