A mutation map for human glycoside hydrolase genes.

Glycoside hydrolases (GHs) are found in all domains of life, and at least 87 distinct genes encoding proteins related to GHs are found in the human genome. GHs serve diverse functions from digestion of dietary polysaccharides to breakdown of intracellular oligosaccharides, glycoproteins, proteoglycans and glycolipids. Congenital disorders of GHs (CDGHs) represent more than 30 rare diseases caused by mutations in one of the GH genes. We previously used whole-exome sequencing of a homogenous Danish population of almost 2000 individuals to probe the incidence of deleterious mutations in the human glycosyltransferases (GTs) and developed a mutation map of human GT genes (GlyMAP-I). While deleterious disease-causing mutations in the GT genes were very rare, and in many cases lethal, we predicted deleterious mutations in GH genes to be less rare and less severe given the higher incidence of CDGHs reported worldwide. To probe the incidence of GH mutations, we constructed a mutation map of human GH-related genes (GlyMAP-II) using the Danish WES data, and correlating this with reported disease-causing mutations confirmed the higher prevalence of disease-causing mutations in several GH genes compared to GT genes. We identified 76 novel nonsynonymous single-nucleotide variations (nsSNVs) in 32 GH genes that have not been associated with a CDGH phenotype, and we experimentally validated two novel potentially damaging nsSNVs in the congenital sucrase-isomaltase deficiency gene, SI. Our study provides a global view of human GH genes and disease-causing mutations and serves as a discovery tool for novel damaging nsSNVs in CDGHs.

S. aureus endocarditis: Clinical aspects and experimental approaches.

Infective endocarditis (IE) is a life-threatening disease, caused by septic vegetations and inflammatory foci on the surface of the endothelium and the valves. Due to its complex and often indecisive presentation the mortality rate is still about 30%. Most frequently bacterial microorganisms entering the bloodstream are the underlying origin of the intracardiac infection. While the disease was primarily restricted to younger patients suffering from rheumatic heart streptococci infections, new at risk categories for Staphylococcus (S.) aureus infections arose over the last years. Rising patient age, increasing drug resistance, intensive treatment conditions such as renal hemodialysis, immunosuppression and long term indwelling central venous catheters but also the application of modern cardiac device implants and valve prosthesis have led to emerging incidences of S. aureus IE in health care settings and community. The aetiologic change has impact on the pathophysiology of IE, the clinical presentation and the overall patient management. Despite intensive research on appropriate in vitro and in vivo models of IE and gained knowledge about the fundamental mechanisms in the formation of bacterial vegetations and extracardiac complications, improved understanding of relevant bacterial virulence factors and triggered host immune responses is required to help developing novel antipathogenic treatment strategies and pathogen specific diagnostic markers. In this review, we summarize and discuss the two main areas affected by the changing patient demographics and provide first, recent knowledge about the pathogenic strategies of S. aureus in the induction of IE, including available experimental models of IE used to study host-pathogen interactions and diagnostic and therapeutic targets. In a second focus we present diagnostic (imaging) regimens for patients with S. aureus IE according to current guidelines as well as treatment strategies and surgical recommendations.

Impact of perioperative liver dysfunction on in-hospital mortality and long-term survival in infective endocarditis patients.

PURPOSE: Infective endocarditis (IE) is often associated with multiorgan dysfunction and mortality. The impact of perioperative liver dysfunction (LD) on outcome remains unclear and little is known about factors leading to postoperative LD. METHODS: We performed a retrospective, single-center analysis on 285 patients with left-sided IE without pre-existing chronic liver disease referred to our center between 2007 and 2013 for valve surgery. Sequential organ failure assessment (SOFA) score was used to evaluate organ dysfunction. Chi-square, Cox regression, and multivariate analyses were used for evaluation. RESULTS: Preoperative LD (Bilirubin >20 µmol/L) was present in 68 of 285 patients. New, postoperative LD occurred in 54 patients. Hypoxic hepatitis presented the most common origin of LD, accompanied with high short-term mortality. In-hospital mortality was higher in patients with preoperative and postoperative LD compared to patients without LD (51.5, 24.1, and 10.4%, respectively, p < 0.001). 5-year survival was worse in patients with pre- or postoperative LD compared to patients without LD (20.1, 37.1, and 57.0% respectively). A landmark analysis revealed similar 5-year survival between groups after patient discharge. Quality of life was similar between groups when patients survived the perioperative period. Logistic regression analysis identified duration of cardiopulmonary bypass and S. aureus infection as independent predictors of postoperative LD. CONCLUSIONS: Perioperative liver dysfunction in patients with infective endocarditis is an independent predictor of short- and long-term mortalities. After surviving the hospital stay, 5-year prognosis is not different and quality of life is not affected by LD. S. aureus and duration of cardiopulmonary bypass represent risk factors for postoperative LD.

Metabolomic profiling in patients undergoing Off-Pump or On-Pump coronary artery bypass surgery.

BACKGROUND: Coronary artery bypass surgery can be performed without (Off-Pump) or with cardiopulmonary bypass (On-Pump). Extracorporeal circulation and cardioplegic arrest may cause alterations in the plasma metabolome. We assessed metabolomic changes in patients undergoing On-Pump or Off-Pump coronary artery bypass surgery. METHODS: We assessed five analyte classes (41 acylcarnitines, 14 amino acids, 92 glycerophospholipids, 15 sphingolipids, sugars, lactate) using a mass-spectrometry-based kit (Biocrates AbsoluteIDQ® p150) in paired arterial and coronary sinus blood obtained from 10 consecutive On-Pump and 10 Off-Pump patients. Cardioplegia for On-Pump was warm blood Calafiore. On-Pump outcomes were corrected for hemodilution through crystalloid priming. RESULTS: Demographic data were equal in both groups with normal ejection fraction, renal and liver function. Patients received 2.25 ± 0.64 bypass grafts. All postoperative courses were uneventful. Of 164 measured metabolites, only 13 (7.9%) were altered by cardiopulmonary bypass. We found more long-chain acylcarnitines Off-Pump and more short-chain acylcarnitines On-Pump. Glycerophospholipids showed lower concentrations On-Pump and arginine (as the only different amino acid) Off-Pump. Interestingly, plasma arginine (nitric oxide precursor) concentration at the end of surgery correlated inversely with postoperative vasopressor need (r = -0.7; p < 0.001). Assessing arterial/venous differences revealed phosphatidylcholine-production and acylcarnitine-consumption. These findings were unaffected by cardiopulmonary bypass, cardioplegia or temporary vessel occlusion during Off-Pump surgery. CONCLUSIONS: Cardiopulmonary bypass and warm blood cardioplegia cause only minor changes to the metabolomic profile of patients undergoing coronary artery bypass surgery. The observed changes affected mainly acylcarnitines. In addition, there appears to be a relationship between arginine and vasopressor need after bypass surgery.

Fasciola gigantica fatty acid binding protein (FABP) as a prophylactic agent against Schistosoma mansoni infection in CD1 mice.

Although schistosomicidal drugs and other control measures exist, the advent of an efficacious vaccine remains the most potentially powerful means for controlling this disease. In this study, native fatty acid binding protein (FABP) from Fasciola gigantica was purified from the adult worm's crude extract by saturation with ammonium sulphate followed by separation on DEAE-Sephadex A-50 anion exchange chromatography and gel filtration using Sephacryl HR-100, respectively. CD1 mice were immunized with the purified, native F. gigantica FABP in Freund's adjuvant and challenged subcutaneously with 120 Schistosoma mansoni cercariae. Immunization of CD1 mice with F. gigantica FABP has induced heterologous protection against S. mansoni, evidenced by the significant reduction in mean worm burden (72.3%), liver and intestinal egg counts (81.3% and 80.8%, respectively), and hepatic granuloma counts (42%). Also, it elicited mixed IgG(1)/IgG(2b) immune responses with predominant IgG1 isotype, suggesting that native F. gigantica FABP is mediated by a mixed Th1/Th2 response. However, it failed to induce any significant differences in the oogram pattern or in the mean granuloma diameter. This indicated that native F. gigantica FABP could be a promising vaccine candidate against S. mansoni infection.

Investigating clinicians' determinants of antimicrobial prescribing behaviour using the Theoretical Domains Framework.

AIM: To identify and quantify potential determinants of antimicrobial prescribing behaviour, using the Theoretical Domains Framework (TDF). METHODS: A cross-sectional survey involving doctors (∼4000) and pharmacists (∼400) working within Hamad Medical Corporation (HMC), Qatar. An online questionnaire, developed with reference to the TDF, included: personal and practice demographics, and Likert statements on potential determinants of antimicrobial prescribing practice. Analysis included principal component analysis (PCA), descriptive and inferential statistics. RESULTS: In total, 535 responses were received, 339 (63.4%) from doctors. Respondents were predominantly male, 346 (64.7%). Just over half (N = 285, 53.3%) had ≤5 years' experience. PCA showed a three-component (C) solution: 'Guidelines compliance' (C1), 'Influences on practice' (C2) and 'Self-efficacy' (C3). The scales derived for each component had high internal consistency (Cronbach's alphas >0.7), indicating statistical appropriateness for developing scales. Respondents generally scored highly for 'Guidelines compliance' and 'Self-efficacy'. The lowest levels of positive scores were in relation to the items within the 'Influences on practice' component, with particular focus on TDF domains, environmental context and resources, and social influences. Inferential analysis comparing component scores across demographic characteristics showed that doctors, the more qualified and those with greater experience, were more likely to be positive in responses. CONCLUSIONS: This study has identified that environmental context and resources, and social influences, with an emphasis on pharmacists and early career clinicians, may be useful targets for behaviour change interventions to improve clinicians' antimicrobial prescribing, thereby reducing antimicrobial resistance rates. Such interventions should focus on appropriate linked behaviour-change techniques.

Severe pathogenic variants of intestinal sucrase-isomaltase interact avidly with the wild type enzyme and negatively impact its function and trafficking.

Sucrase-isomaltase (SI) is the major disaccharidase of the small intestine, exhibiting a broad α-glucosidase activity profile. The importance of SI in gut health is typified by the development of sucrose and starch maldigestion in individuals carrying mutations in the SI gene, like in congenital sucrase-isomaltase deficiency (CSID). Common and rare defective SI gene variants (SIGVs) have also been shown to increase the risk of irritable bowel syndrome (IBS) with symptoms and clinical features similar to CSID and also in symptomatic heterozygote carriers. Here, we investigate the impact of the most abundant and highly pathogenic SIGVs that occur in heterozygotes on wild type SI (SIWT) by adapting an in vitro system that recapitulates SI gene heterozygosity. Our results demonstrate that pathogenic SI mutants interact avidly with SIWT, negatively impact its enzymatic function, alter the biosynthetic pattern and impair the trafficking behavior of the heterodimer. The in vitro recapitulation of a heterozygous state demonstrates potential for SIGVs to act in a semi-dominant fashion, by further reducing disaccharidase activity via sequestration of the SIWT copy into an inactive form of the enzymatic heterodimer. This study provides novel insights into the potential role of heterozygosity in the pathophysiology of CSID and IBS.

Differential Effects of Sucrase-Isomaltase Mutants on Its Trafficking and Function in Irritable Bowel Syndrome: Similarities to Congenital Sucrase-Isomaltase Deficiency.

Congenital sucrase-isomaltase deficiency (CSID) is a rare metabolic intestinal disorder with reduced or absent activity levels of sucrase-isomaltase (SI). Interestingly, the main symptoms of CSID overlap with those in irritable bowel syndrome (IBS), a common functional gastrointestinal disorder with unknown etiology. Recent advances in genetic screening of IBS patients have revealed rare SI gene variants that are associated with IBS. Here, we investigated the biochemical, cellular and functional phenotypes of several of these variants. The data demonstrate that the SI mutants can be categorized into three groups including immature, mature but slowly transported, and finally mature and properly transported but with reduced enzymatic activity. We also identified SI mutant phenotypes that are deficient but generally not as severe as those characterized in CSID patients. The variable effects on the trafficking and function of the mutations analyzed in this study support the view that both CSID and IBS are heterogeneous disorders, the severity of which is likely related to the biochemical phenotypes of the SI mutants as well as the environment and diet of patients. Our study underlines the necessity to screen for SI mutations in IBS patients and to consider enzyme replacement therapy as an appropriate therapy as in CSID.

Injection safety at primary health care level in south-western Saudi Arabia.

In a study of injection safety in Abha health district, Saudi Arabia, data were collected from 47 physicians and 85 nurses at 24 primary health care centres, using an observation checklist and an interview questionnaire. All centres used individually packed disposable syringes and puncture-proof containers to collect used needles. Needlestick injury in the previous year was reported by 14.9% of physicians and 16.5% of nurses (0.21 and 0.38 injuries/person/year respectively). Logistic regression analysis identified recapping the needle after use (physicians and nurses) and bending the needle before disposal (physicians) as significant risk factors for injury.

Congenital Lactase Deficiency: Mutations, Functional and Biochemical Implications, and Future Perspectives.

Congenital lactase deficiency (CLD) is a severe autosomal recessive genetic disorder that affects the functional capacity of the intestinal protein lactase-phlorizin hydrolase (LPH). This disorder is diagnosed already during the first few days of the newborn's life due to the inability to digest lactose, the main carbohydrate in mammalian milk. The symptoms are similar to those in other carbohydrate malabsorption disorders, such as congenital sucrase-isomaltase deficiency, and include severe osmotic watery diarrhea. CLD is associated with mutations in the translated region of the LPH gene that elicit loss-of-function of LPH. The mutations occur in a homozygote or compound heterozygote pattern of inheritance and comprise missense mutations as well as mutations that lead to complete or partial truncations of crucial domains in LPH, such as those linked to the folding and transport-competence of LPH and to the catalytic domains. Nevertheless, the identification of the mutations in CLD is not paralleled by detailed genotype/protein phenotype analyses that would help unravel potential pathomechanisms underlying this severe disease. Here, we review the current knowledge of CLD mutations and discuss their potential impact on the structural and biosynthetic features of LPH. We also address the question of whether heterozygote carriers can be symptomatic for CLD and whether genetic testing is needed in view of the severity of the disease.

Isolation and Quantification of Sphingosine and Sphinganine from Rat Serum Revealed Gender Differences.

Sphingolipids are an important group of lipids that play crucial roles in living cells, facilitating cell recognition, signal transduction and endocytosis. The concentration of sphingosine and some of its derivatives like sphinganine may serve as a biomarker for the diagnosis of sphingolipidoses or be used for further research into similar diseases. In this study, a sphingolipid extraction and a high resolution detection method specific for sphingosine and sphinganine was adapted and tested. Lipids were extracted from rats' serum, coupled to o-phthalaldehyde and detected with a fluorescence detector after running through a silica gel column in a high performance liquid chromatography system. With this method, we analysed 20 male and 20 female rat serum samples and compared the concentrations of sphingosine and sphinganine. The results showed a significant difference between the sphingosine concentrations in the male and female rats. The sphingosine concentration in female rats was 805 ng/mL (standard deviation, SD ± 549), while that in males was significantly lower at (75 ng/mL (SD ± 40)). Furthermore, the sphingosine:sphinganine ratio was almost 15-fold higher in the females' samples. The method presented here facilitates the accurate quantification of sphingosine and sphinganine concentrations down to 2.6 ng and 3.0 ng, respectively, and their ratio in small amounts of rat serum samples to study the sphingolipid metabolism and its potential modulation due to gene mutations or the effect of prevalent toxins.

Heterozygotes Are a Potential New Entity among Homozygotes and Compound Heterozygotes in Congenital Sucrase-Isomaltase Deficiency.

Congenital sucrase-isomaltase deficiency (CSID) is an autosomal recessive disorder of carbohydrate maldigestion and malabsorption caused by mutations in the sucrase-isomaltase (SI) gene. SI, together with maltase-glucoamylase (MGAM), belongs to the enzyme family of disaccharidases required for breakdown of -glycosidic linkages in the small intestine. The effects of homozygote and compound heterozygote inheritance trait of SI mutations in CSID patients have been well described in former studies. Here we propose the inclusion of heterozygote mutation carriers as a new entity in CSID, possibly presenting with milder symptoms. The hypothesis is supported by recent observations of heterozygote mutation carriers among patients suffering from CSID or patients diagnosed with functional gastrointestinal disorders. Recent studies implicate significant phenotypic heterogeneity depending on the character of the mutation and call for more research regarding the correlation of genetics, function at the cellular and molecular level and clinical presentation. The increased importance of SI gene variants in irritable bowel syndrome (IBS) or other functional gastrointestinal disorders FGIDs and their available symptom relief diets like fermentable oligo-, di-, mono-saccharides and polyols FODMAPs suggest that the heterozygote mutants may affect the disease development and treatment.

Polymer complexes. LXXV. Characterization of quinoline polymer complexes as potential bio-active and anti-corrosion agents.

The Cu2+, Co2+, Ni2+ and UO22+ polymer complexes of 5-(2,3-dimethyl-1-phenylpyrazol-5-one azo)-8-hydroxyquinoline (HL) ligand were prepared and characterized. Elemental analyses, IR spectra, X-ray diffraction analysis and thermal analysis studies have been used to confirm the structure of the prepared polymer complexes. The chemical structure of metal chelates commensurate that the ligand acts as a neutral bis(bidentate) by through four sites of coordination (azo dye nitrogen, carbonyl oxygen, phenolic oxygen and hetero nitrogen from pyridine ring). The molecular and electronic structures of the hydrogen bond conformers of HL ligand were optimized theoretically and the quantum chemical parameters were calculated. Elemental analysis data suggested that the polymer complexes have composition of octahedral geometry for all the polymer complexes. Molecular docking of the binding between HL and the receptors of prostate cancer (PDB code 2Q7L Hormone) and the breast cancer (PDB code 1JNX Gene regulation) was studied. The interaction between HL and its polymer complexes with the calf thymus DNA (CT-DNA) was determined by absorption spectra. The antimicrobial activity of HL and its Cu2+, Co2+, Ni2+ and UO22+ polymer complexes were investigated; only Cu(II) polymer complex (1) was specifically active against Aspergillus niger. It inhibited the fungal sporulation and distorted the fungal mycelia, which became squashed at a concentration of 150 µg/ml; transmission electron microscope (TEM) also showed a deactivation of autophagy in the treated A. niger cells via accumulation of autophagic bodies in vacuoles. The inhibition process of the prepared ligand (HL) against the corrosion of carbon steel in 2 M HCl solution was determined by various methods (weight loss, potentiodynamic polarization, electrochemical impedance spectroscopy (EIS) and electrochemical frequency modulation (EFM) techniques) are found to be in reasonable agreement. The mechanism of inhibition in presence of HL in carbon steel corrosion obeys Friendlish adsorption isotherm.

Prevalence of celiac disease in Egyptian children disputes the east-west agriculture-dependent spread of the disease.

OBJECTIVES: Celiac disease (CD) seems to be a common disorder in north Africa; however, to our knowledge no data are yet available on its prevalence in Egypt. This study was undertaken to investigate the frequency of CD in Egyptian children. PATIENTS AND METHODS: We investigated a sample of the general pediatric population (1500 individuals, 656 girls and 844 boys, age range 7 months to 18 years, median age 8.0 years) (group A); 150 children (age range 6 months to 13 years, median age 16 months) admitted for diarrhea or failure to thrive (group B); and 250 children and adolescents with type 1 diabetes (group C). The screening test was serum class A anti-transglutaminase (anti-tTG) antibody; immunoglobulin A (IgA) antiendomysium, total IgA, and IgG anti-tTG, and small bowel biopsy was performed for confirmation of diagnosis. RESULTS: In group A, 8 of 1500 children fulfilled the criteria for CD diagnosis; the prevalence of CD was at least 1 in 187 individuals (0.53%; 95% CI 0.17%-0.89%). In group B, 7 of 150 children had CD (4.7%, 95% CI 1.4-7.9). In group C, 16 of 250 sera showed positive results to both the IgA anti-tTG and the IgA antiendomysium test (6.4%; 95% CI 3.4-9.4). CONCLUSIONS: Celiac disease is a frequent disorder among Egyptian children, both in the general population and in at-risk groups. Therefore, our data do not support the theory of a Middle East-Europe CD prevalence gradient secondary to the pattern of agriculture spreading from the so-called Fertile Crescent.

Primary health care emergency services in Abha district of southwestern Saudi Arabia.

To study emergency services delivery in all 30 primary health care centres in Abha district of Asir region, Saudi Arabia, data were collected about equipment and facilities, physicians' practices and attitudes, and patients' utilization of and satisfaction with emergency services. Two centres had no devoted place for emergency services. Lack of some essential equipment and drugs was evident. The greatest continuing medical education need for doctors was the management of cardiovascular emergencies (72.3%). Many doctors (40.4%) did not consider the majority of cases as true emergencies. Many patients (43.7%) used the centres for emergency services, the most common being trauma, burns and orthopaedics (47.8%). Most patients were satisfied overall with emergency services (82.2%).

Operative treatment of calcaneal fractures: improved outcomes and low complications rates with a strict management protocol.

This is a retrospective review of 80 intra-articular calcaneal fractures treated with open reduction and internal fixation by a specialist team under supervision of a single surgeon in a tertiary centre between 2005 and 2014. The fractures were evaluated with plain radiography and computed tomography, and graded using the Eastwood-Atkins classification. A lateral approach was used and all fractures were fixed with calcaneal plates. All patients had clinical and radiological follow-up. Clinical assessment included foot and ankle disability index, SF-36® and Kerr-Atkins scores. The mean follow-up duration was 72 months (range: 12-130 months). The mean age of patients was 49 years (range: 17-73 years). There were three open fractures and eight patients had other injuries. The mean Bohler's angle improved from 6° preoperatively to 26° postoperatively. The mean foot and ankle disability index score was 78.62, the mean SF-36® scores were 45.5 (physical component) and 52.6 (mental component), and the mean Kerr-Atkins score was 72 (range: 36-100). Early complications included one case of screw protrusion in the subtalar joint (which warranted a repeat procedure), one sural nerve injury and one wound breakdown, which healed with non-operative measures. Twelve patients had symptomatic subtalar joint osteoarthritis. Four of these had subtalar fusion. We believe that our strict protocols of patient selection, intraoperative and postoperative management produced long-term results comparable with those in the peer reviewed literature.