Comparative Proteomic Profiling of Methicillin-Susceptible and Resistant Staphylococcus aureus.

Staphylococcus aureus is a highly successful human pathogen responsible for a wide range of infections. This study provides insights into the virulence, pathogenicity, and antimicrobial resistance determinants of methicillin-susceptible and methicillin-resistant S. aureus (MSSA; MRSA) recovered from non-healthcare environments. Three environmental MSSA and three environmental MRSA are selected for proteomic profiling using isobaric tag for relative and absolute quantitation tandem mass spectrometry (iTRAQ MS/MS). Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway annotation are applied to interpret the functions of the proteins detected. 792 proteins are identified in MSSA and MRSA. Comparative analysis of MRSA and MSSA reveals that 8 of out 792 proteins are upregulated and 156 are downregulated. Proteins that have differences in abundance are predominantly involved in catalytic and binding activity. Among 164 differently abundant proteins, 29 are involved in pathogenesis, antimicrobial resistance, stress response, mismatch repair, and cell wall synthesis. Twenty-two proteins associated with pathogenicity including SPA, SBI, CLFA, and DLT are upregulated in MRSA. Moreover, the upregulated pathogenic protein ENTC2 in MSSA is determined to be a super antigen, potentially capable of triggering toxic shock syndrome in the host. Enhanced pathogenicity, antimicrobial resistance, and stress response are observed in MRSA compared to MSSA.

Effect of berberine on in vitro metabolism of sulfonylureas: A herb-drug interactions study.

Patients with type 2 diabetes may co-ingest herbal and prescription medicines to control their blood sugar levels. Competitive binding of drug and herb may mutually affect their metabolism. This can alter the level of drug and its kinetics in the body, potentially causing toxicities or loss of efficacy. Understanding how the metabolism of sulfonylureas like glyburide and gliclazide can be affected by the presence of berberine and vice versa can provide valuable information on the possible risk of toxicities caused by co-ingestion of drugs. METHODS: Berberine and sulfonylureas (glyburide and gliclazide) were co-incubated with rat liver microsomes in the presence of a NADPH-regenerating system. The metabolites of berberine and sulfonylureas were analysed using liquid chromatography with high-resolution mass spectrometry in the positive ion mode. The role of individual isozymes in the metabolism of berberine, glyburide and gliclazide was investigated by using specific inhibitors. RESULTS: In vitro metabolism of berberine led to the formation of demethyleneberberine (B1a) and its isomer B1b through demethylenation. Berberrubine (B2a) and its isomer B2b were formed through demethylation. The isozymes CYP3A and CYP2D were found to be involved in the metabolism of berberine. In vitro metabolism of glyburide and gliclazide led to the formation of hydroxylated metabolites. The isozymes CYP3A and CYP2C were found to be involved in the metabolism of glyburide. Gliclazide was metabolised by CYP2C. In vitro co-incubation of glyburide or gliclazide with berberine showed that each drug's metabolism was compromised as they share a common isozyme. A strong negative linear correlation of glyburide or gliclazide metabolite levels and the concentration of berberine confirmed the effect of berberine on the metabolism of sulfonylureas. CONCLUSIONS: The metabolism of sulfonylureas and berberine was affected when these compounds were co-incubated with each other. This may be attributable to competitive binding of the herb and drug to the catalytic sites of the same isozymes.

Heme promotes transcriptional and demethylase activities of Gis1, a member of the histone demethylase JMJD2/KDM4 family.

The yeast Gis1 protein is a transcriptional regulator belonging to the JMJD2/KDM4 subfamily of demethylases that contain a JmjC domain, which are highly conserved from yeast to humans. They have important functions in histone methylation, cellular signaling and tumorigenesis. Besides serving as a cofactor in many proteins, heme is known to directly regulate the activities of proteins ranging from transcriptional regulators to potassium channels. Here, we report a novel mechanism governing heme regulation of Gis1 transcriptional and histone demethylase activities. We found that two Gis1 modules, the JmjN + JmjC domain and the zinc finger (ZnF), can bind to heme specifically in vitro. In vivo functional analysis showed that the ZnF, not the JmjN + JmjC domain, promotes heme activation of transcriptional activity. Likewise, measurements of the demethylase activity of purified Gis1 proteins showed that full-length Gis1 and the JmjN + JmjC domain both possess demethylase activity. However, heme potentiates the demethylase activity of full-length Gis1, but not that of the JmjN + JmjC domain, which can confer heme activation of transcriptional activity in an unrelated protein. These results demonstrate that Gis1 represents a novel class of multi-functional heme sensing and signaling proteins, and that heme binding to the ZnF stimulates Gis1 demethylase and transcriptional activities.

BET Proteins Are Required for Transcriptional Activation of the Senescent Islet Cell Secretome in Type 1 Diabetes.

Type 1 diabetes (T1D) results from the progressive loss of pancreatic beta cells as a result of autoimmune destruction. We recently reported that during the natural history of T1D in humans and the female nonobese diabetic (NOD) mouse model, beta cells acquire a senescence-associated secretory phenotype (SASP) that is a major driver of disease onset and progression, but the mechanisms that activate SASP in beta cells were not explored. Here, we show that the SASP in islet cells is transcriptionally controlled by Bromodomain ExtraTerminal (BET) proteins, including Bromodomain containing protein 4 (BRD4). A chromatin analysis of key beta cell SASP genes in NOD islets revealed binding of BRD4 at active regulatory regions. BET protein inhibition in NOD islets diminished not only the transcriptional activation and secretion of SASP factors, but also the non-cell autonomous activity. BET protein inhibition also decreased the extent of SASP induction in human islets exposed to DNA damage. The BET protein inhibitor iBET-762 prevented diabetes in NOD mice and also attenuated SASP in islet cells in vivo. Taken together, our findings support a crucial role for BET proteins in the activation of the SASP transcriptional program in islet cells. These studies suggest avenues for preventing T1D by transcriptional inhibition of SASP.

Analysis of procainamide-derivatised heparan sulphate disaccharides in biological samples using hydrophilic interaction liquid chromatography mass spectrometry.

Glycosaminoglycans (GAGs) are a family of linear heteropolysaccharides made up of repeating disaccharide units that are found on the surface and extracellular matrix of animal cells. They are known to play a critical role in a wide range of cellular processes including proliferation, differentiation and invasion. To elucidate the mechanism of action of these molecules, it is essential to quantify their disaccharide composition. Analytical methods that have been reported involve either chemical or enzymatic depolymerisation of GAGs followed by separation of non-derivatised (native) or derivatised disaccharide subunits and detection by either UV/fluorescence or MS. However, the measurement of these disaccharides is challenging due to their hydrophilic and labile nature. Here we report a pre-column LC-MS method for the quantification of GAG disaccharide subunits. Heparan sulphate (HS) was extracted from cell lines using a combination of molecular weight cutoff and anion exchange spin filters and digested using a mixture of heparinases I, II and III. The resulting subunits were derivatised with procainamide, separated using hydrophilic interaction liquid chromatography and detected using electrospray ionisation operated in positive ion mode. Eight HS disaccharides were separated and detected together with an internal standard. The limit of detection was found to be in the range 0.6-4.9 ng/mL. Analysis of HS extracted from all cell lines tested in this study revealed a significant variation in their composition with the most abundant disaccharide being the non-sulphated ∆UA-GlcNAc. Some structural functional relationships are discussed demonstrating the viability of the pre-column method for studying GAG biology. Graphical abstract Extraction and HILIC UPLC-MS analysis of procainamide-labelled heparan sulphate disaccharides.

Heparan sulfate disaccharide measurement from biological samples using pre-column derivatization, UPLC-MS and single ion monitoring.

Glycosaminoglycans are a heterogeneous family of linear polysaccharides comprised of repeating disaccharide subunits that mediate many effects at the cellular level. There is increasing evidence that the nature of these effects is determined by differences in disaccharide composition. However, the determination of GAG disaccharide composition in biological samples remains challenging and time-consuming. We have developed a method that uses derivatization and selected ion recording and RP-UPLCMS resulting in rapid separation and quantification of twelve heparin/heparin sulfate disaccharides from 5 µg GAG. Limits of detection and quantitation were 0.02-0.15 and 0.07-0.31 µg/ml respectively. We have applied this method to the novel analysis of disaccharide levels extracted from heparan sulfate and human cancer cell lines. Heparan sulfate disaccharides extracted from biological samples following actinase and heparinase incubation and derivatized using reductive amination with 2-aminoacridone. Derivatized disaccharides were analyzed used UPLC-MS with single ion monitoring. Eight HS disaccharide subunits were separated and quantified from HS and cell lines in eleven minutes per sample. In all samples the most abundant subunits present were the unsulfated ΔUA-GlcNAc, ΔUA-GlcNAc,6S and ΔUA,2S-GlcNS,6S. There was considerable variation in the proportions and concentrations of disaccharides between different cell lines. Further studies are needed to examine the significance of these differences.

Factors determining the balance between the wish to die and the wish to live in older adults.

BACKGROUND: The "Internal Struggle Hypothesis" (Kovacs and Beck, ) suggests that suicidal persons may have both a wish to live (WTL) and a wish to die (WTD). The current study investigates whether the three-group typology - "WTL", "ambivalent (AMB)", and "WTD" - is determined by common correlates of suicidality and whether these groups can be ordinally ranked. METHODS: The sample comprised 113 older inpatients. Discriminant analysis was used to create two functions (combining social, psychiatric, psychological, and somatic variables) to predict the assignment of older inpatients into the groups WTL, AMB, and WTD. RESULTS: The functions "Subjective Well-being" and "Social Support" allowed us to assign patients into these three distinct groups with good accuracy (66.1%). "Subjective Well-being" contrasted the groups WTD and WTL and "Social Support" discriminated between the groups WTD and AMB. "Social Support" was highest in the AMB group. CONCLUSIONS: Our results suggest a simultaneous presence of a WTL and a WTD in older inpatients, and also that the balance between them is determined by "Subjective Well-being" and "Social Support". Unexpectedly, the AMB group showed the highest scores on "Social Support". We hypothesize that higher social support might function as an important determinant of a remaining WTL when a WTD is present because of a lower sense of well-being. The study suggests that the groups WTL-AMB-WTD can not situated on a one-dimensional continuum. Copyright © 2016 John Wiley & Sons, Ltd.

Are religiousness and death attitudes associated with the wish to die in older people?

BACKGROUND: A wish to die is common in older persons and is associated with increased mortality. Several risk factors have been identified, but the association between religiousness and a wish to die in older adults has been underexplored, and the association between death attitudes and the presence of a wish to die has not been investigated yet. The aim of this study is to explore the relationship between religiousness and death attitudes on the one hand and wish to die on the other hand, adjusting for clinical factors such as the presence of depression or somatic disorder. METHODS: The sample comprised 113 older inpatients (from a psychiatric and somatic ward) with a mean age of 74 years. Psychiatric diagnoses were assessed by the Structured Clinical Interview for DSM-IV Disorders, and logistic regression analyses estimated the unique contribution of religiousness and death attitudes to the wish to die, controlling for socio-demographic variables, depressive disorder, and somatic symptoms. RESULTS: Both religiousness and death attitudes were associated with a wish to die in univariate models. Adding these variables in a multivariate logistic hierarchical model, death attitudes remained significant predictors but religiousness did not; 55% of the pseudovariance of the wish to die was explained by these variables, with an effective size of 0.89. Major depressive episode, somatic symptoms, Fear of Death, and Escape Acceptance were the most important predictors of the wish to die. CONCLUSIONS: This study suggests that how older adults perceive death partly determines whether they have a wish to die. There may be a clinical, patient-oriented benefit in discussing with older patients about how they perceive death, as this can play a role in the early detection (and prevention) of death or suicide ideation and associated behaviors in older adults.

Suicide rates in five-year age-bands after the age of 60 years: the international landscape.

BACKGROUND: There is paucity of studies examining suicide rates in narrow five-year age-bands after the age of 60 years. This study examined suicide rates in eight five-year age-bands between the age of 60 and 99 years because this will allow more precise comparison between the young old (60-79 years) and the oldest old (80+ years) age groups. METHODS: Data on the number of suicides (International Classification of Diseases - ICD-10 codes, X60-84) in each of the eight five-year age-bands between the age-bands 60-64 years and 95-99 years in both gender for as many years as possible from 2000 were ascertained from three sources: colleagues with access to national data, national statisics office websites and email contact with the national statistics offices. The population size for the corresponding years and age-bands was estimated for each country using data provided by the United Nations website. RESULTS: In men, suicide rates continued to increase for each of the seven five-year age-bands from 60-64 years to 90-94 years age-band, and then declined slightly for the 95-99 year age-band. In women, suicide rates continued to increase for each of the six five-year age-bands from 60-64 years to 85-89 years age-bands, and then declined slightly for the 90-94 years and 95-99 years age-bands. CONCLUSIONS: The overall global suicide rates for each of the eight five-year age-bands are sufficiently large for them to constitute a public health concern. This is especially important given the ongoing rise in the elderly population size and the paucity of data on risk and protective factors for suicide in the five-year age-bands after the age of 60 years.

Are elite cricketers more prone to suicide? A psychological autopsy study of Test cricketer suicides.

OBJECTIVES: It has been hypothesised that the very nature of the game predisposes elite cricketers to higher rates of suicide. AIM: We aim to estimate the suicide rate of male Test cricketers and to determine the reasons for suicide. METHODS: The suicide rate in male Test cricketers was determined. A psychological autopsy was conducted using published biographical data. RESULTS: Twenty suicides amongst 2794 male Test cricketers from 1877 to 2014 yielded a suicide rate of 715.4 per 100,000 for that period. Health, financial and relationship issues were prominent; depression and alcohol misuse were common. CONCLUSIONS: Most suicides in Test cricketers occurred post-retirement in mid to late life with similar correlates to those found in the general male population. The idiosyncrasies of cricket are unlikely to contribute to suicide; however, the post-retirement welfare of Test cricketers should remain a focus of concern and the greater supports available to contemporary Test cricketers needs to extend beyond retirement.

Are gender and life attitudes associated with the wish to die in older psychiatric and somatic inpatients? An explorative study.

BACKGROUND: Death wishes are not uncommon in older persons, and to date, several risk factors have been identified. The presence of these risk factors is insufficient to fully understand why some older people, who are exposed to them, develop a wish to die and why others do not. The purpose of the study was to explore whether Purpose in Life as well as other life attitudes are associated with a death wish in older males and females. METHODS: The sample comprised 113 older inpatients (from a psychiatric and somatic ward) with a mean age of 74 years. Psychiatric diagnoses were assessed by the SCID-II. Logistic regression analyses estimated the unique contribution of (the interaction between) life attitudes and gender to the wish to die, controlling for sociodemographic variables, depressive disorder, and somatic symptoms. RESULTS: We observed a statistically significant relationship between life attitudes and the wish to die. Purpose in Life and the Purpose in Life*Gender interaction explained significant additional variance in the prediction of the wish to die. Purposelessness in life might therefore be an important correlate of a wish to die, especially in older men, independently from sociodemographic and clinical features. CONCLUSIONS: In assessing a wish to die in older adults, life attitudes need to be taken into account, besides the presence of a depressive disorder and/or somatic health. More specifically, finding or maintaining a purpose in later life might be an important feature in the prevention of the wish to die, especially in male persons.

Suicide in centenarians: the international landscape.

BACKGROUND: The elderly population size is growing worldwide due increased life expectancy and decreased mortality in the elderly. This has lead to an increase in the number of centenarians, and their numbers are predicted to increase further. Little is known about suicide rates in centenarians. METHODS: Data on the number of suicides (ICD-10 codes, X60-84) in entenarians of both gender for as many years as possible from 2000 were ascertained from three sources: colleagues, national statisics office websites and e-mail contact with the national statistics offices of as many countries as possible. The number of centernarians for the corresponding years was estimated for each country using data provided by the United Nations website. RESULTS: Data were available from 17 countries. The suicide rate was 57 (95% confidence interval 45-69) per 100, 000 person years in men and 6.8 (95% confidence interval 5.1-8.5) per 100,000 person years in women. CONCLUSIONS: Suicide rates were sufficiently large amongst centenarians for there to constitute a public health concern given the anticipated rise in the centenarian population and the paucity of data on risk and protective factors for suicide in this age group.

Disaccharide compositional analysis of chondroitin sulphate using WAX HILIC-MS with pre-column procainamide labelling; application to the placenta in pre-eclampsia.

Chondroitin sulphate (CS) and dermatan sulphate are negatively charged linear heteropolysaccharides. These glycosaminoglycans (GAG) are involved in cellular signalling via binding to growth factors. CS is expressed in a range of tissue and biological fluids and is highly expressed in the placenta. There is evidence that decorin; a CS proteoglycan is significantly decreased in pre-eclampsia and fetal growth restriction. It is considered that GAG chain composition may influence cellular processes that are altered in pre-eclampsia. The goal of the present study was to develop an LC-MS method with precolumn procainamide labelling for the disaccharide compositional analysis of CS. The method was used to investigate whether the disaccharide composition of placenta-extracted CS is altered in pre-eclampsia. The study revealed differential disaccharide compositions of placental chondroitin sulphate between pre-eclampsia and other pregnancy conditions. This suggests that the method may have diagnostic potential for pregnancy disorders. Furthermore, the findings suggest that CS sulphation might play a significant role in maternal labour.

The relationship between NHS performance indicators and suicide rates.

AIMS AND METHOD: As mental health services have an important role in prevention of suicides, the relationship between Care Quality Commission performance ratings of mental health trusts and the local suicide rates were examined with the null hypothesis that there will be no relationship between them. Data on suicide rates for men and women aged 16-65 for each district covered by a mental health trust were ascertained from the Office of National Statistics for the year 2009. Data on performance ratings of mental health trusts for the same year were obtained from the Care Quality Commission Report. RESULTS: There was no significant relationship between suicide rates in the districts covered by 60 mental health trusts in England and Wales and their performance ratings. CLINICAL IMPLICATIONS: The negative findings suggest that the performance rating of mental health trusts do not influence local suicide rates.

Hypoxia elicits broad and systematic changes in protein subcellular localization.

Oxygen provides a crucial energy source in eukaryotic cells. Hence, eukaryotes ranging from yeast to humans have developed sophisticated mechanisms to respond to changes in oxygen levels. Regulation of protein localization, like protein modifications, can be an effective mechanism to control protein function and activity. However, the contribution of protein localization in oxygen signaling has not been examined on a genomewide scale. Here, we examine how hypoxia affects protein distribution on a genomewide scale in the model eukaryote, the yeast Saccharomyces cerevisiae. We demonstrate, by live cell imaging, that hypoxia alters the cellular distribution of 203 proteins in yeast. These hypoxia-redistributed proteins include an array of proteins with important functions in various organelles. Many of them are nuclear and are components of key regulatory complexes, such as transcriptional regulatory and chromatin remodeling complexes. Under hypoxia, these proteins are synthesized and retained in the cytosol. Upon reoxygenation, they relocalize effectively to their normal cellular compartments, such as the nucleus, mitochondria, endoplasmic reticulum, and cell periphery. The resumption of the normal cellular locations of many proteins can occur even when protein synthesis is inhibited. Furthermore, we show that the changes in protein distribution induced by hypoxia follow a slower trajectory than those induced by reoxygenation. These results show that the regulation of protein localization is a common and potentially dominant mechanism underlying oxygen signaling and regulation. These results may have broad implications in understanding oxygen signaling and hypoxia responses in higher eukaryotes such as humans.

Deletion of a subgroup of ribosome-related genes minimizes hypoxia-induced changes and confers hypoxia tolerance.

Hypoxia is a widely occurring condition experienced by diverse organisms under numerous physiological and disease conditions. To probe the molecular mechanisms underlying hypoxia responses and tolerance, we performed a genome-wide screen to identify mutants with enhanced hypoxia tolerance in the model eukaryote, the yeast Saccharomyces cerevisiae. Yeast provides an excellent model for genomic and proteomic studies of hypoxia. We identified five genes whose deletion significantly enhanced hypoxia tolerance. They are RAI1, NSR1, BUD21, RPL20A, and RSM22, all of which encode functions involved in ribosome biogenesis. Further analysis of the deletion mutants showed that they minimized hypoxia-induced changes in polyribosome profiles and protein synthesis. Strikingly, proteomic analysis by using the iTRAQ profiling technology showed that a substantially fewer number of proteins were changed in response to hypoxia in the deletion mutants, compared with the parent strain. Computational analysis of the iTRAQ data indicated that the activities of a group of regulators were regulated by hypoxia in the wild-type parent cells, but such regulation appeared to be diminished in the deletion strains. These results show that the deletion of one of the genes involved in ribosome biogenesis leads to the reversal of hypoxia-induced changes in gene expression and related regulators. They suggest that modifying ribosomal function is an effective mechanism to minimize hypoxia-induced specific protein changes and to confer hypoxia tolerance. These results may have broad implications in understanding hypoxia responses and tolerance in diverse eukaryotes ranging from yeast to humans.

Deprivation of Liberty Safeguards in England: implementation costs.

BACKGROUND: The Deprivation of Liberty Safeguards (DoLS), introduced into the Mental Capacity Act 2005, were fully implemented on 1 April 2009 in England and Wales. The government estimated 20 000 assessments for DoLS at a cost of £600 per assessment. Aims To estimate the costs likely to be incurred with the implementation of DoLS in England. METHOD: The cost of conducting a single DoLS assessment was estimated using resource-utilisation data ascertained from 37 professionals, secretarial staff and independent mental capacity advocates involved with DoLS assessments in six diverse local DoLS offices. RESULTS: The estimated average cost of a single DoLS assessment was £1277. CONCLUSIONS: The estimated average cost of a single DoLS assessment was significantly higher than the £600 estimated by the government. However, the allocated budget, based on 20 000 estimated DoLS assessments in the first year of its implementation, is likely to be adequate because a significantly lower number of assessments (only 5200) were conducted in the first 9 months after its implementation.

Suicides by country of birth groupings in England and Wales: age-associated trends and standardised mortality ratios.

INTRODUCTION: Suicide rates in England and Wales have declined in recent years. A better understanding of age-associated trends in different ethnic groups may inform strategies to sustain this decline. MATERIALS AND METHODS: This study examines suicide rates and age-associated trends in England and Wales by country of birth (used as a proxy for ethnicity) using the latest available national mortality data. RESULTS: The main findings were (a) suicide rates were generally higher in males than females in all age bands in all country of birth groups except the China group, where suicides rates were higher in females than males in the older age bands; (b) male suicide rates increased with ageing in the Indian sub-continent group and female suicide rates increased with ageing in the Africa and China groups; (c) male standardised mortality ratios (SMRs) were generally higher in the younger age bands in the Eastern Europe and Caribbean groups and generally lower in the Australasian, Middle East and Western Europe groups; (d) male SMRs were generally higher in the older age bands in Eastern Europe, Caribbean, Australasian and Western Europe groups and lower in all age bands in the Indian sub-continent group, and (e) female SMRs were generally higher in the older age bands in the China, Africa and Caribbean groups. CONCLUSION: There is a need for epidemiological data on suicides in BME groups, including age-associated trends, trends over time, risk and protective factors and methods of suicide to inform suicide prevention strategies.