Molecular fingerprints for a novel enzyme family in actinobacteria with glucosamine kinase activity

Actinobacteria have long been the main source of antibiotics, secondary metabolites with tightly controlled biosynthesis by environmental and physiological factors. Phosphorylation of exogenous glucosamine has been suggested as a mechanism for incorporation of this extracellular material into secondary metabolite biosynthesis, but experimental evidence of specific glucosamine kinases in Actinobacteria is lacking. Here, we present the molecular fingerprints for the identification of a unique family of actinobacterial glucosamine kinases. Structural and biochemical studies on a distinctive kinase from the soil bacterium Streptacidiphilus jiangxiensis unveiled its preference for glucosamine and provided structural evidence of a phosphoryl transfer to this substrate. Conservation of glucosamine-contacting residues across a large number of uncharacterized actinobacterial proteins unveiled a specific glucosamine binding sequence motif. This family of kinases and their genetic context may represent the missing link for the incorporation of environmental glucosamine into the antibiotic biosynthesis pathways in Actinobacteria and can be explored to enhance antibiotic production. IMPORTANCE The discovery of novel enzymes involved in antibiotic biosynthesis pathways is currently a topic of utmost importance. The high levels of antibiotic resistance detected worldwide threaten our ability to combat infections and other 20th-century medical achievements, namely, organ transplantation or cancer chemotherapy. We have identified and characterized a unique family of enzymes capable of phosphorylating glucosamine to glucosamine-6-phosphate, a crucial molecule directly involved in the activation of antibiotic production pathways in Actinobacteria, nature’s main source of antimicrobials. The consensus sequence identified for these glucosamine kinases will help establish a molecular fingerprint to reveal yet-uncharacterized sequences in antibiotic producers, which should have an important impact in biotechnological and biomedical applications, including the enhancement and optimization of antibiotic production.We acknowledge the European Synchrotron Radiation Facility (Grenoble, France) for provision of synchrotron radiation facilities and thank their staff for help with data collection. Part of these experiments were performed at beamline BL13-XALOC of ALBA Synchrotron (Cerdanyola del Vallès, Spain), with the collaboration of ALBA staff and CALIPSOplus (grant 730872) funding. The support of the X-ray Crystallography Scientific Platform of i3S (Porto, Portugal) is also acknowledged. We thank Pedro Lamosa from CERMAX, ITQB-NOVA (Oeiras, Portugal), for acquiring and interpreting the NMR data. This work was supported by the Structured Program on Bioengineered Therapies for Infectious Diseases and Tissue Regeneration (Norte-01-0145-FEDER-‏000‏012), funded by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through Fundo Europeu de Desenvolvimento Regional (FEDER) and by FEDER through the COMPETE 2020-Operational Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT-Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of project Institute for Research and Innovation in Health Sciences (POCI-01-0145-FEDER-007274) and also by grants UID/NEU/04539/2019 and POCI-01-0145-FEDER-029221. D.N.-C. acknowledges the European Regional Development Fund (CENTRO-01-0145-FEDER-000012-ealthyAging2020) for a research fellowship and FCT for Ph‏D f‏ellowship SFRH/BD/117777/2016

Prevalence of asthma and its association with rhinitis in the elderly

BACKGROUND: Asthma and rhinitis are frequent respiratory diseases in children and adults. Despite the increase in the aging population, there are few epidemiologic data on both diseases in the elderly. So far, no population-based study has analyzed the association between asthma and rhinitis symptoms and severity in this age group. This study aimed to estimate the prevalence of physician-diagnosed asthma in the population aged ≥65 years in mainland Portugal and to evaluate its association with the presence and classification of rhinitis according to ARIA recommendations, in this age group. METHODS: A cross-sectional, nationwide, population-based survey of individuals aged ≥65 years, living in mainland Portugal was performed. RESULTS: Data were obtained from 3678 respondents. The prevalence of physician-diagnosed asthma was 10.9% (95% confidence interval (95%CI) 9.9-11.9). The frequency of asthma diagnosis increased with the number of nasal symptoms (p < 0.001). A strong association between asthma and rhinitis was found (odds ratio (OR) 13.86 (95%CI 10.66-18.02)). The strength of this association increased with the persistence and severity of rhinitis, being particularly high in elderly subjects with moderate-severe persistent rhinitis (OR 39.9 (95%CI 27.5-58.0)). CONCLUSIONS: Asthma is common in the elderly and strongly associated with rhinitis. The OR for asthma is especially high in persistent and severe ARIA classification rhinitis types. This study strengthens the need for an integrated assessment of asthma together with rhinitis in the elderl

Prevalence and classification of rhinitis in the elderly: a nationwide survey in Portugal

BACKGROUND: Nationwide epidemiologic data on rhinitis in the elderly do not exist. This study aimed to estimate the prevalence of rhinitis in the population aged 65 years or above in mainland Portugal and to characterize and classify rhinitis in this age group. METHODS: Cross-sectional, nationwide, population-based survey of citizens aged 65 years or above, living in mainland Portugal. Current rhinitis (CR) was defined as the presence of at least two symptoms: 'repeated sneezing and itchy nose', 'blocked nose for more than one whole hour', or 'runny nose when not having a cold or flu', either usually or in the last 12 months. Rhinitis severity was assessed using a visual analogue scale; rhinitis was classified according to ARIA. RESULTS: Data were obtained from 3678 responders (92.5% response rate). The prevalence of CR was 29.8% (95% confidence interval (CI): 28.4%-31.3%): 49.1% had mild intermittent, 7.0% mild persistent, 27.5% moderate-severe intermittent, and 16.4% moderate-severe persistent rhinitis. Only 38.6% of patients with CR had been physician diagnosed and 38.7% were under treatment for this disease in the previous year. Allergic conjunctivitis symptoms were referred by 68.6% of subjects with CR (rhinoconjunctivitis population prevalence, 20.5% (95% CI: 19.2%-21.8%)). CONCLUSIONS: Rhinitis and rhinoconjunctivitis are common but underdiagnosed and undertreated diseases in the geriatric population. This was the first nationwide epidemiological survey classifying rhinitis according to ARIA guidelines in this age group. More than 40% of old-age patients presented moderate-severe disease

Patients’ use of information about medicine side effects in relation to experiences of suspected adverse drug reactions

Background Adverse drug reactions (ADRs) are common, and information about medicines is increasingly widely available to the public. However, relatively little work has explored how people use medicines information to help them assess symptoms that may be suspected ADRs. Objective Our objective was to determine how patients use patient information leaflets (PILs) or other medicines information sources and whether information use differs depending on experiences of suspected ADRs. Method This was a cross-sectional survey conducted in six National Health Service (NHS) hospitals in North West England involving medical in-patients taking at least two regular medicines prior to admission. The survey was administered via a questionnaire and covered use of the PIL and other medicines information sources, perceived knowledge about medicines risks/ADRs, experiences of suspected ADRs, plus demographic information. Results Of the 1,218 respondents to the survey, 18.8 % never read the PIL, whilst 6.5 % only do so if something unexpected happens. Educational level was related to perceived knowledge about medicines risks, but not to reading the PIL or seeking further information about medicines risks. Over half the respondents (56.0 %) never sought more information about possible side effects of medicines. A total of 57.2 % claimed they had experienced a suspected ADR. Of these 85.9 % were either very sure or fairly sure this was a reaction to a medicine. Over half of those experiencing a suspected ADR (53.8 %) had read the PIL, of whom 36.2 % did so before the suspected ADR occurred, the remainder afterwards. Reading the PIL helped 84.8 % of these respondents to decide they had experienced an ADR. Educational level, general knowledge of medicines risks and number of regular medicines used all increased the likelihood of experiencing an ADR. Conclusion More patients should be encouraged to read the PIL supplied with medicines. The results support the view that most patients feel knowledgeable about medicines risks and suspected ADRs and value information about side effects, but that reading about side effects in PILs or other medicines information sources does not lead to experiences of suspected ADRs

Single dose systemic methotrexate versus expectant management for treatment of tubal ectopic pregnancy: A placebo-controlled randomised trial

OBJECTIVE: Methotrexate is routinely used worldwide for medical treatment of clinically stable women with tubal ectopic pregnancies. This is despite the lack of robust evidence to show its superior effectiveness over expectant management. The aim of our multicentre randomised trial study was to compare the success rates of methotrexate with placebo for the conservative treatment of tubal ectopic pregnancies. METHODS: The study was multicentre; it took place in three UK early pregnancy units between January 2006 and June 2014. Inclusion criteria were clinically stable women with a conclusive ultrasound diagnosis of a tubal ectopic pregnancy presenting with low serum β-hCG <1500IU/l. Women were randomly assigned to single systemic injection of methotrexate 50mg/m(2) or placebo. The primary outcome of the study was a binary indicator for success of conservative management, defined as resolution of clinical symptoms and decline of serum β-hCG to <20IU/l or negative urine pregnancy test without the need for any additional medical intervention. An intention to treat analysis was followed. RESULTS: We recruited a total of 80 women: 42 to methotrexate and 38 to placebo. The two arms of the study were balanced in terms of age, ethnicity, obstetric histories, pregnancy characteristics and serum β-hCG and progesterone. The proportions of successes were similar: 83% with methotrexate and 76% with placebo. On univariate analysis, this difference was not statistically significant (χ2(1df) = 0.53; P = 0.23). On multivariate logistic regression, β-hCG was the only covariate which was significantly associated with outcome. The odds of failure increased by 0.15% for each unit increase in β-hCG (OR=1.0015; 95% CI 1.0002 to 1.003; P = 0.02). In 14 women presenting with serum hCG 1000-1500IU/l the success of expectant management was 33% compared to 62% in the methotrexate arm. Although this result was not statistically significant a larger sample size would give us greater power to detect a difference in this subgroup of women, In women with successful conservative management there was no significant difference in median resolution times between methotrexate and placebo arms [17.5 days (IQR 14 - 28.0) (n = 30)] vs [14 days (IQR 7 - 29.5) (n = 25)] (P = 0.73) CONCLUSION: The results of our study do not support routine use of methotrexate for the treatment of clinically stable women diagnosed with tubal ectopic pregnancies presenting with low serum hCG <1500IU/l. Further work is required to identify a subgroup of women with tubal ectopic pregnancies and hCG≥1500IU/l in whom methotrexate may offer a safe and cost-effective alternative to surgery

Addressing quality and usability of surface water bodies in semi-arid regions with mining influences

Water resources management has considerable importance, specifically in the context of climate change. This subject has introduced new challenges in semi-arid regions with water quality problems, such as the Iberian Pyrite Belt, which is one of the largest metallogenetic provinces in the world and one of the driest regions in Europe. Positioned in the Mediterranean context, the region has a high density of polymetallic sulphide mines that promote the degradation of water systems. The present study aims to assess the water quality in the Pyrite Belt, considering a total of 34 surface water bodies, including constructed reservoirs, permanent and ephemeral streams, and mining facilities with accumulated water (e.g., pit lakes and mining dams). The water samples were analysed for physico-chemical properties, including field parameters (pH, electrical conductivity), alkalinity/acidity, hardness, anions, and potential toxic elements. The results were used for hydrochemical classifications and the assessment of suitability for public uses. Statistical methods, such as hierarchical cluster analysis and nearest centroid classifier, were used for grouping and evaluating the similarity between water bodies. Two groups were generated from the analysis: i) constructed lakes with alkaline and sodium signatures; and ii) waters suffering from the influence of mining wastes, e.g., showing high acidity, sulphate and metal contents. Therefore, the loss of water quality in the vicinity of mines reflects the impact of acid mine drainage. The methodological approach used may be applied to the integrated management of water resources in regions with mining influences and where it is necessary to combat drought and water scarcity scenarios.Patricia Gomes acknowledge FCT (Science and Technology Foundation, Portugal) by the research fellowship under the POCH (Programa Operacional Capital Humano) supported by the European Social Fund and National Funds of MCTES (Ministerio da Ciencia, Tecnologia e Ensino Superior) with reference SFRH/BD/108887/2015. This work was co-funded by the European Union through the European Regional Development Fund, based on COMPETE 2020 (Programa Operacional da Competitividade e Internacionalizacao) - project ICT (UID/GEO/04683/2013) with reference POCI-01-0145-FEDER-007690 and project Nano-MINENV number 029259

Functional cartography of complex metabolic networks

High-throughput techniques are leading to an explosive growth in the size of biological databases and creating the opportunity to revolutionize our understanding of life and disease. Interpretation of these data remains, however, a major scientific challenge. Here, we propose a methodology that enables us to extract and display information contained in complex networks. Specifically, we demonstrate that one can (i) find functional modules in complex networks, and (ii) classify nodes into universal roles according to their pattern of intra- and inter-module connections. The method thus yields a ``cartographic representation'' of complex networks. Metabolic networks are among the most challenging biological networks and, arguably, the ones with more potential for immediate applicability. We use our method to analyze the metabolic networks of twelve organisms from three different super-kingdoms. We find that, typically, 80% of the nodes are only connected to other nodes within their respective modules, and that nodes with different roles are affected by different evolutionary constraints and pressures. Remarkably, we find that low-degree metabolites that connect different modules are more conserved than hubs whose links are mostly within a single module.Comment: 17 pages, 4 figures. Go to http://amaral.northwestern.edu for the PDF file of the reprin

Cartilage restoration of patellofemoral lesions: a systematic review

Purpose This study aimed to systematically analyze the postoperative clinical, functional, and imaging outcomes, complications, reoperations, and failures following patellofemoral cartilage restoration surgery. Methods This review was conducted according to the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). PubMed, EMBASE, and Cochrane Library databases were searched up to August 31, 2018, to identify clinical studies that assessed surgical outcomes of patellofemoral cartilage restoration surgery. The Methodological Index for Non-Randomized Studies (MINORS) was used to assess study quality. Results Forty-two studies were included comprising 1,311 knees (mean age of 33.7 years and 56% males) and 1,309 patellofemoral defects (891 patella, 254 trochlear, 95 bipolar, and 69 multiple defects, including the patella or trochlea) at a mean follow-up of 59.2 months. Restoration techniques included autologous chondrocyte implantation (56%), particulated juvenile allograft cartilage (12%), autologous matrix-induced chondrogenesis (9%), osteochondral autologous transplantation (9%), and osteochondral allograft transplantation (7%). Significant improvement in at least one score was present in almost all studies and these surpassed the minimal clinically important difference threshold. There was a weighted 19%, 35%, and 6% rate of reported complications, reoperations, and failures, respectively. Concomitant patellofemoral surgery (51% of patients) mostly did not lead to statistically different postoperative outcomes. Conclusion Numerous patellofemoral restoration techniques result in significant functional improvement with a low rate of failure. No definitive conclusions could be made to determine the best surgical technique since comparative studies on this topic are rare, and treatment choice should be made according to specific patient and defect characteristics

Identification and characterization of a novel non-structural protein of bluetongue virus

Bluetongue virus (BTV) is the causative agent of a major disease of livestock (bluetongue). For over two decades, it has been widely accepted that the 10 segments of the dsRNA genome of BTV encode for 7 structural and 3 non-structural proteins. The non-structural proteins (NS1, NS2, NS3/NS3a) play different key roles during the viral replication cycle. In this study we show that BTV expresses a fourth non-structural protein (that we designated NS4) encoded by an open reading frame in segment 9 overlapping the open reading frame encoding VP6. NS4 is 77–79 amino acid residues in length and highly conserved among several BTV serotypes/strains. NS4 was expressed early post-infection and localized in the nucleoli of BTV infected cells. By reverse genetics, we showed that NS4 is dispensable for BTV replication in vitro, both in mammalian and insect cells, and does not affect viral virulence in murine models of bluetongue infection. Interestingly, NS4 conferred a replication advantage to BTV-8, but not to BTV-1, in cells in an interferon (IFN)-induced antiviral state. However, the BTV-1 NS4 conferred a replication advantage both to a BTV-8 reassortant containing the entire segment 9 of BTV-1 and to a BTV-8 mutant with the NS4 identical to the homologous BTV-1 protein. Collectively, this study suggests that NS4 plays an important role in virus-host interaction and is one of the mechanisms played, at least by BTV-8, to counteract the antiviral response of the host. In addition, the distinct nucleolar localization of NS4, being expressed by a virus that replicates exclusively in the cytoplasm, offers new avenues to investigate the multiple roles played by the nucleolus in the biology of the cell

HIPK2 and extrachromosomal histone H2B are separately recruited by Aurora-B for cytokinesis

Cytokinesis, the final phase of cell division, is necessary to form two distinct daughter cells with correct distribution of genomic and cytoplasmic materials. Its failure provokes genetically unstable states, such as tetraploidization and polyploidization, which can contribute to tumorigenesis. Aurora-B kinase controls multiple cytokinetic events, from chromosome condensation to abscission when the midbody is severed. We have previously shown that HIPK2, a kinase involved in DNA damage response and development, localizes at the midbody and contributes to abscission by phosphorylating extrachromosomal histone H2B at Ser14. Of relevance, HIPK2-defective cells do not phosphorylate H2B and do not successfully complete cytokinesis leading to accumulation of binucleated cells, chromosomal instability, and increased tumorigenicity. However, how HIPK2 and H2B are recruited to the midbody during cytokinesis is still unknown. Here, we show that regardless of their direct (H2B) and indirect (HIPK2) binding of chromosomal DNA, both H2B and HIPK2 localize at the midbody independently of nucleic acids. Instead, by using mitotic kinase-specific inhibitors in a spatio-temporal regulated manner, we found that Aurora-B kinase activity is required to recruit both HIPK2 and H2B to the midbody. Molecular characterization showed that Aurora-B directly binds and phosphorylates H2B at Ser32 while indirectly recruits HIPK2 through the central spindle components MgcRacGAP and PRC1. Thus, among different cytokinetic functions, Aurora-B separately recruits HIPK2 and H2B to the midbody and these activities contribute to faithful cytokinesis