Assessment of databases to determine the validity of beta- and gamma-carbonic anhydrase sequences from vertebrates

BackgroundThe inaccuracy of DNA sequence data is becoming a serious problem, as the amount of molecular data is multiplying rapidly and expectations are high for big data to revolutionize life sciences and health care. In this study, we investigated the accuracy of DNA sequence data from commonly used databases using carbonic anhydrase (CA) gene sequences as generic targets. CAs are ancient metalloenzymes that are present in all unicellular and multicellular living organisms. Among the eight distinct families of CAs, including alpha, beta, gamma, delta, zeta, eta, theta, and iota, only alpha -CAs have been reported in vertebrates.ResultsBy an in silico analysis performed on the NCBI and Ensembl databases, we identified several beta- and gamma -CA sequences in vertebrates, including Homo sapiens, Mus musculus, Felis catus, Lipotes vexillifer, Pantholops hodgsonii, Hippocampus comes, Hucho hucho, Oncorhynchus tshawytscha, Xenopus tropicalis, and Rhinolophus sinicus. Polymerase chain reaction (PCR) analysis of genomic DNA persistently failed to amplify positive beta- or gamma -CA gene sequences when Mus musculus and Felis catus DNA samples were used as templates. Further BLAST homology searches of the database-derived "vertebrate" beta- and gamma -CA sequences revealed that the identified sequences were presumably derived from gut microbiota, environmental microbiomes, or grassland ecosystems.ConclusionsOur results highlight the need for more accurate and fast curation systems for DNA databases. The mined data must be carefully reconciled with our best knowledge of sequences to improve the accuracy of DNA data for publication.Peer reviewe

EXPRESSION OF cAMP AND CREB IN THE HUMAN PENIS

The aim of this study is to investigate the expression of adenosine 3',5'-cyclic monophosphate (cAMP) and cAMP-response element-binding protein (CREB) in the human penis as it is known that luteinizing hormone (LH) regulates cellular function mostly through the cAMP signaling pathway and LH receptors are expressed by the penile endothelium. Penile tissue was obtained from three patients during partial or total penectomy due to a rectal cancer with secondary penile metastasis or squamous cell carcinoma of the penis. Immunohistochemistry was used for the detection of cAMP and CREB. Positive immunoreaction for cAMP was present in most cells of superficial, intermedial, and basal layer of urethral epithelium and in fibroblast-like cells (FLC) of interstitial tissue and endothelial cells (EC) of cavernous spaces in corpus spongiosum penis. Positive staining for cAMP was also visible in EC of cavernous spaces and in FLC of interstitial tissue in corpus cavernosum penis. Positive immunoreaction for CREB was present in the superficial and intermedial layer of urethral epithelium, and some positive immunoreaction was also noticed in EC of cavernous spaces and in FLC of interstitial tissue in corpus spongiosum penis. Positive staining was also visible in the EC of cavernous spaces and in fibroplast-like cells of the interstitial tissue in the corpus cavernosum penis. Our results show the presence of cAMP and CREB in the human penis. While LH exerts its actions through cAMP signaling system and our previous studies have shown the expression of luteinizing hormone/choriogonadotropin (LHCG) receptor in the mouse and human penis, this finding may support the hypothesis that LH could affect the spongious and cavernous tissue of the human penis and thereby influence the development of erectile dysfunction among aging men.Peer reviewe

Ascaris lumbricoides β carbonic anhydrase: A potential target enzyme for treatment of ascariasis

BACKGROUND: A parasitic roundworm, Ascaris lumbricoides, is the causative agent of ascariasis, with approximately 760 million cases around the world. Helminthic infections occur with a high prevalence mostly in tropical and developing xcountries. Therefore, design of affordable broad-spectrum anti-helminthic agents against a variety of pathogens, including not only A. lumbricoides but also hookworms and whipworms, is desirable. Beta carbonic anhydrases (β-CAs) are considered promising targets of novel anthelminthics because these enzymes are present in various parasites, while completely absent in vertebrates. METHODS: In this study, we identified an A. lumbricoides β-CA (AIBCA) protein from protein sequence data using bioinformatics tools. We used computational biology resources and methods (including InterPro, CATH/Gene3D, KEGG, and METACYC) to analyze AlBCA and define potential roles of this enzyme in biological pathways. The AlBCA gene was cloned into pFastBac1, and recombinant AIBCA was produced in sf-9 insect cells. Kinetics of AlBCA were analyzed by a stopped-flow method. RESULTS: Multiple sequence alignment revealed that AIBCA contains the two sequence motifs, CXDXR and HXXC, typical for β-CAs. Recombinant AIBCA showed significant CA catalytic activity with k(cat) of 6.0 × 10(5) s(−1) and k(cat)/K(M) of 4.3 × 10(7) M(−1) s(−1). The classical CA inhibitor, acetazolamide, showed an inhibition constant of 84.1 nM. Computational modeling suggests that the molecular architecture of AIBCA is highly similar to several other known β-CA structures. Functional predictions suggest that AIBCA might play a role in bicarbonate-mediated metabolic pathways, such as gluconeogenesis and removal of metabolically produced cyanate. CONCLUSIONS: These results open new avenues to further investigate the precise functions of β-CAs in parasites and suggest that novel β-CA specific inhibitors should be developed and tested against helminthic diseases. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-015-1098-5) contains supplementary material, which is available to authorized users

beta carbonic anhydrase is required for female fertility in Drosophila melanogaster

Background: Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous enzymes that catalyze the reversible hydration reaction of carbon dioxide. CAs are present as six structurally divergent enzyme families: alpha, beta, gamma, delta, zeta and eta. beta-CAs have a wide distribution across different species including invertebrates. Previously, we showed that Drosophila melanogaster beta-CA is a highly active mitochondrial enzyme. In this study, we investigated the function of Drosophila beta-CA by silencing the expression of the beta-CA gene using UAS/GAL4-based RNA interference (RNAi) in Drosophila in vivo. Results: Crossing beta-CA RNAi lines over ubiquitous Actin driver flies did not produce any viable progeny, indicating that beta-CA expression is required for fly development. RNAi silencing of beta-CA ubiquitously in adult flies did not affect their survival rate or function of mitochondrial electron transport chain. Importantly, beta-CA RNAi led to impaired reproduction. All beta-CA knockdown females were sterile, and produced few or no eggs. Whole ovaries of knockdown females looked normal but upon cadherin staining, there was an apparent functional defect in migration of border cells, which are considered essential for normal fertilization. Conclusions: These results indicate that although Drosophila beta-CA is dispensable for survival of adult flies, it is essential for female fertility.Peer reviewe

Cloning, purification, kinetic and anion inhibition studies of a recombinant beta-carbonic anhydrase from the Atlantic salmon parasite platyhelminth Gyrodactylus salaris

A beta-class carbonic anhydrase (CA, EC 4.2.1.1) was cloned from the genome of the Monogenean platyhelminth Gyrodactylus salaris, a parasite of Atlantic salmon. The new enzyme, GsaCA beta has a significant catalytic activity for the physiological reaction, CO2 + H2O (sic) HCO3- + H+ with a k(cat) of 1.1 x 10(5) s(-1) and a k(cat)/K-m of 7.58 x 10(6) M-1 x s(-1). This activity was inhibited by acetazolamide (K-I of 0.46 mu M), a sulphonamide in clinical use, as well as by selected inorganic anions and small molecules. Most tested anions inhibited GsaCA beta at millimolar concentrations, but sulfamide (K-I of 81 mu M), N,N-diethyldithiocarbamate (K-I of 67 mu M) and sulphamic acid (K-I of 6.2 mu M) showed a rather efficient inhibitory action. There are currently very few non-toxic agents effective in combating this parasite. GsaCA beta is subsequently proposed as a new drug target for which effective inhibitors can be designed.Peer reviewe

Cloning, purification, kinetic and anion inhibition studies of a recombinant β-carbonic anhydrase from the Atlantic salmon parasite platyhelminth Gyrodactylus salaris

A β-class carbonic anhydrase (CA, EC 4.2.1.1) was cloned from the genome of the Monogenean platyhelminth Gyrodactylus salaris, a parasite of Atlantic salmon. The new enzyme, GsaCAβ has a significant catalytic activity for the physiological reaction, CO2 + H2O ⇋ HCO3− + H+ with a kcat of 1.1 × 105 s−1 and a kcat/Km of 7.58 × 106 M−1 × s−1. This activity was inhibited by acetazolamide (KI of 0.46 µM), a sulphonamide in clinical use, as well as by selected inorganic anions and small molecules. Most tested anions inhibited GsaCAβ at millimolar concentrations, but sulfamide (KI of 81 µM), N,N-diethyldithiocarbamate (KI of 67 µM) and sulphamic acid (KI of 6.2 µM) showed a rather efficient inhibitory action. There are currently very few non-toxic agents effective in combating this parasite. GsaCAβ is subsequently proposed as a new drug target for which effective inhibitors can be designed.</p

Gene Expression Profiles in Human and Mouse Primary Cells Provide New Insights into the Differential Actions of Vitamin D-3 Metabolites

1α,25-Dihydroxyvitamin D3 (1α,25(OH)2D3) had earlier been regarded as the only active hormone. The newly identified actions of 25-hydroxyvitamin D3 (25(OH)D3) and 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) broadened the vitamin D3 endocrine system, however, the current data are fragmented and a systematic understanding is lacking. Here we performed the first systematic study of global gene expression to clarify their similarities and differences. Three metabolites at physiologically comparable levels were utilized to treat human and mouse fibroblasts prior to DNA microarray analyses. Human primary prostate stromal P29SN cells (hP29SN), which convert 25(OH)D3 into 1α,25(OH)2D3 by 1α-hydroxylase (encoded by the gene CYP27B1), displayed regulation of 164, 171, and 175 genes by treatment with 1α,25(OH)2D3, 25(OH)D3, and 24R,25(OH)2D3, respectively. Mouse primary Cyp27b1 knockout fibroblasts (mCyp27b1−/−), which lack 1α-hydroxylation, displayed regulation of 619, 469, and 66 genes using the same respective treatments. The number of shared genes regulated by two metabolites is much lower in hP29SN than in mCyp27b1−/−. By using DAVID Functional Annotation Bioinformatics Microarray Analysis tools and Ingenuity Pathways Analysis, we identified the agonistic regulation of calcium homeostasis and bone remodeling between 1α,25(OH)2D3 and 25(OH)D3 and unique non-classical actions of each metabolite in physiological and pathological processes, including cell cycle, keratinocyte differentiation, amyotrophic lateral sclerosis signaling, gene transcription, immunomodulation, epigenetics, cell differentiation, and membrane protein expression. In conclusion, there are three distinct vitamin D3 hormones with clearly different biological activities. This study presents a new conceptual insight into the vitamin D3 endocrine system, which may guide the strategic use of vitamin D3 in disease prevention and treatment.Peer reviewe

Etänä tehtävä hoidon tarpeen arviointi sairaanhoitajan kokemana : kuvaileva kirjallisuuskatsaus

Hoidon tarpeen arviointia tehdään tulevaisuudessa yhä enemmän etänä sekä digitaalisia palveluja hyödyntäen. Myös terveydenhuoltolain muutos lisää entisestään tarvetta kehittää etänä tehtävää hoidon tarpeen arviointia, ja sairaanhoitajia tullaan tarvitsemaan yhä enemmän tähän työhön. Tämän opinnäytetyön tarkoituksena oli selvittää sairaanhoitajien kokemuksia etänä tehtävästä hoidon tarpeen arvioinnista. Opinnäytetyön tavoitteena oli kirjallisuuskatsauksen avulla lisätä tietoisuutta etänä tehtävän hoidon tarpeen arvioinnista sairaanhoitajan kokemana. Kirjallisuuskatsauksen tiedonhaku tehtiin sähköisistä tietokannoista, ja mukaan valikoitui 15 alkuperäistutkimusta. Valitut tutkimukset analysoitiin induktiivisella sisällön analyysilla, minkä avulla muodostettiin seitsemän yläluokkaa sairaanhoitajien kokemuksista etänä tehtävästä hoidon tarpeen arvioinnista Opinnäytetyön tuloksissa ilmeni sekä hoidon tarpeen arviointia vaikeuttavia että edistäviä tekijöitä. Vaikeuttavia tekijöitä olivat esimerkiksi näköyhteyden puuttuminen ja potilaiden epäasiallinen käytös. Edistäväksi tekijäksi koettiin sanattoman viestinnän hyödyntäminen. Kommunikaatiotaitojen tärkeys nousi vahvasti esiin, kuten myös työhön ohjattujen resurssien vähyys sekä työn vaikutukset henkiseen hyvinvointiin. Sairaanhoitajat kokivat usein epävarmuutta tehdessään hoidon tarpeen arviointia etänä. Lisäksi organisaation tuki ja odotukset vaikuttivat sairaanhoitajien kokemuksiin etänä tehtävästä hoidon tarpeen arvioinnista. Kliinisten päätöksenteon tukijärjestelmien hyödyntämisestä saatiin vaihtelevia tuloksia. Ne koettiin erittäin hyödyllisiksi apuvälineiksi, mutta toisaalta monimutkaisiksi ja työtä rajoittaviksi. Tuloksia on mahdollista hyödyntää etänä hoidon tarvetta arvioivien sairaanhoitajien työn sujuvuuden ja työhyvinvoinnin kehittämisessä ja parantamisessa. Sairaanhoitajien kokemuksia on syytä tutkia Suomessa lisää. Jatkotutkimusehdotuksena esitetään myös sairaanhoitajien kokemuksien selvittämistä chat-palvelun käytöstä hoidon tarpeen arvioinnissa.There will be increased need for remote assessment of the need for care. The recent changes in healthcare law will further increase the need for nurses and assessing the need for care remotely. The purpose of this study was to gather information about the nurses’ experiences of remote assessment of the need for care. The aim was to increase awareness of remote assessment of the need for care as experienced by the nurses. The study was conducted as a descriptive literature review. The data were collected from electronic databases and collected data were analyzed by inductive content analysis. The results of the study revealed both factors that hinder and promote the assessment of the need for care. For example, the lack of visual information or calls from agitated or rude callers was perceived as a factor complicating the assessment of the need for care. Effective communication skills were identified as a crucial component of remote care assessment. The work was found to affect the nurses’ mental well-being and nurses often felt uncertainty. The shortage of allocated resources was perceived as a hindering factor. The outcomes of computer decision support systems exhibited variability, being viewed as valuable tools by some but also as complicating and potentially restrictive in their work. The results can be used to develop and improve workflow and well-being of nurses who assess the need for care remotely. There is increased need to study nurses’ experiences of remote assessment of the need for care in Finland. Further research is needed to explore nurses' experiences with chat services in assessment of the need for care

Assessment of databases to determine the validity of β- and γ-carbonic anhydrase sequences from vertebrates

Abstract Background The inaccuracy of DNA sequence data is becoming a serious problem, as the amount of molecular data is multiplying rapidly and expectations are high for big data to revolutionize life sciences and health care. In this study, we investigated the accuracy of DNA sequence data from commonly used databases using carbonic anhydrase (CA) gene sequences as generic targets. CAs are ancient metalloenzymes that are present in all unicellular and multicellular living organisms. Among the eight distinct families of CAs, including α, β, γ, δ, ζ, η, θ, and ι, only α-CAs have been reported in vertebrates. Results By an in silico analysis performed on the NCBI and Ensembl databases, we identified several β- and γ-CA sequences in vertebrates, including Homo sapiens, Mus musculus, Felis catus, Lipotes vexillifer, Pantholops hodgsonii, Hippocampus comes, Hucho hucho, Oncorhynchus tshawytscha, Xenopus tropicalis, and Rhinolophus sinicus. Polymerase chain reaction (PCR) analysis of genomic DNA persistently failed to amplify positive β- or γ-CA gene sequences when Mus musculus and Felis catus DNA samples were used as templates. Further BLAST homology searches of the database-derived “vertebrate” β- and γ-CA sequences revealed that the identified sequences were presumably derived from gut microbiota, environmental microbiomes, or grassland ecosystems. Conclusions Our results highlight the need for more accurate and fast curation systems for DNA databases. The mined data must be carefully reconciled with our best knowledge of sequences to improve the accuracy of DNA data for publication