RESUMO
BACKGROUND: Multimorbidity is a common phenomenon among patients treated in general practices. Key challenges within this group include functional difficulties, polypharmacy, treatment burden, fragmentation of care, reduced quality of life and increased health care utilization. These problems cannot be solved in the short consultation time of a general practitioner (GP) since there is an increasing shortage of GPs. In many countries, advanced practice nurses (APNs) are successfully integrated into primary health care for multimorbid patients. The objective of this study is to examine whether the integration of APNs in the primary care of multimorbid patients in Germany leads to optimized care of the target group and to a reduction in the workload of the GPs. METHODS: The intervention includes the integration of APNs into the care for multimorbid patients in general practice for twelve months. Qualifications for APNs include a master's level academic degree and 500 hours of project-specific training. Their tasks include in-depth assessment, preparation, implementation, monitoring and evaluation of a person-centred and evidence-based care plan. In this nonrandomized controlled trial, a prospective multicentre mixed methods study will be performed. The main inclusion criterion was the cooccurrence of three chronic diseases. For data collection in the intervention group (n = 817), routine data from health insurance companies and association of statutory health insurance physicians (ASHIP) will be used, as well as qualitative interviews. In addition, the intervention will be assessed through documentation of the care process and standardized questionnaires using a longitudinal design. The control group (n = 1634) will receive standard care. For the evaluation, routine data from health insurance companies are matched at a ratio of 1:2. Outcomes will be measured using emergency contacts and GP visits, treatment costs, health status of the patients and the satisfaction of parties involved. The statistical analyses will include Poisson regression to compare outcomes between the intervention and control groups. Descriptive and analytical statistical methods will be used in the longitudinal analysis of the intervention group data. Cost analysis will compare total costs and subgroup costs between the intervention and control groups. Qualitative data will be analysed using content analysis. DISCUSSION: Challenges to this protocol could include the political and strategic environment as well as the planned number of participants. TRIAL REGISTRATION: DRKS00026172 on DRKS.
Assuntos
Medicina Geral , Qualidade de Vida , Humanos , Multimorbidade , Estudos Prospectivos , Custos de Cuidados de Saúde , Inquéritos e QuestionáriosRESUMO
INTRODUCTION: Solid-organ transplantation (SOT) is a well-known risk factor for invasive pulmonary aspergillosis (IPA). We report on the epidemiology and outcome of SOT patients with IPA in an intensive care unit (ICU) setting. METHODS: This is a secondary study based on a subset of SOT patients from a prospective observational multicenter cohort (the AspICU project) including ICU patients with at least one Aspergillus spp. positive culture. Cases were classified as proven, probable, or putative IPA, or as Aspergillus-colonized. Mortality was reported at 12 weeks. RESULTS: The study included 52 SOT patients (of which 18 lung, 17 liver, 12 kidney, and five heart transplants). Sixteen patients had proven IPA, 28 were categorized as putative IPA (of which only five reached a probable IPA diagnosis according to the European Organization for Research and Treatment of Cancer/Mycosis Study Group and Research Consortium criteria), and eight as Aspergillus-colonization. Among patients with IPA, 20 (45.5%) developed IPA during their ICU stay following transplantation whereas 24 patients (54.5%) had a medical ICU admission. Regarding medical imaging, nearly all IPA cases presented with non-specific findings as only nine demonstrated robust findings suggestive for invasive fungal disease. Overall, severity of the disease was reflected by a high prevalence of underlying conditions and acute organ derangements. Mortality among patients with IPA was 68%. Lung transplantation was associated with better survival (50%). CONCLUSION: IPA in SOT patients in the ICU develops in the presence of overall high severity of the disease. It rarely presents with suggestive medical imaging thereby hampering diagnosis. IPA in ICU patients with SOT carries a grim prognosis.
Assuntos
Aspergilose Pulmonar Invasiva , Transplante de Órgãos , Aspergillus , Estudos de Coortes , Humanos , Unidades de Terapia Intensiva , Aspergilose Pulmonar Invasiva/diagnóstico por imagem , Aspergilose Pulmonar Invasiva/epidemiologia , Transplante de Órgãos/efeitos adversos , Estudos RetrospectivosRESUMO
BACKGROUND: Inducible high-level expression is favoured for recombinant protein production in Pichia pastoris. Therefore, novel regulated promoters are desired, ideally repressing heterologous gene expression during initial growth and enabling it in the production phase. In a typical large scale fed-batch culture repression is desired during the batch phase where cells grow on a surplus of e.g. glycerol, while heterologous gene expression should be active in the feed phase under carbon (e.g. glucose) limitation. RESULTS: DNA microarray analysis of P. pastoris wild type cells growing in glycerol-based batch and glucose-based fed batch was used for the identification of genes with both, strong repression on glycerol and high-level expression in the feed phase. Six novel glucose-limit inducible promoters were successfully applied to express the intracellular reporter eGFP. The highest expression levels together with strong repression in pre-culture were achieved with the novel promoters P(G1) and P(G6). Human serum albumin (HSA) was used to characterize the promoters with an industrially relevant secreted protein. A P(G1) clone with two gene copies reached about 230% of the biomass specific HSA titer in glucose-based fed batch fermentation compared to a P(GAP) clone with identical gene copy number, while P(G6) only achieved 39%. Two clones each carrying eleven gene copies, expressing HSA under control of P(G1) and P(G6) respectively were generated by post-transformational vector amplification. They produced about 1.0 and 0.7 g L(-1) HSA respectively in equal fed batch processes. The suitability in production processes was also verified with HyHEL antibody Fab fragment for P(G1) and with porcine carboxypeptidase B for P(G6). Moreover, the molecular function of the gene under the control of P(G1) was determined to encode a high-affinity glucose transporter and named GTH1. CONCLUSIONS: A set of novel regulated promoters, enabling induction without methanol, was successfully identified by using DNA microarrays and shown to be suitable for high level expression of recombinant proteins in glucose-based protein production processes.
Assuntos
Pichia/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Animais , Técnicas de Cultura Celular por Lotes , Biomassa , Reatores Biológicos , Carboxipeptidase B/genética , Carboxipeptidase B/metabolismo , Dosagem de Genes , Glucose/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Metanol/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Recombinantes/genética , Albumina Sérica/genética , Albumina Sérica/metabolismo , SuínosRESUMO
Matching both the construction of a recombinant strain and the process design with the characteristics of the target protein has the potential to significantly enhance bioprocess performance, robustness, and reproducibility. The factors affecting the physiological state of recombinant Pichia pastoris Mut(+) (methanol utilization-positive) strains and their cell membranes were quantified at the individual cell level using a combination of staining with fluorescent dyes and flow cytometric enumeration. Cell vitalities were found to range from 5 to 95% under various process conditions in high-cell-density fed-batch cultures, with strains producing either porcine trypsinogen or horseradish peroxidase extracellularly. Impaired cell vitality was observed to be the combined effect of production of recombinant protein, low pH, and high cell density. Vitality improved when any one of these stress factors was excluded. At a pH value of 4, which is commonly applied to counter proteolysis, recombinant strains exhibited severe physiological stress, whereas strains without heterologous genes were not affected. Physiologically compromised cells were also found to be increasingly sensitive to methanol when it accumulated in the culture broth. The magnitude of the response varied when different reporters were combined with either the native AOX1 promoter or its d6* variant, which differ in both strength and regulation. Finally, the quantitative assessment of the physiology of individual cells enables the implementation of innovative concepts in bioprocess development. Such concepts are in contrast to the frequently used paradigm, which always assumes a uniform cell population, because differentiation between the individual cells is not possible with methods commonly used.
Assuntos
Citometria de Fluxo/métodos , Corantes Fluorescentes/metabolismo , Pichia/fisiologia , Proteínas Recombinantes/biossíntese , Biotecnologia/métodos , Proliferação de Células , Contagem de Colônia Microbiana , Meios de Cultura , Metanol/metabolismo , Fenótipo , Pichia/genética , Pichia/crescimento & desenvolvimento , Pichia/metabolismo , Proteínas Recombinantes/genéticaRESUMO
The nitrile hydratase (NHase, EC 4.2.1.84) genes (alpha and beta subunit) and the corresponding activator gene from Rhodococcus equi TG328-2 were cloned and sequenced. This Fe-type NHase consists of 209 amino acids (alpha subunit, M(r) 23 kDa) and 218 amino acids (beta subunit, M(r) 24 kDa) and the NHase activator of 413 amino acids (M(r) 46 kDa). Various combinations of promoter, NHase and activator genes were constructed to produce active NHase enzyme recombinantly in E. coli. The maximum enzyme activity (844 U/mg crude cell extract towards methacrylonitrile) was achieved when the NHase activator gene was separately co-expressed with the NHase subunit genes in E. coli BL21 (DE3). The overproduced enzyme was purified with 61% yield after French press, His-tag affinity chromatography, ultrafiltration and lyophilization and showed typical Fe-type NHase characteristics: besides aromatic and heterocyclic nitriles, aliphatic ones were hydrated preferentially. The purified enzyme had a specific activity of 6,290 U/mg towards methacrylonitrile. Enantioselectivity was observed for aromatic compounds only with E values ranging 5-17. The enzyme displayed a broad pH optimum from 6 to 8.5, was most active at 30 degrees C and showed the highest stability at 4 degrees C in thermal inactivation studies between 4 degrees C and 50 degrees C.
Assuntos
Clonagem Molecular , Escherichia coli/genética , Hidroliases/genética , Hidroliases/metabolismo , Rhodococcus equi/enzimologia , Rhodococcus equi/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Sequência de Bases , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Genes Bacterianos , Hidroliases/isolamento & purificação , Concentração de Íons de Hidrogênio , Metacrilatos/metabolismo , Nitrilas/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Espectrofotometria Ultravioleta , Estereoisomerismo , Especificidade por Substrato , TemperaturaRESUMO
Different members of the nitrilase superfamily (D-carbamoylases, Nit-Fhit proteins, amidases, cyanide dihydratases and nitrilases) were compared by multiple sequence alignments and a long carboxy-terminal extension (about 50 amino acids) identified in all nitrilases and cyanide dihydratases which was not present in other members of the nitrilase superfamily. The function of this C-terminal part was experimentally analysed in the arylacetonitrilase of Pseudomonas fluorescens EBC191 by the construction of various deletion mutants, chimeric enzymes with other bacterial nitrilases and site-specific mutagenesis. The enzyme variants were tested with the substrates 2-phenylpropionitrile and mandelonitrile and compared regarding specific activities, degree of amide formation and enantioselectivity. The enzyme variants containing deletions up to 32 amino acids did not show significant differences in comparison with the wild-type enzyme. Deletion mutants with 47-67 amino acids missing generally demonstrated reduced enzyme activities, increased amounts of amide formation and increased proportions of the (R)-enantiomers of the amides and acids formed. Also certain exchanges of H296 in the C-terminal motif DpvGHY led to enzyme variants with a similar phenotype. Chimeric enzymes which contained up to 59 amino acids deriving from the nitrilases of Rhodococcus rhodochrous NCIMB11216 or Alcaligenes faecalis ATCC8750 were active and resembled, with respect to the enantioselectivity and degree of amide formation, the wild-type enzyme of P.fluorescens.
Assuntos
Aminoidrolases , Mutação , Pseudomonas fluorescens/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminoidrolases/química , Aminoidrolases/genética , Aminoidrolases/metabolismo , Sequência de Bases , Sítios de Ligação , Catálise , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Dimerização , Estabilidade Enzimática , Escherichia coli/genética , Congelamento , Ligação de Hidrogênio , Cinética , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Estereoisomerismo , Especificidade por SubstratoRESUMO
Sphingomonas xenophaga BN6 degrades various (substituted) naphthalenesulfonates to the corresponding (substituted) salicylates. A gene cluster was identified on the plasmid pBN6 which coded for several enzymes participating in the degradative pathway for naphthalenesulfonates. A DNA fragment of 16 915 bp was sequenced which contained 17 ORFs. The genes encoding the 1,2-dihydroxynaphthalene dioxygenase, 2-hydroxychromene-2-carboxylate isomerase, and 2'-hydroxybenzalpyruvate aldolase of the naphthalenesulfonate pathway were identified on the DNA fragment and the encoded proteins heterologously expressed in Escherichia coli. Also, the genes encoding the ferredoxin and ferredoxin reductase of a multi-component, ring-hydroxylating naphthalenesulfonate dioxygenase were identified by insertional inactivation. The identified genes generally demonstrated the highest degree of homology to enzymes encoded by the phenanthrene-degrading organism Sphingomonas sp. P2, or the megaplasmid pNL1 of the naphthalene- and biphenyl-degrading strain Sphingomonas aromaticivorans F199. The genes of S. xenophaga BN6 participating in the degradation of naphthalenesulfonates also shared the same organization in three different transcriptional units as the genes involved in the degradation of naphthalene, biphenyl, and phenanthrene previously found in Sphingomonas sp. P2 and S. aromaticivorans F199. The genes were flanked in S. xenophaga BN6 by ORFs which specify proteins that show the highest homologies to proteins of mobile genetic elements.
Assuntos
Genes Bacterianos , Naftalenossulfonatos/metabolismo , Sphingomonas/genética , Sequência de Bases , Dados de Sequência Molecular , Fases de Leitura Aberta , Sphingomonas/metabolismoRESUMO
The gene encoding an enantioselective arylacetonitrilase was identified on a 3.8 kb DNA fragment from the genomic DNA of Pseudomonas fluorescens EBC191. The gene was isolated, sequenced and cloned into the L-rhamnose-inducible expression vector pJOE2775. The nitrilase was produced in large quantities and purified as a histidine-tagged enzyme from crude extracts of L-rhamnose-induced cells of Escherichia coli JM109. The purified nitrilase was significantly stabilized during storage by the addition of 1 M ammonium sulfate. The temperature optimum (50 degrees C), pH optimum (pH 6.5), and specific activity of the recombinant nitrilase were similar to those of the native enzyme from P. fluorescens EBC191. The enzyme hydrolysed various phenylacetonitriles with different substituents in the 2-position and also heterocyclic and bicyclic arylacetonitriles to the corresponding carboxylic acids. The conversion of most arylacetonitriles was accompanied by the formation of different amounts of amides as by-products. The relative amounts of amides formed from different nitriles increased with an increasing negative inductive effect of the substituent in the 2-position. The acids and amides that were formed from chiral nitriles demonstrated in most cases opposite enantiomeric excesses. Thus mandelonitrile was converted by the nitrilase preferentially to R-mandelic acid and S-mandelic acid amide. The nitrilase gene is physically linked in the genome of P. fluorescens with genes encoding the degradative pathway for mandelic acid. This might suggest a natural function of the nitrilase in the degradation of mandelonitrile or similar naturally occurring hydroxynitriles.
Assuntos
Aminoidrolases/biossíntese , Clonagem Molecular , Escherichia coli/enzimologia , Regulação Bacteriana da Expressão Gênica , Pseudomonas fluorescens/enzimologia , Proteínas Recombinantes , Aminoidrolases/química , Aminoidrolases/genética , Aminoidrolases/isolamento & purificação , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Pseudomonas fluorescens/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Especificidade por Substrato , TemperaturaRESUMO
A systematic survey for the presence of plasmids in 17 different xenobiotic-degrading Sphingomonas strains was performed. In almost all analyzed strains, two to five plasmids with sizes of about 50 to 500 kb were detected by using pulsed-field gel electrophoresis. A comparison of plasmid preparations untreated or treated with S1 nuclease suggested that, in general, Sphingomonas plasmids are circular. Hybridization experiments with labeled gene probes suggested that large plasmids are involved in the degradation of dibenzo-p-dioxin, dibenzofuran, and naphthalenesulfonates in S. wittichii RW1, Sphingomonas sp. HH69, and S. xenophaga BN6, respectively. The plasmids which are responsible for the degradation of naphthalene, biphenyl, and toluene by S. aromaticivorans F199 (pNL1) and of naphthalenesulfonates by S. xenophaga BN6 (pBN6) were site-specifically labeled with a kanamycin resistance cassette. The conjugative transfer of these labeled plasmids was attempted with various bacterial strains as putative recipient strains. Thus, a conjugative transfer of plasmid pBN6 from S. xenophaga BN6 to a cured mutant of strain BN6 and to Sphingomonas sp. SS3 was observed. The conjugation experiments with plasmid pNL1 suggested a broader host range of this plasmid, because it was transferred without any obvious structural changes to S. yanoikuyae B1, Sphingomonas sp. SS3, and S. herbicidovorans. In contrast, major plasmid rearrangements were observed in the transconjugants after the transfer of plasmid pNL1 to Sphingomonas sp. HH69 and of pBN6 to Sphingomonas sp. SS3. No indications for the transfer of a Sphingomonas plasmid to bacteria outside of the Sphingomonadaceae were obtained.
Assuntos
Conjugação Genética , Plasmídeos/genética , Sphingomonas/genética , Xenobióticos/metabolismo , Biodegradação Ambiental , DNA Bacteriano/genética , Dioxinas/metabolismo , Transferência Genética Horizontal , Naftalenossulfonatos/metabolismo , Sphingomonas/metabolismoRESUMO
During aerobic degradation of naphthalene-2-sulfonate (2NS), Sphingomonas xenophaga strain BN6 produces redox mediators which significantly increase the ability of the strain to reduce azo dyes under anaerobic conditions. It was previously suggested that 1,2-dihydroxynaphthalene (1,2-DHN), which is an intermediate in the degradative pathway of 2NS, is the precursor of these redox mediators. In order to analyze the importance of the formation of 1,2-DHN, the dihydroxynaphthalene dioxygenase gene (nsaC) was disrupted by gene replacement. The resulting strain, strain AKE1, did not degrade 2NS to salicylate. After aerobic preincubation with 2NS, strain AKE1 exhibited much higher reduction capacities for azo dyes under anaerobic conditions than the wild-type strain exhibited. Several compounds were present in the culture supernatants which enhanced the ability of S. xenophaga BN6 to reduce azo dyes under anaerobic conditions. Two major redox mediators were purified from the culture supernatants, and they were identified by high-performance liquid chromatography-mass spectrometry and comparison with chemically synthesized standards as 4-amino-1,2-naphthoquinone and 4-ethanolamino-1,2-naphthoquinone.
Assuntos
Proteínas de Bactérias/metabolismo , Naftalenossulfonatos/metabolismo , Sphingomonas/metabolismo , Anaerobiose , Proteínas de Bactérias/genética , Cromatografia Líquida , Espectrometria de Massas , Mutação , Oxirredução , Sphingomonas/genéticaRESUMO
An important goal is to identify the direct activation domain (AD)-interacting components of the transcriptional machinery within the context of native complexes. Toward this end, we first demonstrate that the multisubunit TFIID, SAGA, mediator, and Swi/Snf coactivator complexes from transcriptionally competent whole-cell yeast extracts were all capable of specifically interacting with the prototypic acidic ADs of Gal4 and VP16. We then used hexahistidine tags as genetically introduced activation domain-localized cross-linking receptors. In combination with immunological reagents against all subunits of TFIID and SAGA, we systematically identified the direct AD-interacting subunits within the AD-TFIID and AD-SAGA coactivator complexes enriched from whole-cell extracts and confirmed these results using purified TFIID and partially purified SAGA. Both ADs directly cross-linked to TBP and to a subset of TFIID and SAGA subunits that carry histone-fold motifs.