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1.
Nat Methods ; 19(1): 81-89, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34949807

RESUMEN

CD4+ T cells are central mediators of adaptive and innate immune responses and constitute a major reservoir for human immunodeficiency virus (HIV) in vivo. Detailed investigations of resting human CD4+ T cells have been precluded by the absence of efficient approaches for genetic manipulation limiting our understanding of HIV replication and restricting efforts to find a cure. Here we report a method for rapid, efficient, activation-neutral gene editing of resting, polyclonal human CD4+ T cells using optimized cell cultivation and nucleofection conditions of Cas9-guide RNA ribonucleoprotein complexes. Up to six genes, including HIV dependency and restriction factors, were knocked out individually or simultaneously and functionally characterized. Moreover, we demonstrate the knock in of double-stranded DNA donor templates into different endogenous loci, enabling the study of the physiological interplay of cellular and viral components at single-cell resolution. Together, this technique allows improved molecular and functional characterizations of HIV biology and general immune functions in resting CD4+ T cells.


Asunto(s)
Linfocitos T CD4-Positivos/fisiología , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Infecciones por VIH/genética , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/virología , Proteína 9 Asociada a CRISPR/genética , Movimiento Celular/genética , Células Cultivadas , ADN , Técnicas de Inactivación de Genes , Infecciones por VIH/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Resistencia a Mixovirus/genética , Proteínas de Resistencia a Mixovirus/metabolismo , ARN Guía de Kinetoplastida , Proteína 1 que Contiene Dominios SAM y HD/genética , Transgenes , Factores de Escisión y Poliadenilación de ARNm/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismo
2.
Proc Natl Acad Sci U S A ; 118(15)2021 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-33876744

RESUMEN

Stabilization of messenger RNA is an important step in posttranscriptional gene regulation. In the nucleus and cytoplasm of eukaryotic cells it is generally achieved by 5' capping and 3' polyadenylation, whereas additional mechanisms exist in bacteria and organelles. The mitochondrial mRNAs in the yeast Saccharomyces cerevisiae comprise a dodecamer sequence element that confers RNA stability and 3'-end processing via an unknown mechanism. Here, we isolated the protein that binds the dodecamer and identified it as Rmd9, a factor that is known to stabilize yeast mitochondrial RNA. We show that Rmd9 associates with mRNA around dodecamer elements in vivo and that recombinant Rmd9 specifically binds the element in vitro. The crystal structure of Rmd9 bound to its dodecamer target reveals that Rmd9 belongs to the family of pentatricopeptide (PPR) proteins and uses a previously unobserved mode of specific RNA recognition. Rmd9 protects RNA from degradation by the mitochondrial 3'-exoribonuclease complex mtEXO in vitro, indicating that recognition and binding of the dodecamer element by Rmd9 confers stability to yeast mitochondrial mRNAs.


Asunto(s)
Proteínas de la Membrana/metabolismo , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Regiones no Traducidas 3' , Genes Mitocondriales , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Motivos de Nucleótidos , Unión Proteica , Dominios Proteicos , ARN Mensajero/química , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
3.
BMC Womens Health ; 23(1): 526, 2023 10 06.
Artículo en Inglés | MEDLINE | ID: mdl-37803287

RESUMEN

BACKGROUND: Resistance training (RT) is effective in counteracting the age- and menopause-related loss of muscle mass (MM) and strength in middle-aged women (40-60 years). Research on RT with free weights is limited in pre- and post-menopausal women. Based on this, a 20-week training intervention was conducted with this population to investigate the effects of systematic RT with free weights on strength capacity and body composition. METHOD: Forty-one healthy women (52.0 ± 3.6 years) participated in this study. After 10-week control phase (no RT, T0-T1) followed by a 10-week intervention phase (T1-T2) with RT twice a week and 6-8 sets of each muscle per week. Subjects were randomly assigned to a low-intensity (50% 1-RM) or moderate-intensity (75% 1-RM) RT group and divided into pre-menopausal and post-menopausal according to their hormone profile. Fat-free mass (FFM), MM, fat mass (FM), muscle thickness (Vastus lateralis (VL), Rectus femoris (RF), Triceps brachii (TB)), grip strength, 1-RM squat and bench press were assessed before and after each phase. Statistical analysis was performed using a linear mixed model to account for fixed (time and group) and random (individual) effects. RESULTS: A total of 31 women successfully completed the study. No injuries occurred during the intervention. Significant increases in 1-RM squat and bench press were observed in all groups. No interaction effect was observed for the strength parameters. In pre-menopausal women, FFM, MM and RF muscle thickness increased significantly, while VL showed a trend. These effects were not present in post-menopausal women regardless of RT intensity. CONCLUSION: RT with free weight is safe and effective for middle-aged women to increase 1-RM. Hypertrophy effects were found exclusively in pre-menopausal women. To achieve hypertrophy and/or body composition changes in post-menopausal women, larger training volumes (> 6-8 sets/muscle per week) are likely required.


Asunto(s)
Fuerza Muscular , Entrenamiento de Fuerza , Persona de Mediana Edad , Humanos , Femenino , Fuerza Muscular/fisiología , Músculo Esquelético/fisiología , Composición Corporal , Menopausia , Hipertrofia
4.
Pflege ; 36(5): 259-268, 2023.
Artículo en Alemán | MEDLINE | ID: mdl-36325985

RESUMEN

Complexity of outpatient intensive care for ventilated people: Cross-mapping into the standardised NNN-taxonomy Abstract. Background: In Germany, free text is the preferred method for recording the nursing process in outpatient intensive care, although classification systems could enable a more precise description. Research question: How is nursing care for people with outpatient ventilation represented by the NNN-taxonomy and what are the recommendations for nursing practice? Methods: A qualitative "multiple case" design was applied. Using deductive content analysis (data sources: nursing documentation and secondary analysis of interviews with affected persons), several cases, both individually and across all cases were linked to the NNN-taxonomy (cross-mapping). Results: In total, the nursing documentation of 16 invasively ventilated persons with a mean age of 58.4 years (SD = 16.3) was analysed. Seven persons additionally contributed interview data. Documentation was mainly based on the "Strukturmodell" (14/16) with a moderate to high accuracy (D-Catch Score: 16.6; SD = 4.1). Cross-mapping resulted in 4016 codes: 618 nursing diagnoses, 1956 interventions and 1442 outcomes. Documentation was strongly measure-oriented, not very person-centred and with a lack of differentiation between diagnosis and intervention. Conclusions: To improve nursing practice, a person-centred attitude and the ability to differentiate between nursing diagnoses, interventions and outcomes should be promoted.


Asunto(s)
Proceso de Enfermería , Pacientes Ambulatorios , Humanos , Persona de Mediana Edad , Registros de Enfermería , Diagnóstico de Enfermería , Cuidados Críticos
5.
BMC Nurs ; 21(1): 224, 2022 Aug 11.
Artículo en Inglés | MEDLINE | ID: mdl-35953810

RESUMEN

BACKGROUND: People on home mechanical ventilation (HMV) belong to a heterogeneous population with complex care needs. In Germany, outpatient intensive care is provided in people's private home (PH) or in shared living communities (SLC). Increasing patient numbers have led to criticism of the quality of care in recent years. Since quality deficits from the perspective of those affected are largely unclear, the following research question emerged: How do interviews with ventilated individuals and family caregivers explain any differences or similarities in the quality of care between PH and SLC? METHODS: This study used a mixed-methods convergent parallel design, where quantitative and qualitative components were separately collected and analysed. The quantitative component (structured interviews and online survey) included ventilation characteristics, health-related resource use, health-related quality of life (HRQL) measured with the Severe Respiratory Insufficiency Questionnaire (SRI; range 0-100; higher scores indicated higher HRQL) and the Burden Scale of the Family Caregivers short version (BSFC-s; range 0-30; higher scores indicated higher burden). The qualitative component (semi-structured interviews) focused on people's experience of person-centred care. Data were merged using a weaving method and the Picker framework of Person-Centred Care. RESULTS: The quantitative component revealed that ventilated individuals living in PHs were on average 20 years younger than participants living in SLCs (n = 46; PH: 46.86 ±15.40 years vs. SLC: 65.07 ±11.78 years; p = .001). HRQL (n = 27; PH: 56.62 ±16.40 vs. SLC: 55.35 ±12.72; p > .999) and the burden of family caregivers (n = 16; PH: 13.20 ±10.18 vs. SLC: 12.64 ±8.55; p > .999) were not significantly different between living situation. The qualitative component revealed that person-centred care is possible in both care settings (ventilated individuals: n = 13; family caregivers: n = 18). CONCLUSION: This study describes a care situation that is as heterogeneous as the population of people with HMV. HRQL and the burden of family caregivers are highly individual and, like person-centred care, independent of the living situation. Policy decisions that facilitate person-centred care need to recognise that quality of care is highly individual and starts with the free choice of the care setting.

6.
Artículo en Inglés | MEDLINE | ID: mdl-33289627

RESUMEN

During a study investigating the microbiota of raw milk and its semi-finished products, strains WS 5106T and WS 5096 were isolated from cream and skimmed milk concentrate. They could be assigned to the genus Pseudomonas by their 16S rRNA sequences, but not to any validly named species. In this work, a polyphasic approach was used to characterize the novel strains and to investigate their taxonomic status. Examinations based on the topology of core genome phylogenomy as well as average nucleotide identity (ANIm) comparisons suggested a novel Pseudomonas species within the Pseudomonas fluorescens subgroup. With pairwise ANIm values of 90.1 and 89.8 %, WS 5106T was most closely related to Pseudomonas nabeulensis CECT 9765T and Pseudomonas kairouanensis CECT 9766T. The G+C content of strain WS 5106T was 60.1 mol%. Morphologic analyses revealed Gram-stain-negative, aerobic, catalase and oxidase positive, rod-shaped and motile cells. Proteolysis on skimmed milk agar as well as lipolysis on tributyrin agar occurred at both 28 and 6 °C. Tolerated growth conditions were temperatures between 4 and 34 °C, pH values between 6.0 and 8.0, and salt concentrations of up to 5 %. Fatty acid profiles showed a pattern typical for Pseudomonas, with C16 : 0 as the dominant component. The major cellular polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol and the dominating quinone was Q-9. Based on these results, it is proposed to classify the strains as a novel species, Pseudomonas cremoris sp. nov., with WS 5106T (=DSM 111143T=LMG 31863T) as type strain and WS 5096 (=DSM 111129=LMG 31864) as an additional strain.


Asunto(s)
Leche/microbiología , Filogenia , Pseudomonas/clasificación , Animales , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Ácidos Grasos/química , Genes Bacterianos , Alemania , Hibridación de Ácido Nucleico , Fosfolípidos/química , Proteolisis , Pseudomonas/aislamiento & purificación , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Ubiquinona/química
7.
Appl Microbiol Biotechnol ; 105(4): 1693-1708, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33527148

RESUMEN

The heat-stable peptidase AprX, secreted by psychrotolerant Pseudomonas species in raw milk, is a major cause of destabilization and premature spoilage of ultra-high temperature (UHT) milk and milk products. To enable rapid detection and quantification of seven frequent and proteolytic Pseudomonas species (P. proteolytica, P. gessardii, P. lactis, P. fluorescens, P. protegens, P. lundensis, and P. fragi) in raw milk, we developed two triplex qPCR assays taking into account species-dependent differences in AprX activity. Besides five species-specific hydrolysis probes, targeting the aprX gene, a universal rpoB probe was included in the assay to determine the total Pseudomonas counts. For all six probes, linear regression lines between Cq value and target DNA concentration were obtained in singleplex as well as in multiplex approaches, yielding R2 values of > 0.975 and amplification efficiencies of 85-97%. Moreover, high specificity was determined using genomic DNA of 75 Pseudomonas strains, assigned to 57 species, and 40 other bacterial species as templates in the qPCR. Quantification of the target species and total Pseudomonas counts resulted in linear detection ranges of approx. 103-107 cfu/ml, which correspond well to common Pseudomonas counts in raw milk. Application of the assay using 60 raw milk samples from different dairies showed good agreement of total Pseudomonas counts calculated by qPCR with cell counts derived from cultivation. Furthermore, a remarkably high variability regarding the species composition was observed for each milk sample, whereby P. lundensis and P. proteolytica/P. gessardii were the predominant species detected. KEY POINTS: • Multiplex qPCR for quantification of seven proteolytic Pseudomonas species and total Pseudomonas counts in raw milk • High specificity and sensitivity via hydrolysis probes against aprX and rpoB • Rapid method to determine Pseudomonas contamination in raw milk and predict spoilage potential.


Asunto(s)
Leche , Pseudomonas , Animales , Calor , Leche/metabolismo , Péptido Hidrolasas/metabolismo , Proteolisis , Pseudomonas/genética , Pseudomonas/metabolismo
8.
Appl Microbiol Biotechnol ; 105(11): 4761-4773, 2021 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-34059942

RESUMEN

The highly complex raw milk matrix challenges the sample preparation for amplicon-sequencing due to low bacterial counts and high amounts of eukaryotic DNA originating from the cow. In this study, we optimized the extraction of bacterial DNA from raw milk for microbiome analysis and evaluated the impact of cycle numbers in the library-PCR. The selective lysis of eukaryotic cells by proteinase K and digestion of released DNA before bacterial lysis resulted in a high reduction of mostly eukaryotic DNA and increased the proportion of bacterial DNA. Comparative microbiome analysis showed that a combined enzymatic and mechanical lysis procedure using the DNeasy® PowerFood® Microbial Kit with a modified protocol was best suitable to achieve high DNA quantities after library-PCR and broad coverage of detected bacterial biodiversity. Increasing cycle numbers during library-PCR systematically altered results for species and beta-diversity with a tendency to overrepresentation or underrepresentation of particular taxa. To limit PCR bias, high cycle numbers should thus be avoided. An optimized DNA extraction yielding sufficient bacterial DNA and enabling higher PCR efficiency is fundamental for successful library preparation. We suggest that a protocol using ethylenediaminetetraacetic acid (EDTA) to resolve casein micelles, selective lysis of somatic cells, extraction of bacterial DNA with a combination of mechanical and enzymatic lysis, and restriction of PCR cycles for analysis of raw milk microbiomes is optimal even for samples with low bacterial numbers. KEY POINTS: • Sample preparation for high-throughput 16S rRNA gene sequencing of raw milk microbiota. • Reduction of eukaryotic DNA by enzymatic digestion. • Shift of detected microbiome caused by high cycle numbers in library-PCR.


Asunto(s)
Microbiota , Leche , Animales , Bovinos , ADN Bacteriano/genética , Femenino , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética
9.
BMC Health Serv Res ; 21(1): 774, 2021 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-34353315

RESUMEN

BACKGROUND: The rapid increase in the use of home mechanical ventilation (HMV) for people with chronic respiratory failure poses extreme challenges for the healthcare system. People on HMV have complex care needs and require support from an interprofessional team. In Germany, HMV is criticised for inadequate quality standards, particularly in outpatient intensive care practice. The objective of this study was to describe the quality of care for people on outpatient HMV in Germany, Bavaria and provide recommendations for improvement from the perspective of healthcare professionals (HCPs). METHODS: Semi-structured qualitative telephone interviews with HCPs (i.e., nurses, equipment providers, therapists, and physicians) were analysed using the framework method. The quality framework of Health Improvement Scotland (HIS), which aims to improve the quality of person-centred care, was used to build a deductive analysis matrix. The framework includes the three key areas: (1) Outcomes and impact, (2) Service delivery, and (3) Vision and leadership. The domains (meta-codes) and quality indicators (sub-codes) of the quality framework were used for deductive coding. RESULTS: Overall, 87 HCPs (51 female, mean age of 44.3 years, mean professional experience in HMV of 9.4 years) were interviewed (mean duration of 31 min). There was a complex interaction between the existing health care system (Outcomes and impact, 955 meaning units), the delivery of outpatient intensive care (Service delivery, 939 meaning units), and improvement-focused leadership (Vision and leadership, 70 meaning units) that influenced the quality of care for people on HMV. The main barriers were an acceleration in transition management, a neglect of weaning potential, a shortage of qualified professionals and missing quality criteria. The central recommendations for promoting person-centred care were training and supervision of staff and an inspiring leadership. An integrated care structure supporting medical home visits and outpatient rehabilitation should be developed. CONCLUSION: This study describes a heterogeneous and partly deficient care situation for people on HMV, but demonstrates that high quality care is possible if person-centred care is successfully implemented in all areas of service provision. The recommendations of this study could inform the development of a person-centred integrated care structure for people on HMV.


Asunto(s)
Personal de Salud , Respiración Artificial , Adulto , Atención a la Salud , Femenino , Humanos , Investigación Cualitativa , Calidad de la Atención de Salud
10.
Euro Surveill ; 26(43)2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34713795

RESUMEN

BackgroundIn the SARS-CoV-2 pandemic, viral genomes are available at unprecedented speed, but spatio-temporal bias in genome sequence sampling precludes phylogeographical inference without additional contextual data.AimWe applied genomic epidemiology to trace SARS-CoV-2 spread on an international, national and local level, to illustrate how transmission chains can be resolved to the level of a single event and single person using integrated sequence data and spatio-temporal metadata.MethodsWe investigated 289 COVID-19 cases at a university hospital in Munich, Germany, between 29 February and 27 May 2020. Using the ARTIC protocol, we obtained near full-length viral genomes from 174 SARS-CoV-2-positive respiratory samples. Phylogenetic analyses using the Auspice software were employed in combination with anamnestic reporting of travel history, interpersonal interactions and perceived high-risk exposures among patients and healthcare workers to characterise cluster outbreaks and establish likely scenarios and timelines of transmission.ResultsWe identified multiple independent introductions in the Munich Metropolitan Region during the first weeks of the first pandemic wave, mainly by travellers returning from popular skiing areas in the Alps. In these early weeks, the rate of presumable hospital-acquired infections among patients and in particular healthcare workers was high (9.6% and 54%, respectively) and we illustrated how transmission chains can be dissected at high resolution combining virus sequences and spatio-temporal networks of human interactions.ConclusionsEarly spread of SARS-CoV-2 in Europe was catalysed by superspreading events and regional hotspots during the winter holiday season. Genomic epidemiology can be employed to trace viral spread and inform effective containment strategies.


Asunto(s)
COVID-19 , Infección Hospitalaria , Infección Hospitalaria/epidemiología , Genoma Viral , Genómica , Alemania/epidemiología , Hospitales , Humanos , Filogenia , SARS-CoV-2
11.
J Biol Chem ; 294(18): 7528-7536, 2019 05 03.
Artículo en Inglés | MEDLINE | ID: mdl-30910813

RESUMEN

The genes in mitochondrial DNA code for essential subunits of the respiratory chain complexes. In yeast, expression of mitochondrial genes is controlled by a group of gene-specific translational activators encoded in the nucleus. These factors appear to be part of a regulatory system that enables concerted expression of the necessary genes from both nuclear and mitochondrial genomes to produce functional respiratory complexes. Many of the translational activators are believed to act on the 5'-untranslated regions of target mRNAs, but the molecular mechanisms involved in this regulation remain obscure. In this study, we used a combination of in vivo and in vitro analyses to characterize the interactions of one of these translational activators, the pentatricopeptide repeat protein Pet111p, with its presumed target, COX2 mRNA, which encodes subunit II of cytochrome c oxidase. Using photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation analysis, we found that Pet111p binds directly and specifically to a 5'-end proximal region of the COX2 transcript. Further, we applied in vitro RNase footprinting and mapped two binding targets of the protein, of which one is located in the 5'-untranslated leader and the other is within the coding sequence. Combined with the available genetic data, these results suggest a plausible mechanism of translational activation, in which binding of Pet111p may prevent inhibitory secondary structures from forming in the translation initiation region, thus rendering the mRNA available for interaction with the ribosome.


Asunto(s)
Ciclooxigenasa 2/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de la Membrana/genética , Proteínas Mitocondriales/genética , Factores de Iniciación de Péptidos/genética , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
12.
Int J Syst Evol Microbiol ; 70(2): 935-943, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31730030

RESUMEN

A polyphasic approach was used to investigate the taxonomic status of two bacterial strains, WS 5072T and WS 5092, isolated from skimmed milk concentrate and raw cow's milk. The 16S rRNA and rpoD gene sequences affiliated the strains to the same, hitherto unknown, Pseudomonas species. Further examinations of the draft genomes based on multilocus sequence analysis and average nucleotide identity confirmed the presence of a novel Pseudomonas species. It was most closely related to Pseudomonas fragi DSM 3456T with 86.3 % ANIm. The DNA G+C content of strain WS 5072T was 56.3 mol%. Cells were aerobic, Gram-negative, catalase and oxidase positive, rod-shaped and motile. Growth occurred at 4-34 °C, pH 5.5-8.0 and with salt concentrations of up to 7 %. The major cellular polar lipids were phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The dominating quinone was Q-9 with 94 %, with noticeable amounts of Q-8 (5 %) and traces of Q-7 and Q-10. Fatty acid profiles showed a composition common for Pseudomonas with the major component C16 : 0. Based on these results, the novel species Pseudomonas saxonica sp. nov. is proposed, with the type strain WS 5072T (=DSM 108989T=LMG 31234T) and the additional strain WS 5092 (=DSM 108990=LMG 31235).


Asunto(s)
Microbiología de Alimentos , Leche/microbiología , Filogenia , Pseudomonas/clasificación , Animales , Técnicas de Tipificación Bacteriana , Composición de Base , Bovinos , ADN Bacteriano/genética , Ácidos Grasos/química , Femenino , Genes Bacterianos , Alemania , Tipificación de Secuencias Multilocus , Hibridación de Ácido Nucleico , Fosfolípidos/química , Pseudomonas/aislamiento & purificación , Quinonas/química , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN
13.
Int J Syst Evol Microbiol ; 70(4): 2339-2347, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-32043958

RESUMEN

Two strains, WS 5063T and WS 5067, isolated from raw cow's milk and skimmed milk concentrate, could be affiliated as members of the same, hitherto unknown, Pseudomonas species by 16S rRNA and rpoD gene sequences. Multilocus sequence and average nucleotide identity (ANIm) analyses based on draft genome sequences confirmed the discovery of a novel Pseudomonas species. It was most closely related to Pseudomonas synxantha DSM 18928T with an ANIm of 91.4 %. The DNA G+C content of WS 5063T was 60.0 mol %. Phenotypic characterizations showed that the isolates are rod-shaped, motile, catalase- and oxidase-positive, and aerobic. Growth occurred at 4-34 °C and at pH values of pH 5.5-8.0. Both strains showed strong ß-haemolysis on blood agar. The major cellular polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The dominant quinone was Q-9 (90 %), but noticeable amounts of Q-8 (9 %) and traces of Q-7 were also detected. Fatty acid profiles were typical for Pseudomonas species and exhibited C16 : 0 as a major component. Based on these results, we conclude that both strains belong to a novel species, for which the name Pseudomonas haemolytica sp. nov. is proposed. The type strain is WS 5063T (=DSM 108987T=LMG 31232T) and an additional strain is WS 5067 (=DSM 108988=LMG 31233).


Asunto(s)
Microbiología de Alimentos , Leche/microbiología , Filogenia , Pseudomonas/clasificación , Animales , Técnicas de Tipificación Bacteriana , Composición de Base , Bovinos , ADN Bacteriano/genética , Ácidos Grasos/química , Genes Bacterianos , Hibridación de Ácido Nucleico , Fosfolípidos/química , Pseudomonas/aislamiento & purificación , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Vitamina K 2/análogos & derivados , Vitamina K 2/química
14.
Appl Microbiol Biotechnol ; 104(12): 5519-5533, 2020 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-32296906

RESUMEN

Monoterpenoids are widely used in industrial applications, e.g. as active ingredients in pharmaceuticals, in flavor and fragrance compositions, and in agriculture. Severe toxic effects are known for some monoterpenoids making them challenging compounds for biotechnological production processes. Some strains of the bacterium Pseudomonas putida show an inherent extraordinarily high tolerance towards solvents including monoterpenoids. An understanding of the underlying factors can help to create suitable strains for monoterpenoids de novo production or conversion. In addition, knowledge about tolerance mechanisms could allow a deeper insight into how bacteria can oppose monoterpenoid containing drugs, like tea tree oil. Within this work, the resistance mechanisms of P. putida GS1 were investigated using selected monoterpenoid-hypertolerant mutants. Most of the mutations were found in efflux pump promoter regions or associated transcription factors. Surprisingly, while for the tested monoterpenoid alcohols, ketone, and ether high efflux pump expression increased monoterpenoid tolerance, it reduced the tolerance against geranic acid. However, an increase of geranic acid tolerance could be gained by a mutation in an efflux pump component. It was also found that increased monoterpenoid tolerance can counteract efficient biotransformation ability, indicating the need for a fine-tuned and knowledge-based tolerance improvement for production strain development.Key points• Altered monoterpenoid tolerance mainly related to altered activity of efflux pumps.• Increased tolerance to geranic acid surprisingly caused by decreased export activity. • Reduction of export activity can be beneficial for biotechnological conversions.


Asunto(s)
Farmacorresistencia Bacteriana/genética , Monoterpenos/farmacología , Pseudomonas putida/efectos de los fármacos , Pseudomonas putida/metabolismo , Biotecnología , Biotransformación , Monoterpenos/metabolismo , Mutación , Pseudomonas putida/genética , Terpenos/farmacología , Factores de Transcripción
15.
J Immunol ; 197(5): 1567-76, 2016 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-27439515

RESUMEN

Contact hypersensitivity (CHS) of murine skin serves as a model of allergic contact dermatitis. Hapten-specific CD8 T cells and neutrophils represent the major effector cells driving this inflammatory reaction whereas Foxp3(+) regulatory T cells (Tregs) control the severity of inflammation. However, whether in vivo expansion of endogenous Tregs can downregulate CHS-mediated inflammation remains to be elucidated. In this study, we addressed this issue by using injection of an IL-2/anti-IL-2 mAb JES6-1 complex (IL-2/JES6-1) as a means of Treg induction in 2,4,6-trinitrochlorobenzene-induced CHS. IL-2/JES6-1 injection before or after hapten sensitization led to a considerable reduction of skin inflammation, even when rechallenged up to 3 wk after the last treatment. Conversely, Treg depletion re-established the CHS response in IL-2/JES6-1-treated mice. IL-2/JES6-1 injection resulted in increased frequencies of natural and peripheral Tregs in spleen and draining lymph nodes (LNs), elevated IL-10 and TGF-ß production by CD4 T cells, reduced CD86 expression by dendritic cells, and led to lower numbers of hapten-specific IFN-γ-producing CD8 T effector cells in LNs. Neutrophil and CD8 T cell infiltration was reduced in inflamed ear tissue, whereas CTLA-4(+)Foxp3(+) Treg frequencies were augmented. Adoptive transfer of LN cells of sensitized mice into recipients treated with IL-2/JES6-1 showed impaired CHS. Our results show that in vivo Treg expansion results in a prolonged CHS suppression, a sustained reduction of hapten-specific CD8 T cells, and a decrease in effector cell influx in inflamed tissue.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Dermatitis por Contacto/inmunología , Inflamación/inmunología , Activación de Linfocitos , Piel/inmunología , Linfocitos T Reguladores/inmunología , Traslado Adoptivo , Animales , Linfocitos T CD4-Positivos/inmunología , Haptenos/inmunología , Inflamación/tratamiento farmacológico , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Interleucina-10/inmunología , Interleucina-2/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Neutrófilos/inmunología , Piel/patología
16.
Biochim Biophys Acta Mol Basis Dis ; 1863(2): 560-568, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-27932059

RESUMEN

Pulmonary fibrosis (PF) is a chronic progressive lung disease without effective medical treatment options leading to respiratory failure and death within 3-5years of diagnosis. The pathological process of PF is driven by aberrant wound-healing involving fibroblasts and myofibroblasts differentiated by secreted profibrotic transforming growth factor ß (TGF-ß1). Classical transient receptor potential 6 (TRPC6), a Na+- and Ca2+-permeable cation channel, is able to promote myofibroblast conversion of primary rat cardiac and human dermal fibroblasts and TRPC6-deficiency impaired wound healing after injury. To study a potential role of TRPC6 in the development of PF we analyzed lung function, gene and protein expression in wild-type (WT) and TRPC6-deficient (TRPC6-/-) lungs utilizing a bleomycin-induced PF-model. Fibrotic WT-mice showed a significant higher death rate while bleomycin-treated TRPC6-deficient mice were partly protected from fibrosis as a consequence of a lower production of collagen and an almost normal function of the respiratory system (reduced resistance and elastance compared to fibrotic WT-mice). On a molecular level TGF-ß1 induced TRPC6 up-regulation, increased Ca2+ influx and nuclear NFAT localization in WT primary murine lung fibroblasts (PMLFs) resulting in higher stress fiber formation and accelerated contraction rates as compared to treated TRPC6-deficient fibroblasts. Therefore, we conclude that TRPC6 is an important determinant for TGF-ß1-induced myofibroblast differentiation during fibrosis and specific channel inhibitors might be beneficial in a future treatment of PF.


Asunto(s)
Pulmón/patología , Miofibroblastos/patología , Fibrosis Pulmonar/metabolismo , Canales Catiónicos TRPC/metabolismo , Animales , Diferenciación Celular , Transdiferenciación Celular , Células Cultivadas , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Eliminación de Gen , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Miofibroblastos/metabolismo , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/patología , Canales Catiónicos TRPC/genética , Canal Catiónico TRPC6 , Factor de Crecimiento Transformador beta1/metabolismo , Regulación hacia Arriba
17.
J Immunol ; 195(5): 2417-28, 2015 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-26202982

RESUMEN

Trehalose-6,6-dimycolate (TDM), the mycobacterial cord factor, is an abundant cell wall glycolipid and major virulence factor of Mycobacterium tuberculosis. Its synthetic analog trehalose-6,6-dibehenate (TDB) is a new adjuvant currently in phase I clinical trials. In rodents, the C-type lectin receptors Mincle and Mcl bind TDB/TDM and activate macrophages and dendritic cells (DC) through the Syk-Card9 pathway. However, it is unknown whether these glycolipids activate human innate immune cells through the same mechanism. We performed in vitro analysis of TDB/TDM-stimulated primary human monocytes, macrophages, and DC; determined C-type lectin receptor expression; and tested the contribution of SYK, MINCLE, and MCL by small interfering RNA knockdown and genetic complementation. We observed a robust chemokine and cytokine release in response to TDB or TDM. MCSF-driven macrophages secreted higher levels of IL-8, IL-6, CCL3, CCL4, and CCL2 after stimulation with TDM, whereas DC responded more strongly to TDB and GM-CSF-driven macrophages were equally responsive to TDB and TDM. SYK kinase and the adaptor protein CARD9 were essential for glycolipid-induced IL-8 production. mRNA expression of MINCLE and MCL was high in monocytes and macrophages, with MINCLE and MCL proteins localized intracellularly under resting conditions. Small interfering RNA-mediated MINCLE or MCL knockdown caused on average reduced TDB- or TDM-induced IL-8 production. Conversely, retroviral expression in murine Mincle-deficient DC revealed that human MINCLE, but not MCL, was sufficient to confer responsiveness to TDB/TDM. Our study demonstrates that SYK-CARD9 signaling plays a key role in TDB/TDM-induced activation of innate immune cells in man as in mouse, likely by engagement of MINCLE.


Asunto(s)
Factores Cordón/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Lectinas Tipo C/inmunología , Proteínas Tirosina Quinasas/inmunología , Receptores Inmunológicos/inmunología , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/metabolismo , Animales , Western Blotting , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Adaptadoras de Señalización CARD/metabolismo , Células Cultivadas , Quimiocinas/inmunología , Quimiocinas/metabolismo , Factores Cordón/química , Factores Cordón/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Citometría de Flujo , Expresión Génica/inmunología , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones Noqueados , Monocitos/inmunología , Monocitos/metabolismo , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/inmunología , Unión Proteica/inmunología , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Interferencia de ARN , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/inmunología , Quinasa Syk
18.
J Immunol ; 193(7): 3779-91, 2014 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-25187663

RESUMEN

The Citrobacter rodentium model mimics the pathogenesis of infectious colitis and requires sequential contributions from different immune cell populations, including innate lymphoid cells (ILCs) and CD4(+) lymphocytes. In this study, we addressed the role of STAT3 activation in CD4(+) cells during host defense in mice against C. rodentium. In mice with defective STAT3 in CD4(+) cells (Stat3(ΔCD4)), the course of infection was unchanged during the innate lymphoid cell-dependent early phase, but significantly altered during the lymphocyte-dependent later phase. Stat3(ΔCD4) mice exhibited intestinal epithelial barrier defects, including downregulation of antimicrobial peptides, increased systemic distribution of bacteria, and prolonged reduction in the overall burden of C. rodentium infection. Immunomonitoring of lamina propria cells revealed loss of virtually all IL-22-producing CD4(+) lymphocytes, suggesting that STAT3 activation was required for IL-22 production not only in Th17 cells, but also in Th22 cells. Notably, the defective host defense against C. rodentium in Stat3(∆CD4) mice could be fully restored by specific overexpression of IL-22 through a minicircle vector-based technology. Moreover, expression of a constitutive active STAT3 in CD4(+) cells shaped strong intestinal epithelial barrier function in vitro and in vivo through IL-22, and it promoted protection from enteropathogenic bacteria. Thus, our work indicates a critical role of STAT3 activation in Th17 and Th22 cells for control of the IL-22-mediated host defense, and strategies expanding STAT3-activated CD4(+) lymphocytes may be considered as future therapeutic options for improving intestinal barrier function in infectious colitis.


Asunto(s)
Citrobacter rodentium/inmunología , Colitis/inmunología , Infecciones por Enterobacteriaceae/inmunología , Interleucinas/inmunología , Mucosa Intestinal/inmunología , Factor de Transcripción STAT3/inmunología , Células Th17/inmunología , Animales , Colitis/genética , Colitis/microbiología , Colitis/patología , Infecciones por Enterobacteriaceae/genética , Infecciones por Enterobacteriaceae/patología , Interleucinas/genética , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Ratones , Ratones Transgénicos , Células Th17/patología , Interleucina-22
20.
Am J Respir Crit Care Med ; 188(12): 1451-9, 2013 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-24251695

RESUMEN

RATIONALE: Pulmonary hypertension (PH) is a life-threatening disease, characterized by pulmonary vascular remodeling. Abnormal smooth muscle cell proliferation is a primary hallmark of chronic hypoxia-induced PH. Essential for cell growth are alterations in the intracellular Ca(2+) homeostasis. Classical transient receptor potential (TRPC) proteins have been suggested to contribute to PH development, as TRPC1 and TRPC6 are predominantly expressed in precapillary pulmonary arterial smooth muscle cells (PASMC). Studies in a TRPC6-deficient mouse model revealed an essential function of TRPC6 in acute but not in chronic hypoxia. OBJECTIVES: We aimed to identify the importance of TRPC1 in the pathogenesis of chronic hypoxia-induced PH in mice. METHODS: TRPC1 expression analysis was performed using real-time polymerase chain reaction. TRPC1 function was assessed by in vivo experiments in TRPC1(-/-) animals as well as in isolated precapillary murine PASMC after TRPC1 knockdown by TRPC1-specific small interfering RNAs. MEASUREMENTS AND MAIN RESULTS: Only TRPC1 mRNA was up-regulated under hypoxia in isolated murine PASMC (1% O2 for 72 h). Hypoxia-induced proliferation of murine PASMC was attenuated in cells treated with small interfering RNA against TRPC1 and in cells isolated from TRPC1(-/-) animals compared with untreated and wild-type cells. TRPC1(-/-) mice did not develop PH in response to chronic hypoxia (FI(O2) 0.10 for 21 d) and had less vascular muscularization but a similar degree of right ventricular hypertrophy compared with wild-type mice. CONCLUSIONS: Our results indicate an important role of TRPC1 in pulmonary vascular remodeling underlying the development of hypoxia-induced PH.


Asunto(s)
Hipertensión Pulmonar/metabolismo , Hipoxia/complicaciones , Canales Catiónicos TRPC/metabolismo , Animales , Biomarcadores/metabolismo , Western Blotting , Células Cultivadas , Enfermedad Crónica , Femenino , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/patología , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Canales Catiónicos TRPC/deficiencia , Regulación hacia Arriba
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