DMSO and Its Role in Differentiation Impact Efficacy of Human Adenovirus (HAdV) Infection in HepaRG Cells.

Differentiated HepaRG cells are popular in vitro cell models for hepatotoxicity studies. Their differentiation is usually supported by the addition of dimethyl sulfoxide (DMSO), an amphipathic solvent widely used in biomedicine, for example, in potential novel therapeutic drugs and cryopreservation of oocytes. Recent studies have demonstrated drastic effects, especially on epigenetics and extracellular matrix composition, induced by DMSO, making its postulated inert character doubtful. In this work, the influence of DMSO and DMSO-mediated modulation of differentiation on human adenovirus (HAdV) infection of HepaRG cells was investigated. We observed an increase in infectivity of HepaRG cells by HAdVs in the presence of 1% DMSO. However, this effect was dependent on the type of medium used for cell cultivation, as cells in William's E medium showed significantly stronger effects compared with those cultivated in DMEM. Using different DMSO concentrations, we proved that the impact of DMSO on infectability was dose-dependent. Infection of cells with a replication-deficient HAdV type demonstrated that the mode of action of DMSO was based on viral entry rather than on viral replication. Taken together, these results highlight the strong influence of the used cell-culture medium on the performed experiments as well as the impact of DMSO on infectivity of HepaRG cells by HAdVs. As this solvent is widely used in cell culture, those effects must be considered, especially in screening of new antiviral compounds.

Receptor transfer between immune cells by autoantibody-enhanced, CD32-driven trogocytosis is hijacked by HIV-1 to infect resting CD4 T cells.

Immune cell phenotyping frequently detects lineage-unrelated receptors. Here, we report that surface receptors can be transferred from primary macrophages to CD4 T cells and identify the Fcγ receptor CD32 as driver and cargo of this trogocytotic transfer. Filamentous CD32+ nanoprotrusions deposit distinct plasma membrane patches onto target T cells. Transferred receptors confer cell migration and adhesion properties, and macrophage-derived membrane patches render resting CD4 T cells susceptible to infection by serving as hotspots for HIV-1 binding. Antibodies that recognize T cell epitopes enhance CD32-mediated trogocytosis. Such autoreactive anti-HIV-1 envelope antibodies can be found in the blood of HIV-1 patients and, consistently, the percentage of CD32+ CD4 T cells is increased in their blood. This CD32-mediated, antigen-independent cell communication mode transiently expands the receptor repertoire and functionality of immune cells. HIV-1 hijacks this mechanism by triggering the generation of trogocytosis-promoting autoantibodies to gain access to immune cells critical to its persistence.

Resistance training alters body composition in middle-aged women depending on menopause - A 20-week control trial.

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.

SUMO Modification of Hepatitis B Virus Core Mediates Nuclear Entry, Promyelocytic Leukemia Nuclear Body Association, and Efficient Formation of Covalently Closed Circular DNA.

Persistence of hepatitis B virus (HBV) infection is due to a nuclear covalently closed circular DNA (cccDNA), generated from the virion-borne relaxed circular DNA (rcDNA) genome in a process likely involving numerous cell factors from the host DNA damage response (DDR). The HBV core protein mediates rcDNA transport to the nucleus and likely affects stability and transcriptional activity of cccDNA. Our study aimed at investigating the role of HBV core protein and its posttranslational modification (PTM) with SUMO (small ubiquitin-like modifiers) during the establishment of cccDNA. HBV core protein SUMO PTM was analyzed in His-SUMO-overexpressing cell lines. The impact of HBV core SUMOylation on association with cellular interaction partners and on the HBV life cycle was determined using SUMOylation-deficient mutants of the HBV core protein. Here, we show that the HBV core protein is posttranslationally modified by the addition of SUMO and that this modification impacts nuclear import of rcDNA. By using SUMOylation-deficient HBV core mutants, we show that SUMO modification is a prerequisite for the association with specific promyelocytic leukemia nuclear bodies (PML-NBs) and regulates the conversion of rcDNA to cccDNA. By in vitro SUMOylation of HBV core, we obtained evidence that SUMOylation triggers nucleocapsid disassembly, providing novel insights into the nuclear import process of rcDNA. HBV core protein SUMOylation and subsequent association with PML bodies in the nucleus constitute a key step in the conversion of HBV rcDNA to cccDNA and therefore a promising target for inhibiting formation of the HBV persistence reservoir. IMPORTANCE HBV cccDNA is formed from the incomplete rcDNA involving several host DDR proteins. The exact process and the site of cccDNA formation are poorly understood. Here, we show that HBV core protein SUMO modification is a novel PTM regulating the function of HBV core. A minor specific fraction of the HBV core protein resides with PML-NBs in the nuclear matrix. SUMO modification of HBV core protein mediates its recruitment to specific PML-NBs within the host cell. Within HBV nucleocapsids, SUMOylation of HBV core induces HBV capsid disassembly and is a prerequisite for nuclear entry of HBV core. SUMO HBV core protein association with PML-NBs is crucial for efficient conversion of rcDNA to cccDNA and for the establishment of the viral persistence reservoir. HBV core protein SUMO modification and the subsequent association with PML-NBs might constitute a potential novel target in the development of drugs targeting the cccDNA.

Combinatory Effects of Training and Nutritive Administration of Carbohydrates and Protein via Food on Strength in Postmenopausal Women, and Old Men and Women.

The age-related loss of muscle mass promotes many impairments. Training and protein supplementation are suggested to prevent muscle wasting, but recommendations for all populations are not based on scientific evidence. This study combines protein/carbohydrate supplementation (PCS) and training for seniors and postmenopausal women. Project A: 51 postmenopausal women (PMW, 57.3 ± 3.0 years old) underwent health-oriented training (12 weeks, moderate-strength training + moderate-endurance training). The intervention group (IG) additionally received 110 g sour milk cheese (SMC) and toast. Project B: 25 women and 6 men (65.9 ± 4.9 years old) performed intense sling training (12 weeks). The IG additionally received 110 g SMC, toast, and buttermilk. Strength was tested before and after in both studies. Project A: there was significant increase in strength, no additional effect of PCS, and a reduction in body fat in the controls. Project B: there was significant increase in strength, significant additional effects of PCS for trunk strength, and a significant reduction in body weight. Combining training and PCS may counteract strength loss. Combined endurance/resistance training is recommended to PMW for whom the benefits of PCS are restricted. Aged subjects may benefit from PCS when training intensely, but these benefits may be strongly individual.

[Complexity of outpatient intensive care for ventilated people: Cross-mapping into the standardised NNN-taxonomy]. / Komplexität außerstationärer Intensivpflege beatmeter Menschen.

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.

Comparing the quality of care for long-term ventilated individuals at home versus in shared living communities: a convergent parallel mixed-methods study.

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.

SARS-CoV-2 variants of concern display enhanced intrinsic pathogenic properties and expanded organ tropism in mouse models.

SARS-CoV-2 variants of concern (VOCs) display enhanced transmissibility and resistance to antibody neutralization. Comparing the early 2020 isolate EU-1 to the VOCs Alpha, Beta, and Gamma in mice transgenic for human ACE2 reveals that VOCs induce a broadened scope of symptoms, expand systemic infection to the gastrointestinal tract, elicit the depletion of natural killer cells, and trigger variant-specific cytokine production patterns. Gamma infections result in accelerated disease progression associated with increased immune activation and inflammation. All four SARS-CoV-2 variants induce pDC depletion in the lungs, paralleled by reduced interferon responses. Remarkably, VOCs also use the murine ACE2 receptor for infection to replicate in the lungs of wild-type animals, which induce cellular and innate immune responses that apparently curtail the spread of overt disease. VOCs thus display distinct intrinsic pathogenic properties with broadened tissue and host range. The enhanced pathogenicity of VOCs and their potential for reverse zoonotic transmission pose challenges to clinical and pandemic management.

Rapid, efficient and activation-neutral gene editing of polyclonal primary human resting CD4+ T cells allows complex functional analyses.

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.

Genomic epidemiology reveals multiple introductions of SARS-CoV-2 followed by community and nosocomial spread, Germany, February to May 2020.

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.

How to improve the quality of care for people on home mechanical ventilation from the perspective of healthcare professionals: a qualitative study.

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.

Full-Length SSU rRNA Gene Sequencing Allows Species-Level Detection of Bacteria, Archaea, and Yeasts Present in Milk.

Full-length SSU rRNA gene sequencing allows species-level identification of the microorganisms present in milk samples. Here, we used bulk-tank raw milk samples of two German dairies and detected, using this method, a great diversity of bacteria, archaea, and yeasts within the samples. Moreover, the species-level classification was improved in comparison to short amplicon sequencing. Therefore, we anticipate that this approach might be useful for the detection of possible mastitis-causing species, as well as for the control of spoilage-associated microorganisms. In a proof of concept, we showed that we were able to identify several putative mastitis-causing or mastitis-associated species such as Streptococcusuberis, Streptococcusagalactiae, Streptococcusdysgalactiae, Escherichiacoli and Staphylococcusaureus, as well as several Candida species. Overall, the presented full-length approach for the sequencing of SSU rRNA is easy to conduct, able to be standardized, and allows the screening of microorganisms in labs with Illumina sequencing machines.

Amplicon-sequencing of raw milk microbiota: impact of DNA extraction and library-PCR.

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.

Thermal Inactivation of Carcasses of Mice and Rabbits Infected with Pathogens of Risk Groups Two to Four.

Pathogenesis of viruses or other agents that are infectious to humans is frequently studied in vivo using natural or genetically modified animals. Depending on the risk group of the pathogen, the majority of such experimental studies are performed at least under biosafety level 2 (BSL-2) conditions. Biosafety considerations are therefore critical at all steps of research involving potentially infectious pathogens. Inactivation of pathogens studied using in vitro experiments is usually performed using moist heat sterilization. However, few standardized and validated protocols are currently available for the thermal inactivation of carcasses from laboratory animals infected with such human pathogens. To comply with laboratory biologic safety rules and requirements imposed by regulatory authorities, documentation of appropriate inactivation conditions or use of a validated procedure according to national or international standards is critical. In the current study, we evaluated inactivation protocols in a standard laboratory autoclave for carcasses of either frozen mice or recently terminated rabbits, which were placed inside autoclave bags with bedding material in stainless steel containers. Temperature sensors were placed into different tissues of the carcasses to continuously record temperature in situ and in real-time, and a reference sensor was placed in the autoclave. To achieve pathogen inactivation, autoclaving protocols had to be optimized for both species. Frozen mice required 2 different fractionated prevacuum stages, whereas recently terminated rabbits required 3 different fractionated prevacuum stages. This study provides a template for an evaluation procedure to safely and effectively inactivate mice and rabbits infected with risk group 2 to 4 pathogens.

The pentatricopeptide repeat protein Rmd9 recognizes the dodecameric element in the 3'-UTRs of yeast mitochondrial mRNAs.

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.

Simultaneous quantification of the most common and proteolytic Pseudomonas species in raw milk by multiplex qPCR.

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.

Pseudomonas cremoris sp. nov., a novel proteolytic species isolated from cream.

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.

IL-2-Agonist-Induced IFN-γ Exacerbates Systemic Anaphylaxis in Food Allergen-Sensitized Mice.

Food allergies are common, costly and potentially life-threatening disorders. They are driven by Th2, but inhibited by Th1 reactions. There is also evidence indicating that IL-2 agonist treatment inhibits allergic sensitization through expansion of regulatory T cells. Here, we tested the impact of an IL-2 agonist in a novel model for food allergy to hen´s egg in mice sensitized without artificial adjuvants. Prophylactic IL-2 agonist treatment expanded Treg populations and inhibited allergen-specific sensitization. However, IL-2 agonist treatment of already sensitized mice increased mast cell responses and allergic anaphylaxis upon allergen re-challenge. These effects depended on allergen-specific IgE and were mediated through IFN-γ, as shown by IgE transfer and blockade of IFN-γ with monoclonal antibodies. These results suggest that although shifting the allergic reaction toward a Treg/Th1 response inhibits allergic sensitization, the prototypic Th1 cytokine IFN-γ promotes mast cell activation and allergen-induced anaphylaxis in individuals that are already IgE-sensitized. Hence, while a Th1 response can prevent the development of food allergy, IFN-γ has the ability to exacerbate already established food allergy.

Investigation of monoterpenoid resistance mechanisms in Pseudomonas putida and their consequences for biotransformations.

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.