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Currently, plastic materials are an integral part of our lives, but their production mostly bases on fossil fuels or derivatives, which resources are decreasing. Extraction and processing of non-renewable resources have also negative impact on environment. One of the most promising and environmentally friendly approaches is use of microorganism. This PhD dissertation presents the non-conventional yeast Arxula adeninivorans as a host for production of bio-based and biodegradable poly(hydroxyalkanoates) plastics poly(hydroxybutyrate) and co-polymer poly(hydroxybutyrate-co-hydroxyvalerate). Additionally, the constructed yeast strain was able to secrete enantiomerically pure (R)-3-hydroxybutyric acid.
The production of PHAs requires three enzymes: β-ketothiolase, acetoacetyl-CoA reductase and PHA synthase. The strategy followed in this project was divided into two parts. While all three enzymes are responsible for intracellular production of PHA polymer, first two only lead to secretion of (R)-3-HB into culture media, which was used in a first stage of work to establish and optimize polymer production. Both, different bacterial strains and yeast A. adeninivorans were taken into account in screening of the genes encoding aforementioned enzymes. Bacterial genes were chemically synthesized using codon optimization pattern and endogenous genes were obtained using PCR and genomic DNA template from A. adeninivorans LS3 wild-type strain. Each gene was cloned into Xplor2 vector between TEF1 constitutive promoter and PHO5 terminator. Vector containing both thiolase and reductase genes was used for A. adeninivorans transformation.
The best combination of heterologous genes was overexpression of β-ketothiolase gene from Clostridium acetobutylicum and acetoacetyl-CoA reductase gene from Cupriavidus necator which led to secretion of 4.84 g L−1 (R)-3-HB, at a rate of 0.023 g L−1 h−1 over 214 h in shaking flask cultivation. Further optimization by fed-batch culturing with glucose as a carbon source did not improve (R)-3-HB secretion, but the rate of production was doubled to 0.043 g L−1 h−1 [3.78 g L−1 of (R)-3-HB at 89 h].
The product of acetoacetyl-CoA reductase is (R)-3-HB-CoA and further removing of CoA moiety is needed for acid secretion into culture media. A. adeninivorans is able to conduct this process without any additional modification but the conversion rate is unknown. Two thioesterases, cytosolic TesBp encoded by TesB gene from E. coli and mitochondrial ATes1p encoded by ATES1 gene from A. adeninivorans, were analysed to enhance secretion process. Additionally, a cytosolic version of ATES1 gene (ATES1cyt) was tested. All three genes were expressed in A. adeninivorans cells under TEF1 constitutive promoter together with thiolase and reductase genes. Despite detected enzymatic activity the yield of (R)-3-HB synthesis and secretion was not increased. Moreover, overexpressed thioesterases negatively influenced cell growth, indicating that they act on other metabolic components. The results provided two sets of information, first, the endogenous secretion system is sufficient for (R)-3-HB production; second, further screening of suitable genes needs to be performed.
Based on optimization of (R)-3-HB synthesis, thiolase gene (thl) from C. acetobutylicum and reductase gene (phaB) from C. necator were chosen to combine with PHA synthase gene (phaC) for creating the PHB-V producing strain. The PHA synthase expression module, containing TEF1 promoter and PHO5 terminator, was cloned into Xplor2 vector together with thiolase and reductase expression modules and used for A. adeninivorans transformation. The engineered strain accumulated up to 7.47% PHB of dcw. During the set of cells passaging A. adeninivorans lost the ability to accumulate polymer with maximal 23.1 % of primary accumulation level. Additionally, use of a vector including hygromycin B antibiotic resistance marker (instead of auxotrophic marker in Xplor2) did not improve polymer accumulation and stability.
To counteract the effect of loss of accumulation stability, phasin gene (phaP1), originated from C. necator, was introduce together with PHA pathway genes. First screening cultivations resulted in stabilizing of polymer production reaching 9.58 % PHB of dcw and only 12.0 % loss of production ability. Further experiments increased PHB content with 19.9% PHB of dcw (3.85 g L-1) after 180 h of cultivation using rich medium. Use of another thiolase gene, the second thiolase from C. necator (bktB), which theoretically should induce production of PHBV copolymer, led to accumulation only 11.4% PHB of dcw after 139 h and no PHV fraction was detected.
Variation of the ratio between flask volume and amount of media influences the level of aeration. Importantly, decrease of aeration level significantly increased polymer synthesis. Additionally, PHB-V copolymer accumulation has been induced by use of different carbon source co-substrates. Use of rich media supplemented with ethanol allow the strain with thl thiolase to accumulate up to 42.9 % PHB of dcw without PHV fraction and with bktB thiolase to 30.5 % PHB of dcw. Nevertheless, despite of lower total amount of polymer, supplementation with 1-propanol allow both strains to accumulate PHB-V copolymer with 7.30 %mol and 22.5 %mol of PHV for thl and bktB strains, respectively.
Optimization based on genetic engineering further enhanced polymer production yield led to exceeding of 50 % PHB-V of dcw. For doubling the gene dosage, PHA synthesizing strains of A. adeninivorans were again transformed with Xplor2 vector containing PHA pathway genes. Resulting strains exhibited twice the level of enzymatic activities of thiolase and reductase compared with strains transformed once with expression vector. In a shaking flask experiment the strain transformed twice with vector containing bktB thiolase reached after 240 h 52.1% PHB-V of dcw (10.8 g L-1) with 12.3 %mol of PHV fraction which is the highest level found in yeast. As another genetic approach, a fusion strain has been created. Two different strains have been established and merged using protoplast fusion technique. Doubling of genetic material resulted in similar level of copolymer produced by Arxula as in former experiments (50.2% of dcw, 10.7 g L-1).
Culture conditions were optimized in controllable cultivation using fed-batch mode. Although optimal oxygen and pH level and continuous carbon source and nitrogen feeding were maintained, final polymer level in % of dry mass was around three times lower than for shaking flask experiment. Nevertheless, efficient growth of Arxula in fed-batch mode led to increase of total copolymer level in g L-1 (16.5 g L-1 compare to 10.8 g L-1 for shaking flasks) showing the feasibility of using Arxula strain for up-scaling production of copolymer.
Acetyl-CoA is a main precursor in synthesis of PHB-V copolymer and change of its pool was investigated. ATP citrate lyase is a cytosolic enzyme converting citrate into oxaloacetate and acetyl-CoA, supporting the biosynthesis of fatty acids. Two genes encoding Acl subunits from Aspergillus nidulans (AnAcl1 and AnAcl2) were again cloned into Xplor2 vector and transformed into A. adeninivorans PHA producing strain. Despite of higher enzymatic activity of AnAclp, accumulation of polymer was around three times higher for control without expression of lyase genes. Expectedly, the strain expressing AnAcl1/2 genes accumulated larger amount of each stearic, palmitic and oleic acid in both standard and fatty acid inducing conditions (lower nitrogen level). Thus, overexpression of AnAcl1/2 genes in A. adeninevorans cells may improve biosynthesis of fatty acids but is ineffective for PHB polymer accumulation.
The aim of the project was use of starch-based media, manufactured as by-products, for polymer production. Genetically engineered Arxula strains were cultivated using these media instead of glucose-based media. Although yeast cells were both able to secrete (R)-3-HB and to accumulate PHB, the yield was lower than for previous media. Additionally, only trace of PHV was found at the end of cultivation time when 1-propanol was supplemented. Obtained results showed that use of cheaper media is a promising approach to decrease production costs but further optimization needs to be performed especially for extended scale of production.
Determination of produced copolymer has been done based on microscopic analysis and studies of physical and chemical properties. Results revealed that Arxula accumulated PHA polymer in cytosolic granules with a similar size range compared to the ones produced by bacteria. The physicochemical study showed that produced polymer exhibited slightly different properties in comparison to bacterial polymer with similar content of PHV, i.e. very-low molecular mass, higher melting and glass transition temperature.
All above results showed that A. adeninivorans is a promising host for PHB-V production. Expression of phasin greatly increased production and stability of polymer, which led to an accumulation level never found before in yeast. Further optimization in higher production scale using cheap starch-based media may establish Arxula strain as a valuable tool for industrial production of PHB-V copolymer.
Optomechanical (om) systems are characterized by their nonlinear light-matter interaction. This is responsible for unique dynamic properties and allows the detection of a variety of classical and quantum mechanical phenomena on a microscopic as well as on a macroscopic scale. In this work we have studied the dynamic behavior of two laser-driven om systems, the single om cell ("cavity optomechanics / membrane-in-the-middle setup") and a two-dimensional hexagonal array of these cells ("om graphene"). The first case was motivated by the possibility to detect the transition from quantum mechanics to classical mechanics directly on the basis of the dynamic behavior. For this we focus on multistability effects of the optical and mechanical degrees of freedom, that are modeled by harmonic oscillators. Our description is based on the quantum optical master equation, which takes into account the environmental interaction assuming a vanishing temperature. As a consequence of decoherence, the dynamics occur near the semiclassical limit, i.e. it is characterized by quantum fluctuations. The quantum-to-classical transition is realized formally by rescaling the equations of motion. In the classical limit, quantum fluctuations disappear and the mean field equations were evaluated by analytical and numerical methods. We found that classical multistability is characterized by stationary signatures on the route to chaos, as well as by the coexistence of single-periodic orbits for the mechanical degree of freedom. The latter point was extensively evaluated by means of a self-consistent approach. For the dynamics in the quantum regime quantum fluctuations cannot be neglected. For this purpose, the master equation was solved by means of a numerical implementation of the Quantum State Diffusion (QSD) method. Based on Wigner and autocorrelation functions, we were able to show that quantum multistability is a dynamic effect: chaotic dynamics is suppressed and there is a time-dependent distribution of the phase space volume on classical simple-periodic orbits. The results can be interpreted within a semiclassical picture, which makes use of the single QSD quantum trajectory. Accordingly, the quantum-classical transition is explained as a time-scale effect, which is determined by tunneling probabilities in an effective mean-field potential. The subject of the second part of the work is the transport of low-energy Dirac quasiparticles in om graphene, propagating as light and sound waves. For this purpose, we investigated the scattering of a plane light wave by laser-induced photon-phonon coupling planar and circular barriers. The starting point is the om Dirac equation, which results from the continuum approximation of the Hamiltonian description of the two-dimensional array near the semiclassical limit. This work was motivated by the rich and interesting relativistic transport and tunneling phenomena found for electrons in graphene, which now appear in a new way. The reason is the presence of the new spin degree of freedom, which distinguishes the optical and mechanical excitations. In this spin space, the om interaction can be understood as a potential, which in our analysis consists of a time-independent and a time-dependent sinusoidal part. For the first case of a static barrier, the transport is elastic and is characterized by stationary scattering signatures. After solving the scattering problem via continuity conditions we were able to identify different scattering regimes depending on scattering parameters. In addition to relativistic phenomena such as Klein tunneling, simple parameter variation allows to use the barrier as a resonant light-sound interconverter and angle-dependent emitter. For the oscillating barrier, the transport is inelastic and is characterized by dynamic scattering signatures. To solve the time-periodic scattering problem, we have applied the Floquet theory for an effective two-level system. As a result of the barrier oscillation, photons and phonons can get and give away energy portions in the form of integer multiples of the oscillation frequency. The interference of short (classical) and long-wave (quantum) components leads to mixing of the scattering regimes. This allows to use the barrier as a time-periodic light-sound interconverter with interesting radiation characteristics. In addition, we have argued that the oscillating barrier provides the necessary energetic conditions for detecting zitterbewegung.
Objectives: We aimed to update the 2010 evidence- and consensus-based national clinical guideline on the diagnosis and management of uncomplicated urinary tract infections (UTIs) in adult patients. Materials and Methods: An interdisciplinary group consisting of 17 representatives of 12 medical societies and a patient representative was formed. Systematic literature searches were conducted in MEDLINE, EMBASE, and the Cochrane Library to identify literature published in 2010–2015. Results: We provide 75 recommendations and 68 statements in the updated evidence- and consensus-based national clinical guideline. The diagnostics part covers practical recommendations on cystitis and pyelonephritis for each defined patient group. Clinical examinations, as well as laboratory testing and microbiological pathogen assessment, are addressed. Conclusion: In accordance with the global antibiotic stewardship initiative and considering new insights in scientific research, we updated our German clinical UTI guideline to promote a responsible antibiotic use and to give clear hands-on recommendations for the diagnosis and management of UTIs in adults in Germany for healthcare providers and patients.
Significance of Hyperbaric Oxygenation in the Treatment of Fournier’s Gangrene: A Comparative Study
(2018)
Introduction: Hyperbaric oxygenation (HBO), in addition to anti-infective and surgical therapy, seems to be a key treatment point for Fournier’s gangrene. The aim of this study was to investigate the influence of HBO therapy on the outcome and prognosis of Fournier’s gangrene. Patients and Methods: In the present multicenter, retrospective observational study, we evaluated the data of approximately 62 patients diagnosed with Fournier’s gangrene between 2007 and 2017. For comparison, 2 groups were distinguished: patients without HBO therapy (group A, n = 45) and patients with HBO therapy (group B, n = 17). The analysis included sex, age, comorbidities, clinical symptoms, laboratory and microbiological data, debridement frequency, wound dressing, antibiotic use, outcome and prognosis. The statistical analysis was performed with GraphPad Prism 7® (GraphPad Software, Inc., La Jolla, USA). Results: Demographic data showed no significant differences. The laboratory parameters C-reactive protein and urea were significantly higher in group B (group B: 301.7 vs. 140.6 mg/dL; group A: 124.8 vs. 54.7 mg/dL). Sepsis criteria were fulfilled in 77.8 and 100% of the patients in groups A and B respectively. Treatment in the intensive care unit (ICU) was therefore indicated in 69% of the patients in group A and 100% of the patients in group B. The mean ICU stay was 9 and 32 days for patients in groups A and B respectively. The wound debridement frequency and hospitalization stay were significantly greater in group B (13 vs. 5 debridement and 40 vs. 22 days). Initial antibiosis was test validated in 80% of the patients in group A and 76.5% of the patients in group B. Mortality was 0% in group B and 4.4% in the group A. Conclusion: The positive influence of HBO on the treatment of Fournier’s gangrene can be estimated only from the available data. Despite poorer baseline findings with comparable risk factors, mortality was 0% in the HBO group. The analysis of a larger patient cohort is desirable to increase the significance of the results.
Background: We aimed to update the 2010 evidence- and consensus-based national clinical guideline on the diagnosis and management of uncomplicated urinary tract infections (UTIs) in adult patients. Results are published in 2 parts. Part 1 covers methods, the definition of patient groups, and diagnostics. This second publication focuses on treatment of acute episodes of cystitis and pyelonephritis as well as on prophylaxis of recurrent UTIs. Materials and Methods: An interdisciplinary group consisting of 17 representatives of 12 medical societies and a patient representative was formed. Systematic literature searches were conducted in MEDLINE, EMBASE, and the Cochrane Library to identify literature published in 2010–2015. Results: For the treatment of acute uncomplicated cystitis (AUC), fosfomycin-trometamol, nitrofurantoin, nitroxoline, pivmecillinam, and trimethoprim (depending on the local rate of resistance) are all equally recommended. Cotrimoxazole, fluoroquinolones, and cephalosporins are not recommended as antibiotics of first choice, for concern of an unfavorable impact on the microbiome. Mild to moderate uncomplicated pyelonephritis should be treated with oral cefpodoxime, ceftibuten, ciprofloxacin, or levofloxacin. For AUC with mild to moderate symptoms, instead of antibiotics symptomatic treatment alone may be considered depending on patient preference after discussing adverse events and outcomes. Primarily non-antibiotic options are recommended for prophylaxis of recurrent urinary tract infection. Conclusion: In accordance with the global antibiotic stewardship initiative and considering new insights in scientific research, we updated our German clinical UTI guideline to promote a responsible antibiotic use and to give clear hands-on recommendations for the diagnosis and management of UTIs in adults in Germany for healthcare providers and patients.
Significance of Hyperbaric Oxygenation in the Treatment of Fournier’s Gangrene: A Comparative Study
(2018)
Introduction: Hyperbaric oxygenation (HBO), in addition to anti-infective and surgical therapy, seems to be a key treatment point for Fournier’s gangrene. The aim of this study was to investigate the influence of HBO therapy on the outcome and prognosis of Fournier’s gangrene. Patients and Methods: In the present multicenter, retrospective observational study, we evaluated the data of approximately 62 patients diagnosed with Fournier’s gangrene between 2007 and 2017. For comparison, 2 groups were distinguished: patients without HBO therapy (group A, n = 45) and patients with HBO therapy (group B, n = 17). The analysis included sex, age, comorbidities, clinical symptoms, laboratory and microbiological data, debridement frequency, wound dressing, antibiotic use, outcome and prognosis. The statistical analysis was performed with GraphPad Prism 7® (GraphPad Software, Inc., La Jolla, USA). Results: Demographic data showed no significant differences. The laboratory parameters C-reactive protein and urea were significantly higher in group B (group B: 301.7 vs. 140.6 mg/dL; group A: 124.8 vs. 54.7 mg/dL). Sepsis criteria were fulfilled in 77.8 and 100% of the patients in groups A and B respectively. Treatment in the intensive care unit (ICU) was therefore indicated in 69% of the patients in group A and 100% of the patients in group B. The mean ICU stay was 9 and 32 days for patients in groups A and B respectively. The wound debridement frequency and hospitalization stay were significantly greater in group B (13 vs. 5 debridement and 40 vs. 22 days). Initial antibiosis was test validated in 80% of the patients in group A and 76.5% of the patients in group B. Mortality was 0% in group B and 4.4% in the group A. Conclusion: The positive influence of HBO on the treatment of Fournier’s gangrene can be estimated only from the available data. Despite poorer baseline findings with comparable risk factors, mortality was 0% in the HBO group. The analysis of a larger patient cohort is desirable to increase the significance of the results.
Rabies virus (RABV) is an ancient, highly neurotropic rhabdovirus that causes lethal encephalitis. Most RABV pathogenesis determinants have been identified with laboratory-adapted or attenuated RABVs, but details of natural RABV pathogenesis and attenuation mechanisms are still poorly understood. To provide a deeper insight in the cellular mechanism of pathogenies of field RABV, this work was performed to assess virus strain specific differences in intra-neuronal virus transport, to identify cell culture adaptive mutations in recombinant field viruses and to explore shRNA-expressing RABVs as research tools for targeted host manipulation in infected cells.
Comparison of chimeric RABVs with glycoprotein (G) ecto-domains of different lyssaviruses, together with field RABVs from dog and fox in dorsal root ganglion (DRG) neurons revealed no detectable differences in the axonal accumulation of the viruses. This indicates that previously described G-dependent transport of newly formed RABV in axons can occur both in laboratory-adapted and field RABV. Moreover, partial overlap of nucleoprotein (N) and G protein particles in field virus infected DRG axons supported the hypothesis of the “separate model” for anterograde RABV transport.
Serial passages of recombinant dog and fox field clones in different cell lines led to the identification of general (D266N) and cell line specific (K444N) adaptive mutations in the G ecto-domain of both viruses. In BHK cells, synergistic effects of D226N, K444N and A417T on field dog virus G protein surface localization led to the loss of endoplasmic reticulum (ER) retention of G and increased virus titers in the supernatant, indicating that limited virus release by ER retention is a major bottleneck in cell culture adaptation. In addition, selection of mutations within the C-terminus of the RABV phosphoprotein (P) (R293H and R293C in fox and dog viruses, respectively) led to the hypothesis of altered binding affinities to nucleoprotein and RNP complexes. Identification of the above mentioned amino acid substitutions together with alterations in a suboptimal transcription stop signal in the P/M gene border indicated that adaptation to cell culture replication occurs on both levels, RNA transcription/replication and virus release.
To evaluate the possibility of an expression of a functional microRNA-adapted short-hairpin RNAs (miR-shRNA) expressing RABV, recombinant RABVs encoding miR-shRNAs against cellular Dynein Light Chain 1 (DYNLL1) and Acidic Nuclear Phosphoprotein 32 family member B (ANP32B) were generated. In spite of cytoplasmic transcription of the respective mRNAs, downregulation of DYNLL1 and ANP32B mRNA and respective protein levels in infected cells revealed correct processing to functional shRNAs. Specific downregulation of the cellular genes at 2, 3 and 4 days post infection further demonstrated feasibility of the approach in standard cell lines. However, it remained open whether miR-shRNA expressing RABV can be used to study neuro-infection in vivo. Since first attempts in primary rat neuron cultures failed, it has to be clarified in further experiments whether this strategy can be used in mature, non-dividing neurons or whether breakdown of the nucleus in the course of cell division is a requirement for the processing of cytoplasmically expressed miR-RNA by nuclear RNases.
By providing novel insights in axonal RABV transport and cell culture adaptive mutations this work extends the current understanding of RABV pathogenesis in natural and non-natural cell environments. Moreover, it provides a basis for further pathogenicity studies in which the impact of cell culture adaptation through increased virus release on RABV virulence can be investigated. With successful expression of functional miR-shRNAs from RABV vectors, this work also provides a tool for RABV gene targeting in infected cell lines and thus may contribute to the further investigation of RABV-host-cell-interactions.
Climate Change-Induced Shift of Tree Growth Sensitivity at a Central Himalayan Treeline Ecotone
(2018)
Introduction
We retrospectively evaluated the efficacy and toxicity of gross tumor volume (GTV) mean dose optimized stereotactic body radiation therapy (SBRT) for primary and secondary lung tumors with and without robotic real-time motion compensation.
Materials and methods
Between 2011 and 2017, 208 patients were treated with SBRT for 111 primary lung tumors and 163 lung metastases with a median GTV of 8.2 cc (0.3–174.0 cc). Monte Carlo dose optimization was performed prioritizing GTV mean dose at the potential cost of planning target volume (PTV) coverage reduction while adhering to safe normal tissue constraints. The median GTV mean biological effective dose (BED)10 was 162.0 Gy10 (34.2–253.6 Gy10) and the prescribed PTV BED10 ranged 23.6–151.2 Gy10 (median, 100.8 Gy10). Motion compensation was realized through direct tracking (44.9%), fiducial tracking (4.4%), and internal target volume (ITV) concepts with small (≤5 mm, 33.2%) or large (>5 mm, 17.5%) motion. The local control (LC), progression-free survival (PFS), overall survival (OS), and toxicity were analyzed.
Results
Median follow-up was 14.5 months (1–72 months). The 2-year actuarial LC, PFS, and OS rates were 93.1, 43.2, and 62.4%, and the median PFS and OS were 18.0 and 39.8 months, respectively. In univariate analysis, prior local irradiation (hazard ratio (HR) 0.18, confidence interval (CI) 0.05–0.63, p = 0.01), GTV/PTV (HR 1.01–1.02, CI 1.01–1.04, p < 0.02), and PTV prescription, mean GTV, and maximum plan BED10 (HR 0.97–0.99, CI 0.96–0.99, p < 0.01) were predictive for LC while the tracking method was not (p = 0.97). For PFS and OS, multivariate analysis showed Karnofsky Index (p < 0.01) and tumor stage (p ≤ 0.02) to be significant factors for outcome prediction. Late radiation pneumonitis or chronic rip fractures grade 1–2 were observed in 5.3% of the patients. Grade ≥3 side effects did not occur.
Conclusion
Robotic SBRT is a safe and effective treatment for lung tumors. Reducing the PTV prescription and keeping high GTV mean doses allowed the reduction of toxicity while maintaining high local tumor control. The use of real-time motion compensation is strongly advised, however, well-performed ITV motion compensation may be used alternatively when direct tracking is not feasible.
Background
The role of platelets for mediating closure of the ductus arteriosus in human preterm infants is controversial. Especially, the effect of low platelet counts on pharmacological treatment failure is still unclear.
Methods
In this retrospective study of 471 preterm infants [<1,500 g birth weight (BW)], who were treated for a patent ductus arteriosus (PDA) with indomethacin or ibuprofen, we investigated whether platelet counts before or during pharmacological treatment had an impact on the successful closure of a hemodynamically significant PDA. The effects of other factors, such as sepsis, preeclampsia, gestational age, BW, and gender, were also evaluated.
Results
Platelet counts before initiation of pharmacological PDA treatment did not differ between infants with later treatment success or failure. However, we found significant associations between low platelet counts during pharmacological PDA therapy and treatment failure (p < 0.05). Receiver operating characteristic (ROC) curve analysis showed that platelet counts after the first, and before and after the second cyclooxygenase inhibitor (COXI) cycle were significantly associated with treatment failure (area under the curve of >0.6). However, ROC curve analysis did not reveal a specific platelet cutoff-value that could predict PDA treatment failure. Multivariate logistic regression analysis showed that lower platelet counts, a lower BW, and preeclampsia were independently associated with COXI treatment failure.
Conclusion
We provide further evidence for an association between low platelet counts during pharmacological therapy for symptomatic PDA and treatment failure, while platelet counts before initiation of therapy did not affect treatment outcome.
Tuberculosis (TB) has tremendous public health relevance. It most frequently affects the lung and is characterized by the development of unique tissue lesions, termed granulomas. These lesions encompass various immune populations, with macrophages being most extensively investigated. Myeloid derived suppressor cells (MDSCs) have been recently identified in TB patients, both in the circulation and at the site of infection, however their interactions with Mycobacterium tuberculosis (Mtb) and their impact on granulomas remain undefined. We generated human monocytic MDSCs and observed that their suppressive capacities are retained upon Mtb infection. We employed an in vitro granuloma model, which mimics human TB lesions to some extent, with the aim of analyzing the roles of MDSCs within granulomas. MDSCs altered the structure of and affected bacterial containment within granuloma-like structures. These effects were partly controlled through highly abundant secreted IL-10. Compared to macrophages, MDSCs activated primarily the NF-κB and MAPK pathways and the latter largely contributed to the release of IL-10 and replication of bacteria within in vitro generated granulomas. Moreover, MDSCs upregulated PD-L1 and suppressed proliferation of lymphocytes, albeit with negligible effects on Mtb replication. Further comprehensive characterization of MDSCs in TB will contribute to a better understanding of disease pathogenesis and facilitate the design of novel immune-based interventions for this deadly infection.
Medical education research has focused almost entirely on the education of future physicians. In comparison, findings on other health-related occupations, such as medical assistants, are scarce. With the current study, we wanted to examine the knowledge-is-power hypothesis in a real life educational setting and add to the sparse literature on medical assistants. Acquisition of vocational knowledge in vocational education and training (VET) was examined for medical assistant students (n = 448). Differences in domain-specific vocational knowledge were predicted by crystallized and fluid intelligence in the course of VET. A multiple matrix design with 3 year-specific booklets was used for the vocational knowledge tests of the medical assistants. The unique and joint contributions of the predictors were investigated with structural equation modeling. Crystallized intelligence emerged as the strongest predictor of vocational knowledge at every stage of VET, while fluid intelligence only showed weak effects. The present results support the knowledge-is-power hypothesis, even in a broad and more naturalistic setting. This emphasizes the relevance of general knowledge for occupations, such as medical assistants, which are more focused on learning hands-on skills than the acquisition of academic knowledge.