Intermittent hypoxia treatments cause cellular priming in human microglia.

Obstructive sleep apnoea syndrome (OSAS) is a sleep-disordered breathing characterized by nocturnal collapses of the upper airway resulting in cycles of blood oxygen partial pressure oscillations, which lead to tissue and cell damage due to intermittent hypoxia (IH) episodes. Since OSAS-derived IH may lead to cognitive impairment through not fully cleared mechanisms, herein we developed a new in vitro model mimicking IH conditions to shed light on its molecular effects on microglial cells, with particular attention to the inflammatory response. The in vitro model was set-up and validated by measuring the hypoxic state, HIF-1α levels, oxidative stress by ROS production and mitochondrial activity by MTS assay. Then, the mRNA and protein levels of certain inflammatory markers (NF-κB and interleukin 6 (IL-6)) after different IH treatment protocols were investigated. The IH treatments followed by a normoxic period were not able to produce a high inflammatory state in human microglial cells. Nevertheless, microglia appeared to be in a state characterized by increased expression of NF-κB and markers related to a primed phenotype. The microglia exposed to IH cycles and stimulated with exogenous IL-1ß resulted in an exaggerated inflammatory response with increased NF-κB and IL-6 expression, suggesting a role for primed microglia in OSAS-driven neuroinflammation.

The current state of transplant advanced practice providers: results of the advanced practice provider practice survey.

Advanced practice providers (APPs) are trained, licensed health care providers. The American Society of Transplant APP community of practice developed an electronic survey to investigate transplant APP demographics, scope of practice, and academic activities. We defined the top of scope of practice as delivering health care to the fullest extent of APP education and training as allowed by state laws and regulations. From July 11, 2020, to August 31, 2020, 307 invitations were e-mailed and survey links were distributed electronically on the community of practice hub and social media. Two hundred fifty-three APPs responded. APPs practice in inpatient and outpatient settings. Among the respondent APPs, 11.5% assist in the operating room (OR), 46.3% of inpatient and 46.6% of outpatient APPs perform procedures, and 17.8% run specialized APP clinics. 26.2% feel they do not function at the top of their scope of practice and 29.7% were expected to function as a coordinator some or all of the time. Forty-three percent gave invited lectures, 41.5% have published, and 69.2% teach physician trainees. 74.7% and 35.1%, respectively, would like to participate in research and teach but are limited by time, opportunity, and experience. APPs should practice at the top of their scope of practice. Clinical workloads and lack of time limit the ability of APP to teach and contribute to evidence-based practice.

Macrophage phenotype transitions in a stochastic gene-regulatory network model.

Polarization is the process by which a macrophage cell commits to a phenotype based on external signal stimulation. To know how this process is affected by random fluctuations and events within a cell is of utmost importance to better understand the underlying dynamics and predict possible phenotype transitions. For this purpose, we develop a stochastic modeling approach for the macrophage polarization process. We classify phenotype states using the Robust Perron Cluster Analysis and quantify transition pathways and probabilities by applying Transition Path Theory. Depending on the model parameters, we identify four bistable and one tristable phenotype configuration. We find that bistable transitions are fast but their states less robust. In contrast, phenotype transitions in the tristable situation have a comparatively long time duration, which reflects the robustness of the states. The results indicate parallels in the overall transition behavior of macrophage cells with other heterogeneous and plastic cell types, such as cancer cells. Our approach allows for a probabilistic interpretation of macrophage phenotype transitions and biological inference on phenotype robustness. In general, the methodology can easily be adapted to other systems where random state switches are known to occur.

Bifurcation and sensitivity analysis reveal key drivers of multistability in a model of macrophage polarization.

In this paper, we present and analyze a mathematical model for polarization of a single macrophage which, despite its simplicity, exhibits complex dynamics in terms of multistability. In particular, we demonstrate that an asymmetry in the regulatory mechanisms and parameter values is important for observing multiple phenotypes. Bifurcation and sensitivity analyses show that external signaling cues are necessary for macrophage commitment and emergence to a phenotype, but that the intrinsic macrophage pathways are equally important. Based on our numerical results, we formulate hypotheses that could be further investigated by laboratory experiments to deepen our understanding of macrophage polarization.

Thyroid hormone replacement therapy in pregnancy and motor function, communication skills, and behavior of preschool children: The Norwegian Mother, Father, and Child Cohort Study.

PURPOSE: Limited research has focused on the association between prenatal thyroid hormone replacement therapy (THRT) and motor function, communication skills, and behavior in preschool children. Here, we estimated the association between THRT during pregnancy and the first trimester and these developmental outcomes. METHODS: This study was based on the Norwegian Mother, Father, and Child Cohort Study (MoBa) and other national registries. We included mother-child pairs exposed to THRT during pregnancy (n = 663), after delivery (n = 728), or unexposed (n = 28 040). Exposure to THRT was defined according to filled prescriptions. Child outcomes, presented as T-score differences, were parent-reported using the Ages and Stages Questionnaire, Strengths and Difficulties Questionnaire, and Child Behavior Checklist. RESULTS: Of 29 431 mother-child pairs, 2.3% were prenatally exposed to THRT. We found no difference between prenatally exposed and unexposed children in regards to gross motor function (ß: 0.17, 95% CI -1.19, 1.54), fine motor function (ß: -0.17, 95% CI -1.14, 0.80), communication (ß: -0.31, 95% CI -1.58, 0.96), externalizing (ß: -0.03, 95% CI -1.07, 1.01), internalizing (ß: 0.89, 95% CI -0.20, 1.97), or social behaviors (ß: -0.04, 95% CI -0.92, 0.84). Somatic complaints were higher in THRT-exposed children (ß: 0.98, 95% CI 0.08, 1.87), and children whose mothers were exposed after delivery had more sleep problems than unexposed children (ß: 0.99, 95% CI 0.24, 1.74). CONCLUSIONS: Children prenatally exposed to THRT have developmental outcomes as positive as unexposed children on motor function, communication, and behavior. The association with somatic complaints and sleep were not clinically relevant.

Closing the Gap for Electronic Short-Circuiting: Photosystem†I Mixed Monolayers Enable Improved Anisotropic Electron Flow in Biophotovoltaic Devices.

Well-defined assemblies of photosynthetic protein complexes are required for an optimal performance of semi-artificial energy conversion devices, capable of providing unidirectional electron flow when light-harvesting proteins are interfaced with electrode surfaces. We present mixed photosystem I (PSI) monolayers constituted of native cyanobacterial PSI trimers in combination with isolated PSI monomers from the same organism. The resulting compact arrangement ensures a high density of photoactive protein complexes per unit area, providing the basis to effectively minimize short-circuiting processes that typically limit the performance of PSI-based bioelectrodes. The PSI film is further interfaced with redox polymers for optimal electron transfer, enabling highly efficient light-induced photocurrent generation. Coupling of the photocathode with a [NiFeSe]-hydrogenase confirms the possibility to realize light-induced H2 evolution.

Differential effects of selective- and pan-PPAR agonists on experimental steatohepatitis and hepatic macrophages☆.

BACKGROUND & AIMS: Peroxisome proliferator-activated receptors (PPARs) are essential regulators of whole-body metabolism, but also modulate inflammation in immune cells, notably macrophages. We compared the effects of selective PPAR agonists to those of the pan-PPAR agonist lanifibranor in non-alcoholic fatty liver disease (NAFLD), and studied isoform-specific effects on hepatic macrophage biology. METHODS: Lanifibranor or selective PPARα (fenofibrate), PPARγ (pioglitazone) and PPARδ (GW501516) agonists were therapeutically administered in choline-deficient, amino acid-defined high-fat diet (CDAA-HFD)- and Western diet (WD)-fed mouse models of NAFLD. Acute liver injury was induced by carbon tetrachloride (CCl4). The role of PPARs on macrophage functionality was studied in isolated hepatic macrophages, bone marrow-derived macrophages stimulated with palmitic acid, and circulating monocytes from patients with NAFLD. RESULTS: Lanifibranor improved all histological features of steatohepatitis in CDAA-HFD-fed mice, including liver fibrosis, thereby combining and exceeding specific effects of the single PPAR agonists. Its potent anti-steatotic efficacy was confirmed in a 3D liver biochip model with primary cells. Infiltrating hepatic monocyte-derived macrophages were reduced following PPAR agonist administration, especially with lanifibranor, even after short-term treatment, paralleling improved steatosis and hepatitis. Lanifibranor similarly decreased steatosis, liver injury and monocyte infiltration in the WD model. In the acute CCl4 model, neither single nor pan-PPAR agonists directly affected monocyte recruitment. Hepatic macrophages isolated from WD-fed mice displayed a metabolically activated phenotype. Lanifibranor attenuated the accompanying inflammatory activation in both murine palmitic acid-stimulated bone marrow-derived macrophages, as well as patient-derived circulating monocytes, in a PPARδ-dependent fashion. CONCLUSION: Pan-PPAR agonists combine the beneficial effects of selective PPAR agonists and may counteract inflammation and disease progression more potently. PPARδ agonism and lanifibranor directly modulate macrophage activation, but not infiltration, thereby synergizing with beneficial metabolic effects of PPARα/γ agonists. LAY SUMMARY: Peroxisome proliferated-activated receptors (PPARs) are essential regulators of metabolism and inflammation. We demonstrated that the pan-PPAR agonist lanifibranor ameliorated all aspects of non-alcoholic fatty liver disease in independent experimental mouse models. Non-alcoholic fatty liver disease and fatty acids induce a specific polarization status in macrophages, which was altered by lanifibranor to increase expression of lipid handling genes, thereby decreasing inflammation. PPAR isoforms have differential therapeutic effects on fat-laden hepatocytes, activated hepatic stellate cells and inflammatory macrophages, supporting the clinical development of pan-PPAR agonists.

microRNA 193a-5p Regulates Levels of Nucleolar- and Spindle-Associated Protein 1 to Suppress Hepatocarcinogenesis.

BACKGROUND & AIMS: We performed an integrated analysis to identify microRNAs (miRNAs) and messenger RNAs (mRNAs) with altered expression in liver tumors from 3 mouse models of hepatocellular carcinoma (HCC) and human tumor tissues. METHODS: We analyzed miRNA and mRNA expression profiles of liver tissues from mice with diethylnitrosamine-induced hepatocarcinogenesis, conditional expression of lymphotoxin alpha and lymphotoxin beta, or inducible expression of a Myc transgene (Tet-O-Myc mice), as well as male C57BL/6 mice (controls). miRNA mimics were expressed and miRNAs and mRNAs were knocked down in human (Huh7, Hep3B, JHH2) hepatoma cell lines; cells were analyzed for viability, proliferation, apoptosis, migration, and invasion. Cells were grown as xenograft tumors in nude mice and analyzed. We combined in silico target gene prediction with mRNA profiles from all 3 mouse models. We quantified miRNA levels in 146 fresh-frozen tissues from patients (125 HCCs, 17 matched nontumor tissues, and 4 liver samples from patients without cancer) and published human data sets and tested correlations with patient survival times using Kaplan-Meier curves and the log-rank test. Levels of NUSAP1 mRNA were quantified in 237 HCCs and 5 nontumor liver samples using the TaqMan assay. RESULTS: Levels of the miRNA 193a-5p (MIR193A-5p) were reduced in liver tumors from all 3 mouse tumor models and in human HCC samples, compared with nontumor liver tissues. Expression of a MIR193A-5p mimic in hepatoma cells reduced proliferation, survival, migration, and invasion and their growth as xenograft tumors in nude mice. We found nucleolar and spindle-associated protein 1 (NUSAP1) to be a target of MIR193A-5p; HCC cells and tissues with low levels of MIR193A-5p had increased expression of NUSAP1. Increased levels of NUSAP1 in HCC samples correlated with shorter survival times of patients. Knockdown of NUSAP1 in Huh7 cells reduced proliferation, survival, migration, and growth as xenograft tumors in nude mice. Hydrodynamic tail-vein injections of a small hairpin RNA against NUSAP1 reduced growth of Akt1-Myc-induced tumors in mice. CONCLUSIONS: MIR193A-5p appears to prevent liver tumorigenesis by reducing levels of NUSAP1. Levels of MIR193A-5p are reduced in mouse and human HCC cells and tissues, leading to increased levels of NUSAP1, associated with shorter survival times of patients. Integrated analyses of miRNAs and mRNAs in tumors from mouse models can lead to identification of therapeutic targets in humans. The currently reported miRNA and mRNA profiling data have been submitted to the Gene Expression Omnibus (super-series accession number GSE102418).

A two-step process for capture and purification of human basic fibroblast growth factor from E. coli homogenate: Yield versus endotoxin clearance.

A two-step purification process for human basic fibroblast growth factor (FGF-2) from clarified E. coli homogenate has been developed in which the impurity level after the second step is below the limit of quantification. Endotoxin content is cleared to 0.02 EU/µg FGF-2 and the overall yield is 67%. The performance of the cation exchanger Carboxymethyl-Sepharose Fast Flow (CM-SFF) was compared to the affinity resin Heparin-SFF regarding the impurity profile and product quality in the elution peak. The CM-SFF eluate was further purified using hydrophobic interaction resin Toyopearl-Hexyl-650C. The relative amounts of target product, host cell proteins (HCPs), dsDNA, endotoxin, monomer content, and high molecular weight impurities differed along the elution peak depending on the applied method. The bioactive monomer (>99%) was obtained with a yield of 48% for CM-SFF and 68% for Heparin-SFF. A half-load reduction in CM-SFF increased the yield up to 67% without deterioration of the impurity content. Assuming a dose of 400 µg FGF-2, endotoxin was reduced to 188 EU/dose, dsDNA <10 ng/dose, and HCP <2 ppm/dose using the cation exchanger. In the pooled eluate fractions, dsDNA was removed 4-fold (291 ng/mL) and endotoxin 14-fold (0.47 EU/µg FGF-2) more efficiently by CM-SFF than by affinity chromatography. In contrast, HCP clearance was 3-fold (13 ppm) more efficient with Heparin-SFF than CM-SFF. In contrast to process monitoring by UV280nm or SDS-PAGE, this characterization is the basis for a Process Analytical Technology attempt when correlated with online monitored signals, as it enables knowledge-based pooling according to defined quality criteria.

Risk factors for discontinuation of thyroid hormone replacement therapy in early pregnancy: a study from the Norwegian Mother and Child Cohort Study and the Medical Birth Registry of Norway.

INTRODUCTION: Approximately 3-5% of pregnant women have hypothyroidism. Despite the potential impact of untreated hypothyroidism on infant neurodevelopment, few studies have investigated the risk factors associated with discontinuation of thyroid hormone replacement therapy (THRT) in pregnancy. We aimed to identify such factors in a population of women using THRT prior to pregnancy. MATERIAL AND METHODS: Data from the Norwegian Mother and Child Cohort Study were linked to records in the Medical Birth Registry of Norway. Pregnant women with hypothyroidism prior to pregnancy were categorized as discontinuers or continuers of THRT in pregnancy. The main analysis used generalized estimating equations based on multiply imputed data. RESULTS: Of 86 848 enrolled pregnant women, 2720 (3.2%) had a medically confirmed thyroid disorder and/or reported use of thyroid therapy. More than half (n = 1587; 57.8%) used THRT prior to pregnancy; of these, 207 (13.0%) discontinued and 1380 (86.9%) continued THRT during early pregnancy. Having a non-medicated mental disorder [odds ratio (OR) 1.64, 95% CI 1.03-2.63] and non-compliance with recommended nutritional supplementation (OR 2.51, 95% CI 1.82-3.47) increased the odds of discontinuing THRT. Women medicated for somatic comorbidities (OR 0.56, 95% CI 0.33-0.98) had a 44% decreased odds of discontinuing THRT. CONCLUSIONS: In Norway, around 13% of women with hypothyroidism discontinue THRT in early pregnancy. For discontinuers, non-medicated mental comorbidity and non-compliance with nutritional supplements presented increased risk, whereas having a medicated somatic disorder was protective. Health professionals advising women with hypothyroidism should be aware of risk factors associated with THRT discontinuation.

A biomimetic assembly of folded photosystem I monolayers for an improved light utilization in biophotovoltaic devices.

In the fabrication of photosystem I (PSI)-based biodevices, the use of multilayered architectures aims to maximize the absorption of incident light that can be converted into high-energy electrons. The challenge in this strategy is to overcome the large driving force imposed by the photoinduced potential difference between the two terminal redox centers that are located at opposite sides of PSI, which translates into charge recombination resulting in sub-optimal performance of commonly implemented systems. The integration of PSI monolayers with electrodes using the Langmuir-Blodgett technique enables a preferential anisotropic orientation of PSI in a tightly packed structure, which minimizes short-circuiting processes and aids to improve the performance of PSI-based biodevices. However, the practical application of PSI monolayer-based biodevices is limited due to the small loading of immobilized PSI molecules, leading to overall low utilization of incident light. Inspired by the stacked arrangements of thylakoids in nature, we demonstrate the fabrication of biomimetic structures using multiple PSI monolayers assembled into a folded architecture to improve light absorption and with that the performance of the overall photoelectrode.

Removal of chromatin by salt-tolerant endonucleases for production of recombinant measles virus.

Host cell DNA is a critical impurity in downstream processing of enveloped viruses. Especially, DNA in the form of chromatin is often neglected. Endonuclease treatment is an almost mandatory step in manufacturing of viral vaccines. In order to find the optimal performer, four different endonucleases, two of them salt tolerant, were evaluated in downstream processing of recombinant measles virus. Endonuclease treatment was performed under optimal temperature conditions after clarification and before the purification by flow-through chromatography with a core shell chromatography medium: Capto™ Core 700. Virus infectivity was measured by TCID50. DNA and histone presence in process and purified samples was determined using PicoGreen™ assay and Western blot analysis using an anti-histone antibody, respectively. All tested endonucleases allowed the reduction of DNA content improving product purity. The salt-tolerant endonucleases SAN and M-SAN were more efficient in the removal of chromatin compared with the non-salt-tolerant endonucleases Benzonase® and DENARASE®. Removal of chromatin using M-SAN was also possible without the addition of extra salt to the cell culture supernatant. The combination of the endonuclease treatment, using salt-tolerant endonucleases with flow-through chromatography, using core-shell particles, resulted in high purity and purification efficiency. This strategy has all features for a platform downstream process of recombinant measles virus and beyond.

Pump-less, recirculating organ-on-a-chip (rOoC) platform.

We developed a novel, pump-less directional flow recirculating organ-on-a-chip (rOoC) platform that creates controlled unidirectional gravity-driven flow by a combination of a 3D-tilting system and an optimized microfluidic layout. The rOoC platform was assembled utilizing a layer-to-layer fabrication technology based on thermoplastic materials. It features two organoid compartments supported by two independent perfusion channels and separated by a hydrogel barrier. We developed a computational model to predict wall shear stress values and then measured the flow rate in the microfluidic channels with micro-Particle-Image-Velocimetry (µPIV). The suitability of the rOoC for functional culture of endothelial cells was tested using HUVECs seeded in the perfusion channels. HUVECs aligned in response to the directional flow, formed a barrier and were able to sprout into the organoid compartments. Next, we demonstrated the viability of human stem-cell derived liver organoids in the organoid compartments. Finally, we show the possibility to circulate immune cells in the microfluidic channels that retain viability without being trapped or activated. The rOoC platform allows growing and connecting of two or more tissue or organ representations on-chip with the possibility of applying gradients, endothelial barriers, microvasculature and circulating cells independent of external tubing and support systems.

Structural and chemical characterization of MoO2/MoS2 triple-hybrid materials using electron microscopy in up to three dimensions.

This work presents the synthesis of MoO2/MoS2 core/shell nanoparticles within a carbon nanotube network and their detailed electron microscopy investigation in up to three dimensions. The triple-hybrid core/shell material was prepared by atomic layer deposition of molybdenum oxide onto carbon nanotube networks, followed by annealing in a sulfur-containing gas atmosphere. High-resolution transmission electron microscopy together with electron diffraction, supported by chemical analysis via energy dispersive X-ray and electron energy loss spectroscopy, gave proof of a MoO2 core covered by few layers of a MoS2 shell within an entangled network of carbon nanotubes. To gain further insights into this complex material, the analysis was completed with 3D electron tomography. By using Z-contrast imaging, distinct reconstruction of core and shell material was possible, enabling the analysis of the 3D structure of the material. These investigations showed imperfections in the nanoparticles which can impact material performance, i.e. for faradaic charge storage or electrocatalysis.

Cryo-EM structure of a functional monomeric Photosystem I from Thermosynechococcus elongatus reveals red chlorophyll cluster.

A high-resolution structure of trimeric cyanobacterial Photosystem I (PSI) from Thermosynechococcus elongatus was reported as the first atomic model of PSI almost 20 years ago. However, the monomeric PSI structure has not yet been reported despite long-standing interest in its structure and extensive spectroscopic characterization of the loss of red chlorophylls upon monomerization. Here, we describe the structure of monomeric PSI from Thermosynechococcus elongatus BP-1. Comparison with the trimer structure gave detailed insights into monomerization-induced changes in both the central trimerization domain and the peripheral regions of the complex. Monomerization-induced loss of red chlorophylls is assigned to a cluster of chlorophylls adjacent to PsaX. Based on our findings, we propose a role of PsaX in the stabilization of red chlorophylls and that lipids of the surrounding membrane present a major source of thermal energy for uphill excitation energy transfer from red chlorophylls to P700.

Cryo-EM photosystem I structure reveals adaptation mechanisms to extreme high light in Chlorella ohadii.

Photosynthesis in deserts is challenging since it requires fast adaptation to rapid night-to-day changes, that is, from dawn's low light (LL) to extreme high light (HL) intensities during the daytime. To understand these adaptation mechanisms, we purified photosystem I (PSI) from Chlorella ohadii, a green alga that was isolated from a desert soil crust, and identified the essential functional and structural changes that enable the photosystem to perform photosynthesis under extreme high light conditions. The cryo-electron microscopy structures of PSI from cells grown under low light (PSILL) and high light (PSIHL), obtained at 2.70 and 2.71 Å, respectively, show that part of light-harvesting antenna complex I (LHCI) and the core complex subunit (PsaO) are eliminated from PSIHL to minimize the photodamage. An additional change is in the pigment composition and their number in LHCIHL; about 50% of chlorophyll b is replaced by chlorophyll a. This leads to higher electron transfer rates in PSIHL and might enable C. ohadii PSI to act as a natural photosynthesiser in photobiocatalytic systems. PSIHL or PSILL were attached to an electrode and their induced photocurrent was determined. To obtain photocurrents comparable with PSIHL, 25 times the amount of PSILL was required, demonstrating the high efficiency of PSIHL. Hence, we suggest that C. ohadii PSIHL is an ideal candidate for the design of desert artificial photobiocatalytic systems.

Scalable Fabrication of Biophotoelectrodes by Means of Automated Airbrush Spray-Coating.

The fabrication and electrochemical evaluation of transparent photoelectrodes consisting of Photosystem I (PSI) or Photosystem II (PSII) is described, which are embedded and electrically wired by a redox polymer. The fabrication process is performed by an automated airbrush-type spray coating system, which ensures controlled and scalable electrode preparation. As proof of concept, electrodes with a surface area of up to 25 cm2 were prepared. The macro-porous structure of the indium tin oxide electrodes allows a high loading of the photoactive protein complexes leading to enhanced photocurrents, which are essential for potentially technologically relevant solar-powered devices. In addition, we show that unpurified crude PSII extracts, which can be provided in comparatively high yields for electrode modification, are suitable for photoelectrode fabrication with comparable photocurrent densities.

Production of Circularly Permuted Caspase-2 for Affinity Fusion-Tag Removal: Cloning, Expression in Escherichia coli, Purification, and Characterization.

Caspase-2 is the most specific protease of all caspases and therefore highly suitable as tag removal enzyme creating an authentic N-terminus of overexpressed tagged proteins of interest. The wild type human caspase-2 is a dimer of heterodimers generated by autocatalytic processing which is required for its enzymatic activity. We designed a circularly permuted caspase-2 (cpCasp2) to overcome the drawback of complex recombinant expression, purification and activation, cpCasp2 was constitutively active and expressed as a single chain protein. A 22 amino acid solubility tag and an optimized fermentation strategy realized with a model-based control algorithm further improved expression in Escherichia coli and 5.3 g/L of cpCasp2 in soluble form were obtained. The generated protease cleaved peptide and protein substrates, regardless of N-terminal amino acid with high activity and specificity. Edman degradation confirmed the correct N-terminal amino acid after tag removal, using Ubiquitin-conjugating enzyme E2 L3 as model substrate. Moreover, the generated enzyme is highly stable at -20 °C for one year and can undergo 25 freeze/thaw cycles without loss of enzyme activity. The generated cpCasp2 possesses all biophysical and biochemical properties required for efficient and economic tag removal and is ready for a platform fusion protein process.

Remodeling of photosynthetic electron transport in Synechocystis sp. PCC 6803 for future hydrogen production from water.

Photosynthetic microorganisms such as the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) can be exploited for the light-driven synthesis of valuable compounds. Thermodynamically, it is most beneficial to branch-off photosynthetic electrons at ferredoxin (Fd), which provides electrons for a variety of fundamental metabolic pathways in the cell, with the ferredoxin-NADP+ Oxido-Reductase (FNR, PetH) being the main target. In order to re-direct electrons from Fd to another consumer, the high electron transport rate between Fd and FNR has to be reduced. Based on our previous in vitro experiments, corresponding FNR-mutants at position FNR_K190 (Wiegand, K., et al.: "Rational redesign of the ferredoxin-NADP-oxido-reductase/ferredoxin-interaction for photosynthesis-dependent H2-production". Biochim Biophys Acta, 2018) have been generated in Synechocystis cells to study their impact on the cellular metabolism and their potential for a future hydrogen-producing design cell. Out of two promising candidates, mutation FNR_K190D proved to be lethal due to oxidative stress, while FNR_K190A was successfully generated and characterized: The light induced NADPH formation is clearly impaired in this mutant and it shows also major metabolic adaptations like a higher glucose metabolism as evidenced by quantitative mass spectrometric analysis. These results indicate a high potential for the future use of photosynthetic electrons in engineered design cells - for instance for hydrogen production. They also show substantial differences of interacting proteins in an in vitro environment vs. physiological conditions in whole cells.

Economic burden of incident interstitial lung disease (ILD) and the impact of comorbidity on costs of care.

INTRODUCTION: Evidence about the economic burden related to interstitial lung diseases (ILDs) and the cost-driving factors is sparse. In the knowledge that distinct comorbidities affect the clinical course of ILDs, our study investigates their impact on costs of care within first year after diagnosis. METHODS: Using claims data of individuals diagnosed with Idiopathic Interstitial Pneumonia (IIP) (n = 14 453) or sarcoidosis (n = 9106) between 2010 and 2013, we calculated total and ILD-associated mean annual per capita costs adjusted by age, sex and comorbidity burden via Generalized Linear Gamma models. Then, we assessed the cost impact of chronic obstructive pulmonary disease (COPD), diabetes, coronary artery disease, depression, gastro-esophageal reflux disease, pulmonary hypertension (PH), obstructive sleep apnoea syndrome (OSAS) and lung cancer using the model-based parameter estimates. RESULTS: Total mean annual per capita costs were €11 131 in the pooled cohort, €12 111 in IIP and €8793 in sarcoidosis, each with a 1/3 share of ILD-associated cost. Most comorbidities had a significant cost-driving effect, which was most pronounced for lung cancer in total (1.989 pooled, 2.491 sarcoidosis, 1.696 IIP) and for PH in ILD-associated costs (2.606 pooled, 2.347 IIP, 3.648 sarcoidosis). The lung-associated comorbidities COPD, PH, OSAS more strongly affected ILD-associated than total costs. CONCLUSION: Comorbidities increase the already substantial costs of care in ILDs. To support patient-centred ILD care, not only highly cost-driving conditions that are inherent with high mortality themselves require systematic management. Moreover, conditions that are more rather restricting the patient's activities of daily living should be addressed - despite a low-cost impact.