Characterization of the carboxysomal carbonic anhydrase CsoSCA from Halothiobacillus neapolitanus

In cyanobacteria and many chemolithotrophic bacteria, the CO(2)-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is sequestered into polyhedral protein bodies called carboxysomes. The carboxysome is believed to function as a microcompartment that enhances the catalytic efficacy of RubisCO by providing the enzyme with its substrate, CO(2), through the action of the shell protein CsoSCA, which is a novel carbonic anhydrase. In the work reported here, the biochemical properties of purified, recombinant CsoSCA were studied, and the catalytic characteristics of the carbonic anhydrase for the CO(2) hydration and bicarbonate dehydration reactions were compared with those of intact and ruptured carboxysomes. The low apparent catalytic rates measured for CsoSCA in intact carboxysomes suggest that the protein shell acts as a barrier for the CO(2) that has been produced by CsoSCA through directional dehydration of cytoplasmic bicarbonate. This CO(2) trap provides the sequestered RubisCO with ample substrate for efficient fixation and constitutes a means by which microcompartmentalization enhances the catalytic efficiency of this enzyme.     

Eukaryotic protein production in designed storage organelles

Background  

Protein bodies (PBs) are natural endoplasmic reticulum (ER) or vacuole plant-derived organelles that stably accumulate large amounts of storage proteins in seeds. The proline-rich N-terminal domain derived from the maize storage protein γ zein (Zera) is sufficient to induce PBs in non-seed tissues of Arabidopsis and tobacco. This Zera property opens up new routes for high-level accumulation of recombinant proteins by fusion of Zera with proteins of interest. In this work we extend the advantageous properties of plant seed PBs to recombinant protein production in useful non-plant eukaryotic hosts including cultured fungal, mammalian and insect cells.     

Nitrous oxide and methane fluxes of a pristine slope mire in the German National Park Harz Mountains

Pristine peatlands covered by Histosols (bogs and fens) with high water table and a restricted oxygen (O₂) availability are known to have low emissions of nitrous oxide (N₂O) but may be a significant source for atmospheric methane (CH₄) which are both important greenhouse gases. For the first time N₂O and CH₄ fluxes of a pristine slope mire in the German Harz Mountains have been monitored. Previously reported peatlands are characterised by anaerobic conditions due to high water table levels. Slope mires monitored here receive O₂ through slope water inflow. Gas fluxes have been monitored deploying closed chamber method on a central non-forested area and a forested area at the periphery of the slope mire. By means of groundwater piezometers water table levels, ammonium and nitrate contents as well as hydro-chemical variables like oxygen content and redox potential of the mire pore water have been concurrently measured with trace gas fluxes at both monitoring sites of the slope mire. The slope mire took up small amounts of atmospheric methane at a rate of −0.02 ± 0.01 kg C ha⁻¹ year⁻¹ revealing no significant difference between the forested and non-forested site. Higher uptake rates were observed during low water table level. In contrast to pristine peatlands influx of oxygen containing pore water into slope mire does limit reduction processes and resultant CH₄ emission. N₂O fluxes of the forested and non-forested sites of the slope mire did not differ and amounted to 0.25 ± 0.44 kg N ha⁻¹ year⁻¹. Higher emissions were observed at low water table levels and during thawing periods. In spite of favourable conditions N₂O fluxes of the slope mire have been comparable to those of pristine peatlands.     

Amplified immunohistochemical detection of PrPsc in animal transmissible spongiform encephalopathies using streptomycin.

Due to its sensitivity, immunohistochemistry (IHC) of abnormal prion protein (PrPsc) is used to study experimental and natural cases of transmissible spongiform encephalopathies (TSEs) such as Creutzfeldt-Jakob disease in humans or scrapie and bovine spongiform encephalopathy (BSE) in animals. The limits of detection are particularly critical when PrPsc IHC is used for diagnostic purposes. In this article, we describe for the first time the use of streptomycin sulfate in IHC, providing a novel original and easy way to amplify specifically PrPsc immunohistochemical detection in natural cases of BSE and scrapie, as well as in experimental TSEs in mice models using two different PrP antibodies.     

Mature but not immature Fas ligand (CD95L)-transduced human monocyte-derived dendritic cells are protected from Fas-mediated apoptosis and can be used as killer APC

Several in vitro and animal studies have been performed to modulate the interaction of APCs and T cells by Fas (CD95/Apo-1) signaling to delete activated T cells in an Ag-specific manner. However, due to the difficulties in vector generation and low transduction frequencies, similar studies with primary human APC are still lacking. To evaluate whether Fas ligand (FasL/CD95L) expressing killer APC could be generated from primary human APC, monocyte-derived dendritic cells (DC) were transduced using the inducible Cre/Loxp adenovirus vector system. Combined transduction of DC by AdLoxpFasL and AxCANCre, but not single transduction with these vectors, resulted in dose- and time-dependent expression of FasL in >70% of mature DC (mDC), whereas <20% of immature DC (iDC) expressed FasL. In addition, transduction by AdLoxpFasL and AxCANCre induced apoptosis in >80% of iDC, whereas FasL-expressing mDC were protected from FasL/Fas (CD95/Apo-1)-mediated apoptosis despite coexpression of Fas. FasL-expressing mDC eliminated Fas(+) Jurkat T cells as well as activated primary T cells by apoptosis, whereas nonactivated primary T cells were not deleted. Induction of apoptosis in Fas(+) target cells required expression of FasL in DC and cell-to-cell contact between effector and target cell, and was not dependent on soluble FasL. Induction of apoptosis in Fas(+) target cells required expression of FasL in DC, cell-to-cell contact between effector and target cell, and was not dependent on soluble FasL. The present results demonstrate that FasL-expressing killer APC can be generated from human monocyte-derived mDC using adenoviral gene transfer. Our results support the strategy to use killer APCs as immunomodulatory cells for the treatment of autoimmune disease and allograft rejection.     

Algorithm-based modular psychotherapy vs. cognitive-behavioral therapy for patients with depression,psychiatric comorbidities and early trauma: a proof-of-concept randomized controlled trial

Effect sizes of psychotherapies currently stagnate at a low-to-moderate level. Personalizing psychotherapy by algorithm-based modular procedures promises improved outcomes, greater flexibility, and a better fit between research and practice. However, evidence for the feasibility and efficacy of modular-based psychotherapy, using a personalized treatment algorithm, is lacking. This proof-of-concept randomized controlled trial was conducted in 70 adult outpatients with a primary DSM-5 diagnosis of major depressive disorder, a score higher than 18 on the 24-item Hamilton Rating Scale for Depression (HRSD-24), at least one comorbid psychiatric diagnosis according to the Structured Clinical Interview for DSM-5 (SCID-5), a history of at least “moderate to severe” childhood maltreatment on at least one domain of the Childhood Trauma Questionnaire (CTQ), and exceeding the cut-off value on at least one of three measures of early trauma-related transdiagnostic mechanisms: the Rejection Sensitivity Questionnaire (RSQ), the Interpersonal Reactivity Index (IRI), and the Difficulties in Emotion Regulation Scale-16 (DERS-16). Patients were randomized to 20 sessions of either standard cognitive-behavioral therapy alone (CBT) or CBT plus transdiagnostic modules according to a mechanism-based treatment algorithm (MoBa), over 16 weeks. We aimed to assess the feasibility of MoBa, and to compare MoBa vs. CBT with respect to participants’ and therapists’ overall satisfaction and ratings of therapeutic alliance (using the Working Alliance Inventory - Short Revised, WAI-SR), efficacy, impact on early trauma-related transdiagnostic mechanisms, and safety. The primary outcome for efficacy was the HRSD-24 score at post-treatment. Secondary outcomes included, among others, the rate of response (defined as a reduction of the HRSD-24 score by at least 50% from baseline and a score <16 at post-treatment), the rate of remission (defined as a HRSD-24 score ≤8 at post-treatment), and improvements in early trauma-related mechanisms of social threat response, hyperarousal, and social processes/empathy. We found no difficulties in the selection of the transdiagnostic modules in the individual patients, applying the above-mentioned cut-offs, and in the implementation of MoBa. Both participants and therapists reported higher overall satisfaction and had higher WAI-SR ratings with MoBa than CBT. Both approaches led to major reductions of depressive symptoms at post-treatment, with a non-significant superiority of MoBa over CBT. Patients randomized to MoBa were nearly three times as likely to experience remission at the end of therapy (29.4% vs. 11.4%; odds ratio, OR = 3.2, 95% CI: 0.9-11.6). Among mechanism-based outcomes, MoBa patients showed a significantly higher post-treatment effect on social processes/empathy (p<0.05) compared to CBT patients, who presented an exacerbation on this domain at post-treatment. Substantially less adverse events were reported for MoBa compared to CBT. These results suggest the feasibility and acceptability of an algorithm-based modular psychotherapy complementing CBT in depressed patients with psychiatric comorbidities and early trauma. While initial evidence of efficacy was observed, potential clinical advantages and interindividual heterogeneity in treatment outcomes will have to be investigated in fully powered confirmation trials.     

SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients

Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no apparent “gold standard”. Here we present a novel qPCR protocol that meets the conditions of extreme sensitivity and specificity that are required for detection and quantification of tumor DNA. By consecutive application of two polymerases, one of them designed for extreme base-specificity, the method reaches unprecedented sensitivity and specificity. Three qPCR assays were tested with spike-in experiments, specific for point mutations BRAF V600E, PTEN T167A and NRAS Q61L of melanoma cell lines. It was possible to detect down to one copy of tumor DNA per reaction (Poisson distribution), at a background of up to 200 000 wild type DNAs. To prove its clinical applicability, the method was successfully tested on a small cohort of BRAF V600E positive melanoma patients.