Chloroplasts require glutathione reductase to balance reactive oxygen species and maintain efficient photosynthesis

Thiol‐based redox‐regulation is vital for coordinating chloroplast functions depending on illumination and has been throroughly investigated for thioredoxin‐dependent processes. In parallel, glutathione reductase (GR) maintains a highly reduced glutathione pool, enabling glutathione‐mediated redox buffering. Yet, how the redox cascades of the thioredoxin and glutathione redox machineries integrate metabolic regulation and detoxification of reactive oxygen species remains largely unresolved because null mutants of plastid/mitochondrial GR are embryo‐lethal in Arabidopsis thaliana. To investigate whether maintaining a highly reducing stromal glutathione redox potential (E_GSH) via GR is necessary for functional photosynthesis and plant growth, we created knockout lines of the homologous enzyme in the model moss Physcomitrella patens. In these viable mutant lines, we found decreasing photosynthetic performance and plant growth with increasing light intensities, whereas ascorbate and zeaxanthin/antheraxanthin levels were elevated. By in vivo monitoring stromal E_GSH dynamics, we show that stromal E_GSH is highly reducing in wild‐type and clearly responsive to light, whereas an absence of GR leads to a partial glutathione oxidation, which is not rescued by light. By metabolic labelling, we reveal changing protein abundances in the GR knockout plants, pinpointing the adjustment of chloroplast proteostasis and the induction of plastid protein repair and degradation machineries. Our results indicate that the plastid thioredoxin system is not a functional backup for the plastid glutathione redox systems, whereas GR plays a critical role in maintaining efficient photosynthesis.     

Canadian polar bear population structure using genome‐wide markers

Predicting the consequences of environmental changes, including human‐mediated climate change on species, requires that we quantify range‐wide patterns of genetic diversity and identify the ecological, environmental, and historical factors that have contributed to it. Here, we generate baseline data on polar bear population structure across most Canadian subpopulations (n = 358) using 13,488 genome‐wide single nucleotide polymorphisms (SNPs) identified with double‐digest restriction site‐associated DNA sequencing (ddRAD). Our ddRAD dataset showed three genetic clusters in the sampled Canadian range, congruent with previous studies based on microsatellites across the same regions; however, due to a lack of sampling in Norwegian Bay, we were unable to confirm the existence of a unique cluster in that subpopulation. These data on the genetic structure of polar bears using SNPs provide a detailed baseline against which future shifts in population structure can be assessed, and opportunities to develop new noninvasive tools for monitoring polar bears across their range.     

Different effects of carbohydrate binding modules on the viscoelasticity of nanocellulose gels

Many cellulose degrading and modifying enzymes have distinct parts called carbohydrate binding modules (CBMs). The CBMs have been shown to increase the concentration of enzymes on the insoluble substrate and thereby enhance catalytic activity. It has been suggested that CBMs also have a role in disrupting or dispersing the insoluble cellulose substrate, but dispute remains and explicit evidence of such a mechanism is lacking. We produced the isolated CBMs from two major cellulases (Cel6A and Cel7A) from Trichoderma reesei as recombinant proteins in Escherichia coli. We then studied the viscoelastic properties of native unmodified cellulose nanofibrils (CNF) in combination with the highly purified CBMs to detect possible functional effects of the CBMs on the CNF. The two CBMs showed clearly different effects on the viscoelastic properties of CNF. The difference in effects is noteworthy, yet it was not possible to conclude for example disruptive effects. We discuss here the alternative explanations for viscoelastic effects on CNF caused by CBMs, including the effect of ionic cosolutes.     

Architectural model for muscle growth during maturation

Biomechanics and Modeling in Mechanobiology - Muscle architecture, which includes parameters like fascicle length, pennation angle, and physiological cross-sectional area, strongly influences...     

Sensitive detection of integrated and free transcripts in chimeric antigen receptor T-cell manufactured cell products using droplet digital polymerase chain reaction

Background AimsViral vectors are commonly used to introduce chimeric antigen receptor (CAR) constructs into cell therapy products for the treatment of human disease. They are efficient at gene delivery and integrate into the host genome for subsequent replication but also carry risks if replication-competent lentivirus (RCL) remains in the final product. An optimal CAR T-cell product should contain sufficient integrated viral material and no RCL. Current product testing methods include cell-based assays with slow turnaround times and rapid quantitative polymerase chain reaction (PCR)-based assays that suffer from high result variability. The authors describe the development of a droplet digital PCR (ddPCR) method for detection of the vesicular stomatitis virus G glycoprotein envelope sequence, required for viral assembly, and the replication response element to measure integration of the CAR construct.MethodsAssay validation included precision, linearity, sensitivity, specificity and reproducibility over a range of low to high concentrations.ResultsThe limit of detection was 10 copies/μL, whereas negative samples showed <1.3 copies/μL. Within and between assay imprecision coefficients of variation across the reportable range (10–10 000 copies/μL) were <25%. Accuracy and linearity were verified by comparing known copy numbers with measured copy numbers (R² >0.9985, slope ~0.9). Finally, serial measurements demonstrated very good long-term reproducibility (>95% of replicate results within the originally established ± two standard deviations).ConclusionsDDPCR has excellent reproducibility, linearity, specificity and sensitivity for detecting RCL and assuring the safety of patient products in a rapid manner. The technique can also likely be adapted for the rapid detection of other targets during cell product manufacturing, including purity, potency and sterility assays.     

HPV DNA/RNA detection in various oral and oropharyngeal biomaterials identifies active HPV infections also in non-neoplastic tonsils

Previous studies describe a correlation between HPV-positivity and non-smoking in TSCC; p16^INK4A-expression as surrogate-marker for HPV-DNA/RNA-positivity is discussed controversially. In the present study, these parameters are assessed prospectively. HPV-status of sputum and tonsillar-swabs was analyzed to determine their validity as surrogate-marker for tissue-HPV-status.TSCC- (n = 52) and non-neoplastic tonsillar tissue (n = 163) were analyzed. HPV-DNA- and HPV-RNA-status of total sputum, cellular fraction and supernatants, tonsillar-swabs and -tissue was determined by (RT)-PCR. Immunohistochemistry determined p16^INK4A-expression.23/163 (14.2%) non-neoplastic tonsils were HPV-DNA-positive; five patients (3 HPV16, 2 HPV11) had active HPV-infections (HPV-RNA-positive), in all biomaterials. 140/163 (85.9%) patients were either HPV-DNA-positive or HPV-DNA-negative in all samples. 21/52 (40.4%) TSCC-tonsils were HPV-DNA-positive; 17 patients were HPV-RNA-positive (14 HPV16; 4 HPV18). 40/52 (76.9%) TSCC-patients were congruent in all biomaterials. p16^INK4A-expression alone would have misclassified the HPV-status of 14/52 (26.2%) TSCC-patients.This prospective study confirms the discrepancy between HPV-status and p16^INK4A-expression and the significant correlation between non-smoking and HPV-DNA-positivity. HPV-sputum- and/or swab-results do not consistently match tissue-results, possibly having (detrimental) consequences if those were used to assess tissue-HPV-status. In the 5 patients with active HPV infection in the non-neoplasitic tonsils, tonsillectomy likely prevented subsequent development of TSCC.     

Three Dimensional Modeling of the Cerebrospinal Fluid Dynamics and Brain Interactions in the Aqueduct of Sylvius

A computational fluid dynamics (CFD) method is presented to investigate the flow of cerebro-spinal fluid (CSF) in the cerebral aqueduct. In addition to former approaches exhibiting a rigid geometry, we propose a model which includes a deformable membrane as the wall of this flow channel. An anatomical shape of the aqueduct was computed from magnetic resonance images (MRI) and the resulting meshing was immersed in a marker-and-cell (MAC) staggered grid for to take into account fluid–structure interactions. The time derivatives were digitized using the Crank–Nicolson scheme. The equation of continuity was modified by introducing an artificial compressibility and digitized by a finite difference scheme.

Calculations were validated with the simulation of laminar flow in a rigid tube. Then, comparisons were made between simulations of a rigid aqueduct and a deformable one. We found that the deformability of the walls has a strong influence on the pressure drop for a given flow.     

SOD3 R231G polymorphism associated with coronary artery disease and myocardial infarction. The Ludwigshafen Risk and Cardiovascular Health (LURIC) study

This study examined the superoxide dismutase 3 (SOD3) R231G polymorphism in relation to the severity of coronary artery disease (CAD) and the risk of myocardial infarction (MI) in 3211 individuals; 94.4% of study participants were homozygous for SOD3 231RR and 5.5% were heterozygous for SOD3 231RG. The odds ratios of the RG and GG genotype (adjusted for age, gender and for conventional cardiovascular risk factors) were 2.02 (95% CI, 1.23–3.33, p=0.005) for the highest vs the lowest Friesinger coronary score and 1.40 (95% CI, 1.02–1.92, p=0.037) for MI, respectively. Further the SOD3 RG and GG genotype was associated with lower alpha-tocopherol levels than the wild type SOD3 RR genotype. It is concluded that the SOD3 231RG and GG genotype is associated with lower alpha-tocopherol levels and the severity of CAD and the risk of MI.