Dimer–Dimer Interaction of the Bacterial Selenocysteine Synthase SelA Promotes Functional Active-Site Formation and Catalytic Specificity

The 21st amino acid, selenocysteine (Sec), is incorporated translationally into proteins and is synthesized on its specific tRNA (tRNA^Sec). In Bacteria, the selenocysteine synthase SelA converts Ser-tRNA^Sec, formed by seryl-tRNA synthetase, to Sec-tRNA^Sec. SelA, a member of the fold-type-I pyridoxal 5′-phosphate-dependent enzyme superfamily, has an exceptional homodecameric quaternary structure with a molecular mass of about 500 kDa. Our previously determined crystal structures of Aquifex aeolicus SelA complexed with tRNA^Sec revealed that the ring-shaped decamer is composed of pentamerized SelA dimers, with two SelA dimers arranged to collaboratively interact with one Ser-tRNA^Sec. The SelA catalytic site is close to the dimer–dimer interface, but the significance of the dimer pentamerization in the catalytic site formation remained elusive. In the present study, we examined the quaternary interactions and demonstrated their importance for SelA activity by systematic mutagenesis. Furthermore, we determined the crystal structures of “depentamerized” SelA variants with mutations at the dimer–dimer interface that prevent pentamerization. These dimeric SelA variants formed a distorted and inactivated catalytic site and confirmed that the pentamer interactions are essential for productive catalytic site formation. Intriguingly, the conformation of the non-functional active site of dimeric SelA shares structural features with other fold-type-I pyridoxal 5′-phosphate-dependent enzymes with native dimer or tetramer (dimer-of-dimers) quaternary structures.     

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.     

Complete genome sequence of the orange-red pigmented, radioresistant Deinococcus proteolyticus type strain (MRP(T))

Deinococcus proteolyticus (ex Kobatake et al. 1973) Brook and Murray 1981 is one of currently 47 species in the genus Deinococcus within the family Deinococcaceae. Strain MRP(T) was isolated from feces of Lama glama and possesses extreme radiation resistance, a trait is shares with various other species of the genus Deinococcus, with D. proteolyticus being resistant up to 1.5 Mrad of gamma radiation. Strain MRP(T) is of further interest for its carotenoid pigment. The genome presented here is only the fifth completed genome sequence of a member of the genus Deinococcus (and the forth type strain) to be published, and will hopefully contribute to a better understanding of how members of this genus adapted to high gamma- or UV ionizing-radiation. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 2,886,836 bp long genome with its four large plasmids of lengths 97 kbp, 132 kbp, 196 kbp and 315 kbp harbors 2,741 protein-coding and 58 RNA genes and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Spectrophotometric measurements of human tissues for the detection of subjacent blood vessels in an endonasal endoscopic surgical approach

Thin slices of human tissues are characterized concerning reflection and transmission in a wavelength range from 400 to 1700 nm. The results are primarily useful to find a wavelength for the detection of subjacent blood vessels during surgical procedures, especially neurological surgery. The measurements have been conducted using a customized measuring station, utilizing two halogen bulb lamps and two spectrometers. This paper focuses on creating a data base with the optical properties of artery, brain, bone, nasal mucosa, and nerve. The spectral distributions are compared among each other, similarities and differences are pointed out. Each tissue has got unique spectral characteristics, whereas typical absorption bands can be found in the overall tissues, especially hemoglobin and water absorption bands. The reflectivity maxima are typically located in the red or near‐infrared. All the transmission maxima are located between 1075 nm and 1100 nm. The measurements have been conducted at the Institute of Anatomy at the University of Leipzig. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Structural and functional insights into the modulation of the activity of a flax cytokinin oxidase by flax rust effector AvrL567-A

During infection, plant pathogens secrete effector proteins to facilitate colonization. In comparison with our knowledge of bacterial effectors, the current understanding of how fungal effectors function is limited. In this study, we show that the effector AvrL567-A from the flax rust fungus Melampsora lini interacts with a flax cytosolic cytokinin oxidase, LuCKX1.1, using both yeast two-hybrid and in planta bimolecular fluorescence assays. Purified LuCKX1.1 protein shows catalytic activity against both N6-(Δ2-isopentenyl)-adenine (2iP) and trans-zeatin (tZ) substrates. Incubation of LuCKX1.1 with AvrL567-A results in increased catalytic activity against both substrates. The crystal structure of LuCKX1.1 and docking studies with AvrL567-A indicate that the AvrL567 binding site involves a flexible surface-exposed region that surrounds the cytokinin substrate access site, which may explain its effect in modulating LuCKX1.1 activity. Expression of AvrL567-A in transgenic flax plants gave rise to an epinastic leaf phenotype consistent with hormonal effects, although no difference in overall cytokinin levels was observed. We propose that, during infection, plant pathogens may differentially modify the levels of extracellular and intracellular cytokinins.     

Plant cell packs: a scalable platform for recombinant protein production and metabolic engineering

Industrial plant biotechnology applications include the production of sustainable fuels, complex metabolites and recombinant proteins, but process development can be impaired by a lack of reliable and scalable screening methods. Here, we describe a rapid and versatile expression system which involves the infusion of Agrobacterium tumefaciens into three‐dimensional, porous plant cell aggregates deprived of cultivation medium, which we have termed plant cell packs (PCPs). This approach is compatible with different plant species such as Nicotiana tabacum BY2, Nicotiana benthamiana or Daucus carota and 10‐times more effective than transient expression in liquid plant cell culture. We found that the expression of several proteins was similar in PCPs and intact plants, for example, 47 and 55 mg/kg for antibody 2G12 expressed in BY2 PCPs and N. tabacum plants respectively. Additionally, the expression of specific enzymes can either increase the content of natural plant metabolites or be used to synthesize novel small molecules in the PCPs. The PCP method is currently scalable from a microtiter plate format suitable for high‐throughput screening to 150‐mL columns suitable for initial product preparation. It therefore combined the speed of transient expression in plants with the throughput of microbial screening systems. Plant cell packs therefore provide a convenient new platform for synthetic biology approaches, metabolic engineering and conventional recombinant protein expression techniques that require the multiplex analysis of several dozen up to hundreds of constructs for efficient product and process development.     

Development of Eco-factors for the European Union based on the Ecological Scarcity Method

The International Journal of Life Cycle Assessment - Weighting as an optional step in life cycle impact assessment (LCIA) has recently gained momentum through increased policy requirements in the...     

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.     

Targeting age‐specific changes in CD4+ T cell metabolism ameliorates alloimmune responses and prolongs graft survival

Age impacts alloimmunity. Effects of aging on T‐cell metabolism and the potential to interfere with immunosuppressants have not been explored yet. Here, we dissected metabolic pathways of CD4⁺ and CD8⁺ T cells in aging and offer novel immunosuppressive targets. Upon activation, CD4⁺ T cells from old mice failed to exhibit adequate metabolic reprogramming resulting into compromised metabolic pathways, including oxidative phosphorylation (OXPHOS) and glycolysis. Comparable results were also observed in elderly human patients. Although glutaminolysis remained the dominant and age‐independent source of mitochondria for activated CD4⁺ T cells, old but not young CD4⁺ T cells relied heavily on glutaminolysis. Treating young and old murine and human CD4⁺ T cells with 6‐diazo‐5‐oxo‐l‐norleucine (DON), a glutaminolysis inhibitor resulted in significantly reduced IFN‐γ production and compromised proliferative capacities specifically of old CD4⁺ T cells. Of translational relevance, old and young mice that had been transplanted with fully mismatched skin grafts and treated with DON demonstrated dampened Th1‐ and Th17‐driven alloimmune responses. Moreover, DON diminished cytokine production and proliferation of old CD4⁺ T cells in vivo leading to a significantly prolonged allograft survival specifically in old recipients. Graft prolongation in young animals, in contrast, was only achieved when DON was applied in combination with an inhibition of glycolysis (2‐deoxy‐d‐glucose, 2‐DG) and OXPHOS (metformin), two alternative metabolic pathways. Notably, metabolic treatment had not been linked to toxicities. Remarkably, immunosuppressive capacities of DON were specific to CD4⁺ T cells as adoptively transferred young CD4⁺ T cells prevented immunosuppressive capacities of DON on allograft survival in old recipients. Depletion of CD8⁺ T cells did not alter transplant outcomes in either young or old recipients. Taken together, our data introduce an age‐specific metabolic reprogramming of CD4⁺ T cells. Targeting those pathways offers novel and age‐specific approaches for immunosuppression.