Pelota interacts with HAX1, EIF3G and SRPX and the resulting protein complexes are associated with the actin cytoskeleton

Background  

Pelota (PELO) is an evolutionary conserved protein, which has been reported to be involved in the regulation of cell proliferation and stem cell self-renewal. Recent studies revealed the essential role of PELO in the No-Go mRNA decay, by which mRNA with translational stall are endonucleotically cleaved and degraded. Further, PELO-deficient mice die early during gastrulation due to defects in cell proliferation and/or differentiation.     

Spectrophotometric Analysis of Pigments: A Critical Assessment of a High-Throughput Method for Analysis of Algal Pigment Mixtures by Spectral Deconvolution

The Gauss-peak spectra (GPS) method represents individual pigment spectra as weighted sums of Gaussian functions, and uses these to model absorbance spectra of phytoplankton pigment mixtures. We here present several improvements for this type of methodology, including adaptation to plate reader technology and efficient model fitting by open source software. We use a one-step modeling of both pigment absorption and background attenuation with non-negative least squares, following a one-time instrument-specific calibration. The fitted background is shown to be higher than a solvent blank, with features reflecting contributions from both scatter and non-pigment absorption. We assessed pigment aliasing due to absorption spectra similarity by Monte Carlo simulation, and used this information to select a robust set of identifiable pigments that are also expected to be common in natural samples. To test the method’s performance, we analyzed absorbance spectra of pigment extracts from sediment cores, 75 natural lake samples, and four phytoplankton cultures, and compared the estimated pigment concentrations with concentrations obtained using high performance liquid chromatography (HPLC). The deviance between observed and fitted spectra was generally very low, indicating that measured spectra could successfully be reconstructed as weighted sums of pigment and background components. Concentrations of total chlorophylls and total carotenoids could accurately be estimated for both sediment and lake samples, but individual pigment concentrations (especially carotenoids) proved difficult to resolve due to similarity between their absorbance spectra. In general, our modified-GPS method provides an improvement of the GPS method that is a fast, inexpensive, and high-throughput alternative for screening of pigment composition in samples of phytoplankton material.     

Loss of Cytotoxicity and Gain of Cytokine Production in Murine Tumor-Activated NK Cells

NK cells are able to form a functional memory suggesting that some NK cells are surviving the activation process. We hypothesized that NK cell activation causes the development of a distinct NK cell subset and studied the fate of murine post-activation NK cells. Activation was achieved by in vivo and in vitro exposures to the melanoma tumor cell line B16 that was followed by differentiation in IL-2. When compared with control NK cells, post-activation CD25⁺ NK cells expressed little granzyme B or perforin and had low lysis activity. Post-activation NK cells expressed CD27, CD90, CD127, and were low for CD11b suggesting that tumor-induced activation is restricted to an early NK cell subset. Activation of NK cells led to decreases of CD16, CD11c and increases of CD62L and the IL-18 receptor. In vivo activated but not control NK cells expressed a variety of cytokines that included IFNγ, TNFα, GM-CSF and IL-10. These data suggest that the exposure of a subset of peripheral NK cells to the B16 tumor environment caused an exhaustion of their cytolytic capacity but also a gain in their ability to produce cytokines.     

Angiogenesis Is Induced and Wound Size Is Reduced by Electrical Stimulation in an Acute Wound Healing Model in Human Skin

Angiogenesis is critical for wound healing. Insufficient angiogenesis can result in impaired wound healing and chronic wound formation. Electrical stimulation (ES) has been shown to enhance angiogenesis. We previously showed that ES enhanced angiogenesis in acute wounds at one time point (day 14). The aim of this study was to further evaluate the role of ES in affecting angiogenesis during the acute phase of cutaneous wound healing over multiple time points. We compared the angiogenic response to wounding in 40 healthy volunteers (divided into two groups and randomised), treated with ES (post-ES) and compared them to secondary intention wound healing (control). Biopsy time points monitored were days 0, 3, 7, 10, 14. Objective non-invasive measures and H&E analysis were performed in addition to immunohistochemistry (IHC) and Western blotting (WB). Wound volume was significantly reduced on D7, 10 and 14 post-ES (p = 0.003, p = 0.002, p<0.001 respectively), surface area was reduced on days 10 (p = 0.001) and 14 (p<0.001) and wound diameter reduced on days 10 (p = 0.009) and 14 (p = 0.002). Blood flow increased significantly post-ES on D10 (p = 0.002) and 14 (p = 0.001). Angiogenic markers were up-regulated following ES application; protein analysis by IHC showed an increase (p<0.05) in VEGF-A expression by ES treatment on days 7, 10 and 14 (39%, 27% and 35% respectively) and PLGF expression on days 3 and 7 (40% on both days), compared to normal healing. Similarly, WB demonstrated an increase (p<0.05) in PLGF on days 7 and 14 (51% and 35% respectively). WB studies showed a significant increase of 30% (p>0.05) on day 14 in VEGF-A expression post-ES compared to controls. Furthermore, organisation of granulation tissue was improved on day 14 post-ES. This randomised controlled trial has shown that ES enhanced wound healing by reduced wound dimensions and increased VEGF-A and PLGF expression in acute cutaneous wounds, which further substantiates the role of ES in up-regulating angiogenesis as observed over multiple time points. This therapeutic approach may have potential application for clinical management of delayed and chronic wounds.     

Selective high throughput protein quantification based on UV absorption spectra

The application of high throughput experimentation (HTE) in protein purification process development has created an analytical bottleneck. Recently, a new label‐free and non‐invasive methodology for analyzing multicomponent protein mixtures by means of spectral measurements was presented. Analytics based on the methodology was shown to increase analytical throughput for selective protein quantification significantly, however this was only demonstrated for one particular protein combination. In this work, the possibilities and limitations of the analytical method are investigated further. Principal component analysis (PCA) was performed on a broad range of absorption spectra to investigate their common characteristics and differences. The PCA was used both for cluster analysis and to define a measure for spectral similarity. For binary protein combinations, the calibration precision was shown to decrease exponentially with the defined spectral similarity factor. Knowledge of this correlation can be used to determine a priori whether a calibration will be successful or not. Calibration robustness was investigated by applying the analytics to liquid chromatography performed in HTE mode. Further it was shown, that a spectral difference of 0.6% was sufficient to sucessfully preform a spectral based calibration of two IgG1 monoclonals. Biotechnol. Bioeng. 2013; 110: 448–460. © 2012 Wiley Periodicals, Inc.     

Abnormal collagen in cultures of fibroblasts from human beings with diabetes mellitus

Synthesis of collagen by fibroblasts from diabetic human beings was investigated to determine if abnormal synthesis might be responsible for an apparent acceleration of collagen aging observed previously in diabetics. Increased material was synthesized by cells from diabetics, and the material was abnormal in that it contained a very high molecular weight material and lacked a presumed procollagen dimer. Alpha chains did not appear altered. It is suggested that cultures of cells from diabetics yield a non-helical peptide of procollagen that contains abnormal reducible cross links and lacks normal non-reducible cross links.     

On the systematic position of some Asian enigmatic genera of Asclepiadoideae (Apocynaceae)

The phylogenetic structure of Asclepiadoideae (Apocynaceae) has been elucidated at the tribal and subtribal levels in the last two decades. However, to date, the systematic positions of seven Asian genera, Cosmostigma, Graphistemma, Holostemma, Pentasachme, Raphistemma, Seshagiria and Treutlera, have not been investigated. In this study, we examine the evolutionary relationships among these seven small enigmatic Asian genera and clarify their positions in Asclepiadoideae, using a combination of plastid sequences of rbcL, rps16, trnL and trnL‐ F regions. Cosmostigma and Treutlera are resolved as members of the non‐Hoya clade of Marsdenieae with strong support (maximum parsimony bootstrap support value BS_MP = 96, maximum likelihood bootstrap support value BS_ML = 98, Bayesian‐inferred posterior probability PP = 1.0). Pentasachme is resolved as sister of Stapeliinae to Ceropegieae with moderate support (BS_MP = 64, BS_ML = 66, PP = 0.94). Graphistemma, Holostemma, Raphistemma and Seshagiria are all nested in the Asclepiadeae–Cynanchinae clade (BS_MP = 97, BS_ML = 100, PP = 1.0). The study confirms the generally accepted tribal and subtribal structure of the subfamily. One exception is Eustegia minuta, which is placed here as sister to all Asclepiadeae (BS_MP = 58, BS_ML = 76, PP = 0.99) and not as sister to the Marsdenieae + Ceropegieae clade. The weak support and conflicting position indicate the need for a placement of Eustegia as an independent tribe. In Asclepiadeae, a sister group position of Cynanchinae to the Asclepiadinae + Tylophorinae clade is favoured (BS_MP = 84, BS_ML = 88, PP = 1.0), whereas Schizostephanus is retrieved as unresolved. Oxystelma appears as an early‐branching member of Asclepiadinae with weak support (BS_MP = 52, BS_ML = 74, PP = 0.69). Calciphila and Solenostemma are also associated with Asclepiadinae with weak support (BS_MP = 37, BS_ML = 45, PP = 0.79), but all alternative positions are essentially without support. The position of Indian Asclepiadoideae in the family phylogeny is discussed. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174 , 601–619.