Destruction‐and‐diffraction by X‐ray free‐electron laser

It is common knowledge that macromolecular crystals are damaged by the X‐rays they are exposed to during conventional data collection. One of the claims made about the crystallographic data collection now being collected using X‐ray free‐electron lasers (XFEL) is that they are unaffected by radiation damage. XFEL data sets are assembled by merging data obtained from a very large number of crystals, each of which is exposed to a single femtosecond pulse of radiation, the duration of which is so short that diffraction occurs before the damage done to the crystal has time to become manifest, i.e. “diffraction‐before‐destruction.” However, recent theoretical studies have shown that many of the elemental electronic processes that ultimately result in the destruction of such crystals occur during a single pulse. It is predicted that the amplitudes of atomic scattering factor could be reduced by as much as 75% within the first 5 femtoseconds of such pulses, and that different atoms will respond in different ways. Experimental evidence is provided here that these predictions are correct.     

Regulation of the sperm‐to‐oocyte transition in Caenorhabditis briggsae hermaphrodites by the Cbr‐met‐2 and Cbr‐fem‐3 genes

In Caenorhabditis briggsae hermaphrodites, spermatogenesis begins in the L4 larval stage and persists into early adulthood. Oogenesis begins after spermatogenesis; the sperm‐to‐oocyte transition is irreversible. The timing of this transition is believed to have evolved in response to selection to maximize the intrinsic growth rate. Sperm‐to‐oocyte transitions occurred early in Cbr‐met‐2 and Cbr‐fem‐3 mutants. These early transitions resulted in reduced brood sizes, but had little or no impact on the intrinsic growth rate. In Cbr‐met‐2; Cbr‐fem‐3 doubly mutant hermaphrodites, the transition to oogenesis occurred even earlier and brood size was further reduced, indicating that Cbr‐met‐2 and Cbr‐fem‐3 regulate the sperm‐to‐oocyte transition through separate pathways. Mutations in Cbr‐met‐2 also resulted in an increase in the frequency of males in mutant populations. These increased male frequencies were not caused by increased rates of X nondisjunction during oogenesis in mutant hermaphrodites. Rather, increases in the rates of outcrossing in mutant populations likely were an indirect effect of reduced brood sizes derived from self‐fertilization. Based on these observations, it is possible that the timing of the sperm‐to‐oocyte transition in C. briggsae evolved in response to sexual selection on hermaphrodites to limit rates of outcrossing. Mutations in the orthologous Caenorhabditis elegans gene, Cel‐met‐2, did not impact the timing of the sperm‐to‐oocyte transition, consistent with the independent evolution of hermaphroditic reproduction in these species. Although brood sizes were reduced in Cel‐met‐2 mutant strains, increased male frequencies were not observed. Cbr‐ and Cel‐met‐2 mutations also differed in terms of germline mortality, observed in C. elegans, but not in C. briggsae.     

Epigallocatechin‐3‐O‐gallate up‐regulates microRNA‐199a‐3p expression by down‐regulating the expression of cyclooxygenase‐2 in stimulated human osteoarthritis chondrocytes

Osteoarthritis (OA) is a most common form of arthritis worldwide leading to significant disability. MicroRNAs (miRNAs) are non‐coding RNAs involved in various aspects of cartilage development, homoeostasis and pathology. Several miRNAs have been identified which have shown to regulate expression of target genes relevant to OA pathogenesis such as matrix metalloproteinase (MMP)‐13, cyclooxygenase (COX)‐2, etc. Epigallocatechin‐3‐O‐gallate (EGCG), the most abundant and active polyphenol in green tea, has been reported to have anti‐arthritic effects, however, the role of EGCG in the regulation of miRNAs has not been investigated in OA. Here, we showed that EGCG inhibits COX‐2 mRNA/protein expression or prostaglandin E₂ (PGE₂) production via up‐regulating microRNA hsa‐miR‐199a‐3p expression in interleukin (IL)‐1β‐stimulated human OA chondrocytes. This negative co‐regulation of hsa‐miR‐199a‐3p and COX‐2 by EGCG was confirmed by transfection of OA chondrocytes with anti‐miR‐199a‐3p. Transfection of OA chondrocytes with anti‐miR‐199a‐3p significantly enhanced COX‐2 expression and PGE₂ production (P < 0.001), while EGCG treatment significantly inhibited anti‐miR‐199a‐3p transfection‐induced COX‐2 expression or PGE₂ production in a dose‐dependent manner. These results were further re‐validated by co‐treatment of these transfection OA chondrocytes with IL‐1β and EGCG. EGCG treatment consistently up‐regulated the IL‐1β‐decreased hsa‐miR‐199a‐3p expression (P < 0.05) and significantly inhibited the IL‐1β‐induced COX‐2 expression/PGE₂ production (P < 0.05) in OA chondrocytes transfected with anti‐hsa‐miR‐199a‐3p. Taken together, these results clearly indicate that EGCG inhibits COX‐2 expression/PGE₂ production via up‐regulation of hsa‐miR‐199a‐3p expression. These novel pharmacological actions of EGCG on IL‐1β‐stimulated human OA chondrocytes provide new suggestions that EGCG or EGCG‐derived compounds inhibit cartilage breakdown or pain by up‐regulating the expression of microRNAs in human chondrocytes.     

Enhanced detection of in‐gel released N‐glycans by MALDI‐TOF‐MS

Many biologically relevant glycoproteins need to be separated on 1D‐ or 2D‐gels prior to analysis and are available in picomole amounts. Therefore, it is important to have optimized methods to unravel the glycome that combine in‐gel digestions with MALDI‐TOF‐MS. In this technical report, we investigated how the detection of in‐gel released N‐glycans could be improved by MALDI‐TOF‐MS. First, an AnchorChip target was tested and compared to ground steel target using several reference oligosaccharides. The highest signals were obtained with an AnchorChip target and D‐arabinosazone as the matrix; a LOD of 1.3 to 10 fmol was attained. Then, the effect of octyl‐β‐glucopyranoside, a nonionic detergent, was studied during in‐gel peptide‐N⁴‐(acetyl‐ß‐glucosaminyl) asparagine amidase F digestion of standard glycoproteins and during glycan extraction. Octyl‐β‐glucopyranoside increased the intensity and the amount of detected neutral as well as acidic N‐glycans. A LOD of under 7 pmol glycoprotein could be achieved.     

UDP‐Api/UDP‐Xyl synthases affect plant development by controlling the content of UDP‐Api to regulate the RG‐II‐borate complex

Rhamnogalacturonan‐II (RG‐II) is structurally the most complex glycan in higher plants, containing 13 different sugars and 21 distinct glycosidic linkages. Two monomeric RG‐II molecules can form an RG‐II‐borate diester dimer through the two apiosyl (Api) residues of side chain A to regulate cross‐linking of pectin in the cell wall. But the relationship of Api biosynthesis and RG‐II dimer is still unclear. In this study we investigated the two homologous UDP‐D‐apiose/UDP‐D‐xylose synthases (AXSs) in Arabidopsis thaliana that synthesize UDP‐D‐apiose (UDP‐Api). Both AXSs are ubiquitously expressed, while AXS2 has higher overall expression than AXS1 in the tissues analyzed. The homozygous axs double mutant is lethal, while heterozygous axs1/+ axs2 and axs1 axs2/+ mutants display intermediate phenotypes. The axs1/+ axs2 mutant plants are unable to set seed and die. By contrast, the axs1 axs2/+ mutant plants exhibit loss of shoot and root apical dominance. UDP‐Api content in axs1 axs2/+ mutants is decreased by 83%. The cell wall of axs1 axs2/+ mutant plants is thicker and contains less RG‐II‐borate complex than wild‐type Col‐0 plants. Taken together, these results provide direct evidence of the importance of AXSs for UDP‐Api and RG‐II‐borate complex formation in plant growth and development.     

High‐Energy‐Density Foldable Battery Enabled by Zigzag‐Like Design

Flexible batteries, seamlessly compatible with flexible and wearable electronics, attract a great deal of research attention. Current designs of flexible batteries struggle to meet one of the most extreme yet common deformation scenarios in practice, folding, while retaining high energy density. Inspired by origami folding, a novel strategy to fabricate zigzag‐like lithium ion batteries with superior foldability is proposed. The battery structure could approach zero‐gap between two adjacent energy storage segments, achieving an energy density that is 96.4% of that in a conventional stacking cell. A foldable battery thus fabricated demonstrates an energy density of 275 Wh L^?1 and is resilient to fatigue over 45 000 dynamic cycles with a folding angle of 130°, while retaining stable electrochemical performance. Additionally, the power stability and resilience to nail shorting of the foldable battery are also examined.     

Enhancement in Thermoelectric Figure‐Of‐Merit of an N‐Type Half‐Heusler Compound by the Nanocomposite Approach

An enhancement in the dimensionless thermoelectric figure‐of‐merit (ZT) of an n‐type half‐Heusler material is reported using a nanocomposite approach. A peak ZT value of 1.0 was achieved at 600 °C–700 °C, which is about 25% higher than the previously reported highest value. The samples were made by ball‐milling ingots of composition Hf_0.75Zr_0.25NiSn_0.99Sb_0.01 into nanopowders and hot‐pressing the powders into dense bulk samples. The ingots were formed by arc‐melting the elements. The ZT enhancement mainly comes from reduction of thermal conductivity due to increased phonon scattering at grain boundaries and crystal defects, and optimization of antimony doping.     

β‐arrestin‐1 mediates the TCR‐triggered re‐routing of distal receptors to the immunological synapse by a PKC‐mediated mechanism

T‐cell receptors (TCR) recognize their antigen ligand at the interface between T cells and antigen‐presenting cells, known as the immunological synapse (IS). The IS provides a means of sustaining the TCR signal which requires the continual supply of new TCRs. These are endocytosed and redirected from distal membrane locations to the IS. In our search for novel cytoplasmic effectors, we have identified β‐arrestin‐1 as a ligand of non‐phosphorylated resting TCRs. Using dominant‐negative and knockdown approaches we demonstrate that β‐arrestin‐1 is required for the internalization and downregulation of non‐engaged bystander TCRs. Furthermore, TCR triggering provokes the β‐arrestin‐1‐mediated downregulation of the G‐protein coupled chemokine receptor CXCR4, but not of other control receptors. We demonstrate that β‐arrestin‐1 recruitment to the TCR, and bystander TCR and CXCR4 downregulation, are mechanistically mediated by the TCR‐triggered PKC‐mediated phosphorylation of β‐arrestin‐1 at Ser163. This mechanism allows the first triggered TCRs to deliver a stop migration signal, and to promote the internalization of distal TCRs and CXCR4 and their translocation to the IS. This receptor crosstalk mechanism is critical to sustain the TCR signal.     

Removal of the 5‐nitro‐2‐pyridine‐sulfenyl protecting group from selenocysteine and cysteine by ascorbolysis

We previously reported on a method for the facile removal of 4‐methoxybenzyl and acetamidomethyl protecting groups from cysteine (Cys) and selenocysteine (Sec) using 2,2′‐dithiobis‐5‐nitropyridine dissolved in trifluoroacetic acid, with or without thioanisole. The use of this reaction mixture removes the protecting group and replaces it with a 2‐thio(5‐nitropyridyl) (5‐Npys) group. This results in either a mixed selenosulfide bond or disulfide bond (depending on the use of Sec or Cys), which can subsequently be reduced by thiolysis. A major disadvantage of thiolysis is that excess thiol must be used to drive the reaction to completion and then removed before using the Cys‐containing or Sec‐containing peptide in further applications. Here, we report a further advancement of this method as we have found that ascorbate at pH 4.5 and 25 °C will reduce the selenosulfide to the selenol. Ascorbolysis of the mixed disulfide between Cys and 5‐Npys is much less efficient but can be accomplished at higher concentrations of ascorbate at pH 7 and 37 °C with extended reaction times. We envision that our improved method will allow for in situ reactions with alkylating agents and electrophiles without the need for further purification, as well as a number of other applications. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

MiR‐376a suppresses the proliferation and invasion of non‐small‐cell lung cancer by targeting c‐Myc

It has been reported that miR‐376a is involved in the formation and progression of several types of cancer. However, the expression and function of miR‐376a is still unknown in non‐small cell lung carcinomas (NSCLC). In this study, the expression of miR‐376a in NSCLC tissues and cell lines were examined by real‐time PCR, the effects of miR‐376a on cell proliferation, apoptosis and invasion were evaluated in vitro. Luciferase reporter assay was performed to identify the targets of miR‐376a. The results showed that miR‐376a was significantly downregulated in NSCLC tissues and cell lines. Restoration of miR‐376a in NSCLC cell line A549 significantly inhibited cell proliferation, increased cell apoptosis and suppressed cell invasion, compared with control‐transfected A549 cells. Luciferase reporter assay showed that c‐Myc, an oncogene that regulating cell survival, angiogenesis and metastasis, was a direct target of miR‐376a. Over‐expression of miR‐376a decreased the mRNA and protein levels of c‐Myc in A549 cells. In addition, upregulation of c‐Myc inhibited miR‐376a‐induced inhibition of cell proliferation and invasion in A549 cells. Therefore, our results indicate a tumor suppressor role of miR‐376a in NSCLC by targeting c‐Myc. miR‐376a may be a promising therapeutic target for NSCLC.     

Visualization of the post‐Golgi vesicle‐mediated transportation of TGF‐β receptor II by quasi‐TIRFM

Transforming growth factor β receptor II (Tβ RII) is synthesized in the cytoplasm and then transported to the plasma membrane of cells to fulfil its signalling duty. Here, we applied live‐cell fluorescence imaging techniques, in particular quasi‐total internal reflection fluorescence microscopy, to imaging fluorescent protein‐tagged Tβ RII and monitoring its secretion process. We observed punctuate‐like Tβ RII‐containing post‐Golgi vesicles formed in MCF7 cells. Single‐particle tracking showed that these vesicles travelled along the microtubules at an average speed of 0.51 μm/s. When stimulated by TGF‐β ligand, these receptor‐containing vesicles intended to move towards the plasma membrane. We also identified several factors that could inhibit the formation of such post‐Golgi vesicles. Although the inhibitory mechanisms still remain unknown, the observed characteristics of Tβ RII‐containing vesicles provide new information on intracellular Tβ RII transportation. It also renders Tβ RII a good model system for studying post‐Golgi vesicle‐trafficking and protein transportation. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Analysis of the rat hypothalamus proteome by data‐independent label‐free LC‐MS/MS

Studies of neuronal, endocrine, and metabolic disorders would be facilitated by characterization of the hypothalamus proteome. Protein extracts prepared from 16 whole rat hypothalami were measured by data‐independent label‐free nano LC‐MS/MS. Peptide features were detected, aligned, and searched against a rat Swiss‐Prot database using ProteinLynx Global Server v.2.5. The final combined dataset comprised 21 455 peptides, corresponding to 622 unique proteins, each identified by a minimum of two distinct peptides. The majority of the proteins (69%) were cytosolic, and 16% were membrane proteins. Important proteins involved in neurological and synaptic function were identified including several members of the Ras‐related protein family and proteins involved in glutamate biosynthesis.     

Cell‐cycle‐dependent PC‐PLC regulation by APC/CCdc20‐mediated ubiquitin‐proteasome pathway

Phosphatidylcholine‐specific phospholipase C (PC‐PLC) is involved in the cell signal transduction, cell proliferation, and apoptosis. The mechanism of its action, however, has not been fully understood, particularly, the role of PC‐PLC in the cell cycle. In the present study, we found that cell division cycle 20 homolog (Cdc20) and PC‐PLC were co‐immunoprecipitated reciprocally by either antibody in rat hepatoma cells CBRH‐7919 as well as in rat liver tissue. Using confocal microscopy, we found that PC‐PLC and Cdc20 were co‐localized in the perinuclear endoplasmic reticulum region (the “juxtanuclear quality control” compartment, JUNQ). The expression level and activities of PC‐PLC changed in a cell‐cycle‐dependent manner and were inversely correlated with the expression of Cdc20. Intriguingly, Cdc20 overexpression altered the subcellular localization and distribution of PC‐PLC, and caused PC‐PLC degradation by the ubiquitin proteasome pathway (UPP). Taken together, our data indicate that PC‐PLC regulation in cell cycles is controlled by APC/C^Cdc20‐mediated UPP. J. Cell. Biochem. 107: 686–696, 2009. © 2009 Wiley‐Liss, Inc.     

Wastewater treatment by the HCR‐process

The increasing requirements in wastewater treatment have led to the development of new wastewater treatment processes based on the know‐how and experience in reaction and process engineering of the chemical industry. Due to their compactness, closed operation and high flexibility, these new processes show a large potential for process integration and significant cost reduction in particular for highly polluted industrial wastewaters. This paper discusses the HCR (h igh‐performance c ompact r eactor) ‐ process, developed at the Mass Transfer Laboratory of the Technical University of Clausthal within the last decade. This process has been realized in more than 30 technical applications with a volume loading of up to 70 kg COD/m³ d and an energy consumption of about 0.4 kWh per kg COD_elim.     

Regulation of with‐no‐lysine kinase signaling by Kelch‐like proteins

In 2001, with‐no‐lysine (WNK) kinases were identified as the genes responsible for the human hereditary hypertensive disease pseudohypoaldosteronism type II (PHAII). It took a further 6 years to clarify that WNK kinases participate in a signaling cascade with oxidative stress‐responsive gene 1 (OSR1), Ste20‐related proline‐alanine‐rich kinase (SPAK), and thiazide‐sensitive NaCl cotransporter (NCC) in the kidney and the constitutive activation of this signaling cascade is the molecular basis of PHAII. Since this discovery, the WNK–OSR1/SPAK–NCC signaling cascade has been shown to be involved not only in PHAII but also in the regulation of blood pressure under normal and pathogenic conditions, such as hyperinsulinemia. However, the molecular mechanisms of WNK kinase regulation by dietary and hormonal factors and by PHAII‐causing mutations remain poorly understood. In 2012, two additional genes responsible for PHAII, Kelch‐like 3 (KLHL3) and Cullin3, were identified. At the time of their discovery, the molecular mechanisms underlying the interaction between these genes and their involvement in PHAII were unknown. Here we review the pathophysiological roles of the WNK signaling cascade clarified to date and introduce a new mechanism of WNK kinase regulation by KLHL3 and Cullin3, which provides insight on previously unknown mechanisms of WNK kinase regulation.