Vanadium K-edge X-ray-absorption spectroscopy of the functioning and thionine-oxidized forms of the VFe-protein of the vanadium nitrogenase from Azotobacter chroococcum.

Vanadium K-edge X-ray-absorption spectra were collected for samples of thionine-oxidized, super-reduced (during enzyme turnover) and dithionite-reduced VFe-protein of the vanadium nitrogenase of Azotobacter chroococcum (Acl*). Both the e.x.a.f.s and the x.a.n.e.s. (X-ray-absorption near-edge structure) are consistent with the vanadium being present as part of a VFeS cluster; the environment of the vanadium is not changed significantly in different oxidation states of the protein. The vanadium atom is bound to three oxygen (or nitrogen), three sulphur and three iron atoms at 0.215(3), 0.231(3) and 0.275(3) nm respectively.     

Resveratrol as a Pan-HDAC Inhibitor Alters the Acetylation Status of Jistone Proteins in Human-Derived Hepatoblastoma Cells

The polyphenolic alcohol resveratrol has demonstrated promising activities for the prevention and treatment of cancer. Different modes of action have been described for resveratrol including the activation of sirtuins, which represent the class III histone deacetylases (HDACs). However, little is known about the activity of resveratrol on the classical HDACs of class I, II and IV, although these classes are involved in cancer development or progression and inhibitors of HDACs (HDACi) are currently under investigation as promising novel anticancer drugs. We could show by in silico docking studies that resveratrol has the chemical structure to inhibit the activity of different human HDAC enzymes. In vitro analyses of overall HDAC inhibition and a detailed HDAC profiling showed that resveratrol inhibited all eleven human HDACs of class I, II and IV in a dose-dependent manner. Transferring this molecular mechanism into cancer therapy strategies, resveratrol treatment was analyzed on solid tumor cell lines. Despite the fact that hepatocellular carcinoma (HCC) is known to be particularly resistant against conventional chemotherapeutics, treatment of HCC with established HDACi already has shown promising results. Testing of resveratrol on hepatoma cell lines HepG2, Hep3B and HuH7 revealed a dose-dependent antiproliferative effect on all cell lines. Interestingly, only for HepG2 cells a specific inhibition of HDACs and in turn a histone hyperacetylation caused by resveratrol was detected. Additional testing of human blood samples demonstrated a HDACi activity by resveratrol ex vivo. Concluding toxicity studies showed that primary human hepatocytes tolerated resveratrol, whereas in vivo chicken embryotoxicity assays demonstrated severe toxicity at high concentrations. Taken together, this novel pan-HDACi activity opens up a new perspective of resveratrol for cancer therapy alone or in combination with other chemotherapeutics. Moreover, resveratrol may serve as a lead structure for chemical optimization of bioavailability, pharmacology or HDAC inhibition.     

The plant host Brassica napus induces in the pathogen Verticillium longisporum the expression of functional catalase peroxidase which is required for the late phase of disease

The devastating soilborne fungal pathogen Verticillium longisporum is host specific to members of the family Brassicaceae, including oilseed rape (Brassica napus) as the economically most important crop. The fungus infects through the roots and causes stunting and early senescence of susceptible host plants and a marked decrease in crop yield. We show here that V. longisporum reacts to the presence of B. napus xylem sap with the production of six distinct upregulated and eight downregulated proteins visualized by two-dimensional gel electrophoresis. Identification of 10 proteins by mass spectrometry revealed that all upregulated proteins are involved in oxidative stress response. The V. longisporum catalase peroxidase (VlCPEA) was the most upregulated protein and is encoded by two isogenes, VlcpeA-1 and VlcpeA-2. Both genes are 98% identical, corroborating the diploid or "amphihaploid" status of the fungus. Knock downs of both VlcpeA genes reduced protein expression by 80% and resulted in sensitivity against reactive oxygen species. Whereas saprophytic growth and the initial phase of the plant infection were phenotypically unaffected, the mutants were not able to perform the late phases of disease. We propose that the catalase peroxidase plays a role in protecting the fungus from the oxidative stress generated by the host plant at an advanced phase of the disease.     

Differential requirements of the C terminus of Nbs1 in suppressing adenovirus DNA replication and promoting concatemer formation

Adenoviruses (Ad) with the early region E4 deleted (E4-deleted virus) are defective for DNA replication and late protein synthesis. Infection with E4-deleted viruses results in activation of a DNA damage response, accumulation of cellular repair factors in foci at viral replication centers, and joining together of viral genomes into concatemers. The cellular DNA repair complex composed of Mre11, Rad50, and Nbs1 (MRN) is required for concatemer formation and full activation of damage signaling through the protein kinases Ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR). The E4orf3 and E4orf6 proteins expressed from the E4 region of Ad type 5 (Ad5) inactivate the MRN complex by degradation and mislocalization, and prevent the DNA damage response. Here we investigated individual contributions of the MRN complex, concatemer formation, and damage signaling to viral DNA replication during infection with E4-deleted virus. Using virus mutants, short hairpin RNA knockdown and hypomorphic cell lines, we show that inactivation of MRN results in increased viral replication. We demonstrate that defective replication in the absence of E4 is not due to concatemer formation or DNA damage signaling. The C terminus of Nbs1 is required for the inhibition of Ad DNA replication and recruitment of MRN to viral replication centers. We identified regions of Nbs1 that are differentially required for concatemer formation and inhibition of Ad DNA replication. These results demonstrate that targeting of the MRN complex explains the redundant functions of E4orf3 and E4orf6 in promoting Ad DNA replication. Understanding how MRN impacts the adenoviral life cycle will provide insights into the functions of this DNA damage sensor.     

A Novel Link between Fic (Filamentation Induced by cAMP)-mediated Adenylylation/AMPylation and the Unfolded Protein Response

The maintenance of endoplasmic reticulum (ER) homeostasis is a critical aspect of determining cell fate and requires a properly functioning unfolded protein response (UPR). We have discovered a previously unknown role of a post-translational modification termed adenylylation/AMPylation in regulating signal transduction events during UPR induction. A family of enzymes, defined by the presence of a Fic (filamentation induced by cAMP) domain, catalyzes this adenylylation reaction. The human genome encodes a single Fic protein, called HYPE (Huntingtin yeast interacting protein E), with adenylyltransferase activity but unknown physiological target(s). Here, we demonstrate that HYPE localizes to the lumen of the endoplasmic reticulum via its hydrophobic N terminus and adenylylates the ER molecular chaperone, BiP, at Ser-365 and Thr-366. BiP functions as a sentinel for protein misfolding and maintains ER homeostasis. We found that adenylylation enhances BiP''s ATPase activity, which is required for refolding misfolded proteins while coping with ER stress. Accordingly, HYPE expression levels increase upon stress. Furthermore, siRNA-mediated knockdown of HYPE prevents the induction of an unfolded protein response. Thus, we identify HYPE as a new UPR regulator and provide the first functional data for Fic-mediated adenylylation in mammalian signaling.     

Expression of a bacterial 3‐dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency

Lignin confers recalcitrance to plant biomass used as feedstocks in agro‐processing industries or as source of renewable sugars for the production of bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically employ gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, new strategies have emerged offering better spatiotemporal control of lignin deposition, including the expression of enzymes that interfere with the normal process for cell wall lignification. In this study, we report that expression of a 3‐dehydroshikimate dehydratase (QsuB from Corynebacterium glutamicum) reduces lignin deposition in Arabidopsis cell walls. QsuB was targeted to the plastids to convert 3‐dehydroshikimate – an intermediate of the shikimate pathway – into protocatechuate. Compared to wild‐type plants, lines expressing QsuB contain higher amounts of protocatechuate, p‐coumarate, p‐coumaraldehyde and p‐coumaryl alcohol, and lower amounts of coniferaldehyde, coniferyl alcohol, sinapaldehyde and sinapyl alcohol. 2D‐NMR spectroscopy and pyrolysis‐gas chromatography/mass spectrometry (pyro‐GC/MS) reveal an increase of p‐hydroxyphenyl units and a reduction of guaiacyl units in the lignin of QsuB lines. Size‐exclusion chromatography indicates a lower degree of lignin polymerization in the transgenic lines. Therefore, our data show that the expression of QsuB primarily affects the lignin biosynthetic pathway. Finally, biomass from these lines exhibits more than a twofold improvement in saccharification efficiency. We conclude that the expression of QsuB in plants, in combination with specific promoters, is a promising gain‐of‐function strategy for spatiotemporal reduction of lignin in plant biomass.     

Role of HRTPT in kidney proximal epithelial cell regeneration: Integrative differential expression and pathway analyses using microarray and scRNA-seq

Damage to proximal tubules due to exposure to toxicants can lead to conditions such as acute kidney injury (AKI), chronic kidney disease (CKD) and ultimately end-stage renal failure (ESRF). Studies have shown that kidney proximal epithelial cells can regenerate particularly after acute injury. In the previous study, we utilized an immortalized in vitro model of human renal proximal tubule epithelial cells, RPTEC/TERT1, to isolate HRTPT cell line that co-expresses stem cell markers CD133 and CD24, and HREC24T cell line that expresses only CD24. HRTPT cells showed most of the key characteristics of stem/progenitor cells; however, HREC24T cells did not show any of these characteristics. The goal of this study was to further characterize and understand the global gene expression differences, upregulated pathways and gene interaction using scRNA-seq in HRTPT cells. Affymetrix microarray analysis identified common gene sets and pathways specific to HRTPT and HREC24T cells analysed using DAVID, Reactome and Ingenuity software. Gene sets of HRTPT cells, in comparison with publicly available data set for CD133+ infant kidney, urine-derived renal progenitor cells and human kidney-derived epithelial proximal tubule cells showed substantial similarity in organization and interactions of the apical membrane. Single-cell analysis of HRTPT cells identified unique gene clusters associated with CD133 and the 92 common gene sets from three data sets. In conclusion, the gene expression analysis identified a unique gene set for HRTPT cells and narrowed the co-expressed gene set compared with other human renal–derived cell lines expressing CD133, which may provide deeper understanding in their role as progenitor/stem cells that participate in renal repair.     

Iron-deficiency anaemia enhances red blood cell oxidative stress

Oxidative stress associated with iron deficiency anaemia in a murine model was studied feeding an iron-deficient diet. Anaemia was monitored by a decrease in hematocrit and haemoglobin. For the 9 week study an increase in total iron binding capacity was also demonstrated. Anaemia resulted in an increase in red blood cells (RBC) oxidative stress as indicated by increased levels of fluorescent heme degradation products (1.24-fold after 5 weeks; 2.1-fold after 9 weeks). The increase in oxidative stress was further confirmed by elevated levels of methemoglobin for mice fed an iron-deficient diet. Increased haemoglobin autoxidation and subsequent generation of ROS can account for the shorter RBC lifespan and other pathological changes associated with iron-deficiency anaemia.     

Comparison of heterologous neutralizing antibody responses of human immunodeficiency virus type 1 (HIV-1)- and HIV-2-infected Senegalese patients: distinct patterns of breadth and magnitude distinguish HIV-1 and HIV-2 infections

Neutralizing antibody responses against heterologous isolates in human immunodeficiency virus type 1 (HIV-1) and HIV-2 infections were compared, and their relationships with established clinical markers of progression were examined. Neutralizing responses against 7 heterologous primary isolates and 1 laboratory strain were compared between 32 untreated HIV-1-infected subjects and 35 untreated HIV-2-infected subjects using a pseudotyped reporter virus assay. The breadth of the neutralizing response, defined as the proportion of panel viruses positively neutralized by patient plasma, was significantly greater among HIV-2-infected subjects than among HIV-1-infected subjects. Notably, for fully one-third of HIV-2 subjects, all viruses were effectively neutralized in our panel. Magnitudes of responses, defined as reciprocal 50% inhibitory concentration (IC(50)) titers for positive reactions, were significantly greater among HIV-1-infected subjects than among HIV-2-infected subjects. When plasma samples from HIV-1 patients were tested for cross-neutralization of HIV-2 and vice versa, we found that these intertype responses are very rare and their prevalences comparable in both HIV-1 and HIV-2 infection. The significantly higher magnitude of heterologous responses for HIV-1 compared to HIV-2 prompted us to examine associations with viremia, which is known to be significantly higher in HIV-1 infection. Importantly, there was a significant positive correlation between the IC(50) titer and viral load within both the HIV-1 and HIV-2 groups, suggesting heterologous antibodies may be driven by viral replication. We conclude that HIV-2 infection is characterized by a broad, low-magnitude intratype neutralization response, while HIV-1 is characterized by a narrower but higher-magnitude intratype response and that a significant positive association between the IC(50) titer and viremia is common to both HIV-1 and HIV-2 infections.     

SSP2 and OSW1, two sporulation-specific genes involved in spore morphogenesis in Saccharomyces cerevisiae

Spore formation in Saccharomyces cerevisiae requires the synthesis of prospore membranes (PSMs) followed by the assembly of spore walls (SWs). We have characterized extensively the phenotypes of mutants in the sporulation-specific genes, SSP2 and OSW1, which are required for spore formation. A striking feature of the osw1 phenotype is asynchrony of spore development, with some spores displaying defects in PSM formation and others spores in the same ascus blocked at various stages in SW development. The Osw1 protein localizes to spindle pole bodies (SPBs) during meiotic nuclear division and subsequently to PSMs/SWs. We propose that Osw1 performs a regulatory function required to coordinate the different stages of spore morphogenesis. In the ssp2 mutant, nuclei are surrounded by PSMs and SWs; however, PSMs and SWs often also encapsulate anucleate bodies both inside and outside of spores. In addition, the SW is not as thick as in wild type. The ssp2 mutant defect is partially suppressed by overproduction of either Spo14 or Sso1, both of which promote the fusion of vesicles at the outer plaque of the SPB early in PSM formation. We propose that Ssp2 plays a role in vesicle fusion during PSM formation.