HisB as novel selection marker for gene targeting approaches in Aspergillus niger

Background For Aspergillus niger, a broad set of auxotrophic and dominant resistance markers is available. However, only few offer targeted modification of a gene of interest into or at a genomic locus of choice, which hampers functional genomics studies. We thus aimed to extend the available set by generating a histidine auxotrophic strain with a characterized hisB locus for targeted gene integration and deletion in A. niger. Results A histidine-auxotrophic strain was established via disruption of the A. niger hisB gene by using the counterselectable pyrG marker. After curing, a hisB - , pyrG - strain was obtained, which served as recipient strain for further studies. We show here that both hisB orthologs from A. nidulans and A. niger can be used to reestablish histidine prototrophy in this recipient strain. Whereas the hisB gene from A. nidulans was suitable for efficient gene targeting at different loci in A. niger, the hisB gene from A. niger allowed efficient integration of a Tet-on driven luciferase reporter construct at the endogenous non-functional hisB locus. Subsequent analysis of the luciferase activity revealed that the hisB locus is tight under non-inducing conditions and allows even higher luciferase expression levels compared to the pyrG integration locus. Conclusion Taken together, we provide here an alternative selection marker for A. niger, hisB, which allows efficient homologous integration rates as well as high expression levels which compare favorably to the well-established pyrG selection marker.EC/FP7/303864/EU/Bridging the world of fungi and dementia/PROFITSTU Berlin, Open-Access-Mittel - 201

The Actin Targeting Compound Chondramide Inhibits Breast Cancer Metastasis via Reduction of Cellular Contractility

Background: A major player in the process of metastasis is the actin cytoskeleton as it forms key structures in both invasion mechanisms, mesenchymal and amoeboid migration. We tested the actin binding compound Chondramide as potential anti-metastatic agent. Methods: In vivo, the effect of Chondramide on metastasis was tested employing a 4T1-Luc BALB/c mouse model. In vitro, Chondramide was tested using the highly invasive cancer cell line MDA-MB-231 in Boyden-chamber assays, fluorescent stainings, Western blot and Pull down assays. Finally, the contractility of MDA-MB-231 cells was monitored in 3D environment and analyzed via PIV analysis. Results: In vivo, Chondramide treatment inhibits metastasis to the lung and the migration and invasion of MDA-MB-231 cells is reduced by Chondramide in vitro. On the signaling level, RhoA activity is decreased by Chondramide accompanied by reduced MLC-2 and the stretch induced guanine nucleotide exchange factor Vav2 activation. At same conditions, EGF-receptor autophosphorylation, Akt and Erk as well as Rac1 are not affected. Finally, Chondramide treatment disrupted the actin cytoskeleton and decreased the ability of cells for contraction. Conclusions: Chondramide inhibits cellular contractility and thus represents a potential inhibitor of tumor cell invasion

The analysis of heterotaxy patients reveals new loss-of-function variants of GRK5

G protein-coupled receptor kinase 5 (GRK5) is a regulator of cardiac performance and a potential therapeutic target in heart failure in the adult. Additionally, we have previously classified GRK5 as a determinant of left-right asymmetry and proper heart development using zebrafish. We thus aimed to identify GRK5 variants of functional significance by analysing 187 individuals with laterality defects (heterotaxy) that were associated with a congenital heart defect (CHD). Using Sanger sequencing we identified two moderately frequent variants in GRK5 with minor allele frequencies <10%, and seven very rare polymorphisms with minor allele frequencies <1%, two of which are novel variants. Given their evolutionarily conserved position in zebrafish, in-depth functional characterisation of four variants (p.Q41L, p.G298S, p.R304C and p.T425M) was performed. We tested the effects of these variants on normal subcellular localisation and the ability to desensitise receptor signalling as well as their ability to correct the left-right asymmetry defect upon Grk5l knockdown in zebrafish. While p.Q41L, p.R304C and p.T425M responded normally in the first two aspects, neither p.Q41L nor p.R304C were capable of rescuing the lateralisation phenotype. The fourth variant, p.G298S was identified as a complete loss-of-function variant in all assays and provides insight into the functions of GRK5

Resources management and effective co-operation fo organisations in Amazonia: a discussion on the social component of recycling in the city of Belém.

This paper alms to undstand the organisation process of recycling in Amazonia through the discussion of the social components of the sorting collection system mainly conducted by former farming families in the city fo Belém

Src Dependent Pancreatic Acinar Injury Can Be Initiated Independent of an Increase in Cytosolic Calcium

Several deleterious intra-acinar phenomena are simultaneously triggered on initiating acute pancreatitis. These culminate in acinar injury or inflammatory mediator generation in vitro and parenchymal damage in vivo. Supraphysiologic caerulein is one such initiator which simultaneously activates numerous signaling pathways including non-receptor tyrosine kinases such as of the Src family. It also causes a sustained increase in cytosolic calcium- a player thought to be crucial in regulating deleterious phenomena. We have shown Src to be involved in caerulein induced actin remodeling, and caerulein induced changes in the Golgi and post-Golgi trafficking to be involved in trypsinogen activation, which initiates acinar cell injury. However, it remains unclear whether an increase in cytosolic calcium is necessary to initiate acinar injury or if injury can be initiated at basal cytosolic calcium levels by an alternate pathway. To study the interplay between tyrosine kinase signaling and calcium, we treated mouse pancreatic acinar cells with the tyrosine phosphatase inhibitor pervanadate. We studied the effect of the clinically used Src inhibitor Dasatinib (BMS-354825) on pervanadate or caerulein induced changes in Src activation, trypsinogen activation, cell injury, upstream cytosolic calcium, actin and Golgi morphology. Pervanadate, like supraphysiologic caerulein, induced Src activation, redistribution of the F-actin from its normal location in the sub-apical area to the basolateral areas, and caused antegrade fragmentation of the Golgi. These changes, like those induced by supraphysiologic caerulein, were associated with trypsinogen activation and acinar injury, all of which were prevented by Dasatinib. Interestingly, however, pervanadate did not cause an increase in cytosolic calcium, and the caerulein induced increase in cytosolic calcium was not affected by Dasatinib. These findings suggest that intra-acinar deleterious phenomena may be initiated independent of an increase in cytosolic calcium. Other players resulting in acinar injury along with the Src family of tyrosine kinases remain to be explored. © 2013 Mishra et al

A novel HSF4 gene mutation (p.R405X) causing autosomal recessive congenital cataracts in a large consanguineous family from Pakistan

<p>Abstract</p> <p>Background</p> <p>Hereditary cataracts are most frequently inherited as autosomal dominant traits, but can also be inherited in an autosomal recessive or X-linked fashion. To date, 12 loci for autosomal recessive cataracts have been mapped including a locus on chromosome 16q22 containing the disease-causing gene <it>HSF4 </it>(Genbank accession number <ext-link ext-link-id="NM_001040667" ext-link-type="gen">NM_001040667</ext-link>). Here, we describe a family from Pakistan with the first nonsense mutation in <it>HSF4 </it>thus expanding the mutational spectrum of this heat shock transcription factor gene.</p> <p>Methods</p> <p>A large consanguineous Pakistani family with autosomal recessive cataracts was collected from Quetta. Genetic linkage analysis was performed for the common known autosomal recessive cataracts loci and linkage to a locus containing <it>HSF4 </it>(OMIM 602438) was found. All exons and adjacent splice sites of the heat shock transcription factor 4 gene (<it>HSF4</it>) were sequenced. A mutation-specific restriction enzyme digest (H<it>ph</it>I) was performed for all family members and unrelated controls.</p> <p>Results</p> <p>The disease phenotype perfectly co-segregated with markers flanking the known cataract gene HSF4, whereas other autosomal recessive loci were excluded. A maximum two-point LOD score with a Zmax = 5.6 at θ = 0 was obtained for D16S421. Direct sequencing of HSF4 revealed the nucleotide exchange c.1213C > T in this family predicting an arginine to stop codon exchange (p.R405X).</p> <p>Conclusion</p> <p>We identified the first nonsense mutation (p.R405X) in exon 11 of <it>HSF4 </it>in a large consanguineous Pakistani family with autosomal recessive cataract.</p

Retarded PDI diffusion and a reductive shift in poise of the calcium depleted endoplasmic reticulum

Background: Endoplasmic reticulum (ER) lumenal protein thiol redox balance resists dramatic variation in unfolded protein load imposed by diverse physiological challenges including compromise in the key upstream oxidases. Lumenal calcium depletion, incurred during normal cell signaling, stands out as a notable exception to this resilience, promoting a rapid and reversible shift towards a more reducing poise. Calcium depletion induced ER redox alterations are relevant to physiological conditions associated with calcium signaling, such as the response of pancreatic cells to secretagogues and neuronal activity. The core components of the ER redox machinery are well characterized; however, the molecular basis for the calcium-depletion induced shift in redox balance is presently obscure. Results: In vitro, the core machinery for generating disulfides, consisting of ERO1 and the oxidizing protein disulfide isomerase, PDI1A, was indifferent to variation in calcium concentration within the physiological range. However, ER calcium depletion in vivo led to a selective 2.5-fold decline in PDI1A mobility, whereas the mobility of the reducing PDI family member, ERdj5 was unaffected. In vivo, fluorescence resonance energy transfer measurements revealed that declining PDI1A mobility correlated with formation of a complex with the abundant ER chaperone calreticulin, whose mobility was also inhibited by calcium depletion and the calcium depletion-mediated reductive shift was attenuated in cells lacking calreticulin. Measurements with purified proteins confirmed that the PDI1A-calreticulin complex dissociated as Ca2+ concentrations approached those normally found in the ER lumen ([Ca2+] K-0.5max = 190 mu M). Conclusions: Our findings suggest that selective sequestration of PDI1A in a calcium depletion-mediated complex with the abundant chaperone calreticulin attenuates the effective concentration of this major lumenal thiol oxidant, providing a plausible and simple mechanism for the observed shift in ER lumenal redox poise upon physiological calcium depletion.Wellcome Trust [Wellcome 084812/Z/08/Z]; European Commission (EU FP7 Beta-Bat) [277713]; Fundacao para a Ciencia e Tecnologia, Portugal [PTDC/QUI-BIQ/119677/2010]info:eu-repo/semantics/publishedVersio

Drosophila KCNQ Channel Displays Evolutionarily Conserved Electrophysiology and Pharmacology with Mammalian KCNQ Channels

Of the five human KCNQ (Kv7) channels, KCNQ1 with auxiliary subunit KCNE1 mediates the native cardiac IKs current with mutations causing short and long QT cardiac arrhythmias. KCNQ4 mutations cause deafness. KCNQ2/3 channels form the native M-current controlling excitability of most neurons, with mutations causing benign neonatal febrile convulsions. Drosophila contains a single KCNQ (dKCNQ) that appears to serve alone the functions of all the duplicated mammalian neuronal and cardiac KCNQ channels sharing roughly 50–60% amino acid identity therefore offering a route to investigate these channels. Current information about the functional properties of dKCNQ is lacking therefore we have investigated these properties here. Using whole cell patch clamp electrophysiology we compare the biophysical and pharmacological properties of dKCNQ with the mammalian neuronal and cardiac KCNQ channels expressed in HEK cells. We show that Drosophila KCNQ (dKCNQ) is a slowly activating and slowly-deactivating K+ current open at sub-threshold potentials that has similar properties to neuronal KCNQ2/3 with some features of the cardiac KCNQ1/KCNE1 accompanied by conserved sensitivity to a number of clinically relevant KCNQ blockers (chromanol 293B, XE991, linopirdine) and opener (zinc pyrithione). We also investigate the molecular basis of the differential selectivity of KCNQ channels to the opener retigabine and show a single amino acid substitution (M217W) can confer sensitivity to dKCNQ. We show dKCNQ has similar electrophysiological and pharmacological properties as the mammalian KCNQ channels, allowing future study of physiological and pathological roles of KCNQ in Drosophila and whole organism screening for new modulators of KCNQ channelopathies

De Novo Missense Mutations in DHX30 Impair Global Translation and Cause a Neurodevelopmental Disorder

DHX30 is a member of the family of DExH-box helicases, which use ATP hydrolysis to unwind RNA secondary structures. Here we identified six different de novo missense mutations in DHX30 in twelve unrelated individuals affected by global developmental delay (GDD), intellectual disability (ID), severe speech impairment and gait abnormalities. While four mutations are recurrent, two are unique with one affecting the codon of one recurrent mutation. All amino acid changes are located within highly conserved helicase motifs and were found to either impair ATPase activity or RNA recognition in different in vitro assays. Moreover, protein variants exhibit an increased propensity to trigger stress granule (SG) formation resulting in global translation inhibition. Thus, our findings highlight the prominent role of translation control in development and function of the central nervous system and also provide molecular insight into how DHX30 dysfunction might cause a neurodevelopmental disorder

The Rare IL22RA2 Signal Peptide Coding Variant rs28385692 Decreases Secretion of IL-22BP Isoform-1, -2 and -3 and Is Associated with Risk for Multiple Sclerosis.

The IL22RA2 locus is associated with risk for multiple sclerosis (MS) but causative variants are yet to be determined. In a single nucleotide polymorphism (SNP) screen of this locus in a Basque population, rs28385692, a rare coding variant substituting Leu for Pro at position 16 emerged significantly (p = 0.02). This variant is located in the signal peptide (SP) shared by the three secreted protein isoforms produced by IL22RA2 (IL-22 binding protein-1(IL-22BPi1), IL-22BPi2 and IL-22BPi3). Genotyping was extended to a Europe-wide case-control dataset and yielded high significance in the full dataset (p = 3.17 × 10-4). Importantly, logistic regression analyses conditioning on the main known MS-associated SNP at this locus, rs17066096, revealed that this association was independent from the primary association signal in the full case-control dataset. In silico analysis predicted both disruption of the alpha helix of the H-region of the SP and decreased hydrophobicity of this region, ultimately affecting the SP cleavage site. We tested the effect of the p.Leu16Pro variant on the secretion of IL-22BPi1, IL-22BPi2 and IL-22BPi3 and observed that the Pro16 risk allele significantly lowers secretion levels of each of the isoforms to around 50%-60% in comparison to the Leu16 reference allele. Thus, our study suggests that genetically coded decreased levels of IL-22BP isoforms are associated with augmented risk for MS