Efficient oligo nucleotide mediated CRISPR-Cas9 gene editing in Aspergilli.

CRISPR-Cas9 technologies are revolutionizing fungal gene editing. Here we show that survival of specific Cas9/sgRNA mediated DNA double strand breaks (DSBs) depends on the non-homologous end-joining, NHEJ, DNA repair pathway and we use this observation to develop a tool, TAPE, to assess protospacer efficiency in Aspergillus nidulans. Moreover, we show that in NHEJ deficient strains, highly efficient marker-free gene targeting can be performed. Indeed, we show that even single-stranded oligo nucleotides efficiently work as repair templates of specific Cas9/sgRNA induced DNA DSBs in A. nidulans, A. niger, and in A. oryzae indicating that this type of repair may be wide-spread in filamentous fungi. Importantly, we demonstrate that by using single-stranded oligo nucleotides for CRISPR-Cas9 mediated gene editing it is possible to introduce specific point mutations as well gene deletions at efficiencies approaching 100%. The efficiency of the system invites for multiplexing and we have designed a vector system with the capacity of delivering Cas9 and multiple sgRNAs based on polymerase III promoters and tRNA spacers. We show that it is possible to introduce two point mutations and one gene insertion in one transformation experiment with a very high efficiency. Our system is compatible with future high-throughput gene-editing experiments.

Structural and Catalytic Properties of S1 Nuclease from Aspergillus oryzae Responsible for Substrate Recognition, Cleavage, Non-Specificity, and Inhibition.

The single-strand-specific S1 nuclease from Aspergillus oryzae is an archetypal enzyme of the S1-P1 family of nucleases with a widespread use for biochemical analyses of nucleic acids. We present the first X-ray structure of this nuclease along with a thorough analysis of the reaction and inhibition mechanisms and of its properties responsible for identification and binding of ligands. Seven structures of S1 nuclease, six of which are complexes with products and inhibitors, and characterization of catalytic properties of a wild type and mutants reveal unknown attributes of the S1-P1 family. The active site can bind phosphate, nucleosides, and nucleotides in several distinguished ways. The nucleoside binding site accepts bases in two binding modes-shallow and deep. It can also undergo remodeling and so adapt to different ligands. The amino acid residue Asp65 is critical for activity while Asn154 secures interaction with the sugar moiety, and Lys68 is involved in interactions with the phosphate and sugar moieties of ligands. An additional nucleobase binding site was identified on the surface, which explains the absence of the Tyr site known from P1 nuclease. For the first time ternary complexes with ligands enable modeling of ssDNA binding in the active site cleft. Interpretation of the results in the context of the whole S1-P1 nuclease family significantly broadens our knowledge regarding ligand interaction modes and the strategies of adjustment of the enzyme surface and binding sites to achieve particular specificity.

A novel presenilin 1 mutation (Ala275Val) as cause of early-onset familial Alzheimer disease.

Mutations in the presenilin 1 (PS1) gene (PSEN1) are associated with familial Alzheimer disease (FAD). Here, we report on a 50-year-old patient presenting with progressive deterioration of his short-term memory and a family history of early-onset dementia. Diagnostic workup included a neuropsychological examination, structural magnetic resonance (MR) imaging, cerebrospinal fluid (CSF) biomarkers including total tau, phosphorylated tau, and Aß42 levels, as well as sequencing relevant fragments of the genes PSEN1, PSEN2, and APP. Additionally, we were able to obtain archival paraffin-embedded cerebellar tissue from the patient's father for cosegregation analysis. Clinical, neuropsychological and MR imaging data were indicative of early-onset Alzheimer disease. Furthermore, CSF biomarkers showed a typical pattern for Alzheimer disease. DNA sequencing revealed a heterozygous nucleotide transition (c.824C>T) in exon 8 of PSEN1, leading to an amino acid change from alanine to valine at codon 275 (Ala275Val). The same mutation was found in an archival brain specimen of the patient's demented father, but not in a blood sample of the non-demented mother. This mutation alters a conserved residue in the large hydrophilic loop of PS1, suggesting pathogenic relevance. Cosegregegation analysis and the structural as well as the presumed functional role of the mutated and highly conserved residue suggest FAD causing characteristics of the novel PSEN1 mutation Ala275Val.

Association between fully automated MRI-based volumetry of different brain regions and neuropsychological test performance in patients with amnestic mild cognitive impairment and Alzheimer's disease.

Fully automated magnetic resonance imaging (MRI)-based volumetry may serve as biomarker for the diagnosis in patients with mild cognitive impairment (MCI) or dementia. We aimed at investigating the relation between fully automated MRI-based volumetric measures and neuropsychological test performance in amnestic MCI and patients with mild dementia due to Alzheimer's disease (AD) in a cross-sectional and longitudinal study. In order to assess a possible prognostic value of fully automated MRI-based volumetry for future cognitive performance, the rate of change of neuropsychological test performance over time was also tested for its correlation with fully automated MRI-based volumetry at baseline. In 50 subjects, 18 with amnestic MCI, 21 with mild AD, and 11 controls, neuropsychological testing and T1-weighted MRI were performed at baseline and at a mean follow-up interval of 2.1 ± 0.5 years (n = 19). Fully automated MRI volumetry of the grey matter volume (GMV) was performed using a combined stereotactic normalisation and segmentation approach as provided by SPM8 and a set of pre-defined binary lobe masks. Left and right hippocampus masks were derived from probabilistic cytoarchitectonic maps. Volumes of the inner and outer liquor space were also determined automatically from the MRI. Pearson's test was used for the correlation analyses. Left hippocampal GMV was significantly correlated with performance in memory tasks, and left temporal GMV was related to performance in language tasks. Bilateral frontal, parietal and occipital GMVs were correlated to performance in neuropsychological tests comprising multiple domains. Rate of GMV change in the left hippocampus was correlated with decline of performance in the Boston Naming Test (BNT), Mini-Mental Status Examination, and trail making test B (TMT-B). The decrease of BNT and TMT-A performance over time correlated with the loss of grey matter in multiple brain regions. We conclude that fully automated MRI-based volumetry allows detection of regional grey matter volume loss that correlates with neuropsychological performance in patients with amnestic MCI or mild AD. Because of the high level of automation, MRI-based volumetry may easily be integrated into clinical routine to complement the current diagnostic procedure.

Genetic risk factors for depression in Alzheimer`s disease patients.

OBJECTIVE: Alzheimer's Disease (AD) is often associated with depressive symptoms developing at any time before and after AD onset. The aetiology of depression in AD has not sufficiently been characterized, but biological aspects due to neurodegeneration and/ or genetic risk factors may play a plausible role and may distinguish it from common depressive disorders. METHOD: To investigate the possible relationship between genetic risk factors and depression in AD, we assessed genetic polymorphisms reported to be associated with depression (MAOA VNTR, ACE 288bp Insertion/ Deletion, 5HTTLPR, COMT Val158Met, BDNF Val66Met, TPH1 A218C, HTR2A T102C, P2RX7 Q460R, FKBP5 rs1360780 and CRHR1 rs242941) in a cross-sectional study on 246 AD patients with or without clinically significant major depressive disorder (MDD) according to DSM-IV. RESULTS: Significant associations between AD and MDD have been found for three polymorphisms mainly in females (TPH1 A218C, MAOA VNTR and BDNF Val66Met) and one polymorphism in the total population only (FKBP5 rs1360780). There was an increased risk of having MDD in homozygous female carriers of the TPH1 A-allele (odds ratio: 4.35) and homozygous carriers of the MAOA VNTR low activity allele 3R (odds ratio: 3.37). CONCLUSION: We detected allelic or genotypic associations of MAOA, TPH1, FKBP5 and BDNF in clinically significant MDD in AD. Odds-ratios were generally higher in female AD-patients, which might be due to the composition of the study population. Further studies on the neurotransmitter systems affected by the genetic polymorphisms found to be associated with MDD in AD may help to elucidate the underlying pathomechanisms of MDD.

Structure-function interactions of correct retrieval in healthy elderly women.

Recent studies have indicated that there are complex interactions between activation changes and structural alterations in aging. To investigate this issue, we combined functional with structural MRI in healthy old and young women. When contrasting correct with incorrect recognition of words, we found decreased right prefrontal as well as increased middle temporal gyrus (MTG) activation in the older adults. Performance was correlated with prefrontal activation in younger and with MTG activation in older subjects, respectively. Furthermore, frontal decrease and MTG increase were associated with reduced gray matter density of the prefrontal cortex (PFC) and the medial temporal lobe (MTL) across young and old subjects. Within older adults, however, left MTG activation and structural integrity were positively correlated. Thus, our data indicates a two-step structure-function interaction of correct retrieval in healthy aging. First, age-related reduced gray matter density is associated with diminished frontal activation, as well as with compensatory activation in the MTG. Second, compensatory utilization of additional resources is dependent upon a certain degree of structural integrity in the PFC and the MTL.

Characterization of the Aspergillus niger prtT, a unique regulator of extracellular protease encoding genes.

Expression of several Aspergillus niger genes encoding major secreted, but not vacuolar, protease genes including the major acid protease gene pepA, was shown to be affected in the previously isolated A. niger protease mutant, AB1.13 [Mattern, I.E., van Noort, J.M., van den Berg, P., Archer, D.A., Roberts, I.N., Hondel, C.A.M.J.J., 1992. Isolation and characterization of mutants of Aspergillus niger deficient in extracellular proteases. Molecular & General Genetics 2, 332-336]. Complementation cloning of the putative protease-regulatory gene affected in this mutant was accomplished using a functional selection approach based on the use of the A. nidulans amdS selection marker driven by the A. niger pepA promoter. As expected the PpepA::amdS selection marker is not expressed in the mutant. Introduction of a self-replicating cosmid library into the mutant strain carrying the PpepA::amdS marker allowed selection of AmdS+ transformants functionally complementing the proposed regulatory mutation. Analysis of complementing cosmid clones revealed that the complementing sequences contained a gene encoding a member of the fungal-specific Zn2Cys6-binuclear cluster protein family. Sequence comparison of the encoded protein, PrtT, showed that it has homologues among different Aspergillus species. The A. oryzae homologue was shown to govern expression of the major alkaline protease AlpA and neutral protease Np1 in this species. In contrast to several other pathway specific regulators, such as AmyR and XlnR, no PrtT orthologues could be found in any other non-Aspergillus (or related) species and surprisingly, also not in Aspergillus nidulans. Interestingly, in all Aspergillus species carrying a prtT orthologue the gene is tightly clustered to a completely syntenous region carrying an amylolytic gene cluster including another Zn2Cys6-binuclear cluster protein, AmyR. Northern analysis of the A. niger prtT gene showed (constitutive) expression from two upstream promoters about 700 bp apart. The presence of several short upstream open reading frames downstream of both the distal and the proximal transcription start point of the prtT gene suggests regulation at the post-translational level. Also regulation at the level of differential splicing is suggested by the fact that several Aspergillus EST databases carry a considerable fraction of clones in which in frame intron sequences are retained.

Asymmetrical dimethylarginine is increased in plasma and decreased in cerebrospinal fluid of patients with Alzheimer's disease.

BACKGROUND: Asymmetrical dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthase and may alter NO production during pathological conditions. Concerning Alzheimer's disease (AD), there are reports on altered cerebral NO metabolism, but only few studies on ADMA concentrations in plasma and cerebrospinal fluid (CSF). METHODS: We assessed plasma ADMA in 80 AD patients and 80 age- and gender-matched controls and CSF ADMA in a subgroup of 53 AD patients and 20 controls. RESULTS: ADMA plasma concentrations were increased, while CSF ADMA concentrations were decreased in AD patients. There was a significant association between decreasing CSF ADMA levels and the severity of cognitive impairment. CONCLUSION: Elevated ADMA in plasma might be a contributing factor for AD through alterations of NO metabolism, for example decreased cerebral microperfusion, while decreased levels of CSF ADMA might lead to a cerebral increase of NO, peroxynitrite production and oxidative protein damage. Our study reveals different mechanisms of plasma and CSF ADMA regulation, both potentially contributing to AD pathology.

Hippocampal-prefrontal encoding activation predicts whether words can be successfully recalled or only recognized.

The main goal of the present fMRI-study was to identify the neural correlates underlying the successful encoding of words which can subsequently be freely recalled or recognized but not recalled. We were particularly interested in common as well as distinct neural substrates of both retrieval modes. To assess qualitatively differently activated brain areas, categorical subsequent memory analyses were applied. In addition, we used linear parametric modulation to detect brain regions associated with "memory-strength". Our findings suggest that the successful verbal encoding of words, which were recognized but not recalled relies on a subset of the regions engaged during successful encoding of freely recalled words. Furthermore, it seems to be dependent on the magnitude of relational binding in a prefrontal-hippocampal circuit whether a word can subsequently be recalled freely or only recognized.

Combining voxel-based morphometry and diffusion tensor imaging to detect age-related brain changes.

The present study combined optimized voxel-based morphometry and diffusion tensor imaging to detect age-related brain changes. We compared grey matter density maps (grey matter voxel-based morphometry) and white matter fractional anisotropy maps (diffusion tensor imaging-voxel-based morphometry) between two groups of 17 younger and 17 older women. Older women exhibited reduced white matter fractional anisotropy as well as decreased grey matter density most prominently in the frontal, limbic, parietal and temporal lobes. A discriminant analysis identified four frontal and limbic grey and white matter areas that separated the two groups most effectively. We conclude that grey matter voxel-based morphometry and diffusion tensor imaging voxel-based morphometry are well suited for the detection of age-related changes and their combination provides high accuracy when detecting the neural correlates of aging.

Cloning, heterologous expression, and enzymatic characterization of a thermostable glucoamylase from Talaromyces emersonii.

The gene encoding a thermostable glucoamylase from Talaromyces emersonii was cloned and, subsequently, heterologously expressed in Aspergillus niger. This glucoamylase gene encodes a 618 amino acid long protein with a calculated molecular weight of 62,827Da. T. emersonii glucoamylase fall into glucoside hydrolase family 15, showing approximately 60% sequence similarity to glucoamylase from A. niger. The expressed enzyme shows high specific activity towards maltose, isomaltose, and maltoheptaose, having 3-6-fold elevated k(cat) compared to A. niger glucoamylase. T. emersonii glucoamylase showed significantly improved thermostability with a half life of 48h at 65 degrees C in 30% (w/v) glucose, compared to 10h for glucoamylase from A. niger. The ability of the glucoamylase to hydrolyse amylopectin at 65 degrees C is improved compared to A. niger glucoamylase, giving a significant higher final glucose yield at elevated temperatures. The increased thermal stability is thus reflected in the industrial performance, allowing T. emersonii glucoamylase to operate at a temperature higher than the A. niger enzyme.