Characterization of the Temperature-Sensitive Mutations un-7 and png-1 in Neurospora crassa

The model filamentous fungus Neurospora crassa has been studied for over fifty years and many temperature-sensitive mutants have been generated. While most of these have been mapped genetically, many remain anonymous. The mutation in the N. crassa temperature-sensitive lethal mutant un-7 was identified by a complementation based approach as being in the open reading frame designated NCU00651 on linkage group I. Other mutations in this gene have been identified that lead to a temperature-sensitive morphological phenotype called png-1. The mutations underlying un-7 result in a serine to phenylalanine change at position 273 and an isoleucine to valine change at position 390, while the mutation in png-1 was found to result in a serine to leucine change at position 279 although there were other conservative changes in this allele. The overall morphology of the strain carrying the un-7 mutation is compared to strains carrying the png-1 mutation and these mutations are evaluated in the context of other temperature-sensitive mutants in Neurospora

Effects of deletion of the Streptococcus pneumoniae lipoprotein diacylglyceryl transferase gene lgt on ABC transporter function and on growth in vivo

Lipoproteins are an important class of surface associated proteins that have diverse roles and frequently are involved in the virulence of bacterial pathogens. As prolipoproteins are attached to the cell membrane by a single enzyme, prolipoprotein diacylglyceryl transferase (Lgt), deletion of the corresponding gene potentially allows the characterisation of the overall importance of lipoproteins for specific bacterial functions. We have used a Δlgt mutant strain of Streptococcus pneumoniae to investigate the effects of loss of lipoprotein attachment on cation acquisition, growth in media containing specific carbon sources, and virulence in different infection models. Immunoblots of triton X-114 extracts, flow cytometry and immuno-fluorescence microscopy confirmed the Δlgt mutant had markedly reduced lipoprotein expression on the cell surface. The Δlgt mutant had reduced growth in cation depleted medium, increased sensitivity to oxidative stress, reduced zinc uptake, and reduced intracellular levels of several cations. Doubling time of the Δlgt mutant was also increased slightly when grown in medium with glucose, raffinose and maltotriose as sole carbon sources. These multiple defects in cation and sugar ABC transporter function for the Δlgt mutant were associated with only slightly delayed growth in complete medium. However the Δlgt mutant had significantly reduced growth in blood or bronchoalveolar lavage fluid and a marked impairment in virulence in mouse models of nasopharyngeal colonisation, sepsis and pneumonia. These data suggest that for S. pneumoniae loss of surface localisation of lipoproteins has widespread effects on ABC transporter functions that collectively prevent the Δlgt mutant from establishing invasive infection

Atomic Layer Deposition of ZnO on Multi-walled Carbon Nanotubes and Its Use for Synthesis of CNT–ZnO Heterostructures

In this article, direct coating of ZnO on PECVD-grown multi-walled carbon nanotubes (MWCNTs) is achieved using atomic layer deposition (ALD). Transmission electron microscopy investigation shows that the deposited ZnO shell is continuous and uniform, in contrast to the previously reported particle morphology. The ZnO layer has a good crystalline quality as indicated by Raman and photoluminescence (PL) measurements. We also show that such ZnO layer can be used as seed layer for subsequent hydrothermal growth of ZnO nanorods, resulting in branched CNT–inorganic hybrid nanostructures. Potentially, this method can also apply to the fabrication of ZnO-based hybrid nanostructures on other carbon nanomaterials

Identification of a Transcription Factor Controlling pH-Dependent Organic Acid Response in Aspergillus niger.

Acid formation in Aspergillus niger is known to be subjected to tight regulation, and the acid production profiles are fine-tuned to respond to the ambient pH. Based on transcriptome data, putative trans-acting pH responding transcription factors were listed and through knock out studies, mutants exhibiting an oxalate overproducing phenotype were identified. The yield of oxalate was increased up to 158% compared to the wild type and the corresponding transcription factor was therefore entitled Oxalic Acid repression Factor, OafA. Detailed physiological characterization of one of the ΔoafA mutants, compared to the wild type, showed that both strains produced substantial amounts of gluconic acid, but the mutant strain was more efficient in re-uptake of gluconic acid and converting it to oxalic acid, particularly at high pH (pH 5.0). Transcriptional profiles showed that 241 genes were differentially expressed due to the deletion of oafA and this supported the argument of OafA being a trans-acting transcription factor. Furthermore, expression of two phosphoketolases was down-regulated in the ΔoafA mutant, one of which has not previously been described in fungi. It was argued that the observed oxalate overproducing phenotype was a consequence of the efficient re-uptake of gluconic acid and thereby a higher flux through glycolysis. This results in a lower flux through the pentose phosphate pathway, demonstrated by the down-regulation of the phosphoketolases. Finally, the physiological data, in terms of the specific oxygen consumption, indicated a connection between the oxidative phosphorylation and oxalate production and this was further substantiated through transcription analysis

A reproducible brain tumour model established from human glioblastoma biopsies

<p>Abstract</p> <p>Background</p> <p>Establishing clinically relevant animal models of glioblastoma multiforme (GBM) remains a challenge, and many commonly used cell line-based models do not recapitulate the invasive growth patterns of patient GBMs. Previously, we have reported the formation of highly invasive tumour xenografts in nude rats from human GBMs. However, implementing tumour models based on primary tissue requires that these models can be sufficiently standardised with consistently high take rates.</p> <p>Methods</p> <p>In this work, we collected data on growth kinetics from a material of 29 biopsies xenografted in nude rats, and characterised this model with an emphasis on neuropathological and radiological features.</p> <p>Results</p> <p>The tumour take rate for xenografted GBM biopsies were 96% and remained close to 100% at subsequent passages <it>in vivo</it>, whereas only one of four lower grade tumours engrafted. Average time from transplantation to the onset of symptoms was 125 days ± 11.5 SEM. Histologically, the primary xenografts recapitulated the invasive features of the parent tumours while endothelial cell proliferations and necrosis were mostly absent. After 4-5 <it>in vivo </it>passages, the tumours became more vascular with necrotic areas, but also appeared more circumscribed. MRI typically revealed changes related to tumour growth, several months prior to the onset of symptoms.</p> <p>Conclusions</p> <p><it>In vivo </it>passaging of patient GBM biopsies produced tumours representative of the patient tumours, with high take rates and a reproducible disease course. The model provides combinations of angiogenic and invasive phenotypes and represents a good alternative to <it>in vitro </it>propagated cell lines for dissecting mechanisms of brain tumour progression.</p

Formation of Sclerotia and Production of Indoloterpenes by Aspergillus niger and Other Species in Section Nigri

Several species in Aspergillus section Nigri have been reported to produce sclerotia on well-known growth media, such as Czapek yeast autolysate (CYA) agar, with sclerotia considered to be an important prerequisite for sexual development. However Aspergillus niger sensu stricto has not been reported to produce sclerotia, and is thought to be a purely asexual organism. Here we report, for the first time, the production of sclerotia by certain strains of Aspergillus niger when grown on CYA agar with raisins, or on other fruits or on rice. Up to 11 apolar indoloterpenes of the aflavinine type were detected by liquid chromatography and diode array and mass spectrometric detection where sclerotia were formed, including 10,23-dihydro-24,25-dehydroaflavinine. Sclerotium induction can thus be a way of inducing the production of new secondary metabolites from previously silent gene clusters. Cultivation of other species of the black aspergilli showed that raisins induced sclerotium formation by A. brasiliensis, A. floridensis A. ibericus, A. luchuensis, A. neoniger, A. trinidadensis and A. saccharolyticus for the first time

Criteria for evaluation of disease extent by 123I-metaiodobenzylguanidine scans in neuroblastoma: a report for the International Neuroblastoma Risk Group (INRG) Task Force

BackgroundNeuroblastoma is an embryonic tumour of the sympathetic nervous system, metastatic in half of the patients at diagnosis, with a high preponderance of osteomedullary disease, making accurate evaluation of metastatic sites and response to therapy challenging. Metaiodobenzylguanidine (mIBG), taken into cells via the norepinephrine transporter, provides a sensitive and specific method of assessing tumour in both soft tissue and bone sites. The goal of this report was to develop consensus guidelines for the use of mIBG scans in staging, response assessment and surveillance in neuroblastoma.MethodsThe International Neuroblastoma Risk Group (INRG) Task Force, including a multidisciplinary group in paediatric oncology of North and South America, Europe, Oceania and Asia, formed a subcommittee on metastatic disease evaluation, including expert nuclear medicine physicians and oncologists, who developed these guidelines based on their experience and the medical literature, with approval by the larger INRG Task Force.ResultsGuidelines for patient preparation, radiotracer administration, techniques of scanning including timing, energy, specific views, and use of single photon emission computed tomography are included. Optimal timing of scans in relation to therapy and for surveillance is reviewed. Validated semi-quantitative scoring methods in current use are reviewed, with recommendations for use in prognosis and response evaluation.ConclusionsMetaiodobenzylguanidine scans are the most sensitive and specific method of staging and response evaluation in neuroblastoma, particularly when used with a semi-quantitative scoring method. Use of the optimal techniques for mIBG in staging and response, including a semi-quantitative score, is essential for evaluation of the efficacy of new therapy

What If Evaluation Takes Place Seated Around the Table Whilst Enjoying a Drink?:A Social Practice View of Evaluation of Development Education

This article critically explores the evaluation practice of practitioners working in an English development education organization on the basis of an in-depth longitudinal case study. Ethnographically collected data include observations, documentary analysis and 26 semi-structured interviews with funders, practitioners and coordinators that capture what they do on a daily basis and how they attribute meaning and value to their practice of evaluation. Social practice theory locates evaluation as an everyday routinized practice. A typology of practitioners' engagement in evaluation practice reveals the importance of the reflective and socio-relational features in evaluation in order to transform its practice into an enjoyable experience

Identification of the CRE-1 Cellulolytic Regulon in Neurospora crassa

Background: In filamentous ascomycete fungi, the utilization of alternate carbon sources is influenced by the zinc finger transcription factor CreA/CRE-1, which encodes a carbon catabolite repressor protein homologous to Mig1 from Saccharomyces cerevisiae. In Neurospora crassa, deletion of cre-1 results in increased secretion of amylase and b-galactosidase. Methodology/Principal Findings: Here we show that a strain carrying a deletion of cre-1 has increased cellulolytic activity and increased expression of cellulolytic genes during growth on crystalline cellulose (Avicel). Constitutive expression of cre-1 complements the phenotype of a N. crassa Dcre-1 strain grown on Avicel, and also results in stronger repression of cellulolytic protein secretion and enzyme activity. We determined the CRE-1 regulon by investigating the secretome and transcriptome of a Dcre-1 strain as compared to wild type when grown on Avicel versus minimal medium. Chromatin immunoprecipitation-PCR of putative target genes showed that CRE-1 binds to only some adjacent 59-SYGGRG-39 motifs, consistent with previous findings in other fungi, and suggests that unidentified additional regulatory factors affect CRE-1 binding to promoter regions. Characterization of 30 mutants containing deletions in genes whose expression level increased in a Dcre-1 strain under cellulolytic conditions identified novel genes that affect cellulase activity and protein secretion