An in vitro model of lissencephaly: expanding the role of DCX during neurogenesis.

Lissencephaly comprises a spectrum of brain malformations due to impaired neuronal migration in the developing cerebral cortex. Classical lissencephaly is characterized by smooth cerebral surface and cortical thickening that result in seizures, severe neurological impairment and developmental delay. Mutations in the X-chromosomal gene DCX, encoding doublecortin, is the main cause of classical lissencephaly. Much of our knowledge about DCX-associated lissencephaly comes from post-mortem analyses of patient's brains, mainly since animal models with DCX mutations do not mimic the disease. In the absence of relevant animal models and patient brain specimens, we took advantage of induced pluripotent stem cell (iPSC) technology to model the disease. We established human iPSCs from two males with mutated DCX and classical lissencephaly including smooth brain and abnormal cortical morphology. The disease was recapitulated by differentiation of iPSC into neural cells followed by expression profiling and dissection of DCX-associated functions. Here we show that neural stem cells, with absent or reduced DCX protein expression, exhibit impaired migration, delayed differentiation and deficient neurite formation. Hence, the patient-derived iPSCs and neural stem cells provide a system to further unravel the functions of DCX in normal development and disease.

Identification of Candida species in vaginal flora using conventional and molecular methods.

Vaginal swab samples were collected both from women with signs of acute vulvovaginal candidiasis (VVC; n=270) and from healthy controls (n=100). The microscopic examination revealed a significant difference in the proportion of positive cultures of Candida between the VVC (33%) and control (21%) groups. To determine the frequency of different species, positive cultures were analyzed using the germ tube test, CHROMagar medium, API 20 AUX System and DNA analysis. CHROMagar and API 20 AUX System tests identified 67% of samples as C. albicans. Out of these, 42% appeared to be C. dubliniensis when the specific primers in the polymerase chain reaction (PCR) were used. We observed the prevalence of Candida species isolated from the vagina in descending order as follows: C. albicans (38-39%), C. glabrata (32-33%), C. dubliniensis (28-29%) and C. tropicalis (0-1%) for both the VVC and the control group.

Use of the Saccharomyces cerevisiae endopolygalacturonase promoter to direct expression in Escherichia coli.

In Saccharomyces cerevisiae, an endopolygalacturonase encoded by the PGL1 gene catalyzes the random hydrolysis of the α-1,4 glycosidic linkages in polygalacturonic acid. To study the regulation of the PGL1 gene, we constructed a reporter vector containing the lacZ gene under the control of PGL1 promoter. Surprisingly, when Escherichia coli DH5α was transformed by this vector, cells harboring the constructed plasmid produced blue colonies. Sequence analysis of this promoter revealed that E. coli consensus sequences required to express an in-frame lacZ alpha product were present. We next decided to investigate how the PGL1 promoter is regulated in E. coli compared to yeast. In this study, we examined the modulation of the PGL1 promoter in E. coli, and the results indicated that its activity is greatly induced by saturated digalacturonic acid and is indirectly regulated by the transcriptional regulators the 2-keto-3-deoxygluconate repressor. Moreover, PGL1 expression is enhanced under aerobic conditions. We found that ß-galactosidase activity in E. coli could reach 180 units, which is 40-fold greater than the activity produced in S. cerevisiae, and greater than recombinant protein expression previously reported by other researchers. We thus demonstrate that this vector can be considered as a dual expression plasmid for both E. coli and S. cerevisiae hosts. So far, no modulation of endoPG promoters expressed in E. coli has been reported.

An efficient method for DNA extraction from Cladosporioid fungi.

We developed an efficient method for DNA extraction from Cladosporioid fungi, which are important fungal plant pathogens. The cell wall of Cladosporioid fungi is often melanized, which makes it difficult to extract DNA from their cells. In order to overcome this we grew these fungi for three days on agar plates and extracted DNA from mycelium mats after manual or electric homogenization. High-quality DNA was isolated, with an A(260)/A(280) ratio ranging between 1.6 and 2.0. Isolated genomic DNA was efficiently digested with restriction enzymes and produced distinct banding patterns on agarose gels for the different Cladosporium species. Clear DNA fragments from the isolated DNA were amplified by PCR using small and large subunit rDNA primers, demonstrating that this method provides DNA of sufficiently high quality for molecular analyses.

Molecular detection of ochratoxigenic Aspergillus species isolated from coffee beans in Saudi Arabia.

Ten fungal isolates from coffee beans were morphologically identified as Aspergillus niger, A. ochraceus and A. carbonari-us (N = 5, 3, and 2, respectively). Only one isolate, morphologically identified as A. niger, was unable to produce ochratoxin A (OTA). This may be a new species in the Aspergillus section Nigri. OTA levels in all the other isolates were above the limit of detection (0.15 mg/kg). Based on microsatellite-primed PCR (MP-PCR) profiles, using three microsatellite primers, three main groups were obtained by UPGMA cluster analysis: A. niger, A. ochraceus and A. carbonarius. A clear-cut association was found between the MP-PCR genotype and the ability to produce OTA. Using the primer pairs OCRA1/OCRA2, a single fragment of about 400 bp was amplified only when genomic DNA from the A. ochraceus isolates was used.

M13-microsatellite PCR and rDNA sequence markers for identification of Trichoderma (Hypocreaceae) species in Saudi Arabian soil.

Seven fungal isolates were identified as pan-global Hypocrea/Trichoderma species, from section Trichoderma, on the basis of their morphology. These species were H. lixii/T. harzianum and H. orientalis/T. longibrachiatum. PCR-based markers with primer M13 (core sequence of phage M13) and internal-transcribed spacer sequences of ribosomal DNA were used to confirm the identity of the two Trichoderma species. Sequence identification was performed using the TrichOKEY version 2.0 barcode program and the multilocus similarity search database TrichoBLAST. Sequences from the ribosomal DNA internal-transcribed spacer regions showed limited variation among the Trichoderma species. This analysis divided the isolates into two main groups. Grouping the isolates based on cluster analysis of their DNA profiles matched the grouping based on morphological taxonomy. Molecular data obtained from analyses of gene sequences are essential to distinguish phonetically cryptic species in this group and to establish phylogenetic relationships.

Effect of neem (Azardirachta indica A. Juss) seeds and leaves extract on some plant pathogenic fungi.

In this study plant pathogenic fungi Alternaria solani, Fusarium oxysporum, Rhizoctonia solani and Sclerotinia sclerotiorum were chosen to study the effect of ethanolic, hexane and methanolic extracts of neem seeds and leaves. Antifungal effects of neem leave and seed extracts obtained by ethanol, hexane and ptrolium ether were examined separately in vitro against Fusarium oxysporum, Rhizoctonia solani, Alternaria solani and Sclerotinia sclerotiorum. Results indicated that seeds and leaves extracts could cause growth inhibition of tested fungi, although the rate of inhibition of tested fungi varied with different extracts and concentrations. But all these extracts and concentrations of extract inhibited the growth of pathogenic fungi at a significant level. Azadirachtin, nimonol and expoxyazdirodione were detected from neem extract by using High Performance Liquid Chromatography (HPLC). We can conclude that neem leave and seed extracts were effective as antifungal against all tested fungi but F. oxysporum and R. solani were the most sensitive fungi.