CRISPR/Cas-derived RNA-guided nucleases (RGNs) that can generate DNA double-strand breaks (DSBs) at a specific sequence are widely used for targeted genome editing by induction of DSB repair in many organisms. The CRISPR/Cas system consists of two components: a single Cas9 nuclease and a single-guide RNA (sgRNA). Therefore, the system for constructing RGNs is simple and efficient, but the utilization of RGNs in filamentous fungi has not been validated. In this study, we established the CRISPR/Cas system in the model filamentous fungus, Pyricularia oryzae, using Cas9 that was codon-optimized for filamentous fungi, and the endogenous RNA polymerase (RNAP) III U6 promoter and a RNAP II fungal promoter for the expression of the sgRNA. We further demonstrated that RGNs could recognize the desired sequences and edit endogenous genes through homologous recombination-mediated targeted gene replacement with high efficiency. Our system will open the way for the development of various CRISPR/Cas-based applications in filamentous fungi.

In this study, it has been clearly demonstrated that prenatal dexamethasone treatment (Dex; 0.05 mg/kg on gestational days 17, 18, and 19) resulted in the significant reductions of 5-hydroxytryptamine (5-HT) turnover in four brain regions, including the neocortex, hippocampus, hypothalamus, and midbrain + pons-medulla (M+P-M) but not in the striatum in the offspring at 3 and 14 wk of life, as well as dopamine turnover in the hypothalamus. [3H]paroxetine binding densities were increased in the hypothalamus and M+P-M at 14 wk of life, which corresponded to increased 5-HT contents in both regions. On the other hand, significantly lower norepinephrine contents in the neocortex and hippocampus were observed in the Dex group compared with the control group at 14 wk of life. In addition, the exposure to new environmental condition elevated blood corticosterone levels and enhanced behavioral activities to a greater extent in the Dex group than in controls at 7 wk of life, suggesting that elevated glucocorticoid levels during the pregnancy mimicked prenatal mild stress, producing developmental alterations in brain monoamine metabolism, endocrine response, and behavior in adult offspring.

IntroductionThe neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is considered to be a potential therapeutic agent for prevention of cerebral ischemia. Ischemia is a most common cause of death after heart attack and cancer causing major negative social and economic consequences. This study was designed to investigate the effect of PACAP38 injection intracerebroventrically in a mouse model of permanent middle cerebral artery occlusion (PMCAO) along with corresponding SHAM control that used 0.9% saline injection.MethodsIschemic and non-ischemic brain tissues were sampled at 6 and 24 hours post-treatment. Following behavioral analyses to confirm whether the ischemia has occurred, we investigated the genome-wide changes in gene and protein expression using DNA microarray chip (4x44K, Agilent) and two-dimensional gel electrophoresis (2-DGE) coupled with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), respectively. Western blotting and immunofluorescent staining were also used to further examine the identified protein factor.ResultsOur results revealed numerous changes in the transcriptome of ischemic hemisphere (ipsilateral) treated with PACAP38 compared to the saline-injected SHAM control hemisphere (contralateral). Previously known (such as the interleukin family) and novel (Gabra6, Crtam) genes were identified under PACAP influence. In parallel, 2-DGE analysis revealed a highly expressed protein spot in the ischemic hemisphere that was identified as dihydropyrimidinase-related protein 2 (DPYL2). The DPYL2, also known as Crmp2, is a marker for the axonal growth and nerve development. Interestingly, PACAP treatment slightly increased its abundance (by 2-DGE and immunostaining) at 6 h but not at 24 h in the ischemic hemisphere, suggesting PACAP activates neuronal defense mechanism early on.ConclusionsThis study provides a detailed inventory of PACAP influenced gene expressions and protein targets in mice ischemic brain, and suggests new targets for thereaupetic interventions.

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Prenatal exposure to chemicals is well known to induce developmental abnormalities in the central nervous system of children. Developmental neurotoxicity (DNT) tests are important to identify neurotoxic agents and prevent neurodevelopmental disorders. We have investigated DNT, focusing on the fetal brain shortly after chemical exposure. To demonstrate a usefulness of a study focusing on the fetal brain in DNT tests, we assessed the fetal brain in a rat valproate-induced autism model. Rats were treated with sodium valproate (VPA, 800 mg/kg) orally on gestational day (GD) 9 or 11 (VPA9 or VPA11), and the fetal brains were examined on GD16 using immunohistochemistry for serotonin (5-HT), tyrosine hydroxylase (TH), and TuJ1 (neuron specific class III beta-tubulin). Hypoplasia of the cortical plate was induced in both VPA9 and VPA11 groups. Abnormal migration of TH-positive and 5-HT neurons, possibly due to the appearance of an abnormally running nerve tract in the pons, was observed only in the VPA11 group. In addition, when we compared the incidence of these abnormalities between pregnant rats mated in our own animal facility (in-house group), and rats purchased pregnant (supplier group), the supplier group was much more sensitive, especially to the pons abnormality. Shipping stress may affect the reproducibility of VPA-induced DNT. The present results demonstrate that examination of the GD16 fetal brain was useful for detecting and characterizing abnormal development of the brain after VPA exposure. Further discussion was made with reference to the findings in children with autism.