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Ras-related Rap1A GTPase is implicated in pancreas β-cell insulin secretion and is stimulated by the cAMP sensor Epac2, a guanine exchange factor and activator of Rap1 GTPase. In this study, we examined the differential proteomic profiles of pancreata from C57BL/6 Rap1A-deficient (Null) and control wild-type (WT) mice with nanoLC-ESI-MS/MS to assess targets of Rap1A potentially involved in insulin regulation. We identified 77 overlapping identifier proteins in both groups, with 8 distinct identifier proteins in Null versus 56 distinct identifier proteins in WT mice pancreata. Functional enrichment analysis showed four of the eight Null unique proteins, ERO1-like protein β (Ero1lβ), triosephosphate isomerase (TP1), 14-3-3 protein γ, and kallikrein-1, were exclusively involved in insulin biogenesis, with roles in insulin metabolism. Specifically, the mRNA expression of Ero1lβ and TP1 was significantly (p < 0.05) increased in Null versus WT pancreata. Rap1A deficiency significantly affected glucose tolerance during the first 15–30 min of glucose challenge but showed no impact on insulin sensitivity. Ex vivo glucose-stimulated insulin secretion (GSIS) studies on isolated Null islets showed significantly impaired GSIS. Furthermore, in GSIS-impaired islets, the cAMP-Epac2-Rap1A pathway was significantly compromised compared to the WT. Altogether, these studies underscore an essential role of Rap1A GTPase in pancreas physiological function. [ABSTRACT FROM AUTHOR]

In vitro and ex-vivo target identification strategies often fail to predict in vivo efficacy, particularly for glioblastoma (GBM), a highly heterogenous tumor rich in resistant cancer stem cells (GSCs). An in vivo screening tool can improve prediction of therapeutic efficacy by considering the complex tumor microenvironment and the dynamic plasticity of GSCs driving therapy resistance and recurrence. This study proposes lipid nanoparticles (LNPs) as an efficient in vivo CRISPR-Cas9 gene editing tool for target validation in mesenchymal GSCs. LNPs co-delivering mRNA (mCas9) and single-guide RNA (sgRNA) were successfully formulated and optimized facilitating both in vitro and in vivo gene editing. In vitro, LNPs achieved up to 67 % reduction in green fluorescent protein (GFP) expression, used as a model target, outperforming a commercial transfection reagent. Intratumoral administration of LNPs in GSCs resulted in ∼80 % GFP gene knock-out and a 2-fold reduction in GFP signal by day 14. This study showcases the applicability of CRISPR-Cas9 LNPs as a potential in vivo screening tool in GSCs, currently lacking effective treatment. By replacing GFP with a pool of potential targets, the proposed platform presents an exciting prospect for therapeutic target validation in orthotopic GSCs, bridging the gap between preclinical and clinical research. [Display omitted] • LNPs an efficient in vitro transfection tool of CRISPR-Cas9 RNAs in GSCs, an alterantive to commercial transfecion reagents. • The CRISPR-Cas9 LNP platform enables efficient in vivo gene editing of a difficult to treat model of mesenchymal GSCs. • Proposing CRISPR-Cas9 LNPs as an in vivo screening tool for therapeutic target identification in GSCs. [ABSTRACT FROM AUTHOR]

Clubroot disease is a severe threat to Brassica crops globally, particularly in western Canada. Genetic resistance, achieved through pyramiding clubroot resistance (CR) genes with different modes of action, is the most important strategy for managing the disease. However, studies on the CR gene functions are quite limited. In this study, we have conducted investigations into the temporal, structural, and interacting features of a newly cloned CR gene, Rcr1, using CRISPR/Cas9 technology. For temporal functionality, we developed a novel CRISPR/Cas9- based binary vector, pHHIGR-Hsp18.2, to deliver Rcr1 into a susceptible canola line (DH12075) and observed that early expression of Rcr1 is critical for conferring resistance. For structural functionality, several independent mutations in specific domains of Rcr1 resulted in loss-offunction, highlighting their importance for CR phenotype. In the study of the interacting features of Rcr1, a cysteine protease gene and its homologous allele in canola were successfully disrupted via CRISPR/Cas9 as an interacting component with Rcr1 protein, resulting in the conversion from clubroot resistant to susceptible in plants carrying intact Rcr1. These results indicated an indispensable role of these two cysteine proteases in Rcr1-mediated resistance response. This study, the first of its kind, provides valuable insights into the functionality of Rcr1. Further, the new vector pHHIGR-Hsp18.2 demonstrated an inducible feature on the removal of add-on traits, which should be useful for functional genomics and other similar research in brassica crops. [ABSTRACT FROM AUTHOR]

CRISPR-Cas9, enable precise DNA manipulation via RNA-guided breaks. KHDC3L and PEG3 genes are vital; CRISPR-Cas9 studies their roles in reproduction, development, and gene regulation. Focusing on KHDC3L's impact on PEG3 promoter in HCT116 cells, insights into gene functions and disease mechanisms emerge, informing potential therapies. This study aims to design sgRNAs for the KHDC3L gene using CRISPR tools involved ranking, off-target evaluation, and cloning. HCT116 cells were cultured, synchronized, and transfected with sgRNAs using lipofectamine. Successful transfections were confirmed by fluorescence microscopy. Clonal expansion followed, with DNA extracted and genotyped using PCR and Sanger sequencing. Bisulfite conversion analyzed DNA methylation, employing restriction enzymes for CpG site analysis. Statistical significance (p≤0.05) was assessed using SPSS software. The neighboring regions exhibited significant genomic changes. The designed sgRNAs were cloned into the PX458 plasmid, directing Cas9 to create double-strand breaks (DSBs) in KHDC3L exon 3. Transfected cells showed around 65% efficiency. Gap-PCR confirmed knock-out in 3 out of 17 clones. COBRA analysis revealed allele-specific CpG island methylation in PEG3, indicating the impact of KHDC3L knock-out on PEG3 promoter methylation and expression. The study demonstrates increased PEG3 promoter methylation upon KHDC3L deletion, indicating its role in modulation. Knockout correlates with reduced cell proliferation and colony formation, suggesting KHDC3L's role in promoting cell growth. The gene's relevance in PEG3 regulation and potential therapeutic implications are underscored, though further mechanistic insights are warranted. [ABSTRACT FROM AUTHOR]

Background: We previously identified Leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1) as a marker of long-term neurogenic stem cells in the lateral wall of the adult mouse brain. The morphology of the stem cells thus identified differed from the canonical B1 type stem cells, raising a question about their cellular origin. Thus, we investigated the development of these stem cells in the postnatal and juvenile brain. Furthermore, because Lrig1 is a known regulator of quiescence, we also investigated the effect(s) of its deletion on the cellular proliferation in the lateral wall. Methods: To observe the development of the Lrig1-lineage stem cells, genetic inducible fate mapping studies in combination with thymidine analog administration were conducted using a previously published Lrig1T2A-iCreERT2 mouse line. To identify the long-term consequence(s) of Lrig1 germline deletion, old Lrig1 knock-out mice were generated using two different Lrig1 null alleles in the C57BL/6J background. The lateral walls from these mice were analyzed using an optimized whole mount immunofluorescence protocol and confocal microscopy. Results: We observed the Lrig1-lineage labeled cells with morphologies consistent with neurogenic stem cell identity in postnatal, juvenile, and adult mouse brains. Interestingly, when induced at postnatal or juvenile ages, morphologically distinct cells were revealed, including cells with the canonical B1 type stem cell morphology. Almost all of the presumptive stem cells labeled were non-proliferative at these ages. In the old Lrig1 germline knock-out mice, increased proliferation was observed compared to wildtype littermates without concomitant increase in apoptosis. Conclusions: Once set aside during embryogenesis, the Lrig1-lineage stem cells remain largely quiescent during postnatal and juvenile development until activation in adult age. The absence of premature proliferative exhaustion in the Lrig1 knock-out stem cell niche during aging is likely due to a complex cascade of effects on the adult stem cell pool. Thus, we suggest that the adult stem cell pool size may be genetically constrained via Lrig1. [ABSTRACT FROM AUTHOR]

Background: Saccharomyces cerevisiae generally consumes glucose to produce ethanol accompanied by the main by-products of glycerol, acetic acid, and lactic acid. The minimization of the formation of by-products in S. cerevisiae was an effective way to improve the economic viability of the bioethanol industry. In this study, S. cerevisiae GPD2, FPS1, ADH2, and DLD3 genes were knocked out by the Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR-Cas9) approach. The mechanism of gene deletion affecting ethanol metabolism was further elucidated based on metabolic flux and transcriptomics approaches. Results: The engineered S. cerevisiae with gene deletion of GPD2, FPS1, ADH2, and DLD3 was constructed by the CRISPR-Cas9 approach. The ethanol content of engineered S. cerevisiae GPD2 Delta FPS1 Delta ADH2 Delta DLD3 Delta increased by 18.58% with the decrease of glycerol, acetic acid, and lactic acid contents by 22.32, 8.87, and 16.82%, respectively. The metabolic flux analysis indicated that the carbon flux rethanol in engineered strain increased from 60.969 to 63.379. The sequencing-based RNA-Seq transcriptomics represented 472 differential expression genes (DEGs) were identified in engineered S. cerevisiae, in which 195 and 277 genes were significantly up-regulated and down-regulated, respectively. The enriched pathways of up-regulated genes were mainly involved in the energy metabolism of carbohydrates, while the down-regulated genes were mainly enriched in acid metabolic pathways. Conclusions: The yield of ethanol in engineered S. cerevisiae increased with the decrease of the by-products including glycerol, acetic acid, and lactic acid. The deletion of genes GPD2, FPS1, ADH2, and DLD3 resulted in the redirection of carbon flux. [ABSTRACT FROM AUTHOR]

Background: Saccharomyces cerevisiae generally consumes glucose to produce ethanol accompanied by the main by-products of glycerol, acetic acid, and lactic acid. The minimization of the formation of by-products in S. cerevisiae was an effective way to improve the economic viability of the bioethanol industry. In this study, S. cerevisiae GPD2, FPS1, ADH2, and DLD3 genes were knocked out by the Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR-Cas9) approach. The mechanism of gene deletion affecting ethanol metabolism was further elucidated based on metabolic flux and transcriptomics approaches. Results: The engineered S. cerevisiae with gene deletion of GPD2, FPS1, ADH2, and DLD3 was constructed by the CRISPR-Cas9 approach. The ethanol content of engineered S. cerevisiae GPD2 Delta FPS1 Delta ADH2 Delta DLD3 Delta increased by 18.58% with the decrease of glycerol, acetic acid, and lactic acid contents by 22.32, 8.87, and 16.82%, respectively. The metabolic flux analysis indicated that the carbon flux rethanol in engineered strain increased from 60.969 to 63.379. The sequencing-based RNA-Seq transcriptomics represented 472 differential expression genes (DEGs) were identified in engineered S. cerevisiae, in which 195 and 277 genes were significantly up-regulated and down-regulated, respectively. The enriched pathways of up-regulated genes were mainly involved in the energy metabolism of carbohydrates, while the down-regulated genes were mainly enriched in acid metabolic pathways. Conclusions: The yield of ethanol in engineered S. cerevisiae increased with the decrease of the by-products including glycerol, acetic acid, and lactic acid. The deletion of genes GPD2, FPS1, ADH2, and DLD3 resulted in the redirection of carbon flux. [ABSTRACT FROM AUTHOR]

[Objective] The regulation of GATA transcription factor Ac-nsdD in Aspergillus cristatus of Fu brick tea was further researched.[Method] The The characteristic of Ac-NsdD protein sequence of A. cristatus was analyzed by genome sequencing and annotation and phylogenetic construction. The Ac-nsdD deletion mutant was constructed by homologous recombination via Agrobacterium tumefaciens-mediated transformation, and the phenotype of the A. cristatus mutant was analyzed. [Result] Based on the genome sequencing and annotation, A. cristatus Ac-nsdD encoded a putative transcription factor with a conserved DNA-binding domain, ZnF GATA. Phylogenetic analysis showed that A. cristatus Ac-nsdD gene was closely related to other homologs from filamentous fungal species. The Ac-nsdD gene deletion mutant strain during in vitro cultivation in M3 Y medium displayed the number of cleistothecium decreased significantly when compared with their wild type.Interestingly, during in vitro cultivation in M3 Y medium containing 5% NaCl, the mutant strain showed the amount of cleistothecium was decreased, the amount of conidiation was significantly increased, growth rate slower, pigment formation in the middle of the medium was significantly increased. Furthermore, during in vitro cultivation in M3 Y medium containing 17% NaCl, the Ac-nsdD gene mutant showed the amount of cleistothecium was reduced to nearly zero, the amount of conidiation was decreased, growth rate slower. [Conclusion]. The Ac-nsdD gene in a salt-free environment might have a positive effect on sexual development in A. cristatus. In response to low salt-induced osmotic stress, Ac-nsdD gene might positively regulate sexual reproduction, negatively regulate asexual development, maintain a developmental balance between asexual and sexual sporulation, be involved in pigment biosynthesis. The Ac-nsdD gene might negatively regulate sexual development, asexual development and vegetative growth in response to high salt-induced osmotic stress. This study provides laboratory generated data support for further study of the signaling pathways and downstream-regulated genes associated with Ac-nsdD gene, and for common and unique signal transduction pathways in sexual and asexual development in filamentous fungi. [ABSTRACT FROM AUTHOR]

The CRISPRi system using dCas9 Sth1 from Streptococcus thermophilus developed for Mycobacterium tuberculosis and M. smegmatis was modified to allow gene knock-out in M. abscessus. Efficacy of the knock-out system was evaluated by applying deletions and insertions to the mps1 gene. A comparative genomic analysis of mutants and wild type validated the target specificity. • Streptococcus thermophilus CRISPR/Cas (Cas9 Sth1) used for gene knock-out in Mycobacterium abscessus. • A large variety of deletion size obtained with CRISPR/Cas9 Sth1 in M. abscessus. • High gene target specificity provided by CRISPR/ Cas9 Sth1 in M. abscessus. [ABSTRACT FROM AUTHOR]

[Display omitted] • CRTS (4-ketolase/3-hydroxylase) was in dicyclic pathway of astaxanthin biosynthesis. • CRTYB (lycopene beta-cyclase) played a role in dicyclic and monocyclic pathway. • CRTS and CRTYB were possible rate-limiting enzymes of carotenoids biosynthesis. Phaffia rhodozyma is a potential industrial source of natural astaxanthin. Dicyclic and monocyclic pathways of carotenoid synthesis are important nodes in Phaffia rhodozyma , but the relationship between the two pathways and astaxanthin production remains unclear. Eight genes related to dicyclic and monocyclic pathways in three different strains of P. rhodozyma were analysed, and the relationship between expression and astaxanthin biosynthesis was explored. Among these genes, CRTS genes (R = 0.69, P < 0.05) and CRTYB (R = 0.75, P < 0.05) showed the closest correlations with carotenoid and astaxanthin biosynthesis, respectively. To further study these relationships in detail, the CRTYB and CRTS genes were knocked out by homologous recombination. After CRTYB knockout, astaxanthin was decreased to an undetectable level. This result suggested that CRTYB plays a role in dicyclic and monocyclic pathways. Meanwhile, the CRTS gene was in the dicyclic pathway of astaxanthin biosynthesis, and its knockout promoted the monocyclic pathway and resulted in a 25 % increase in astaxanthin production at 120 h. The possible rate-limiting enzymes were the enzymes encoding CRTS and CRTYB by regression analysis. Knockout of the CRTS and CRTYB genes was helpful in understanding the synthetic pathway of astaxanthin and significant to the industrial application of astaxanthin production. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the effects of cofD gene knock-out on the synthesis of coenzyme F420 and production of methane in Methanobrevibacter ruminantium (M. ruminantium). The experiment successfully constructed a cofD gene knock-out M. ruminantium via homologous recombination technology. The results showed that the logarithmic phase of mutant M. ruminantium (12 h) was lower than the wild-type (24 h). The maximum biomass and specific growth rate of mutant M. ruminantium were significantly lower (P < 0.05) than those of wild-type, and the maximum biomass of mutant M. ruminantium was approximately half of the wild-type; meanwhile, the proliferation was reduced. The synthesis amount of coenzyme F420 of M. ruminantium was significantly decreased (P < 0.05) after the cofD gene knock-out. Moreover, the maximum amount of H2 consumed and CH4 produced by mutant were 14 and 2% of wild-type M. ruminantium respectively. In conclusion, cofD gene knock-out induced the decreased growth rate and reproductive ability of M. ruminantium. Subsequently, the synthesis of coenzyme F420 was decreased. Ultimately, the production capacity of CH4 in M. ruminantium was reduced. Our research provides evidence that cofD gene plays an indispensable role in the regulation of coenzyme F420 synthesis and CH4 production in M. ruminantium. [ABSTRACT FROM AUTHOR]

Despite its convenience and precision, CRISPR-based gene editing approaches still suffer from off-target effects and low efficiencies, which are partially rooted in Cas9, the nuclease component of the CRISPR/Cas9 system. In this study, we showed how mouse genome editing efficiency can be improved by constitutive and inheritable expression of Cas9 nuclease. For this goal, a transgenic mouse line expressing the Cas9 protein (Cas9-mouse) was generated. For in vitro assessment of gene editing efficiency, the Cas9-mice were crossed with the EGFP-mice to obtain mouse embryonic fibroblasts (MEF) expressing both EGFP and Cas9 (MEFCas9-EGFP). Transfection of these cells with in vitro transcribed (IVT) EGFP sgRNA or phU6-EGFPsgRNA plasmid led to robust decrease of Mean Fluorescent Intensity (MFI) to 8500 ± 1025 a.u. and 13,200 ± 1006 a.u. respectively. However, in the control group, in which the MEFEGFP cells were transfected with a pX330-EGFPsgRNA plasmid, the measured MFI was 16,800 ± 2254 a.u. For in vivo assessment, the Cas9-zygotes at two pronuclei stage (2PN) were microinjected with a phU6-HhexsgRNA vector and the gene mutation efficiency was compared with the wild-type (WT) zygotes microinjected with a pX330-HhexsgRNA plasmid. The analysis of born mice showed that while the injection of Cas9-zygotes resulted in 43.75% Hhex gene mutated mice, it was just 15.79% for the WT zygotes. In conclusion, the inheritable and constitutive expression of Cas9 in mice provides an efficient platform for gene editing, which can facilitate the production of genetically-modified cells and animals. [ABSTRACT FROM AUTHOR]

Background: Trichophyton rubrum is an obligate human parasitic fungus and responsible for approximately 80–90% of dermatomycosis in human. Molecular genetic manipulations of this pathogen are challenging and available tools and protocols are only rudimentary. We adapt molecular genetics methods of well established fungal model organism, to knock out genes in T. rubrum. For the adaptation, crucial modifications are necessary. With the implementation of in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, it is possible to adapt molecular genetic methods, to knock out genes in T. rubrum. Results: The gene knock-out method is based on integration of a selection marker into the target site, to interrupt the gene translation. The target gene gets preassigned by the homologous sequence of the in vitro synthesized Cas9-sgRNA ribonucleoprotein complex. To develop the method, we first isolated and characterized a T. rubrum strain with a high amount of microconidia. Next, we developed a transformation protocol, whereby the Cas9-sgRNA ribonucleoprotein gets delivered into the fungal protoplast by the PEG method. We knocked out the URA3 gene and resulted, as predicted, uracil auxotrophic strains. These strains can be used for specific gene knock-outs by reintegrating the URA3 fragment and selection on uracil lacking cultivation media. Exemplary, we knocked out the TRP3 gene and got the predicted phenotype, tryptophan auxotrophic strains. The mutation had been verified by sequencing. Conclusions: We developed a method, based on in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, for target specific gene knock-outs in T. rubrum. We knocked out the Ura3 gene and resulted uracil auxotrophic strains. These strains were used for target specific gene knock-outs by reintegrating the Ura3 fragment into the target gene site to interrupt the gene transcription. The developed method allows to adapt sophisticate gene manipulation methods of model fungal species to non-model species. [ABSTRACT FROM AUTHOR]

Temporally-regulated maternal RNA translation is essential for embryonic development, with defective degradation resulting in stalled 2-cell embryos. We show that DDX1, a DEAD box protein implicated in RNA transport, may be a key regulator of maternal RNA utilization. DDX1 protein localizes exclusively to cytoplasmic granules in both oocytes and early stage mouse embryos, with DDX1 requiring RNA for retention at these sites. Homozygous knockout of Ddx1 causes stalling of mouse embryos at the 2–4 cell stages. These results suggest a maternal RNA-dependent role for DDX1 in the progression of embryos past the 2–4 cell stage. The change in appearance of DDX1-containing granules in developing embryos further supports a role in temporally-regulated degradation of RNAs. We carried out RNA-immunoprecipitations (RNA-IPs) to identify mRNAs bound to DDX1 in 2-cell embryos, focusing on 16 maternal genes previously shown to be essential for embryonic development past the 1- to 2-cell stages. Five of these RNAs were preferentially bound by DDX1: Ago2 , Zar1 , Tle6, Floped and Tif1α. We propose that DDX1 controls access to subsets of key maternal RNAs required for early embryonic development. • DDX1 localizes to cytoplasmic granules in early stage embryos. • DDX1 knockout embryos stall at the 2- to 4-cell stages. • DDX1 co-localizes with stress granule markers TIA-1 and TIAR. • DDX1 localization to cytoplasmic granules is RNA dependent. • DDX1 binds to maternal RNAs that are essential for early embryonic development. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats associated with Cas9 (CRISPR/Cas9) system is a powerful and efficient tool for molecular modification in the genome using programable RNAs. It holds a wide application in plant and animal biotechnology for enhancing multiple traits of interest. Here, we attempted to gather and review the recent progress and provide an overview of the application benefits of this technology in aquatic animal genomes. In this respect, several studies were performed on different species (marine and freshwater) investigating the growth bioprocess trait, wherein, the majority of them focused on somatic axis suppressors such as mstn (myostatin) knock-outs and their effects on the differentiation and proliferation of somatic cells. Regarding the reproduction feature, the retrieved studies provide insights into germ cells, sex reversal, and fertility mechanisms with more focus on zebrafish and tilapia compared to other species. In turn, a generation of de novo strains more resistant to viral infections has been successfully attended to proving the practical ability of this technology in enhancing the immunity of certain species such as grass carp. Furthermore, multiple experiments were performed to explore the pigmentation particularity and understand the melanin biosynthesis pathway via tyr (tyrosinase) gene knock-out in different freshwater species. To investigate the metabolism processes, researchers attempted to grasp the molecular mechanisms underlying polyunsaturated fatty acids biosynthesis and lipid metabolism in Atlantic salmon and channel catfish. In another aspect, earlier studies were undertaken in cyprinids to produce intramuscular bone-free populations as an important trait of interest. Finally, we examined the possible challenges and perspectives for the future use of this technology in the research field. Overall, the application of CRISPR/Cas9 system is an effective approach to manipulating target genes and enhancing economic traits in different aquatic animal species, thereby strengthening genetic breeding programs. • Inducing mutagenesis to enhance different biological traits via CRISPR/Cas9. • Microinjection is the main used technique for CRISPR/Cas9 delivery. • Challenges and perspectives for the future use of this editing tool in the research field. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (Mtb) protein tyrosine phosphatase (PtpA) has so far been known to control intracellular survival of mycobacteria; whereas the ATP synthase which is essential for mycobacterial growth has recently been contemplated in developing a breakthrough anti-TB drug, diarylquinoline. Since both of these enzymes have been established as validated drug targets; we report a robust and functional relationship between these two enzymes through a series of experiments using Mtb H37Ra. In the present study we report that the mycobacterial ATP synthase alpha subunit is regulated by PtpA. We generated gene knock-out for the enzyme PtpA and subjected to determine the mycobacterial replication and the proteome profile of wild type, mutant (Δ ptpA) and complemented (Δ ptpA : ptpA) strains of Mtb H37Ra. A substantial amount of decrease in the protein level of ATP synthase alpha subunit (AtpA) in case of mutant H37Ra was observed, while the levels of the enzyme were either increased or remained unchanged, in wild type and in the complemented strains. • Tyrosine Phosphatase A (PtpA) increases intracellular survival of Mtb H37Ra. • ATP synthase is the essential enzyme which is regulated by PtpA. • Levels of alpha subunit of ATP synthase is notably reduced in ptpA KO H37Ra. • First report showing that PtpA interacts with alpha subunit of ATP synthase in Mtb H37Ra. [ABSTRACT FROM AUTHOR]

Abstract Type IV secretion system (T4SS) is a specialized nanomachine that is utilized for the pathogenicity of gram-negative bacteria. However, the role of T4SS genes in virulence of rice bacterial brown stripe pathogen Acidovorax oryzae (Ao) strain RS-2 is not clear, which contains T4SS gene cluster based on genome-wide analysis. Here we compared the virulence-related phenotypes between the wild-type strain RS-2 and nine T4SS mutants, which were constructed in this study. Results indicated that mutation of pilT, pilM, pilQ, or pilZ3 genes not only significantly reduced bacterial virulence, but also caused a reduction of 20.4–62.0% in biofilm formation and 37.7–47.7% reduction in motility, but had no effect on exopolysaccharide (EPS) production or extracellular enzymatic activities when compared to the wild type. The four T4SS genes had a differential effect on bacterial growth after 24 h post-incubation. The complemented strains of the four T4SS mutants restored similar virulence symptom as the wild type. In addition, no change was observed in bacterial virulence by mutation of the other five T4SS genes. Totally, these results demonstrated that T4SS played vital roles in bacterial virulence, motility and biofilm formation in plant pathogen Ao strain RS-2. [ABSTRACT FROM AUTHOR]

The Type VI secretion system (T6SS) is a class of macromolecular machine that is required for the virulence of gram-negative bacteria. However, it is still not clear what the role of T6SS in the virulence of rice bacterial brown stripe pathogen Acidovorax avenae subsp. avenae (Aaa) is. The aim of the current study was to investigate the contribution of T6SS in Aaa strain RS2 virulence using insertional deletion mutation and complementation approaches. This strain produced weak virulence but contains a complete T6SS gene cluster based on a genome-wide analysis. Here we compared the virulence-related phenotypes between the wild-type (RS-2) and 25 T6SS mutants, which were constructed using homologous recombination methods. The mutation of 15 T6SS genes significantly reduced bacterial virulence and the secretion of Hcp protein. Additionally, the complemented 7 mutations ΔpppA, ΔclpB, Δhcp, ΔdotU, ΔicmF, ΔimpJ, and ΔimpM caused similar virulence characteristics as RS-2. Moreover, the mutant ΔpppA, ΔclpB, ΔicmF, ΔimpJ and ΔimpM genes caused by a 38.3~56.4% reduction in biofilm formation while the mutants ΔpppA, ΔclpB, ΔicmF and Δhcp resulted in a 37.5~44.6% reduction in motility. All together, these results demonstrate that T6SS play vital roles in the virulence of strain RS-2, which may be partially attributed to the reductions in Hcp secretion, biofilm formation and motility. However, differences in virulence between strain RS-1 and RS-2 suggest that other factors may also be involved in the virulence of Aaa. [ABSTRACT FROM AUTHOR]

Random boolean networks are a model of genetic regulatory networks that has proven able to describe experimental data in biology. Random boolean networks not only reproduce important phenomena in cell dynamics, but they are also extremely interesting from a theoretical viewpoint, since it is possible to tune their asymptotic behaviour from order to disorder. The usual approach characterizes network families as a whole, either by means of static or dynamic measures. We show here that a more detailed study, based on the properties of system's attractors, can provide information that makes it possible to predict with higher precision important properties, such as system's response to gene knock-out. A new set of principled measures is introduced, that explains some puzzling behaviours of these networks. These results are not limited to random Boolean network models, but they are general and hold for any discrete model exhibiting similar dynamical characteristics. [ABSTRACT FROM AUTHOR]

The postharvest pathogen Penicillium digitatum is responsible for green mold decay on citrus fruit causing important economic losses. To examine possible elements involved in fungal pathogenesis/virulence and fungicide resistance, identification and functional characterization of PdSte12, a particular type of C2H2 fungal transcription factor was carried out. PdSte12 was functionally inactivated through homologous recombination. The deletant fungal strains (ΔPdSte12) failed to cause green mold decay on citrus fruit. ΔPdSte12 mutants exhibited reduced growth and impaired conidiogenesis during fungal infection towards citrus fruit. Additionally, PdSte12 was characterized via overexpression transformants, showing higher infectivity rate in a low virulence P. digitatum strain, providing evidence of PdSte12 ’s role in virulence. Moreover, fungicide sensitivity evaluation showed that PdSte12 was not involved in fungicide resistance as other transcription factors are. These results indicate that the PdSte12 transcription factor controls invasive growth and asexual reproduction as the major virulence function. [ABSTRACT FROM AUTHOR]

Aureobasidium melanogenum HN6.2 was found to able to produce an esterase of 208.1 ± 2.7 units per ml within 72 h. A molecular weight of the purified esterase was 60.2 kDa and its PI was 4.86. The optimal pH and temperature of the purified esterase were 8.0 and 40 °C, respectively. The purified esterase was stable at the temperature less than 40 °C and in the pH range of 7.5–8.0. The esterase activity was greatly inhibited in the presence of Zn 2+ , Hg 2+ , Fe 2+ , Ni 2+ and SDS. K m and V max values of the enzyme for ρ-nitrophenyl butyrate were 68.6 μM and 251.4 μM per min, respectively. Mass analysis showed that it belonged to a carboxylesterase. After an esterase gene ( Car-Est ) cloned from the genomic DNA of the marine yeast strain HN6.2 was disrupted, the disruptant Y44 obtained exhibited a significant decrease in esterase activity. Meanwhile, a transcriptional level of the Car-Est gene of the disruptant Y44 was only 5.18% that of the Car-Est gene of its wild type strain HN6.2, confirming that the cloned esterase gene was indeed closely related to the esterase activity of the yeast HN6.2 strain. [ABSTRACT FROM AUTHOR]

Background: CRISPR-Cas9 genome editing and labeling has emerged as an important tool in biologic research, particularly in regards to potential transgenic and gene therapy applications. Delivery of CRISPR-Cas9 plasmids to target cells is typically done by non-viral methods (chemical, physical, and/or electrical), which are limited by low transfection efficiencies or with viral vectors, which are limited by safety and restricted volume size. In this work, a non-viral transfection technology, named lance array nanoinjection (LAN), utilizes a microfabricated silicon chip to physically and electrically deliver genetic material to large numbers of target cells. To demonstrate its utility, we used the CRISPR-Cas9 system to edit the genome of isogenic cells. Two variables related to the LAN process were tested which include the magnitude of current used during plasmid attraction to the silicon lance array (1.5, 4.5, or 6.0 mA) and the number of times cells were injected (one or three times). Results: Results indicate that most successful genome editing occurred after injecting three times at a current control setting of 4.5 mA, reaching a median level of 93.77 % modification. Furthermore, we found that genome editing using LAN follows a non-linear injection-dose response, meaning samples injected three times had modification rates as high as nearly 12 times analogously treated single injected samples. Conclusions: These findings demonstrate the LAN's ability to deliver genetic material to cells and indicate that successful alteration of the genome is influenced by a serial injection method as well as the electrical current settings. [ABSTRACT FROM AUTHOR]

Members of the genus Ranavirus (family Iridoviridae ) are large double-stranded (ds) DNA viruses that are found world-wide infecting fish, amphibian and reptile ectothermic hosts. Ranavirus genomes range from 105 to 155 kbp in length and they are predicted to encode around 90–125 genes. Currently, our knowledge of the function of ∼50% of these genes is known or inferred based on homology to orthologous genes characterized in other systems; however, the function of the remaining open reading frames (ORFS) is unknown. Therefore, in order to begin to uncover the function of unknown ORFs in ranaviruses we developed a standardized approach to generate a recombination cassette for any ORF in Ambystoma tigrinum virus (ATV). Our standardized approach quickly and efficiently assembles recombination cassettes and recombinant ATV. We have used this approach to identify two essential, one semi-essential and two non-essential genes in ATV. [ABSTRACT FROM AUTHOR]

Hepatocarcinoma animal models, such as the induced tumor model, transplanted tumor model, gene animal model, are significant experimental tools for the evaluation of targeting drug delivery system as well as the pre-clinical studies of liver cancer. The application of antitumor drug-targeting models not only furnishes similar biological characteristics to human liver cancer but also offers guarantee of pharmacokinetic indicators of the liver-targeting preparations. In this article, we have reviewed some kinds of antitumor drug-targeting models of hepatoma and speculated that the research on this field would be capable of attaining a deeper level and expecting a superior achievement in the future. [ABSTRACT FROM PUBLISHER]

Objective To investigate the effect of SIRT1 gene knock-out on VEGF/AKT pathway in osteoarthritis mice, and to explore its role in the pathogenesis of cartilage degeneration in osteoarthritis. Methods The mice were divided into 2 groups: SIRT1 + / + mice osteoarthritis group （Group A, n = 6） and SIRT1- /- mice osteoarthritis group （Group B, n = 6）. FQ-PCR was used to detect the mRNA expression of SIRT1. HE staining and Safranin O-Fast Green staining were used to observe the morphological changes of the articular cartilage of the knee. The Mankin's score was used to evaluate the degree of articular cartilage degeneration. Immunohistochemical staining was used to detect the protein expressions of SIRT1, VEGF, and AKT. Results The mRNA expression of SIRT1 in Group B was 2. 24417 ± 1. 316569, which was significantly lower than that in Group A （P < 0. 01）. The HE staining and Safranin O-Fast Green staining showed that the articular cartilage in Group B degenerated obviously, with Mankin's score of 9. 8333 ± 1. 94079, which was significantly higher than that in Group A （P < 0. 05）. The immunohistochemical staining integrals of SIRT1,VEGF, and AKT in Group B were 3. 3333 ± 1. 96638, 10. 0000 ± 2. 44949, and 1. 3333 ± 1. 21106. The protein expression of SIRT1 in Group B was not significantly different from that in Group A （P > 0. 05）. The protein expression of VEGF in Group B was significantly higher than that in Group A （P < 0. 05）, but the protein expression of AKT in Group B was much lower than that in Group A （P < 0. 01）. Conclusion Knocking-out SIRT1 may aggravate cartilage degeneration in osteoarthritis through VEGF/AKT signal pathway activation. Therefore, SIRT1 may play a protective role against osteoarthritis. [ABSTRACT FROM AUTHOR]

Obesity is on the rise in westernized countries, and visceral obesity in particular is associated with enhanced risk of developing metabolic disease and accelerated aging. Various dietary restriction regimens have been shown to extend healthy lifespan in a variety of species. However, identification of alternative approaches that could be more acceptable to humans is actively being pursued. We have shown previously that mice deficient in pregnancy-associated plasma protein-A (PAPP-A) have an extended healthy lifespan on a regular chow diet. In this study, we determined the lifespan of PAPP-A knock-out (KO) and wild-type (WT) littermates fed a high fat diet (HFD) starting at 12 months of age. PAPP-A KO and WT mice had equivalent weight gain as measured over 25 weeks on HFD. However, PAPP-A KO mice on HFD had a significant increase in lifespan ( P = 0.018). Body composition and tissue pathology were assessed in a separate cohort of mice after 30 weeks on HFD. Percent body fat was equivalent in the two groups. However, there was a decrease in visceral fat depot weights and an increase in serum adiponectin levels in PAPP-A KO compared to WT mice. Major pathological differences were seen in kidney, heart and testes, with PAPP-A KO mice having little, if any, evidence of inflammation, mineralization, or degeneration in these tissues compared to WT mice. Thus, PAPP-A is a novel drug target with the potential to promote healthy longevity without a need for dietary restriction. [ABSTRACT FROM AUTHOR]

Comamonas testosteroni ( C. testosteroni ) is a gram negative bacterium which can use steroid as a carbon source and degrade steroid with about 20 special enzymes. Most of the enzymes are inducible enzymes. 3-Oxoacyl-ACP reductase (E.C. 1.1.1.100) alternatively known as β-ketoacyl-ACP reductase (BKR) is involved in fatty acid syntheses. DNA sequence comparison showed that BKR belongs to the short-chain alcohol dehydrogenase (SDR) family. Our results showed that BKR is necessary for the degradation of steroid hormones in C. testosteroni . The DNA fragment of the BKR gene was cloned into an expressional plasmid pET-15b. BKR protein was expressed with 6× His-tag on the N-terminus and the enzyme was purified with Ni-column. Antibodies against BKR were prepared and a new BKR quantitative ELISA was created in our laboratory. The purified BKR is a 30.6 kDa protein on SDS–PAGE. C. testosteroni was induced by testosterone, estradiol, estriol and cholesterol. The expression of BKR was detected with an ELISA. The result showed that the BKR expression could be induced by cholesterol and estriol but not by testosterone and estradiol. BKR gene knock-out mutant (M-C.T.) was prepared by homologous integration. High performance liquid chromatography (HPLC) was used to detect steroid hormone degradation in C. testosteroni ATCC11996 and BKR gene knock-out mutant. We proved that the M-C.T. eliminated of testosterone degradation. Degradations of cholesterol and estradiol were also decreased. We conclude that the novel BKR in C. testosteroni plays an important role in steroid degradation. This work provides some new information of SDR and steroid degradation in C. testosteroni . [ABSTRACT FROM AUTHOR]

Bacillus amyloliquefaciens is an important industrial microorganism, which can be applied as a protein expression host. However, the extracellular protein expression ability of B. amyloliquefaciens is still low. Hence, it is necessary to develop a B. amyloliquefaciens host with a high extracellular protein expression capacity. In this study, B. amyloliquefaciens HZ-12 was the original strain, then seven important extracellular protease genes (epr , nprE , aprE -a, mpr , pbpF , vpr , ykct 1), one important intracellular protease gene (aprX) and two redundant proteins genes (htrB , hag) were deleted in HZ-12, resulting in B. amyloliquefaciens BAX-10. In order to evaluate the extracellular protein expression ability of BAX-10, the alkaline protease gene aprE from Bacillus subtilis D7 was expressed in BAX-10. After fermentation, the alkaline protease activity of BAX-10/ aprE reached 666.82 U/mL, which was 57% higher than that of the control strain HZ/ aprE. Moreover, the deletion of the ten genes had no negative effect on the growth of B. amyloliquefaciens. In summary, B. amyloliquefaciens BAX-10 could be used as a potential platform host for expression of target extracellular proteins. • amyloliquefaciens with a high extracellular protease expression ability was constructed. • Neutral protease and alkaline protease were the main extracellular proteases of HZ12. • The alkaline protease activity of BAX-10/ aprE was 1.57 times than that of HZ/ aprE. • The alkaline protease expression ability of BAX-10/ aprE was higher than that of BL9/ aprE. [ABSTRACT FROM AUTHOR]

Abstract: Trypanosoma cruzi calreticulin (TcCRT) can hijack complement C1, mannan-binding lectin and ficolins from serum thus inhibiting the classical and lectin complement pathway activation respectively. To understand the in vivo biological functions of TcCRT in T. cruzi we generated a clonal cell line lacking one TcCRT allele (TcCRT+/−) and another clone overexpressing it (TcCRT+). Both clones were derived from the TCC T. cruzi strain. As expected, TcCRT+/− epimastigotes showed impairment on TcCRT synthesis, whereas TcCRT+ ones showed increased protein levels. In correlation to this, monoallelic mutant parasites were significantly susceptible to killing by the complement machinery. On the contrary, TcCRT+ parasites showed higher levels of resistance to killing mediate by the classical and lectin but not the alternative pathway. The involvement of surface TcCRT in depleting C1 was demonstrated through restoration of serum killing activity by addition of exogenous C1. In axenic cultures, a reduced propagation rate of TcCRT+/− parasites was observed. Moreover, TcCRT+/− parasites presented a reduced rate of differentiation in in vitro assays. As shown by down- or upregulation of TcCRT expression this gene seems to play a major role in providing T. cruzi with the ability to resist complement system. [Copyright &y& Elsevier]

Zinc-finger nucleases ( ZFNs) are artificial enzymes that create site-specific double-strand breaks and thereby induce targeted genome editing. Here, we demonstrated successful gene disruption in somatic and germ cells of medaka ( Oryzias latipes) using ZFN to target exogenous EGFP genes. Embryos that were injected with an RNA sequence pair coding for ZFNs showed mosaic loss of green fluorescent protein fluorescence in skeletal muscle. A number of mutations that included both deletions and insertions were identified within the ZFN target site in each embryo, whereas no mutations were found at the non-targeted sites. In addition, ZFN-induced mutations were introduced in germ cells and efficiently transmitted to the next generation. The mutation frequency varied (6-100%) in the germ cells from each founder, and a founder carried more than two types of mutation in germ cells. Our results have introduced the possibility of targeted gene disruption and reverse genetics in medaka. [ABSTRACT FROM AUTHOR]

Over the past 25 years, the reverse genetic approach including precise and conditional replacement or loss of gene function at a specific locus was considered possible only in mice due to the absence of embryonic stem (ES) or induced pluripotent stem (iPS) cell lines in other species. Recently, however, stem cell technology in rats has become available for biomedical research. In this paper we overview the recent progress of rat ES and iPS cell technology. Starting from the establishment of rat ES cells, the use of ES cells for foreign gene transfer and endogenous gene knock-out is discussed, followed by the successful establishment of rat iPS cells and the generation of an iPS cell-derived organ via interspecific blastocyst complementation. Finally, the possible contribution of rat stem cell technology to reproductive medicine is described. [ABSTRACT FROM AUTHOR]

Abstract: Phosphoinositide-specific phospholipase C (PI-PLC) is a major regulator of calcium-dependent signal transduction, which has been shown to be important in various processes of the malaria parasite Plasmodium. PI-PLC is generally implicated in calcium liberation from intracellular stores through the action of its product, inositol-(1,4,5)-trisphosphate, and is itself dependent on calcium for its activation. Here we describe the plc genes from Plasmodium species. The encoded proteins contain all domains typically found in PI-PLCs of the δ class but are almost twice as long as their orthologues in mammals. Transcriptional analysis by qRT-PCR of plc during the erythrocytic cycle of P. falciparum revealed steady expression levels that increased at the late schizont stages. Genetic analysis in the P. berghei model revealed that the plc locus was targetable but that plc gene knock-outs could not be obtained, thereby strongly indicating that the gene is essential during blood stage development. Alternatively, we attempted to modify plc expression through a promoter exchange approach but found the gene to be refractory to over-expression indicating that plc expression levels might additionally be tightly controlled. [Copyright &y& Elsevier]

Abstract: Hydroperoxide detoxification in African trypanosomes is achieved by 2-Cys-peroxiredoxin (TXNPx)- and non-selenium glutathione peroxidase (Px)-type enzymes which both obtain their reducing equivalents from the unique trypanothione/tryparedoxin system. Previous RNA interference approaches revealed that the cytosolic TXNPx and the Px-type enzymes are essential for Trypanosoma brucei. Because of partially overlapping in vitro substrate specificities and subcellular localisation the physiological function of the individual enzymes was not yet clear. As shown here, TXNPx and Px are expressed at comparable levels and in their active reduced state. Px-overexpressing parasites were less sensitive toward linoleic acid hydroperoxide but not hydrogen peroxide. Kinetic studies confirmed that Px—but not TXNPx—reduces lipophilic hydroperoxides including phospholipids with high efficiency. Most interestingly, the severe proliferation defect of Px-depleted bloodstream cells could be rescued by Trolox, but not by hydrophilic antioxidants, in the medium. This allowed us to knock-out the three Px genes individually and thus to distinguish their in vivo role. Deletion of the cytosolic Px I and II resulted in extremely fast membrane peroxidation followed by cell lysis. Cells lacking specifically the mitochondrial Px III showed a transient growth retardation and cardiolipin peroxidation but adapted within 24h to normal proliferation. [Copyright &y& Elsevier]

The calY gene, encoding metalloprotease camelysin in the Bacillus thuringiensis acrystalliferous strain XBU001, was amplified and sequenced. The camelysin from the calY sequence was 199 amino acids in size ( c. 22 000 Da). The temperature-sensitive plasmid pKESX was used to construct a metalloprotease camelysin-deficient strain of B. thuringiensis. The calY gene was replaced by an erythromycin-resistant gene in KCTF. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and MS analysis showed that the metalloprotease InhA was not expressed after knocking out the gene calY. The temperature-sensitive plasmid pKPC was used to construct a metalloprotease camelysin complementation strain KCTFC. The InhA protein was found in KCTFC. Analysis of the expression of InhA in the wild-type strain KCTF12, camelysin-deficient and complementation strains indicated that inhA expression depended on camelysin. Although camelysin did not directly regulate the expression of the InhA through binding to the promoter of the inhA, the results suggest that camelysin can positively regulate the expression of the InhA protein. [ABSTRACT FROM AUTHOR]

Abstract: The industrially applied ascomycete Hypocrea jecorina (synonym: Trichoderma reesei) exhibits a low rate of exogenous DNA integration by homologous recombination (HR). This hinders the high-throughput generation of strains by gene replacement and is therefore impeding systematic functional gene analyses towards, e.g. strain improvement for protein or enzyme production. To increase the rate of HR events during fungal transformation we identified and deleted the orthologue of the human KU70 in H. jecorina, which is required for the nonhomologous end joining (NHEJ) pathway and responsible for ectopic DNA integration. The effect of the absence of the H. jecorina tku70 on gene targeting was tested by deletion of two so far uncharacterized genes encoding a short chain dehydrogenase and a fungal specific transcription factor. Efficiency of gene targeting for both genes was >95% in a Δtku70 strain when 1kb homologous flanking regions were used in the deletion construct. This is a significant increase in targeting efficiency compared to the parental – non-tku70 deleted – strain TU-6 where a gene knock-out frequency of only 5–10% was observed. Together with the recently annotated genomic sequence of H. jecorina, this system provides a useful tool for a genome-wide functional gene analysis on a high-throughput scale to improve the biotechnological potential of this fungus. [Copyright &y& Elsevier]

AXL, a member of the TAM family, is a promising therapeutic target due to its elevated expression in advanced hepatocellular carcinoma (HCC), particularly in association with acquired drug resistance. Previously, RNA interference was used to study its role in cancer, and several phenotypic changes, including attenuated cell proliferation and decreased migration and invasion, have been reported. The mechanism of action of AXL in HCC is elusive. We first studied the AXL expression in HCC cell lines by real-time PCR and western blot and showed its stringent association with a mesenchymal phenotype. We then explored the role of AXL in mesenchymal SNU475 cells by CRISPR-Cas9 mediated gene knock-out. AXL-depleted HCC cells displayed drastic phenotypic changes, including increased DNA damage response, prolongation of doubling time, G2 arrest, and polyploidization in vitro and loss of tumorigenicity in vivo. Pharmacological inhibition of AXL by R428 recapitulated G2 arrest and polyploidy phenotype. These observations strongly suggest that acute loss of AXL in some mesenchymal HCC cells is lethal and points out that its inhibition may represent a druggable vulnerability in AXL-high HCC patients. [ABSTRACT FROM AUTHOR]

Abstract: Mycothiol (1d-myo-inosityl 2-[N-acetyl-l-cysteinyl]amido-2-deoxy-α-d-glucopyranoside) is an important microbial thiol present only in actinomycetes. Rhodococcus jostii RHA1 degrades a wide range of xenobiotics, including polychlorinated biphenyls, nitriles and N-nitrosodimethylamine. Analyses revealed that this strain produces two thiols, mycothiol and ergothioneine, found in the other actinomycetes. A mycothiol ligase mutant strain of R. jostii RHA1 deficient in the production of mycothiol was constructed. This mutant has a number of interesting characteristics: (a) it overproduces the intermediate glucosamine-inositol (1-O-(2-amino-1-deoxy-α-d-glucopyranosyl)-d-myo-inositol); (b) it is deficient in the biochemical degradation of a number of xenobiotics metabolized by the parent strain; (c) it shows increased susceptibility to a number of antibiotics; and (d) it shows unusual growth characteristics, exhibiting a long lag phase before normal exponential growth. The diverse phenotypes of the mutant indicate the utility of R. jostii RHA1 as a model for deciphering the various functions of mycothiol. [Copyright &y& Elsevier]

Abstract: The role of wiring molecules in circuit assembly is tested directly in genetically engineered animals, in which the corresponding gene has been selectively mutated. Minor alterations in neuronal circuits in these mutant animals are explained by redundancy and/or adaptive changes of other genes relevant for brain development. There is very little known, however, about the extent and nature of the compensatory molecular mechanisms. Using gene microarrays, we compared gene expression patterns in the somatosensory cortex of wild type and ephrinA5 deficient mice, which exhibit subtle, but highly reproducible alterations of thalamocortical projections and intrinsic cortical circuits. We found that between 2.2%–5.7% of all transcripts (140–373 targets) detected in the somatosensory cortex are differentially expressed in comparing wild type and ephrinA5 mutants. A gene group analysis of the annotated transcripts revealed that a high proportion of the dysregulated genes encode proteins relevant for circuit development. Finer grain analysis by in situ hybridization and quantitative RT-PCR revealed that 20% of the Eph/ephrin family genes expressed in the somatosensory cortex are up-regulated in the mutant. One of these genes, EphB6, was up-regulated in all cortical layers, where it is normally expressed. However, the ephrinA2 and EphA5 were up-regulated only in selected layers in the cortex of the mutant; expression levels in other layers did not change. These findings indicate that there is specificity of adaptive and compensative changes in gene expression after the mutation of a single gene relevant for cortical development. Our results also point to the complexity of interpreting phenotypes of gene knock-out animals. [Copyright &y& Elsevier]

Abstract: In many bacteria, gene replacement can only be achieved using RP4-based conjugative systems where exogenous DNA is delivered to the recipient on conditionally replicating plasmids carrying an oriT RP4. In the commonly used strains SM10 and S17-1 conjugative functions are provided by an RP4 plasmid inserted in the chromosome. Those strains, besides mobilizing oriT RP4-carrying plasmids, also transfer their own chromosomal genes, which can be inconvenient for several reasons. We describe here a new version of an RP4-based conjugative system that allows for efficient transfer of mobilizable plasmids without the transfer of chromosomal genes, providing fully controlled and efficient exogeneous DNA delivery to non-transformable bacteria. [Copyright &y& Elsevier]

Experience-dependent changes in the structure of dendritic spines may contribute to learning and memory. Encoded by three genes, the Shank family of postsynaptic scaffold proteins are abundant and enriched in the postsynaptic density (PSD) of central excitatory synapses. When expressed in cultured hippocampal neurons, Shank promotes the maturation and enlargement of dendritic spines. Recently, Shank3 has been genetically implicated in human autism, suggesting an important role for Shank proteins in normal cognitive development. Here, we report the phenotype of Shank1 knock-out mice. Shank1 mutants showed altered PSD protein composition; reduced size of dendritic spines; smaller, thinner PSDs; and weaker basal synaptic transmission. Standard measures of synaptic plasticity were normal. Behaviorally, they had increased anxiety-related behavior and impaired contextual fear memory. Remarkably, Shank1-deficient mice displayed enhanced performance in a spatial learning task; however, their long-term memory retention in this task was impaired. These results affirm the importance of Shank1 for synapse structure and function in vivo, and they highlight a differential role for Shank1 in specific cognitive processes, a feature that may be relevant to human autism spectrum disorders. [ABSTRACT FROM AUTHOR]

Abstract: To date, the function of MADS-domain transcription factors in non-seed plants remains largely elusive, although a number of genes have been isolated and characterized from a variety of species. In our study we analyzed PPM2, a classical MIKC-type MADS-box gene from the moss Physcomitrella patens, taking advantage of the unique technical properties Physcomitrella offers in terms of efficient homologous recombination. We determined mRNA and protein distribution and performed targeted disruption of the genomic locus for functional analysis of PPM2. Despite weak ubiquitous expression, PPM2 protein is mostly found in male and female gametangia and basal parts of developing sporophytes. Therefore, PPM2 seems to function in both the haploid and the diploid phase of the moss life cycle. This situation reflects an evolutionary transition state of gene recruitment from an ancestral gametophytic generation into a derived sporophytic generation which became dominating in tracheophytes. However, a knock-out of the PPM2 gene did not cause visible phenotypical changes in the respective structures. The implications of our findings for the understanding of the evolutionary history of MADS-box transcription factors in plants are discussed. [Copyright &y& Elsevier]

Abstract: In a previous study it was shown that a simple random Boolean network model, with two input connections per node, can describe with a good approximation (with the exception of the smallest avalanches) the distribution of perturbations in gene expression levels induced by the knock-out of single genes in Saccharomyces cerevisiae. Here we address the reason why such a simple model actually works: we present a theoretical study of the distribution of avalanches and show that, in the case of a Poissonian distribution of outgoing links, their distribution is determined by the value of the Derrida exponent. This explains why the simulations based on the simple model have been effective, in spite of the unrealistic hypothesis about the number of input connections per node. Moreover, we consider here the problem of the choice of an optimal threshold for binarizing continuous data, and we show that tuning its value provides an even better agreement between model and data, valuable also in the important case of the smallest avalanches. Finally, we also discuss the choice of an optimal value of the Derrida parameter in order to match the experimental distributions: our results indicate a value slightly below the critical value 1. [Copyright &y& Elsevier]

To better understand the role of multiple neurotrophin ligands and their receptors in vertebrate brain evolution, we examined the distribution of trk neurotrophin receptors in representatives of several vertebrate classes. Trk receptors are largely expressed in homologous neuronal populations among different species/classes of vertebrates. In many neurons, trkB and trkC receptors are co-expressed. TrkB and trkC receptors are primarily found in neurons with more restricted, specialized dendritic and axonal fields that are thought to be involved in discriminative or ‘analytical’ functions. The neurotrophin receptor trkA is expressed predominantly in neurons with larger, overlapping dendritic fields with more heterogeneous connections (‘integrative’ or ‘modulatory’ systems) such as nociceptive and sympathetic autonomic nervous system, locus coeruleus and cholinergic basal forebrain. Surveys of trk receptor expression and function in the peripheral nervous system of different vertebrate classes reveal trends ranging from dependency on a single neurotrophin to a more complex dependency on increasing numbers of neurotrophins and their receptors, for example, in taste and inner ear innervation. Gene deletion studies in mice provide evidence for a complex regulation of neuronal survival of sensory ganglion cells by different neurotrophins. Although expression of neurotrophins and their receptors is predominantly conserved in most circuits, increasing diversity of neurotrophin ligands and their receptors and a more complex dependency of neurons on neurotrophins might have facilitated the formation of at least some new neuronal entities. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Myelin-forming oligodendrocytes facilitate saltatory nerve conduction and support neuronal functions in the mammalian CNS. Although the processes of oligodendrogliogenesis and differentiation from neural progenitor cells have come to light in recent years, the molecular mechanisms underlying oligodendrocyte myelinogenesis are poorly defined. Herein, we demonstrate the pivotal role of the basic helixloop-helix transcription factor, Olig1, in oligodendrocyte myelinogenesis in brain development. Mice lacking a functional Olig1 gene develop severe neurological deficits and die in the third postnatal week. In the brains of these mice, expression of myelin-specific genes is abolished, whereas the formation of oligodendrocyte progenitors is not affected. Furthermore, multilamellar wrapping of myelin membranes around axons does not occur, despite recognition and contact of axons by oligodendrocytes, and Olig1-null mice develop widespread progressive axonal degeneration and gliosis. In contrast, myelin sheaths are formed in the spinal cord, although the extent of myelination is severely reduced. At the molecular level, we find that Olig1 regulates transcription of the major mydin-specific genes, Mbp,Plp1, and Mag, and suppresses expression of a major astrocyte-specific gene, Gfap. Together, our data indicate that Olig1 is a central regulator of oligodendrocyte myelinogenesis in brain and that axonal recognition and myelination by oligodendrocytes are separable processes. [ABSTRACT FROM AUTHOR]

Mammillary bodies and the mammillothalamic tract are parts of a classic neural circuitry that has been implicated in severe memory disturbances accompanying Korsakoff's syndrome. However, the specific role of mammillary bodies in memory functions remains controversial, often being considered as just an extension of the hippocampal memory system. To study this issue we used mutant mice with a targeted mutation in the transcription factor geneFoxb1.These mice suffer perinatal degeneration of the medial and most of the lateral mammillary nuclei, as well as of the mammillothalamic bundle.Foxb1mutant mice showed no deficits in such hippocampal-dependent tasks as contextual fear conditioning and social transmission of food preference. They were also not impaired in the spatial reference memory test in the radial arm maze. However,Foxb1mutants showed deficits in the task for spatial navigation within the Barnes maze. Furthermore, they showed impairments in spatial working memory tasks such as the spontaneous alternation and the working memory test in the radial arm maze. Thus, our behavioural analysis ofFoxb1mutants suggests that the medial mammillary nuclei and mammillothalamic tract play a role in a specific subset of spatial tasks, which require combined use of both spatial and working memory functions. Therefore, the mammillary bodies and the mammillothalamic tract may form an important route through which the working memory circuitry receives spatial information from the hippocampus. [ABSTRACT FROM AUTHOR]

Vertebrate synapsins are abundant synaptic vesicle phosphoproteins that have been proposed to fine-regulate neurotransmitter release by phosphorylation-dependent control of synaptic vesicle motility. However, the consequences of a total lack of all synapsin isoforms due to a knock-out of all three mouse synapsin genes have not yet been investigated. In Drosophila a single synapsin gene encodes several isoforms and is expressed in most synaptic terminals. Thus the targeted deletion of the synapsin gene of Drosophila eliminates the possibility of functional knock-out complementation by other isoforms. Unexpectedly, synapsin null mutant flies show no obvious defects in brain morphology, and no striking qualitative changes in behaviour are observed. Ultrastructural analysis of an identified ‘model’ synapse of the larval nerve muscle preparation revealed no difference between wild-type and mutant, and spontaneous or evoked excitatory junction potentials at this synapse were normal up to a stimulus frequency of 5 Hz. However, when several behavioural responses were analysed quantitatively, specific differences between mutant and wild-type flies are noted. Adult locomotor activity, optomotor responses at high pattern velocities, wing beat frequency, and visual pattern preference are modified. Synapsin mutant flies show faster habituation of an olfactory jump response, enhanced ethanol tolerance, and significant defects in learning and memory as measured using three different paradigms. Larval behavioural defects are described in a separate paper. We conclude that Drosophila synapsins play a significant role in nervous system function, which is subtle at the cellular level but manifests itself in complex behaviour. [ABSTRACT FROM AUTHOR]

It is shown here how gene knock-out experiments can be simulated in Random Boolean Networks (RBN), which are well-known simplified models of genetic networks. The results of the simulations are presented and compared with those of actual experiments in S. cerevisiae. RBN with two incoming links per node have been considered, and the Boolean functions have been chosen at random among the set of so-called canalizing functions.Genes are knocked-out (i.e. silenced) one at a time, and the variations in the expression levels of the other genes, with respect to the unperturbed case, are considered. Two important variables are defined: (i) avalanches, which measure the size of the perturbation generated by knocking out a single gene, and (ii) susceptibilities, which measure how often the expression of a given gene is modified in these experiments.A remarkable observation is that the distributions of avalanches and susceptibilities are very robust, i.e. they are very similar in different random networks; this should be contrasted with the distribution of other variables that show a high variance in RBN.Moreover, the distribution of avalanches and susceptibilities of the RBN models are close to those observed in actual experiments performed with S. cerevisiae, where the changes in gene expression levels have been recorded with DNA microarrays.These findings suggest that these distributions might be “generic” properties, common to a wide range of genetic models and real genetic networks. The importance of such generic properties is discussed. [Copyright &y& Elsevier]

Histamine is a versatile mediator that, according to in vitro studies, affects the synthesis of inflammatory cytokines and acute-phase proteins. The histidine-decarboxylase knockout (HDC–/–) mouse is a model of in vivo investigation of the physiologic and metabolic integration of the acutephase response. These mice do not synthesise histamine and feeding them with histamine-poor diet they are almost completely histamine-deficient. We compared the serum concentrations of representatives of acute-phase plasma proteins, as well as the levels IL-6 and IL-1α in wild type and HDC–/– mice during local (turpentine-induced) or systemic (LPS-induced) inflammation. The level of some acute-phase proteins significantly differed in wild-type and HDC–/– mice while others remained unaffected. The IL-6 levels are also differ in the wild-type and histamine-deficient animals, suggesting that the effect of histamine is attained through IL-6, although direct effect is not disclosed yet. [ABSTRACT FROM AUTHOR]

Nigrostriatal dopaminergic neurons express many nicotinic acetylcholine receptor (nAChR) subunits capable of forming multiple nAChR subtypes. These subtypes are expressed differentially along the neuron and presumably mediate diverse responses. β3 subunit mRNA has restricted expression but is abundant in the substantia nigra and ventral tegmental areas. To investigate the potential tole(s) of nicotinic receptors containing the β3 subunit in dopaminergic tracts, we generated mice with a null mutation in the β3 gene. We were thereby able to identify a population of β3-dependent α-conotoxin MII-binding nAChRs that modulate striatal dopamine release. Changes were also observed in locomotor activity and prepulse inhibition of acoustic startle, behaviors that are controlled, in part, by nigrostriatal and mesolimbic dopaminergic activity, respectively, suggesting that β3-containing nAChRs modulate these behaviors. [ABSTRACT FROM AUTHOR]

Background/Aims: Almost all circulating insulin-like growth factor-1 (IGF-1) is produced and secreted from the liver. However, the possible role of IGF-1 in local regulation of liver functions including liver growth is unclear. In the present study, we investigated the role of IGF-1 on liver growth in vivo and in hepatic stellate cell function in vitro. Results: Liver-specific knock-out of the IGF-1 gene by use of the cre-loxP system caused enhanced liver growth, possibly reflecting increased growth hormone (GH) secretion due to decreased negative feedback by IGF-1. Studies on cultured rat hepatic stellate cells (HSC) showed that IGF-1 and hepatocyte-conditioned medium (PCcM) time- and dose-dependently increased hepatocyte growth factor (HGF) mRNA and HGF immunoreactivity. IGF-1 and PCcM also enhanced DNA synthesis in the HSC cultures. The PCcM did not contain bioactive IGF-1 and was also able to stimulate proliferation when prepared under serum- and hormone-free conditions. Conclusion: In vivo results show that IGF-1 is not essential for normal growth of the intact liver. The in vitro results indicate that both IGF-1 and IGF- 1-independent factor(s) from hepatocytes can stimulate HGF production by HSC. It remains to be investigated whether these effects are of importance for liver regeneration or pathological conditions.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]