Gene Transfection Efficacies of Novel Cationic Gemini Lipids Possessing Aromatic Backbone and Oxyethylene Spacers

Six novel gemini cationic lipids based on aromatic backbone, bearing n-C14H29 or n-C16H33 hydrocarbon chains, differing in the length of oxyethylene type spacers −CH2-(CH2-O-CH2)m-CH2− between each ammonium headgroups have been synthesized, where m varies from 1 to 3. Each of these lipids formed stable suspensions in aqueous media. Cationic liposomes were prepared from each of these lipids individually and as mixtures of each cationic lipid and DOPE. These were used as nonviral gene delivery agents. Transfection studies showed that among lipids bearing n-C14H29 chains, the transfection efficacies decreased with the increase in the length of the spacer, whereas in case of lipids bearing n-C16H33 chains, the transfection efficacies increased with the increase in the length of the spacer. Lipid bearing n-C16H33 hydrocarbon chains with a [−(CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2)−] spacer was found to be a potent gene transfer agent and its transfection was highly serum compatible even in the presence of 50% serum conditions.

Characterization of the carbonic anhydrase gene family and other key osmoregulatory genes in Australian freshwater crayfish (genus Cherax)

Cherax quadricarinatus (Redclaw), C. destructor (Yabby) and C. cainii (Marron) are a group of economically important freshwater crayfish and have been developed for aquaculture production in many countries. As crayfish are farmed in a wide range of culture conditions, optimisation of water quality parameters, are crucial for their maximum growth performance. Previous reports have shown that fluctuations in water quality can negatively impact on growth of crayfish. Therefore, this project aims to identify and characterize the major genes that enable freshwater crayfish to persist in different water chemistries and evaluate their patterns of expression under different water parameters. Overall, this project found a number of candidate genes in all three species and determined that water chemistry had a strong influence on the expression of candidate genes. This information is important in the optimization of water quality parameters in freshwater crayfish aquaculture production.

Neoadjuvant crizotinib in advanced inflammatory myofibroblastic tumour with ALK gene rearrangement

Background Inflammatory myofibroblastic tumours (IMTs) are rare sarcomas that were first described in the lung. They are composed of myofibroblastic mesenchymal spindle cells accompanied by an inflammatory infiltrate of plasma cells. Complete resection is the treatment of choice. There is currently no standard treatment for inoperable or recurrent disease. Expression of ALK protein triggered by ALK gene rearrangement at chromosome 2p23 has been found in 36%-60% of IMTs. Case report We report a rapid early response to crizotinib as neoadjuvant therapy, enabling surgical excision of a large ALK-translocated IMT, which resulted in complete disease clearance. To the best of our knowledge, this is the first case in the literature of a patient with IMT in whom crizotinib was used successfully in the neoadjuvant or curative setting.

First-Line afatinib versus chemotherapy in patients with non–small cell lung cancer and common epidermal growth factor receptor gene mutations and brain metastases

Introduction Metastatic spread to the brain is common in patients with non–small cell lung cancer (NSCLC), but these patients are generally excluded from prospective clinical trials. The studies, phase III study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations (LUX-Lung 3) and a randomized, open-label, phase III study of BIBW 2992 versus chemotherapy as first-line treatment for patients with stage IIIB or IV adenocarcinoma of the lung harbouring an EGFR activating mutation (LUX-Lung 6) investigated first-line afatinib versus platinum-based chemotherapy in epidermal growth factor receptor gene (EGFR) mutation-positive patients with NSCLC and included patients with brain metastases; prespecified subgroup analyses are assessed in this article. Methods For both LUX-Lung 3 and LUX-Lung 6, prespecified subgroup analyses of progression-free survival (PFS), overall survival, and objective response rate were undertaken in patients with asymptomatic brain metastases at baseline (n = 35 and n = 46, respectively). Post hoc analyses of clinical outcomes was undertaken in the combined data set (n = 81). Results In both studies, there was a trend toward improved PFS with afatinib versus chemotherapy in patients with brain metastases (LUX-Lung 3: 11.1 versus 5.4 months, hazard ratio [HR] = 0.54, p = 0.1378; LUX-Lung 6: 8.2 versus 4.7 months, HR = 0.47, p = 0.1060). The magnitude of PFS improvement with afatinib was similar to that observed in patients without brain metastases. In combined analysis, PFS was significantly improved with afatinib versus with chemotherapy in patients with brain metastases (8.2 versus 5.4 months; HR, 0.50; p = 0.0297). Afatinib significantly improved the objective response rate versus chemotherapy in patients with brain metastases. Safety findings were consistent with previous reports. Conclusions These findings lend support to the clinical activity of afatinib in EGFR mutation–positive patients with NSCLC and asymptomatic brain metastases.

Gene modulation and immunoregulatory roles of Interferon gamma

Interferon-gamma (IFN gamma) is a central regulator of the immune response and signals via the Janus Activated Kinase (JAK)-Signal Transducer and Activator of Transcription (STAT) pathway. Phosphorylated STAT1 homodimers translocate to the nucleus, bind to Gamma Activating Sequence (GAS) and recruit additional factors to modulate gene expression. A bioinformatics analysis revealed that greater number of putative promoters of immune related genes and also those not directly involved in immunity contain GAS compared to response elements (RE) for Interferon Regulatory Factor (IRF)1, Nuclear factor kappa B (NF kappa B) and Activator Protein (AP)1. GAS is present in putative promoters of well known IFN gamma-induced genes, IRF1, GBP1, CXCL10, and other genes identified were TLR3, VCAM1, CASP4, etc. Analysis of three microarray studies revealed that the expression of asubset of only GAS containing immune genes were modulated by IFN gamma. As a significant correlation exists between GAS containing immune genes and IFN gamma-regulated gene expression, this strategy may identify novel IFN gamma-responsive immune genes. This analysis is integrated with the literature on the roles of IFN gamma in mediating a plethoraof functions: anti-microbial responses, antigen processing,inflammation, growth suppression, cell death, tumor immunity and autoimmunity. Overall, this review summarizes our present knowledge onIFN gamma mediated signaling and functions. (C) 2009 Elsevier Ltd. All rights reserved.

Ultra high throughput sequencing excludes MDH1 as candidate gene for RP28-linked retinitis pigmentosa

Purpose: Mutations in IDH3B, an enzyme participating in the Krebs cycle, have recently been found to cause autosomal recessive retinitis pigmentosa (arRP). The MDH1 gene maps within the RP28 arRP linkage interval and encodes cytoplasmic malate dehydrogenase, an enzyme functionally related to IDH3B. As a proof of concept for candidate gene screening to be routinely performed by ultra high throughput sequencing (UHTs), we analyzed MDH1 in a patient from each of the two families described so far to show linkage between arRP and RP28. Methods: With genomic long-range PCR, we amplified all introns and exons of the MDH1 gene (23.4 kb). PCR products were then sequenced by short-read UHTs with no further processing. Computer-based mapping of the reads and mutation detection were performed by three independent software packages. Results: Despite the intrinsic complexity of human genome sequences, reads were easily mapped and analyzed, and all algorithms used provided the same results. The two patients were homozygous for all DNA variants identified in the region, which confirms previous linkage and homozygosity mapping results, but had different haplotypes, indicating genetic or allelic heterogeneity. None of the DNA changes detected could be associated with the disease. Conclusions: The MDH1 gene is not the cause of RP28-linked arRP. Our experimental strategy shows that long-range genomic PCR followed by UHTs provides an excellent system to perform a thorough screening of candidate genes for hereditary retinal degeneration.

Transcription of Human Resistin Gene Involves an Interaction of Sp1 with Peroxisome Proliferator-Activating Receptor Gamma (PPAR gamma)

Background: Resistin is a cysteine rich protein, mainly expressed and secreted by circulating human mononuclear cells. While several factors responsible for transcription of mouse resistin gene have been identified, not much is known about the factors responsible for the differential expression of human resistin.Methodology/Principal Finding: We show that the minimal promoter of human resistin lies within similar to 80 bp sequence upstream of the transcriptional start site (-240) whereas binding sites for cRel, CCAAT enhancer binding protein alpha (C/EBP-alpha), activating transcription factor 2 (ATF-2) and activator protein 1 (AP-1) transcription factors, important for induced expression, are present within sequences up to -619. Specificity Protein 1(Sp1) binding site (-276 to -295) is also present and an interaction of Sp1 with peroxisome proliferator activating receptor gamma (PPAR gamma) is necessary for constitutive expression in U937 cells. Indeed co-immunoprecipitation assay demonstrated a direct physical interaction of Sp1 with PPAR gamma in whole cell extracts of U937 cells. Phorbol myristate acetate (PMA) upregulated the expression of resistin mRNA in U937 cells by increasing the recruitment of Sp1, ATF-2 and PPAR gamma on the resistin gene promoter. Furthermore, PMA stimulation of U937 cells resulted in the disruption of Sp1 and PPAR gamma interaction. Chromatin immunoprecipitation (ChIP) assay confirmed the recruitment of transcription factors phospho ATF-2, Sp1, Sp3, PPAR gamma, chromatin modifier histone deacetylase 1 (HDAC1) and the acetylated form of histone H3 but not cRel, C/EBP-alpha and phospho c-Jun during resistingene transcription.Conclusion: Our findings suggest a complex interplay of Sp1 and PPAR gamma along with other transcription factors that drives the expression of resistin in human monocytic U937 cells.

Stochastic simulations of the origins and implications of long-tailed distributions in gene expression

Gene expression noise results in protein number distributions ranging from long-tailed to Gaussian. We show how long-tailed distributions arise from a stochastic model of the constituent chemical reactions and suggest that, in conjunction with cooperative switches, they lead to more sensitive selection of a subpopulation of cells with high protein number than is possible with Gaussian distributions. Single-cell-tracking experiments are presented to validate some of the assumptions of the stochastic simulations. We also examine the effect of DNA looping on the shape of protein distributions. We further show that when switches are incorporated in the regulation of a gene via a feedback loop, the distributions can become bimodal. This might explain the bimodal distribution of certain morphogens during early embryogenesis.

Characterization of the human SLC22A18 gene promoter and its regulation by the transcription factor Sp1

SLC22A18, a poly-specific organic cation transporter, is paternally imprinted in humans and mice. It shows loss-of-heterozygosity in childhood and adult tumors, and gain-of-imprinting in hepatocarcinomas and breast cancers. Despite the importance of this gene, its transcriptional regulation has not been studied, and the promoter has not yet been characterized. We therefore set out to identify the potential cis-regulatory elements including the promoter of this gene. The luciferase reporter assay in human cells indicated that a region from -120 by to +78 by is required for the core promoter activity. No consensus TATA or CHAT boxes were found in this region, but two Sp1 binding sites were conserved in human, chimpanzee, mouse and rat. Mutational analysis of the two Sp1 sites suggested their requirement for the promoter activity. Chromatin-immunoprecipitation showed binding of Sp1 to the promoter region in vivo. Overexpression of Sp1 in Drosophila Sp1-null SL2 cells suggested that Sp1 is the transactivator of the promoter. The human core promoter was functional in mouse 3T3 and monkey COS7 cells. We found a CpG island which spanned the core promoter and exon 1. COBRA technique did not reveal promoter methylation in 10 normal oral tissues, 14 oral tumors, and two human cell lines HuH7 and A549. This study provides the first insight into the mechanism that controls expression of this imprinted tumor suppressor gene. A COBRA-based assay has been developed to look for promoter methylation in different cancers. The present data will help to understand the regulation of this gene and its role in tumorigenesis. (C) 2008 Elsevier B.V. All rights reserved.

Ultra high throughput sequencing excludes MDH1 as candidate gene for RP28-linked retinitis pigmentosa

Purpose: Mutations in IDH3B, an enzyme participating in the Krebs cycle, have recently been found to cause autosomal recessive retinitis pigmentosa (arRP). The MDH1 gene maps within the RP28 arRP linkage interval and encodes cytoplasmic malate dehydrogenase, an enzyme functionally related to IDH3B. As a proof of concept for candidate gene screening to be routinely performed by ultra high throughput sequencing (UHTs), we analyzed MDH1 in a patient from each of the two families described so far to show linkage between arRP and RP28. Methods: With genomic long-range PCR, we amplified all introns and exons of the MDH1 gene (23.4 kb). PCR products were then sequenced by short-read UHTs with no further processing. Computer-based mapping of the reads and mutation detection were performed by three independent software packages. Results: Despite the intrinsic complexity of human genome sequences, reads were easily mapped and analyzed, and all algorithms used provided the same results. The two patients were homozygous for all DNA variants identified in the region, which confirms previous linkage and homozygosity mapping results, but had different haplotypes, indicating genetic or allelic heterogeneity. None of the DNA changes detected could be associated with the disease.

Characterization of the human SLC22A18 gene promoter and its regulation by the transcription factor Sp1

SLC22A18, a poly-specific organic cation transporter, is paternally imprinted in humans and mice. It shows loss-of-heterozygosity in childhood and adult tumors, and gain-of-imprinting in hepatocarcinomas and breast cancers. Despite the importance of this gene, its transcriptional regulation has not been studied, and the promoter has not yet been characterized. We therefore set out to identify the potential cis-regulatory elements including the promoter of this gene. The luciferase reporter assay in human cells indicated that a region from -120 by to +78 by is required for the core promoter activity. No consensus TATA or CHAT boxes were found in this region, but two Sp1 binding sites were conserved in human, chimpanzee, mouse and rat. Mutational analysis of the two Sp1 sites suggested their requirement for the promoter activity. Chromatin-immunoprecipitation showed binding of Sp1 to the promoter region in vivo. Overexpression of Sp1 in Drosophila Sp1-null SL2 cells suggested that Sp1 is the transactivator of the promoter. The human core promoter was functional in mouse 3T3 and monkey COS7 cells. We found a CpG island which spanned the core promoter and exon 1. COBRA technique did not reveal promoter methylation in 10 normal oral tissues, 14 oral tumors, and two human cell lines HuH7 and A549. This study provides the first insight into the mechanism that controls expression of this imprinted tumor suppressor gene. A COBRA-based assay has been developed to look for promoter methylation in different cancers. The present data will help to understand the regulation of this gene and its role in tumorigenesis. (C) 2008 Elsevier B.V. All rights reserved.

Implications of Bohmian quantum ontology for psychopathology

This article discusses the prospects of quantum psychiatry from a Bohmian point of view, which provides an ontological interpretation of quantum theory, and extends such ontology to include mind. At first, we discuss the more general relevance of quantum theory to psychopathology. The basic idea is that because quantum theory emphasizes the role of wholeness, it might be relevant to psychopathology, where breakdown of unity in the mental domain is a key feature. We then discuss the role of information in psychopathology, and consider the connections with quantum theory in this area. In particular, we discuss David Bohm’s notion of active information, which arises in the ontological interpretation of quantum theory, and is suggested to play a fundamental role as the bridge between mind and matter. Some such bridge is needed if we are to understand how subtle mental properties are able to influence more manifest physical properties in the brain (all the way to the molecular and possibly microtubular level), and how changes in those possibly quantum‐level physical processes are able to influence higher cognitive functions. We also consider the implications of the notion of active information for psychopathology. The prospects of implementing the Bohmian scheme in neuroquantal terms are then briefly considered. Finally, we discuss some possible therapeutic implications of Bohm’s approach to information and the relation of mind and matter.

Array-based gene expression, CGH and tissue data defines a 12q24 gain in neuroblastic tumors with prognostic implication

Neuroblastoma has successfully served as a model system for the identification of neuroectoderm-derived oncogenes. However, in spite of various efforts, only a few clinically useful prognostic markers have been found. Here, we present a framework, which integrates DNA, RNA and tissue data to identify and prioritize genetic events that represent clinically relevant new therapeutic targets and prognostic biomarkers for neuroblastoma.

Implications of Bohmian quantum ontology for psychopathology

This article discusses the prospects of quantum psychiatry from a Bohmian point of view, which provides an ontological interpretation of quantum theory, and extends such ontology to include mind. At first, we discuss the more general relevance of quantum theory to psychopathology. The basic idea is that because quantum theory emphasizes the role of wholeness, it might be relevant to psychopathology, where breakdown of unity in the mental domain is a key feature. We then discuss the role of information in psychopathology, and consider the connections with quantum theory in this area. In particular, we discuss David Bohm’s notion of active information, which arises in the ontological interpretation of quantum theory, and is suggested to play a fundamental role as the bridge between mind and matter. Some such bridge is needed if we are to understand how subtle mental properties are able to influence more manifest physical properties in the brain (all the way to the molecular and possibly microtubular level), and how changes in those possibly quantum‐level physical processes are able to influence higher cognitive functions. We also consider the implications of the notion of active information for psychopathology. The prospects of implementing the Bohmian scheme in neuroquantal terms are then briefly considered. Finally, we discuss some possible therapeutic implications of Bohm’s approach to information and the relation of mind and matter.