With the completion of genome sequences of major model organisms, increasingly sophisticated genetic tools are necessary for investigating the complex and coordinated functions of genes. Here we describe a genetic manipulation system termed ''genomic engineering'' in Drosophila. Genomic engineering is a 2-step process that combines the ends-out (replacement) gene targeting with phage integrase C31-mediated DNA integration. First, through an improved and modified gene targeting method, a founder knockout line is generated by deleting the target gene and replacing it with an integration site of C31. Second, DNA integration by C31 is used to reintroduce modified target-gene DNA into the native locus in the founder knock-out line. Genomic engineering permits directed and highly efficient modifications of a chosen genomic locus into virtually any desired mutant allele. We have successfully applied the genomic engineering scheme on 6 different genes and have generated at their loci more than 70 unique alleles.cell polarity ͉ ends-out targeting ͉ homologous recombination ͉ phiC31 integrase T he development of homologous recombination (HR)-based gene targeting was a major breakthrough in Drosophila genetics (1, 2). At present, in Drosophila as well as in mice, a HR-based approach is virtually the only way to make directed modifications of a target gene (3, 4). However, because the entire targeting process must be repeated for making each allele, the amount of time and labor may become impractical to make more than just a few targeted alleles. In addition, because of the requirement of HR, it can be very difficult to introduce appreciably complicated DNA sequence modifications by gene targeting. The current lack of adequate genetic tools for directed and efficient modifications of the genome presents a major hurdle in Drosophila genetics today. For example, many of the protein pathways that are highly conserved between Drosophila and vertebrates, such as the cell polarity pathway (5), appear to be exceedingly complex. Rigorous genetic dissections of such intricate protein networks can be highly challenging, because in most cases the functions of mutated or modified individual genes of such pathways can only be assayed by artificial over-expression methods, which often lack the requisite controllability and fidelity of gene expression. One ideal solution would be for each protein gene of interest to generate, at the gene's native genomic locus, a set of defined mutant alleles that are strategically designed to test hypotheses about the protein's in vivo functions and interactions. Furthermore, being able to generate any conceivable alleles of a target gene, such as functional fusion alleles of fluorescent proteins/purification tags or alleles with conditional activities, would also offer us unprecedented freedom and opportunities to explore unique experiments of imaging, proteomics, and disease models.To achieve the goal of such directed, efficient, and versatile modifications of the Drosophila genome, we have developed a...

In this report, we describe several approaches to improve the scalability and throughput of major genetic crosses in ends-out gene targeting. We generated new sets of targeting vectors and fly stocks and introduced a novel negative selection marker that drastically reduced the frequency of false-positive targeting candidates.

Pulmonary inflammation, which is characterized by the presence of perivascular macrophages, has been proposed as a key pathogenic driver of pulmonary hypertension (PH), a vascular disease with increasing global significance. However, the mechanisms of expansion of lung macrophages and the role of blood-borne monocytes in PH are poorly understood. Using multicolor flow cytometric analysis of blood in mouse and rat models of PH and patients with PH, an increase in blood monocytes was observed. In parallel, lung tissue displayed increased chemokine transcript expression, including those responsible for monocyte recruitment, such as and, accompanied by an expansion of interstitial lung macrophages. These data indicate that blood monocytes are recruited to lung perivascular spaces and differentiate into inflammatory macrophages. Correspondingly, parabiosis between congenically different hypoxic mice demonstrated that most interstitial macrophages originated from blood monocytes. To define the actions of these cells in PH in vivo, we reduced blood monocyte numbers via genetic deficiency of or in chronically hypoxic male mice and by pharmacologic inhibition of Cxcl1 in monocrotaline-exposed rats. Both models exhibited decreased inflammatory blood monocytes, as well as interstitial macrophages, leading to a substantial decrease in arteriolar remodeling but with a less robust hemodynamic effect. This study defines a direct mechanism by which interstitial macrophages expand in PH. It also demonstrates a pathway for pulmonary vascular remodeling in PH that depends upon interstitial macrophage-dependent inflammation yet is dissociated, at least in part, from hemodynamic consequences, thus offering guidance on future anti-inflammatory therapeutic strategies in this disease.

Background: Stigma toward mentally ill individuals acts as a barrier to accessing care and receiving treatment. Aim: To review current evidence pertaining to stigma toward mental illness in the Middle East in order to inform effective and sustainable interventions in this region. Methods: We conducted a systematic literature search using the PubMed database and evaluated all identified studies according to specific inclusion criteria. Results: Stigma toward individuals with mental illness does exist in the Middle East. Stigmatizing attitudes are particularly high toward culturally proscribed mental illnesses like alcohol abuse and lower for other disorders such as depression and psychosis. Conclusions: We propose the following initiatives to reduce stigma toward mental illness in the Middle East: (a) educate families to enable them to support their affected relatives, (b) increase cooperation between psychiatrists and faith healers and (c) educate young people in schools to increase their awareness and understanding of mental illnesses and to combat negative stereotypes.

Latent infection with neurotropic herpes viruses, such as herpes simplex virus, type 1 (HSV1), has been generally considered benign in most immunocompetent individuals except for rare cases of encephalitis. However, several recent studies have shown impaired cognitive functions among individuals with schizophrenia exposed to HSV1 compared with schizophrenia patients not exposed to HSV1. Such impairments are robust and are prominently observed in working memory, verbal memory, and executive functions. Brain regions that play a key role in the regulation of these domains have shown smaller volumes, along with correlation between these morphometric changes and cognitive impairments in schizophrenia. One study noted temporal decline in executive function and gray matter loss among HSV1-exposed first-episode antipsychotic-naïve schizophrenia patients. Furthermore, a proof-of-concept double-blind placebo-controlled trial indicated improvement in cognitive performance following supplemental anti-herpes-specific medication among HSV1 seropositive schizophrenia patients. Cross-sectional studies have also identified an association between HSV1 exposure and lesser degrees of cognitive impairment among healthy control individuals and patients with bipolar disorder. These studies fulfill several Bradford-Hill criteria, suggesting etiological links between HSV1 exposure and cognitive impairment. Exposure to other human herpes viruses such as cytomegalovirus and herpes simplex virus type 2 (HSV2) may also be associated with cognitive impairment, but the data are less consistent. These studies are reviewed critically and further lines of enquiry recommended. The results are important from a public health perspective, as HSV1 exposure is highly prevalent in many populations.