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There are hundreds of genes typically overexpressed in breast cancer cells and it's often assumed that their overexpression contributes to cancer progression. However, the precise proportion of these overexpressed genes contributing to tumorigenicity remains unclear. To address this gap, we undertook a comprehensive screening of a diverse set of seventy-two genes overexpressed in breast cancer. This systematic screening evaluated their potential for inducing malignant transformation and, concurrently, assessed their impact on breast cancer cell proliferation and viability. Select genes including ALDH3B1, CEACAM5, IL8, PYGO2, and WWTR1, exhibited pronounced activity in promoting tumor formation and establishing gene dependencies critical for tumorigenicity. Subsequent investigations revealed that CEACAM5 overexpression triggered the activation of signaling pathways involving β-catenin, Cdk4, and mTOR. Additionally, it conferred a growth advantage independent of exogenous insulin in defined medium and facilitated spheroid expansion by inducing multiple layers of epithelial cells while preserving a hollow lumen. Furthermore, the silencing of CEACAM5 expression synergized with tamoxifen-induced growth inhibition in breast cancer cells. These findings underscore the potential of screening overexpressed genes for both oncogenic drivers and tumor dependencies to expand the repertoire of therapeutic targets for breast cancer treatment., (© 2024. The Author(s).)

Here we provide a detailed tutorial on CRISPR in vivo screening. Using the mouse as the model organism, we introduce a range of CRISPR tools and applications, delineate general considerations for 'transplantation-based' or 'direct in vivo' screening design, and provide details on technical execution, sequencing readouts, computational analyses and data interpretation. In vivo screens face unique pitfalls and limitations, such as delivery issues or library bottlenecking, which must be counteracted to avoid screening failure or flawed conclusions. A broad variety of in vivo phenotypes can be interrogated such as organ development, hematopoietic lineage decision and evolutionary licensing in oncogenesis. We describe experimental strategies to address various biological questions and provide an outlook on emerging CRISPR applications, such as genetic interaction screening. These technological advances create potent new opportunities to dissect the molecular underpinnings of complex organismal phenotypes., (© 2022. Springer Nature Limited.)

Systemic therapies for pancreatic ductal adenocarcinoma (PDAC) remain unsatisfactory. Clinical prognosis is particularly poor for tumor subtypes with activating aberrations in the MYC pathway, creating an urgent need for novel therapeutic targets. To unbiasedly find MYC-associated epigenetic dependencies, we conducted a drug screen in pancreatic cancer cell lines. Here, we found that protein arginine N-methyltransferase 5 (PRMT5) inhibitors triggered an MYC-associated dependency. In human and murine PDACs, a robust connection of MYC and PRMT5 was detected. By the use of gain- and loss-of-function models, we confirmed the increased efficacy of PRMT5 inhibitors in MYC-deregulated PDACs. Although inhibition of PRMT5 was inducing DNA damage and arresting PDAC cells in the G2/M phase of the cell cycle, apoptotic cell death was executed predominantly in cells with high MYC expression. Experiments in primary patient-derived PDAC models demonstrated the existence of a highly PRMT5 inhibitor-sensitive subtype. Our work suggests developing PRMT5 inhibitor-based therapies for PDAC.

Genetically engineered mouse models (GEMMs) transformed the study of organismal disease phenotypes but are limited by their lengthy generation in embryonic stem cells. Here, we describe methods for rapid and scalable genome engineering in somatic cells of the liver and pancreas through delivery of CRISPR components into living mice. We introduce the spectrum of genetic tools, delineate viral and nonviral CRISPR delivery strategies and describe a series of applications, ranging from gene editing and cancer modeling to chromosome engineering or CRISPR multiplexing and its spatio-temporal control. Beyond experimental design and execution, the protocol describes quantification of genetic and functional editing outcomes, including sequencing approaches, data analysis and interpretation. Compared to traditional knockout mice, somatic GEMMs face an increased risk for mouse-to-mouse variability because of the higher experimental demands of the procedures. The robust protocols described here will help unleash the full potential of somatic genome manipulation. Depending on the delivery method and envisaged application, the protocol takes 3-5 weeks., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

With the genetic portraits of all major human malignancies now available, we next face the challenge of characterizing the function of mutated genes, their downstream targets, interactions and molecular networks. Moreover, poorly understood at the functional level are also non-mutated but dysregulated genomes, epigenomes or transcriptomes. Breakthroughs in manipulative mouse genetics offer new opportunities to probe the interplay of molecules, cells and systemic signals underlying disease pathogenesis in higher organisms. Herein, we review functional screening strategies in mice using genetic perturbation and chemical mutagenesis. We outline the spectrum of genetic tools that exist, such as transposons, CRISPR and RNAi and describe discoveries emerging from their use. Genome-wide or targeted screens are being used to uncover genomic and regulatory landscapes in oncogenesis, metastasis or drug resistance. Versatile screening systems support experimentation in diverse genetic and spatio-temporal settings to integrate molecular, cellular or environmental context-dependencies. We also review the combination of in vivo screening and barcoding strategies to study genetic interactions and quantitative cancer dynamics during tumour evolution. These scalable functional genomics approaches are transforming our ability to interrogate complex biological systems.

The systematic perturbation of genomes using CRISPR/Cas9 deciphers gene function at an unprecedented rate, depth and ease. Commercially available sgRNA libraries typically contain tens of thousands of pre-defined constructs, resulting in a complexity challenging to handle. In contrast, custom sgRNA libraries comprise gene sets of self-defined content and size, facilitating experiments under complex conditions such as in vivo systems. To streamline and upscale cloning of custom libraries, we present CLUE, a bioinformatic and wet-lab pipeline for the multiplexed generation of pooled sgRNA libraries. CLUE starts from lists of genes or pasted sequences provided by the user and designs a single synthetic oligonucleotide pool containing various libraries. At the core of the approach, a barcoding strategy for unique primer binding sites allows amplifying different user-defined libraries from one single oligonucleotide pool. We prove the approach to be straightforward, versatile and specific, yielding uniform sgRNA distributions in all resulting libraries, virtually devoid of cross-contaminations. For in silico library multiplexing and design, we established an easy-to-use online platform at www.crispr-clue.de. All in all, CLUE represents a resource-saving approach to produce numerous high quality custom sgRNA libraries in parallel, which will foster their broad use across molecular biosciences., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In the 2015 Paris Agreement, nations worldwide pledged emissions reductions (Nationally Determined Contributions-NDCs) to avert the threat of climate change, and agreed to periodically review these pledges to strengthen their level of ambition. Previous studies have analyzed NDCs largely in terms of their implied contribution to limit global warming, their implications on the energy sector or on mitigation costs. Nevertheless, a gap in the literature exists regarding the understanding of implications of the NDCs on countries' Energy-Water-Land nexus resource systems. The present paper explores this angle within the regional context of Latin America by employing the Global Change Assessment Model, a state-of-the-art integrated assessment model capable of representing key system-wide interactions among nexus sectors and mitigation policies. By focusing on Brazil, Mexico, Argentina and Colombia, we stress potential implications on national-level water demands depending on countries' strategies to enforce energy-related emissions reductions and their interplays with the land sector. Despite the differential implications of the Paris pledges on each country, increased water demands for crop and biomass irrigation and for electricity generation stand out as potential trade-offs that may emerge under the NDC policy. Hence, this study underscores the need of considering a nexus resource planning framework (known as "Nexus Approach") in the forthcoming NDCs updating cycles as a mean to contribute toward sustainable development., Competing Interests: The authors have declared that no competing interests exist.

RNA molecules are subject to a complex co-transcriptional and post-transcriptional life cycle, controlled at all stages by RNA binding proteins (RBPs) and non-coding RNAs that influence mRNA stability, splicing, localization, and decay. Together with mechanisms regulating the process of transcription itself, non-coding RNAs and RBPs contribute to a model of para-transcriptional coordination of gene expression, which is utilized during normal tissue physiology and cancer development in order to execute complex gene expression programs. Several key regulators of RNA biology, such as certain splice factors, represent bona fide cancer vulnerabilities, but our understanding of these processes is still far away from being comprehensive. Genetic forward screens utilizing technologies such as transposons, RNAi and CRISPR aid the field in rapidly establishing functional phenotypes and genetic cancer cell addictions. This review focuses on four individual regulatory gene expression processes governed by regulators of the RNA life cycle, the impact of functional genomics on streamlining the discovery process and the role of such mechanisms in tumor biology., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Mutated in approximately 30% of human cancers, Ras GTPases are the most common drivers of oncogenesis and render tumors unresponsive to many standard therapies. Despite decades of research, no drugs directly targeting Ras are currently available. We have previously characterized a small protein antagonist of K-Ras, R11.1.6, and demonstrated its direct competition with Raf for Ras binding. Here we evaluate the effects of R11.1.6 on Ras signaling and cellular proliferation in a panel of human cancer cell lines. Through lentiviral transduction, we generated cell lines that constitutively or through induction with doxycycline express R11.1.6 or a control protein YW1 and show specific binding by R11.1.6 to endogenous Ras through microscopy and co-immunoprecipitation experiments. Genetically encoded intracellular expression of this high-affinity Ras antagonist, however, fails to measurably disrupt signaling through either the MAPK or PI3K pathway. Consistently, cellular proliferation was unaffected as well. To understand this lack of signaling inhibition, we quantified the number of molecules of R11.1.6 expressed by the inducible cell lines and developed a simple mathematical model describing the competitive binding of Ras by R11.1.6 and Raf. This model supports a potential mechanism for the lack of biological effects that we observed, suggesting stoichiometric and thermodynamic barriers that should be overcome in pharmacologic efforts to directly compete with downstream effector proteins localized to membranes at very high effective concentrations. Mol Cancer Ther; 17(8); 1773-80. ©2018 AACR ., (©2018 American Association for Cancer Research.)

We investigated the insertion of small potassium (K + ) channel proteins (Kcv MA-1D and Kcv NTS ) into model membranes and the lipid-protein structural interference, combining neutron reflectometry and electrophysiology. Neutron reflectometry experiments showed how the transverse structure and mechanical properties of the bilayer were modified, upon insertion of the proteins in single model-membranes, either supported on solid substrate or floating. Parallel electrophysiology experiments were performed on the same channels reconstituted in free-standing planar lipid bilayers, of both typical composition and matched to the neutron reflectometry experiment, assessing their electrical features. Functional and structural results converge in detecting that the proteins, conical in shape, insert with a directionality, cytosolic side first. Our work addresses the powerful combination of the two experimental approaches. We show here that membrane structure spectroscopy and ion channel electrophysiology can become synergistic tools in the analysis of structural-functional properties of biomimetic complex environment., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Glioblastoma (GBM) is a devastating malignancy with few therapeutic options. We identify PRMT5 in an in vivo GBM shRNA screen and show that PRMT5 knockdown or inhibition potently suppresses in vivo GBM tumors, including patient-derived xenografts. Pathway analysis implicates splicing in cellular PRMT5 dependency, and we identify a biomarker that predicts sensitivity to PRMT5 inhibition. We find that PRMT5 deficiency primarily disrupts the removal of detained introns (DIs). This impaired DI splicing affects proliferation genes, whose downregulation coincides with cell cycle defects, senescence and/or apoptosis. We further show that DI programs are evolutionarily conserved and operate during neurogenesis, suggesting that they represent a physiological regulatory mechanism. Collectively, these findings reveal a PRMT5-regulated DI-splicing program as an exploitable cancer vulnerability., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective: To evaluate secnidazole as a single oral dose treatment for bacterial vaginosis in a phase 2 randomized, double-blind, placebo-controlled study., Methods: In a phase 2, randomized, double-blind, dose-ranging, placebo-controlled study, women with bacterial vaginosis who met all Amsel criteria (discharge; pH 4.7 or greater; 20% or greater clue cells; positive whiff test) were randomized one to one to one at 24 U.S. centers to 1 or 2 g secnidazole compared with placebo. The primary endpoint was clinical cure (normalization of discharge, amine odor, and clue cells) 21-30 days after treatment. Secondary endpoints included microbiologic cure, defined as a Nugent score of 0-3, and therapeutic cure, defined as meeting criteria for both clinical and microbiologic cure. The modified intent to treat was used for efficacy analyses and included all randomized patients who met the enrollment criteria. Assuming a clinical cure rate of 40% in the active groups and 15% in the placebo group, a sample size of 52 patients per group provided approximately 80% power to detect a significant difference between groups (.05 level [two-sided]) using a Cochran-Mantel-Haenszel test., Results: Between May and September 2014, 215 patients were enrolled. In the intent-to-treat population, the clinical cure rate was 65.3% for the 2-g group, 49.3% for the 1-g group, and 19.4% for the placebo group. The modified intent-to-treat population included 188 women (median age 33 years; 32% with four or more bacterial vaginosis episodes in the previous year; 54% black) with baseline Nugent scores 4 or greater. Clinical, microbiologic, and therapeutic cure rates were 67.7%, 40.3%, and 40.3% for 2 g secnidazole and 51.6%, 23.4%, and 21.9% for 1 g secnidazole compared with 17.7%, 6.5%, and 6.5% for placebo, respectively (P<.05 for secnidazole compared with placebo; all endpoints). Both doses were well-tolerated., Conclusion: Oral granules containing 1 and 2 g secnidazole were superior to placebo in bacterial vaginosis treatment (P<.001 for both groups). These data support the development of secnidazole for bacterial vaginosis treatment., Clinical Trial Registration: ClinicalTrials.gov, NCT02147899.

Introduction: Bacterial vaginosis (BV) is a serious infection that is the most common vaginal infection in women of childbearing potential. SYM-1219 is a novel, granule formulation containing 2 g of secnidazole that is being developed as a single, oral dose to treat women with BV. Because many of the women diagnosed with BV use hormonal contraception, the effect of SYM-1219 on the pharmacokinetics (PK) of commonly prescribed oral contraceptive drugs, ethinyl estradiol (EE2), and norethindrone (NET) was evaluated., Methods: This two-period, randomized, open-label study examined effects in 54 healthy female subjects. During the first period of the study, each subject received EE2 0.035-mg/NET 1-mg tablets. During the second period of the study, subjects were randomized to receive either EE2 0.035-mg/NET 1-mg tablets with concomitant 2-g SYM-1219 or 2-g SYM-1219 followed by EE2 0.035-mg/NET 1-mg tablets 1 day later. The PK of EE2 and NET were analyzed for 24 h following administration., Results: Coadministration of SYM-1219 and EE2/NET, either on the same day or 1 day apart, had no clinically relevant effects on the bioavailability of EE2 or NET. The combined use of SYM-1219 with EE2/NET was well tolerated. Taken together, these results indicate that contraceptive efficacy should be maintained during coadministration of SYM-1219 and EE2/NET., Conclusion: SYM-1219 is a valuable single-dose treatment option for women with BV that will not interfere with combined oral contraceptive methods., Funding: Symbiomix Therapeutics.

Introduction: Whereas tumorigenic processes have traditionally been attributed to gene amplification, deletion, or mutation, it is now clear that epigenetic changes represent an additional hallmark of cancer. This review explains the basic principles of epigenetic regulation and therapy, provides an overview of clinically approved drugs, introduces novel targets and compounds, and discusses the potential reasons behind treatment success and failure., Areas Covered: We provide a brief introduction to the concept of epigenetic regulation in general and explain how epigenetic pathways are altered in cancer. Based on this, we go on to explore the rational behind epigenetic cancer therapy, provide an overview of clinical success and failure of specific drugs, describe novel pharmaceutical targets, review epigenetic combination treatment, and finally discuss biological concepts influencing treatment success. Expert commentary: Even though many early epigenetic therapy trials had disappointing results, lessons learned from these studies have heavily influenced the design of modern trials leading to improved therapeutic outcomes. Better preclinical model systems may help to reduce the risk of clinical failure and to identify high-confidence targets for clinical follow-up.

Targeted transcriptional regulation is a powerful tool to study genetic mediators of cellular behavior. Here, we show that catalytically dead Cas9 (dCas9) targeted to genomic regions upstream or downstream of the transcription start site allows for specific and sustainable gene-expression level alterations in tumor cells in vitro and in syngeneic immune-competent mouse models. We used this approach for a high-coverage pooled gene-activation screen in vivo and discovered previously unidentified modulators of tumor growth and therapeutic response. Moreover, by using dCas9 linked to an activation domain, we can either enhance or suppress target gene expression simply by changing the genetic location of dCas9 binding relative to the transcription start site. We demonstrate that these directed changes in gene-transcription levels occur with minimal off-target effects. Our findings highlight the use of dCas9-mediated transcriptional regulation as a versatile tool to reproducibly interrogate tumor phenotypes in vivo.

In normal cells, p53 is activated by DNA damage checkpoint kinases to simultaneously control the G1/S and G2/M cell cycle checkpoints through transcriptional induction of p21(cip1) and Gadd45α. In p53-mutant tumors, cell cycle checkpoints are rewired, leading to dependency on the p38/MK2 pathway to survive DNA-damaging chemotherapy. Here we show that the RNA binding protein hnRNPA0 is the "successor" to p53 for checkpoint control. Like p53, hnRNPA0 is activated by a checkpoint kinase (MK2) and simultaneously controls both cell cycle checkpoints through distinct target mRNAs, but unlike p53, this is through the post-transcriptional stabilization of p27(Kip1) and Gadd45α mRNAs. This pathway drives cisplatin resistance in lung cancer, demonstrating the importance of post-transcriptional RNA control to chemotherapy response.

Glioblastoma (GBM) is often treated with the cytotoxic drug temozolomide, but the disease inevitably recurs in a drug-resistant form after initial treatment. Here, we report that in GBM cells, even a modest decrease in the mismatch repair (MMR) components MSH2 and MSH6 have profound effects on temozolomide sensitivity. RNAi-mediated attenuation of MSH2 and MSH6 showed that such modest decreases provided an unexpectedly strong mechanism of temozolomide resistance. In a mouse xenograft model of human GBM, small changes in MSH2 were sufficient to suppress temozolomide-induced tumor regression. Using The Cancer Genome Atlas to analyze mRNA expression patterns in tumors from temozolomide-treated GBM patients, we found that MSH2 transcripts in primary GBM could predict patient responses to initial temozolomide therapy. In recurrent disease, the absence of microsatellite instability (the standard marker for MMR deficiency) suggests a lack of involvement of MMR in the resistant phenotype of recurrent disease. However, more recent studies reveal that decreased MMR protein levels occur often in recurrent GBM. In accordance with our findings, these reported decreases may constitute a mechanism by which GBM evades temozolomide sensitivity while maintaining microsatellite stability. Overall, our results highlight the powerful effects of MSH2 attenuation as a potent mediator of temozolomide resistance and argue that MMR activity offers a predictive marker for initial therapeutic response to temozolomide treatment., (©2015 American Association for Cancer Research.)

Wiskott-Aldrich-Syndrome (WAS) is a rare X-linked recessive disease caused by mutations of the WAS gene. It is characterized by immunodeficiency, autoimmunity, low numbers of small platelets (microthrombocytopenia) and a high risk of cancer, especially B cell lymphoma and leukemia.

Background: A functional reconstitution of channel proteins in planar lipid bilayers is still very versatile to study structure/function correlates under well-defined conditions at the single protein level., New Method: In this study we present an improved planar lipid bilayer technique in which an air bubble is used for stabilizing unstable/leaky bilayers or for removing excess lipids. The bubble can also be used as a tool for reducing the number of channels in the bilayer with the goal of having only one active channel in the membrane., Results: Stable planar lipid bilayers are formed within seconds to minutes. In the case of multiple channel insertion the air bubble can be used to reduce the number of channels within minutes., Comparison With Existing Method(s): The simple improvement of the classical folding technique guarantees a very fast creation of stable bilayers even with difficult phospholipids in a conventional vertical bilayer set-up; it requires no modifications of the existing set-up., Conclusions: This technique is very easy to handle and guarantees successful single channel recordings for any kind of planar lipid bilayer experiment., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The viral channel KcvNTS belongs to the smallest K(+) channels known so far. A monomer of a functional homotetramer contains only 82 amino acids. As a consequence of the small size the protein is almost fully submerged into the membrane. This suggests that the channel is presumably sensitive to its lipid environment. Here we perform a comparative analysis for the function of the channel protein embedded in three different membrane environments. 1. Single-channel currents of KcvNTS were recorded with the patch clamp method on the plasma membrane of HEK293 cells. 2. They were also measured after reconstitution of recombinant channel protein into classical planar lipid bilayers and 3. into horizontal bilayers derived from giant unilamellar vesicles (GUVs). The recombinant channel protein was either expressed and purified from Pichia pastoris or from a cell-free expression system; for the latter a new approach with nanolipoprotein particles was used. The data show that single-channel activity can be recorded under all experimental conditions. The main functional features of the channel like a large single-channel conductance (80pS), high open-probability (>50%) and the approximate duration of open and closed dwell times are maintained in all experimental systems. An apparent difference between the approaches was only observed with respect to the unitary conductance, which was ca. 35% lower in HEK293 cells than in the other systems. The reason for this might be explained by the fact that the channel is tagged by GFP when expressed in HEK293 cells. Collectively the data demonstrate that the small viral channel exhibits a robust function in different experimental systems. This justifies an extrapolation of functional data from these systems to the potential performance of the channel in the virus/host interaction. This article is part of a Special Issue entitled: Viral Membrane Proteins-Channels for Cellular Networking., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Wiskott-Aldrich syndrome (WAS) is characterized by microthrombocytopenia, immunodeficiency, autoimmunity, and susceptibility to malignancies. In our hematopoietic stem cell gene therapy (GT) trial using a γ-retroviral vector, 9 of 10 patients showed sustained engraftment and correction of WAS protein (WASP) expression in lymphoid and myeloid cells and platelets. GT resulted in partial or complete resolution of immunodeficiency, autoimmunity, and bleeding diathesis. Analysis of retroviral insertion sites revealed >140,000 unambiguous integration sites and a polyclonal pattern of hematopoiesis in all patients early after GT. Seven patients developed acute leukemia [one acute myeloid leukemia (AML), four T cell acute lymphoblastic leukemia (T-ALL), and two primary T-ALL with secondary AML associated with a dominant clone with vector integration at the LMO2 (six T-ALL), MDS1 (two AML), or MN1 (one AML) locus]. Cytogenetic analysis revealed additional genetic alterations such as chromosomal translocations. This study shows that hematopoietic stem cell GT for WAS is feasible and effective, but the use of γ-retroviral vectors is associated with a substantial risk of leukemogenesis.

Therapy-resistant microenvironments represent a major barrier toward effective elimination of disseminated malignancies. Here, we show that select microenvironments can underlie resistance to antibody-based therapy. Using a humanized model of treatment refractory B cell leukemia, we find that infiltration of leukemia cells into the bone marrow rewires the tumor microenvironment to inhibit engulfment of antibody-targeted tumor cells. Resistance to macrophage-mediated killing can be overcome by combination regimens involving therapeutic antibodies and chemotherapy. Specifically, the nitrogen mustard cyclophosphamide induces an acute secretory activating phenotype (ASAP), releasing CCL4, IL8, VEGF, and TNFα from treated tumor cells. These factors induce macrophage infiltration and phagocytic activity in the bone marrow. Thus, the acute induction of stress-related cytokines can effectively target cancer cells for removal by the innate immune system. This synergistic chemoimmunotherapeutic regimen represents a potent strategy for using conventional anticancer agents to alter the tumor microenvironment and promote the efficacy of targeted therapeutics., (Copyright © 2014 Elsevier Inc. All rights reserved.)

BMI1 is a known oncogenic transcriptional repressor in glioblastoma stem-like cells, but its downstream mediators are poorly understood. Recently, in Cancer Cell, Gargiulo et al. (2013) designed a rational in vivo RNAi screen based on BMI1 ChIP-seq from neural progenitors and identified functional tumor suppressor targets, including Atf3 and Cbx7., (Copyright © 2013 Elsevier Inc. All rights reserved.)

During all stages of tumor progression, cancer cells are subjected to inappropriate extracellular matrix environments and must undergo adaptive changes in order to evade growth constraints associated with the loss of matrix attachment. A gain of function screen for genes that enable proliferation independently of matrix anchorage identified a cell adhesion molecule PVRL4 (poliovirus-receptor-like 4), also known as Nectin-4. PVRL4 promotes anchorage-independence by driving cell-to-cell attachment and matrix-independent integrin β4/SHP-2/c-Src activation. Solid tumors frequently have copy number gains of the PVRL4 locus and some have focal amplifications. We demonstrate that the transformation of breast cancer cells is dependent on PVRL4. Furthermore, growth of orthotopically implanted tumors in vivo is inhibited by blocking PVRL4-driven cell-to-cell attachment with monoclonal antibodies, demonstrating a novel strategy for targeted therapy of cancer. DOI:http://dx.doi.org/10.7554/eLife.00358.001.

Primary immunodeficiencies (PIDs) represent exquisite models for studying mechanisms of human host defense. In this study, we report on two unrelated kindreds, with two patients each, who had cryptosporidial infections associated with chronic cholangitis and liver disease. Using exome and candidate gene sequencing, we identified two distinct homozygous loss-of-function mutations in the interleukin-21 receptor gene (IL21R; c.G602T, p.Arg201Leu and c.240&#95;245delCTGCCA, p.C81&#95;H82del). The IL-21R(Arg201Leu) mutation causes aberrant trafficking of the IL-21R to the plasma membrane, abrogates IL-21 ligand binding, and leads to defective phosphorylation of signal transducer and activator of transcription 1 (STAT1), STAT3, and STAT5. We observed impaired IL-21-induced proliferation and immunoglobulin class-switching in B cells, cytokine production in T cells, and NK cell cytotoxicity. Our study indicates that human IL-21R deficiency causes an immunodeficiency and highlights the need for early diagnosis and allogeneic hematopoietic stem cell transplantation in affected children.

Herein we describe the synthesis of the first Thalidomide-biotin analogue in order to initiate investigations into the unknown molecular mode of action of Thalidomide. In this manner we describe the attachment of biotin tether through the Huisgen 1,3-dipolar cycloaddition or "click" synthetic methodology.

A library of new thalidomide C4/5 analogues containing either a phenyl or alkyne tether were synthesized using Sonogashira or Suzuki cross coupling reactions from their aryl halogenated precursors. All thalidomide analogues were tested for their ability to inhibit the expression of the proinflammatory cytokine Tumor Necrosis Factor (TNF). More explicitly the use of a novel reporter system utilizing the promoter region of the TNF gene in a human T-cell line provided a rapid and effective measure of NFkappaB transcriptional activity. Several compounds either containing either an aryl-isobutyl or aryl-isopropoxy group were the most effective in inhibiting TNF expression, and were several times more active than thalidomide itself. Five of the more active derivatives indicated an apoptotic response while one of these compounds, containing an aldehyde tether, showed possible influence of cell cycling effects., (Copyright 2009 Elsevier Ltd. All rights reserved.)

microRNAs provide a novel layer of regulation for gene expression by interfering with the stability and/or translation of specific target mRNAs. Overall levels of microRNAs are frequently down-regulated in cancer cells, and reducing general microRNA processing increases cancerogenesis in transgenic models, suggesting that at least some microRNAs might act as effectors in tumor suppression. Accordingly, the tumor suppressor p53 up-regulates miR-34a, a microRNA that contributes to apoptosis and acute senescence. Here, we used array hybridization to find that p53 induces two additional, mutually related clusters of microRNAs, leading to the up-regulation of miR-192, miR-194, and miR-215. The same microRNAs were detected at high levels in normal colon tissue but were severely reduced in many colon cancer samples. On the other hand, miR-192 and its cousin miR-215 can each contribute to enhanced CDKN1A/p21 levels, colony suppression, cell cycle arrest, and cell detachment from a solid support. These effects were partially dependent on the presence of wild-type p53. Antagonizing endogenous miR-192 attenuated 5-fluorouracil-induced accumulation of p21. Hence, miR-192 and miR-215 can act as effectors as well as regulators of p53; they seem to suppress cancerogenesis through p21 accumulation and cell cycle arrest.

Background: Genetics is a known risk factor for alcoholism, and human alcoholics are known to suffer from a loss of brain function and mass. A 4 day rat binge drinking model is known to cause brain region-specific damage. To investigate the role of genetics in binge-drinking-induced brain damage, we studied bidirectionally selected rat lines, the alcohol-preferring P and the alcohol-nonpreferring NP rat lines., Method: P and NP rats were treated with a 4 day binge ethanol protocol. Animals were killed, transcardially perfused, and fixed, and their brains were removed, sectioned, and stained by using the amino cupric silver stain of de Olmos or by using immunohistochemistry for phospho-extracellular signal regulated kinases and other antigens., Results: Significant brain damage was found in the olfactory bulbs, posterior perirhinal cortex, and entorhinal cortex in both P and NP rats. P rats were found to have significantly greater brain damage, compared with NP rats, in the posterior perirhinal and posterior entorhinal cortexes, 239% +/- 50% (p < 0.02) and 219% +/- 46% (p < 0.01), respectively. Phospho-extracellular signal regulated kinase immunohistochemistry stained prominently in damaged brain areas., Conclusions: The P rat line, a genetic model of alcoholism, shows greater region-specific brain damage due to binge ethanol treatment than its genetic counterpart, the NP rat line. These findings suggest that genetics contribute to susceptibility for binge-induced brain damage.

Background: Inhibition of NMDA receptor function in brain is believed to be an important action of ethanol (EtOH). To investigate EtOH inhibition of NMDA receptor responses in vivo, the interaction of these agents in brain after different routes of administration were investigated by using transcription factor Fos protein expression to follow NMDA receptor activation and EtOH inhibition of this response., Methods: The induction of Fos-like immunoreactivity (Fos-LI) in 38 regions of the rat brain was measured 2 hr after treatment with NMDA, EtOH, or both. To determine the relative contribution of abdominal drug effects on Fos induction, rats received either intraperitoneal (ip) or intragastric (ig) EtOH and ip or intravenous (iv) NMDA. Rats received EtOH (2.5 g/kg ip or 4 g/kg ig) or vehicle 15 min before NMDA (125 mg/kg ip or 60 mg/kg iv) or vehicle., Results: For the 38 forebrain regions examined, ip and iv NMDA significantly induced Fos-LI in 13 and 32 regions, respectively. These effects occurred without elicitation of tonic-clonic seizure activity and were strong after iv NMDA in the frontal, prefrontal, and cingulate cortices, supraoptic nucleus, anterior lateral septum, and dentate gyrus. For EtOH, prominent Fos-LI induction was found in the central amygdala, dorsolateral bed nucleus of the stria terminalis, Edinger-Westphal nucleus, and paraventricular hypothalamus. Despite ip and ig EtOH induction of Fos-LI in these regions, the major effect of EtOH was to block NMDA-induced Fos-LI in 8 of 13 (ip) and 27 of 32 (ig) of the NMDA-positive regions, respectively, including retrosplenial, cingulate, and medial prefrontal cortices, central amygdala, and taenia tecta., Conclusions: These results provide new evidence for the regionally specific functional interactions of EtOH on NMDA receptors in vivo. Moreover, these results support efforts to identify brain region-specific targets for EtOH and EtOH-induced changes in gene expression.

To determine if nutrition plays a role in ethanol withdrawal and alcohol-induced brain damage, the effects of a 4-day ethanol binge treatment using ethanol in a nutritionally complete liquid diet compared to ethanol mixed with water were studied. The nutritionally complete diet group (ETOH-diet) received a complete diet of sugars, proteins and fats with vitamins and minerals with approximately 53% of calories from ethanol while the nutritionally deprived group (ETOH-H2O) received 100% of calories from ethanol. No difference in withdrawal behavior was found between the ETOH-diet and ETOH-H2O groups during the 72-hour period studied. In addition, no difference was seen for serum levels of magnesium and zinc taken at last dose or following 72 h of withdrawal. Serum alanine aminotransferase (ALT) and ammonia were increased in both groups with ETOH-diet showing a greater increase in ALT than ETOH-H2O. Both groups showed damage in the olfactory bulb, perirhinal, agranular insular, piriform and lateral entorhinal cortical areas as well as hippocampal dentate gyrus and CA-3. Interestingly, the ETOH-diet group displayed more damage at last dose in the posterior dentate and CA-3 of hippocampus than did the ETOH-H2O group. This study suggests that nutritional components and total caloric intake do not effect behavior during ethanol withdrawal and that a nutritionally complete diet may increase ethanol-induced brain damage., (Copyright 2001 National Science Council, ROC and S. Karger AG, Basel)

Background: Adolescents respond differently to alcohol than adults. Furthermore, binge drinking in young adolescents is becoming increasingly common., Methods: To determine if the effects of binge drinking on brain damage are different in juveniles compared with adults, the effects of a 4 day binge ethanol treatment (e.g., 4 days of 4 times per day 15% ethanol intragastrically, approximately 9-10 g/kg/day ethanol) were investigated in adolescent-juvenile rats (JVN) 35 days old and compared with adult (ADT) rats 80 to 90 days old. Brain damage was measured by using the amino cupric silver stain of de Olmos et al. (1994)., Results: Significant brain damage was found in both groups. The olfactory bulbs were equally damaged in both groups; however, the associated frontal cortical olfactory regions were damaged only in JVN. The anterior portions of the piriform and perirhinal cortices also were damaged only in JVN rats. Quantitation of silver-stained frontal areas in binge ethanol-treated JVN rats ranged from 400% to 1,260% of control values. For example, in anterior perirhinal cortex, silver stain increased from 48 +/- 14 to 444 +/- 114 (mm2 x 10(3) argyrophilic area; p < 0.01) in JVN control and binge ethanol-treated animals, respectively. In contrast, posterior perirhinal cortex showed greater damage in adults, being 236 +/- 76 vs. 875 +/- 135 (mm2 x 10(3) argyrophilic area; p < 0.005) in JVN and ADT, respectively., Conclusions: The young-adolescent brain shows differential sensitivity to alcohol-induced brain damage compared with adults.

Experiments were designed to test the hypothesis that activation of forebrain 5-HT1A receptors elicits cardiovascular responses. The microinjection of R-(+)-8-hydroxy-2-(di-n-propylamino) tetralin [(+)-8-OH-DPAT], a selective 5-HT1A receptor agonist, in the preoptic area of conscious rats increased blood pressure and heart rate at doses of 0.2-20 nmol; lower doses (0.002 and 0.02 nmol) were ineffective. The concomitant administration of methiothepin, a non-selective 5-HT receptor antagonist, into the preoptic area attenuated the responses. In addition, the tachycardia elicited by (+)-8-OH-DPAT was abolished by the peripheral beta-adrenoceptor antagonist sotalol, but not by atropine methyl nitrate. Finally, the tachycardia, but not the hypertension, was also produced by (+)-8-OH-DPAT in urethane-anesthetized rats. These results suggest that activation of 5-HT1A receptors in the preoptic area or an adjacent region of the forebrain produces: (1) an increase in heart rate consistent with sympathoadrenal activation; and (2) an increase in blood pressure which might be the result of sympathoexcitation or secondary to behavioral arousal.

Roles for H1, H2, and H3 receptor subtypes for drinking elicited by exogenous histamine and drinking elicited by eating was examined in adult male Sprague-Dawley rats. Drinking elicited by SC 5 mg/kg histamine was: (a) inhibited approximately 30% by H1 antagonism using IP 1 mg/kg dexbrompheniramine (DXB); (b) inhibited approximately 30% by H2 antagonism using IP 16 mg/kg cimetidine (C); (c) inhibited approximately 40% by H3 antagonism using SC 10 mg/kg thioperamide (Th); (d) inhibited approximately 80% by combined H1 and H2 antagonism using IP DXB plus IP C; (e) inhibited approximately 85% by combined H1 and H3 antagonism using IP DXB plus SC Th; (f) inhibited approximately 70% by combined H2 and H3 antagonism using IP C plus SC Th; and (g) abolished by combined H1, H2, and H3 antagonism using IP DXB plus IP C plus SC Th. For rats eating pellets and drinking after 24-h food deprivation: (a) systemic injections of DXB, C, and Th, sufficient to abolish drinking elicited by SC histamine, inhibited water/food ratio (W/F) by approximately 20%; (b) ICV injections (through a chronic cannula in a lateral ventricle) of 50 micrograms DXB plus 100 micrograms C plus 60 micrograms Th inhibited W/F by approximately 20%. For rats drinking after IG infusion (through a chronic gastric catheter) of 2 ml 1,800 mOsm/kg NaCl: (a) systemic injections of DXB, C, and Th, sufficient to abolish drinking elicited by SC histamine, inhibited water intake by approximately 70%; (b) IP DXB alone and IP C alone failed to inhibit water intake; (c) IP Th alone inhibited water intake by approximately 20%; (d) IP DXB combined with IP C inhibited water intake by approximately 55%. The results demonstrate the involvement of H1, H2, and H3 receptors for drinking elicited by exogenous histamine, and our findings extend the evidence for a role for endogenous histamine and H1, H2, and H3 receptor subtypes for drinking elicited by eating, including drinking elicited by gastrointestinal osmotic consequences of eating that can increase systemic plasma osmolality.

A role for endogenous angiotensin II and its AT1 and AT2 receptor subtypes for mediating drinking elicited by eating was examined in adult male Sprague-Dawley rats. The ability of pharmacological antagonism of AT1 and/or AT2 receptors to abolish drinking elicited by exogenous angiotensin II was established first. The s.c. injection of the AT1 antagonist losartan (DuP 753) was sufficient to abolish drinking elicited by s.c. angiotensin II. The ICV injection (through a surgically implanted chronic cannula) of losartan inhibited drinking elicited by ICV angiotensin II; the combined ICV injection of losartan plus the AT2 antagonist PD123319 was sufficient to abolish drinking elicited by ICV angiotensin II. For rats drinking and eating after 24-h food deprivation, s.c. losartan plus PD123319 inhibited water to food ratio, but ICV losartan and/or PD123319 failed to inhibit food-related drinking. For nondeprived rats eating a small cracker, s.c. losartan and/or PD123319 attenuated water intake, but only ICV losartan produced statistically significant inhibition of drinking elicited by ingestion of cracker. The IG infusion (through a surgically implanted gastric catheter) of 2 ml 600 or 900 mOsm/kg NaCl, a treatment that is subthreshold for increase in systemic plasma osmolality at the initiation of drinking, elicited drinking that was attenuated by s.c. losartan and/or PD123319 and attenuated by ICV losartan only. The IG infusion of 2 ml 1800 mOsm/kg NaCl, a treatment that is above threshold for increase in systemic plasma osmolality at the initiation of drinking, elicited drinking that was not inhibited by S or ICV losartan and/or PD123319. These results demonstrate that peripheral AT1 and AT2 and central AT1 receptors for angiotensin II contribute to drinking elicited by eating and the gastrointestinal osmotic consequences of eating. These findings extend the evidence demonstrating a renal renin-angiotensin contribution to food-related drinking in rats.

Two supplementary methods for time-dependent droplet sizing, both based on the spectral dependence of light extinction, are applied to an adiabatically expanding vapor in which droplets are formed as a result of heterogeneous condensation. First, by measuring the extinction coefficients at three different wavelengths, we obtain time-dependent values of the modal radius, the size variance, and the droplet number density, with a typical time resolution of 1 µs. The shape of the size-distribution function is investigated by a second method. Using a white-light source in combination with a spectrometer and a CCD array, we obtain the full visible light attenuation spectrum with a time resolution of 1.5 ms. By applying an inversion technique based on trial size distributions, we find that the zeroth-order log-normal distribution describes the fog adequately. Both methods yield the same droplet growth curves and droplet number densities.

The first open reading frame (ORF 1) of potato virus X (PVX) encodes a putative replicase gene. Transgenic tobacco lines expressing ORF 1 are resistant to PVX infection when inoculated with either PVX or PVX RNA. Analyses of lines containing various portions of the ORF 1 gene demonstrated that resistance is conferred to plants by expressing approximately the first half of the ORF 1 gene. One line expressing the untranslated leader and first 674 codons of ORF 1 is highly resistant to PVX infection. Conversely, lines expressing either approximately the third or fourth quarter of the ORF 1 gene, which contain the conserved nucleotide triphosphate (NTP) binding motif and Gly-Asp-Asp (GDD) motif, respectively, are not protected from PVX infection. In the resistant full-length and amino-terminal lines, lower numbers of local lesions were observed, and the virus accumulation in the inoculated and upper leaves was reduced when compared with the nontransformed control. When the performance of the most resistant ORF 1 line was compared with the most resistant coat protein (CP) line in a resistance test, the best ORF 1 line was more resistant to PVX infection than the best transgenic line expressing the PVX CP gene. These findings define a promising new approach for controlling plant viral infection.

Expression of the maize mitochondrial T-urf13 gene results in a sensitivity to a family of fungal pathotoxins and to methomyl, a structurally unrelated systemic insecticide. Similar effects of pathotoxins and methomyl are observed when T-urf13 is cloned and expressed in Escherichia coli. An interaction between these compounds and the membrane-bound URF13 protein permeabilizes the inner mitochondrial and bacterial plasma membranes. To understand the toxin-URF13 effects, we have investigated whether toxin specifically binds to the URF13 protein. Our studies indicate that toxin binds to the URF13 protein in maize mitochondria and in E. coli expressing URF13. Binding analysis in E. coli reveals cooperative toxin binding. A low level of specific toxin binding is also demonstrated in cms-T and cms-T-restored mitochondria; however, binding does not appear to be cooperative in maize mitochondria. Competition and displacement studies in E. coli demonstrate that toxin binding is reversible and that the toxins and methomyl compete for the same, or for overlapping, binding sites. Two toxin-insensitive URF13 mutants display a diminished capability to bind toxin in E. coli, which identifies residues of URF13 important in toxin binding. A third toxin-insensitive URF13 mutant shows considerable toxin binding in E. coli, demonstrating that toxin binding can occur without causing membrane permeabilization. Our results indicate that toxin-mediated membrane permeabilization only occurs when toxin or methomyl is bound to URF13.

The alpha subunit of the F(1)-ATPase complex of maize is a mitochondrial translational product, presumably encoded by the mitochondrial genome. Based on nucleotide and amino acid homology, we have identified a mitochondrial gene, designated atpalpha, that appears to code for the F(1)-ATPase alpha subunit of Zea mays. The atpalpha gene is present as a single copy in the maize. Texas cytoplasm and is actively transcribed. The maize alpha polypeptide has a predicted length of 508 amino acids and a molecular mass of 55,187 daltons. Amino acid homologies between the maize mitochondrial alpha subunit and the tobacco chloroplast CF(1) and Escherichia coli alpha subunits are 54 and 51%, respectively. The origin of the atpalpha gene is discussed.

Integrated inverted repeat (IR) sequences similar to those of the S plasmids have been isolated from the genomes of the normal and S type male-sterile cytoplasms of maize mitochondria. The nucleotide sequences of both the IRs and their flanking regions have distinguished and characterized several different types of repeats. The repeats may be involved in the recombinational process that occurs continuously in the mitochondrial genome. One cloned fragment, derived from a fertile revertant and containing sequences similar to S-2, does not appear to act as a typical transposable element during reversion. Several of the flanking regions examined contain a small repeat of 34 base pairs, in which a nonanucleotide segment is found with similarity to the yeast mitochondrial promoter.

URF13, the product of the mitochondrial T-urf13 gene, confers on Texas cytoplasmic male-steril maize (Zea mays L.) a unique susceptibility to a fungal pathogen (Bipolaris maydis race T) and sensitivity to its pathotoxin. Expression of URF13 in Escherichia coli imparts pathotoxin sensitivity to the bacterium. We show by ion uptake studies in E. coli that a pathotoxin-URF13 interaction causes membrane permeability. Similarly, mitochondrial dysfunction caused by membrane permeabilization probably accounts for increased colonization of maize carrying the Texas cytoplasm by toxin-producing pathogens. Site-directed mutagenesis studies show that approximately one-quarter of the amino acids at the carboxyl end of URF13 can be eliminated without affecting toxin sensitivity. We have identified two dicyclohexylcarbodiimide (DCCD) binding sites in the URF13 protein and show that one of the sites is involved in conferring DCCD protection against the pathotoxin. Substitutional mutations at this DCCD binding site also eliminate toxin sensitivity.