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Ninety-five of 115 patients (83%) had imaging findings, 28/115 (24%) had melanoma ± incidental findings, 6/115 (5%) had only melanoma imaging findings, and 67/115 (58%) had only incidental imaging findings. Pathologic stage III increased 3.21 times the odds of having an imaging finding of any type (95% CI: 1.19-8.67, I P i = 0.02) and 6.81 times the odds of having a melanoma ± incidental imaging result (95% CI: 1.88-24.69, I P i = 0.01). Patients with no imaging findings were at significantly decreased odds of melanoma-related mortality compared to patients with melanoma ± non-melanoma findings (P < 0.01). gl. [Extracted from the article]

To know the incidence, clinical development and perinatal results of cases of AVB. Cases without structural heart disease have a better prognosis, although many of these children end up requiring a pacemaker implant. One patient moved her home to another location, and there, the pregnancy was ended electively at week 34 of gestation by Caesarean section. [Extracted from the article]

Lifestyle Medicine (LM) is a rapidly growing discipline that focuses on the role of lifestyle factors in preventing, managing, and reversing chronic disease. At this point in the field's evolution, there is strong evidence that the 6 pillars of LM-a whole-food, plant-predominant eating pattern, physical activity, restorative sleep, stress management, avoidance of risky substances, and positive social connections-are central in the creation and maintenance of health. Previous publications, many of them randomized controlled studies and meta-analyses, have solidified the evidence base for the use of the 6 pillars within the field of LM. As data emerged, so did its governing body, the American College of Lifestyle Medicine (ACLM), and with it a rich history began to unfold. Several articles have been written on the early history of the ACLM and the growth of the field; however, this review article explores the history and foundation of LM, aiming to provide a comprehensive understanding of its relevance and impact on health care. It underscores landmark studies that have defined the field and provides a road map detailing national and global barriers and areas of potential future growth., Competing Interests: John Kelly, founding president of ACLM; Sley Tanigawa Guimaraes, Hospital Israelita Albert Einstein, International Board of Lifestyle Medicine at the Mexican Lifestyle Medicine Conference 2022, President of the Brazilian College of Lifestyle Medicine; David Lippman, board member on the American Board of Lifestyle Medicine; Dean Ornish, directs PMRI institute has received grants from Ballantine Books, Sharecare, The Preventive Medicine Research Institute, United Airlines, Beyond Meat, Founder, Ornish Lifestyle Medicine; Richard Rosenfeld, director for guidelines and quality ACLM, Treasurer and Board Member ABLM. Mariah Stump, University of Arizona Center for Integrative Medicine lecturer, ACP National meeting speaker, Chair of PCP Advisory Committee to Rhode Island Department of Health; Erica Veazey, no disclosures; David Katz,-no disclosures. The authors report no competing interests., (© 2023 The Authors.)

Bsardet Biedl syndrome (BBS) is a genetic condition associated with various clinical features including cutaneous disorders and certain autoimmune and inflammatory diseases pointing to a potential role of BBS proteins in the regulation of immune function. BBS1 protein, which is a key component of the BBSome, a protein complex involved in the regulation of cilia function and other cellular processes, has been implicated in the immune synapse assembly by promoting the centrosome polarization to the antigen-presenting cells. Here, we assessed the effect of disrupting the BBSome, through Bbs1 gene deletion, in T cells. Interestingly, mice lacking the Bbs1 gene specifically in T cells ( T-BBS1 -/- ) displayed normal body weight, adiposity, and glucose handling, but have smaller spleens. However, T-BBS1 -/- mice had no change in the proportion and absolute number of B cells and T cells in the spleen and lymph nodes. There was also no alteration in the CD4/CD8 lineage commitment or survival in the thymus of T-BBS1 -/- mice. On the other hand, T-BBS1 -/- mice treated with Imiquimod dermally exhibited a significantly higher percentage of CD3-positive splenocytes that was due to CD4 but not CD8 T cell predominance. Notably, we found that T-BBS1 -/- mice had significantly decreased wound closure, an effect that was more pronounced in males indicating that the BBSome plays an important role in T cell-mediated skin repair. Together, these findings implicate the BBSome in the regulation of selective functions of T cells.

Lifestyle medicine (LM) offers future generations of clinicians practical tools to effectively prevent, manage and reverse chronic disease. Due to a variety of factors, introduction of such curricula in medical training has been slow. Until LM becomes more standard in medical schools, electives and tracks are an innovative way to introduce curricula in a time-efficient manner so students can have access to this valuable information during their formative training years. Creating a culture for the acceptance of LM is a critical first step and can be accomplished by collaborating with like-minded faculty as well as developing student interest groups. The latter can also be a strong driver for curricular change. This article provides an overview of several structures that can be implemented within existing curricula to offer students a foundation in LM. Included are offerings during the pre-clinical years, third/fourth year electives, culinary medicine rotations, online opportunities, and the development of a full track. Specific components of each structure are shared as well as examples of successful use of community partnerships, use of pre-existing educational resources, and activities implemented. The authors conclude that implementing electives is a promising avenue for educators to expose medical students to LM and can be molded to work within a variety of current educational structures., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2023 The Author(s).)

Actinic prurigo is a rare pruritic photodermatosis. We report the use of Polypodium leucotomos extract in an 11-year-old female patient with actinic prurigo, resulting in a significant attenuation of her disease without development of adverse effects to date. Polypodium leucotomos exerts a pleiotropic immunomodulatory and antioxidant effect by shifting the balance from pro- to an antiinflammatory cytokine environment. This counteracts the effects of UV-induced cellular damage characteristic of photodermatoses., (© 2021 Wiley Periodicals LLC.)

Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines. We describe findings of this screen, outlining the classes of cancer dependency genes and their relationships to genetic, expression, and lineage features. In addition, we describe robust gene-interaction networks recapitulating both protein complexes and functional cooperation among complexes and pathways. This dataset along with a web portal is provided to the community to assist in the discovery and translation of new therapeutic approaches for cancer., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Selective expression of dominant negative (DN) peroxisome proliferator-activated receptor γ (PPARγ) in vascular smooth muscle cells (SMC) results in hypertension, atherosclerosis, and increased nuclear factor-κB (NF-κB) target gene expression. Mesenteric SMC were cultured from mice designed to conditionally express wild-type (WT) or DN-PPARγ in response to Cre recombinase to determine how SMC PPARγ regulates expression of NF-κB target inflammatory genes. SMC-specific overexpression of WT-PPARγ or agonist-induced activation of endogenous PPARγ blunted tumor necrosis factor α (TNF-α)-induced NF-κB target gene expression and activity of an NF-κB-responsive promoter. TNF-α-induced gene expression responses were enhanced by DN-PPARγ in SMC. Although expression of NF-κB p65 was unchanged, nuclear export of p65 was accelerated by WT-PPARγ and prevented by DN-PPARγ in SMC. Leptomycin B, a nuclear export inhibitor, blocked p65 nuclear export and inhibited the anti-inflammatory action of PPARγ. Consistent with a role in facilitating p65 nuclear export, WT-PPARγ coimmunoprecipitated with p65, and WT-PPARγ was also exported from the nucleus after TNF-α treatment. Conversely, DN-PPARγ does not bind to p65 and was retained in the nucleus after TNF-α treatment. Transgenic mice expressing WT-PPARγ or DN-PPARγ specifically in SMC (S-WT or S-DN) were bred with mice expressing luciferase controlled by an NF-κB-responsive promoter to assess effects on NF-κB activity in whole tissue. TNF-α-induced NF-κB activity was decreased in aorta and carotid artery from S-WT but was increased in vessels from S-DN mice. We conclude that SMC PPARγ blunts expression of proinflammatory genes by inhibition of NF-κB activity through a mechanism promoting nuclear export of p65, which is abolished by DN mutation in PPARγ., (© 2017 American Heart Association, Inc.)

Peroxisome proliferator activated receptor (PPARγ) is a nuclear receptor transcription factor that regulates adipogenesis and energy homeostasis. Recent studies suggest PPARγ may mediate some of its metabolic effects through actions in the brain. We used a Cre-recombinase-dependent (using Nestin Cre ) conditionally activatable transgene expressing either wild-type (WT) or dominant-negative (P467L) PPARγ to examine mechanisms by which PPARγ in the nervous system controls energy balance. Inducible expression of PPARγ was evident throughout the brain. Expression of 2 PPARγ target genes, aP2 and CD36, was induced by WT but not P467L PPARγ in the brain. Surprisingly, Nes Cre /PPARγ-WT mice exhibited severe microcephaly and brain malformation, suggesting that PPARγ can modulate brain development. On the contrary, Nes Cre /PPARγ-P467L mice exhibited blunted weight gain to high-fat diet, which correlated with a decrease in lean mass and tissue masses, accompanied by elevated plasma GH, and depressed plasma IGF-1, indicative of GH resistance. There was no expression of the transgene in the pancreas but both fasting plasma glucose, and fed and fasted plasma insulin levels were markedly decreased. Nes Cre /PPARγ-P467L mice fed either control diet or high-fat diet displayed impaired glucose tolerance yet exhibited increased sensitivity to exogenous insulin and increased insulin receptor signaling in white adipose tissue, liver, and skeletal muscle. These observations support the concept that alterations in PPARγ-driven mechanisms in the nervous system play a role in the regulation of growth and glucose metabolic homeostasis.

Peroxisome proliferator-activated receptor-γ (PPARγ), a master regulator of adipogenesis, was recently shown to affect energy homeostasis through its actions in the brain. Deletion of PPARγ in mouse brain, and specifically in the pro-opiomelanocortin (POMC) neurons, results in resistance to diet-induced obesity. To study the mechanisms by which PPARγ in POMC neurons controls energy balance, we constructed a Cre-recombinase-dependent conditionally activatable transgene expressing either wild-type (WT) or dominant-negative (P467L) PPARγ and the tdTomato reporter. Inducible expression of both forms of PPARγ was validated in cells in culture, in liver of mice infected with an adenovirus expressing Cre-recombinase (AdCre), and in the brain of mice expressing Cre-recombinase either in all neurons (NES(Cre)/PPARγ-P467L) or selectively in POMC neurons (POMC(Cre)/PPARγ-P467L). Whereas POMC(Cre)/PPARγ-P467L mice exhibited a normal pattern of weight gain when fed 60% high-fat diet, they exhibited increased weight gain and fat mass accumulation in response to a 10% fat isocaloric-matched control diet. POMC(Cre)/PPARγ-P467L mice were leptin sensitive on control diet but became leptin resistant when fed 60% high-fat diet. There was no difference in body weight between POMC(Cre)/PPARγ-WT mice and controls in response to 60% high-fat diet. However, POMC(Cre)/PPARγ-WT, but not POMC(Cre)/PPARγ-P467L, mice increased body weight in response to rosiglitazone, a PPARγ agonist. These observations support the concept that alterations in PPARγ-driven mechanisms in POMC neurons can play a role in the regulation of metabolic homeostasis under certain dietary conditions., (Copyright © 2016 the American Physiological Society.)

5-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway. The MTAP gene is frequently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. By interrogating data from a large-scale short hairpin RNA-mediated screen across 390 cancer cell line models, we found that the viability of MTAP-deficient cancer cells is impaired by depletion of the protein arginine methyltransferase PRMT5. MTAP-deleted cells accumulate the metabolite methylthioadenosine (MTA), which we found to inhibit PRMT5 methyltransferase activity. Deletion of MTAP in MTAP-proficient cells rendered them sensitive to PRMT5 depletion. Conversely, reconstitution of MTAP in an MTAP-deficient cell line rescued PRMT5 dependence. Thus, MTA accumulation in MTAP-deleted cancers creates a hypomorphic PRMT5 state that is selectively sensitized toward further PRMT5 inhibition. Inhibitors of PRMT5 that leverage this dysregulated metabolic state merit further investigation as a potential therapy for MTAP/CDKN2A-deleted tumors., (Copyright © 2016, American Association for the Advancement of Science.)

The introduction of MAPK pathway inhibitors paved the road for significant advancements in the treatment of BRAF-mutant (BRAF(MUT)) melanoma. However, even BRAF/MEK inhibitor combination therapy has failed to offer a curative treatment option, most likely because these pathways constitute a codependent signaling network. Concomitant PTEN loss of function (PTEN(LOF)) occurs in approximately 40% of BRAF(MUT) melanomas. In this study, we sought to identify the nodes of the PTEN/PI3K pathway that would be amenable to combined therapy with MAPK pathway inhibitors for the treatment of PTEN(LOF)/BRAF(MUT) melanoma. Large-scale compound sensitivity profiling revealed that PTEN(LOF) melanoma cell lines were sensitive to PI3Kβ inhibitors, albeit only partially. An unbiased shRNA screen (7,500 genes and 20 shRNAs/genes) across 11 cell lines in the presence of a PI3Kβ inhibitor identified an adaptive response involving the IGF1R-PI3Kα axis. Combined inhibition of the MAPK pathway, PI3Kβ, and PI3Kα or insulin-like growth factor receptor 1 (IGF1R) synergistically sustained pathway blockade, induced apoptosis, and inhibited tumor growth in PTEN(LOF)/BRAF(MUT) melanoma models. Notably, combined treatment with the IGF1R inhibitor, but not the PI3Kα inhibitor, failed to elevate glucose or insulin signaling. Taken together, our findings provide a strong rationale for testing combinations of panPI3K, PI3Kβ + IGF1R, and MAPK pathway inhibitors in PTEN(LOF)/BRAF(MUT) melanoma patients to achieve maximal response., (©2015 American Association for Cancer Research.)

Loss of peroxisome proliferator-activated receptor (PPAR)-γ function in the vascular endothelium enhances atherosclerosis and NF-κB target gene expression in high-fat diet-fed apolipoprotein E-deficient mice. The mechanisms by which endothelial PPAR-γ regulates inflammatory responses and protects against atherosclerosis remain unclear. To assess functional interactions between PPAR-γ and inflammation, we used a model of IL-1β-induced aortic dysfunction in transgenic mice with endothelium-specific overexpression of either wild-type (E-WT) or dominant negative PPAR-γ (E-V290M). IL-1β dose dependently decreased IκB-α, increased phospho-p65, and increased luciferase activity in the aorta of NF-κB-LUC transgenic mice. IL-1β also dose dependently reduced endothelial-dependent relaxation by ACh. The loss of ACh responsiveness was partially improved by pretreatment of the vessels with the PPAR-γ agonist rosiglitazone or in E-WT. Conversely, IL-1β-induced endothelial dysfunction was worsened in the aorta from E-V290M mice. Although IL-1β increased the expression of NF-κB target genes, NF-κB p65 inhibitor did not alleviate endothelial dysfunction induced by IL-1β. Tempol, a SOD mimetic, partially restored ACh responsiveness in the IL-1β-treated aorta. Notably, tempol only modestly improved protection in the E-WT aorta but had an increased protective effect in the E-V290M aorta compared with the aorta from nontransgenic mice, suggesting that PPAR-γ-mediated protection involves antioxidant effects. IL-1β increased ROS and decreased the phospho-endothelial nitric oxide synthase (Ser(1177))-to-endothelial nitric oxide synthase ratio in the nontransgenic aorta. These effects were completely abolished in the aorta with endothelial overexpression of WT PPAR-γ but were worsened in the aorta with E-V290M even in the absence of IL-1β. We conclude that PPAR-γ protects against IL-1β-mediated endothelial dysfunction through a reduction of oxidative stress responses but not by blunting IL-1β-mediated NF-κB activity., (Copyright © 2016 the American Physiological Society.)

Loss of peroxisome proliferator-activated receptor-γ (PPARγ) function causes hypertension, whereas its activation lowers blood pressure. Evidence suggests that these effects may be attributable to PPARγ activity in the vasculature. However, the specific transcriptional targets of PPARγ in vessels remain largely unidentified. In this study, we examined the role of smooth muscle PPARγ during salt-sensitive hypertension and investigated its transcriptional targets and functional effect. Transgenic mice expressing dominant-negative PPARγ (S-P467L) in smooth muscle cells were more prone to deoxycorticosterone acetate-salt-induced hypertension and mesenteric arterial dysfunction compared with nontransgenic controls. Despite similar morphometry at baseline, vascular remodeling in conduit and small arteries was enhanced in S-P467L after deoxycorticosterone acetate-salt treatment. Gene expression profiling in aorta and mesenteric arteries revealed significantly decreased expression of tissue inhibitor of metalloproteinase-4 (TIMP-4) in S-P467L. Expression of TIMP-4 was increased by deoxycorticosterone acetate-salt treatment, but this increase was ablated in S-P467L. Interference with PPARγ activity either by treatment with a PPARγ inhibitor, GW9662, or by expressing P467L PPARγ markedly suppressed TIMP-4 in primary smooth muscle cells. PPARγ binds to a PPAR response element (PPRE) in chromatin close to the TIMP-4 gene in smooth muscle cells, suggesting that TIMP-4 is a novel target of PPARγ. The interference with PPARγ and decrease in TIMP-4 were accompanied by an increase in total matrix metalloproteinase activity. PPARγ-mediated loss of TIMP-4 increased, whereas overexpression of TIMP-4 decreased smooth muscle cell migration in a scratch assay. Our findings highlight a protective mechanism induced by PPARγ in deoxycorticosterone acetate-salt treatment, establishing a novel mechanistic link between PPARγ and TIMP-4., (© 2015 American Heart Association, Inc.)

Dysregulation of peroxisome proliferator-activated receptor gamma (PPARγ) activity leads to significant alterations in cardiovascular and metabolic regulation. This is most keenly observed by the metabolic syndrome-like phenotypes exhibited by patients carrying mutations in PPARγ. We will summarize recent findings regarding mechanisms of PPARγ regulation in the cardiovascular and nervous systems focusing largely on PPARγ in the smooth muscle, endothelium, and brain. Canonically, PPARγ exerts its effects by regulating the expression of target genes in these cells, and we will discuss mechanisms by which PPARγ targets in the vasculature regulate cardiovascular function. We will also discuss emerging evidence that PPARγ in the brain is a mediator of appetite and obesity. Finally, we will briefly review how novel PPARγ activators control posttranslational modifications of PPARγ and their prospects to offer new therapeutic options for treatment of metabolic diseases without the adverse side effects of thiazolidinediones which strongly activate transcriptional activity of PPARγ.

This article is the last of a four-part series addressing the use of non-pharmacological interventions for older adults with behavioral and psychological symptoms of dementia (BPSD). These types of interventions are used to prevent, lessen, or eliminate BPSD, thereby reducing patient reliance on psychoactive medications. These interventions are easy to use, cost-effective, and simple to implement. The Centers for Medicare & Medicaid Services' psychoactive medication reduction initiative encourages all staff to use nonpharmacological interventions to manage BPSD. As with any attempt to handle BPSD, health care professionals and staff need a tool-box of interventions, as what works one day may not work the next and what works with one older adult may not work with another. This article describes the categories of diversional and physical nonpharmalogical interventions, presents the evidence supporting their use, and provides information on effective implementation., (Copyright 2015, SLACK Incorporated.)

This article is part three of a four-part series addressing the use of nonpharmacological interventions in place of or in conjunction with psychotropic medications in older adults with cognitive impairment. Acquiring a better understanding of the mechanics for how each intervention works makes selection of an intervention easier at the time it is needed. Selection of the appropriate nonpharmacological intervention is based on person-centered care and how to adapt and implement it for each individual. Selection also depends on target behavior, behavior triggers, and the physical and cognitive functioning of the individual with the behavioral and psychological symptoms of dementia. Nonpharmacological interventions can be implemented by all staff members, not just recreational and activity personnel. The Centers for Medicare & Medicaid Services initiative would like to see all staff involved with these interventions, which can be implemented on the spot, as they are needed, to prevent, reduce, or stop a particular behavior. The current article will describe sensory and nurturing interventions, present the evidence supporting their use, and provide information on effective implementation., (Copyright 2014, SLACK Incorporated.)

The Centers for Medicare and Medicaid Services launched a new initiative aimed at improving behavioral health and safeguarding older adults residing in nursing homes from unnecessary antipsychotic drug use. This article is part two of a four-part series on how caregivers working with older adults can implement nonpharmacological interventions. Many different types of nonpharmacological interventions exist, including staff techniques, communication skills, the identification of basic and medical needs, and actual activities, which may be performed alone, one-on-one, or in small groups. To implement nonpharmacological interventions, a trusting relationship must be established. What is done, what is not done, and how one behaves can all precipitate or prevent agitation, anxiety, depression, and apathy in older adults. This article will address the trusting relationship concept that must be actualized for nonpharmacological interventions to be successful., (Copyright 2014, SLACK Incorporated.)

Acid-sensing ion channel 1A (ASIC1A) is abundant in the nucleus accumbens (NAc), a region known for its role in addiction. Because ASIC1A has been suggested to promote associative learning, we hypothesized that disrupting ASIC1A in the NAc would reduce drug-associated learning and memory. However, contrary to this hypothesis, we found that disrupting ASIC1A in the mouse NAc increased cocaine-conditioned place preference, suggesting an unexpected role for ASIC1A in addiction-related behavior. Moreover, overexpressing ASIC1A in rat NAc reduced cocaine self-administration. Investigating the underlying mechanisms, we identified a previously unknown postsynaptic current during neurotransmission that was mediated by ASIC1A and ASIC2 and thus well positioned to regulate synapse structure and function. Consistent with this possibility, disrupting ASIC1A altered dendritic spine density and glutamate receptor function, and increased cocaine-evoked plasticity, which resemble changes previously associated with cocaine-induced behavior. Together, these data suggest that ASIC1A inhibits the plasticity underlying addiction-related behavior and raise the possibility of developing therapies for drug addiction by targeting ASIC-dependent neurotransmission.

Numerous studies have found excessive or in appropriate use of antipsychotic drugs in nursing home patients with cognitive impairment or perceived behavioral issues. Inappropriately medicating this vulnerable population can lead to several negative outcomes, including failure to have needs met, injury, illness, and even death. In response to recent literature and government reports highlighting this issue, in 2012, the Centers for Medicare and Medicaid Services (CMS) launched an initiative called the National Partnership to Improve Dementia Care. This article discusses the CMS initiative, as well as the feasibility and recent trends in the use of nonpharmacological interventions that could be implemented when working with patients with cognitive impairment and behavioral and psychological symptoms associated with dementia., (Copyright 2014, SLACK Incorporated.)

Neurological deficits caused by H-I (hypoxia-ischaemia) to the perinatal brain are often severely debilitating and lead to motor impairment, intellectual disability and seizures. Perinatal brain injury is distinct from adult brain injury in that the developing brain is undergoing the normal process of neuronal elimination by apoptotic cell death and thus the apoptotic machinery is more easily engaged and activated in response to injury. Thus cell death in response to neonatal H-I brain injury is partially due to mitochondrial dysfunction and activation of the apoptosome and caspase 3. An important regulator of the apoptotic response following mitochondrial dysfunction is XIAP (X-linked inhibitor of apoptosis protein). XIAP inhibits apoptosis at the level of caspase 9 and caspase 3 activation, and lack of XIAP in vitro has been shown to lead to increased apoptotic cell death. In the present study we show that mice lacking the gene encoding the XIAP protein have an exacerbated response to neonatal H-I injury as measured by tissue loss at 7 days following the injury. In addition, when the XIAP-deficient mice were studied at 24 h post-H-I we found that the increase in injury correlates with an increased apoptotic response in the XIAP-deficient mice and also with brain imaging changes in T2-weighted magnetic resonance imaging and apparent diffusion coefficient that correspond to the location of apoptotic cell death. These results identify a critical role of XIAP in regulating neuronal apoptosis in vivo and demonstrate the enhanced vulnerability of neurons to injury in the absence of XIAP in the developing brain.

The beta-catenin pathway is involved in growth, differentiation and tumor formation. Suppression of pathway activity by expressed inhibitors can cause growth arrest or apoptosis in certain colon carcinoma lines. We compare the effects of 2 pathway inhibitors, a VE-cadherin cytoplasmic domain fragment (Cad5CD) and a truncated, dominant-negative Tcf4 (TcfDN), using a microplate assay for cell death and microarray gene expression analysis. The cell-lethal assay shows that Cad5CD, when expressed in HT29 human colon tumor cells and 3 non-colon lines, selectively kills the HT29 cells. Cad5CD overexpression inhibits beta-catenin/Tcf4 transcriptional activity, as determined by results from microarray experiments. Our results support the view that beta-catenin is an attractive anti-cancer target, especially if the Cad5CD binding site or Cad5CD itself can be exploited for drug development. In addition, therapeutically relevant phenotypes such as drug selectivity may be difficult to predict from gene expression analysis alone. Other more specialized phenotypic tests such as cell-lethal assays may be required., (Copyright 2003 Wiley-Liss, Inc.)

Screens for cytostasis/cytoxicity have considerable value for the discovery of therapeutic agents and the investigation of the biology of apoptosis. For instance, genetic screens for proteins, protein fragments, peptides, RNAs, or chemicals that kill tissue culture cells may aid in identifying new cancer therapeutic targets. A microplate assay for cell death is needed to achieve throughputs sufficient to sift through thousands of agents from expression or chemical libraries. The authors describe a homogeneous assay for cell death in tissue culture cells compatible with 96- or 384-well plates. In combination with a previously described system for retroviral packaging and transduction, nearly 6000 expression library clones could be screened per week in a 96-well plate format. The screening system may also prove useful for chemical screens.

Transdominant genetic selections can yield protein fragment and peptide modulators of specific biochemical pathways. In yeast, such screens have been highly successful in targeting the MAP (mitogen-activated protein) kinase growth-control pathway. We performed a similar type of selection aimed at recovery of modulators of the mammalian MAP kinase cascade. Two pathway activators were identified, fragments of the TrkB and Raf-1 kinases. In a second selection directed at the beta-catenin growth-control pathway, three different clones encoding cadherin fragments were recovered. In neither selection were peptide inhibitors observed. We conclude that some transdominant selections in mammalian cells can readily yield high-penetrance protein fragments, but may be less amenable to isolation of peptide inhibitors.

Pseudotyped retroviral vectors combine the advantages of broad host range, high expression, stable chromosomal integration, and ease of preparation. These vectors greatly facilitate delivery into mammalian cells of sequences encoding individual peptide inhibitors-including those with therapeutic utility-and inhibitor libraries. However, retroviral vectors vary in behavior, particularly with respect to expression levels in different cell lines. Expression level is especially important in transdominant experiments because the concentration of an inhibitor (for example, an expressed peptide) is one of the key determinants in the degree of complex formation between the inhibitor and its target. Thus, inhibitor concentration should have an impact on the expressivity and/or penetrance of an induced phenotype. Here, we compare several retroviral vectors and human cell lines for relative expression levels using a green fluorescent protein reporter. We show for a subset of these lines that cellular protein concentrations produced by single-copy vectors range up to about 2 microM. We also examine other variables that contribute to expression level, such as the nature of the expressed protein's carboxy terminus. Finally, we test the effect of increased concentration on phenotype with a nine-amino-acid peptide derived from the human papilloma virus protein E7 which overcomes E7-mediated cell growth.

Purpose: To test prior published observations that patients with brown irides were more susceptible to the oculocardiac reflex., Subjects and Methods: 466 adults and children underwent surgery on an extraocular muscle under general anesthesia without anticholinergic blockade. The first rectus muscle isolated was given a predetermined specific force pull during electrocardiograph monitoring. Iris color was retrieved from the deliberate clinical charting., Results: The occurrence of the oculocardiac reflex did not correlate with age, but did profoundly for the inferior rectus and least for the lateral rectus. Iris color did not influence the incidence of occurrence of moderate or severe oculocardiac reflex., Conclusion: The prediction of oculocardiac reflex propensity remains elusive.

As a means of determining whether there has been selection to conserve the basic pattern of filamentous phage mRNAs, the major mRNAs representing genes II to VIII have been defined for a phage distantly related to the Ff group specific for Escherichia coli hosts bearing F pili. Phage IKe has a genome with 55% identity with the Ff genome and infects E. coli strains bearing N pili. The results reveal a remarkably similar pattern of overlapping polycistronic mRNAs with a common 3' end and unique 5' ends. The IKe mRNAs, like the Ff phage mRNAs, represent a combination of primary transcripts and processed RNAs. However, examination of the sequences containing the RNA endpoint positions revealed that effectively the only highly conserved regulatory element is the rho-independent terminator that generates the common 3' end. Promoters and processing sites have not been maintained in identical positions, but frequently are placed so as to yield RNAs with similar coding function. By conserving the pattern of transcription and processing despite divergence in the regulatory elements and possibly the requirements for host, endoribonucleases, the results argue that the pattern is not simply fortuitous.

The abundant mRNAs used as templates for synthesis of filamentous phage f1 proteins are a combination of primary transcripts and 3' products of processing. The processing steps are mediated by host endoribonucleases. One of the enzymes implicated in f1 mRNA processing is RNase E, the only endonuclease thus far shown to have a global role in mRNA decay. By establishing the temperature-sensitive phenotypes of RNase E mutants and then inducing a transcription unit bearing cloned f1 processing sites, we show that RNase E is required for production of at least three of the processed RNAs. Using in vivo processing assays, we also test directly the regions implicated genetically in previous work to contain the processing sites. The sites function as discrete domains in a number of transcription units, show little influence of translation, but appear to have increased activity at the 5' terminus of an mRNA. From their functional properties, we suggest that the known processing sites from phage f1 that are dependent on RNase E may be representative of relatively late steps in rne-dependent cleavage pathways.

The binding of 5'-end biotinylated DNA, ranging in size from 100 to 5000 base pairs, was studied using streptavidin-coated polystyrene latex particles with diameters between 0.944 and 0.090 micron. The experimental binding constants and forward rate constants of this solid-phase reaction were determined to be several orders of magnitude lower than values for the biotin-streptavidin interaction in solution as expected and were shown to depend on the size of both ligand and substrate. An observed inflection in the binding constant of the biotinylated DNA appeared around 1000 base pairs, possibly indicating different surface orientations of the macroligand above and below this critical size. This effect was more pronounced for the smaller latex particles used in this study and highlighted possible differences in the surface arrangement of streptavidin on the differently sized particles. Diffusion limitation to the binding reaction was found to be significant in all cases. In this present work, an exponential relationship was established between the experimental binding constant and the number of base pairs in the biotinylated DNA. This relationship possibly provides a means to predict capacity and binding speed in cases where adsorption, purification, and release of larger DNA chains are required.