Use of mobile technology to identify behavioral mechanisms linked to mental health outcomes in Kenya: protocol for development and validation of a predictive model.

OBJECTIVE: This study proposes to identify and validate weighted sensor stream signatures that predict near-term risk of a major depressive episode and future mood among healthcare workers in Kenya. APPROACH: The study will deploy a mobile application (app) platform and use novel data science analytic approaches (Artificial Intelligence and Machine Learning) to identifying predictors of mental health disorders among 500 randomly sampled healthcare workers from five healthcare facilities in Nairobi, Kenya. EXPECTATION: This study will lay the basis for creating agile and scalable systems for rapid diagnostics that could inform precise interventions for mitigating depression and ensure a healthy, resilient healthcare workforce to develop sustainable economic growth in Kenya, East Africa, and ultimately neighboring countries in sub-Saharan Africa. This protocol paper provides an opportunity to share the planned study implementation methods and approaches. CONCLUSION: A mobile technology platform that is scalable and can be used to understand and improve mental health outcomes is of critical importance.

Effects of genetics and sex on adolescent behaviors following neonatal ethanol exposure in BXD recombinant inbred strains.

Introduction: Fetal alcohol spectrum disorders (FASD) are the leading preventable neurodevelopmental disorders and two hallmark symptoms of FASD are abnormal behavior, and cognitive and learning deficits. The severity of alcohol's teratogenic effects on the developing brain is influenced by genetics and sex. We previously identified recombinant inbred BXD mouse strains that show differential vulnerability to ethanol-induced cell death in the developing hippocampus, a brain region important in learning and memory. The present study aimed to test the hypothesis that strains with increased vulnerability to ethanol-induced cell death in the hippocampus have concomitant deficits in multiple hippocampal-related behaviors during adolescence. Methods: The current study evaluated the effects of developmental ethanol exposure on adolescent behavior in two BXD strains that show high cell death (BXD48a, BXD100), two that show low cell death (BXD60, BXD71), and the two parental strains (C57BL/6 J (B6), DBA/2 J (D2)). On postnatal day 7, male and female neonatal pups were treated with ethanol (5.0 g/kg) or saline given in two equal doses 2 h apart. Adolescent behavior was assessed across multiple behavioral paradigms including the elevated plus maze, open field, Y-maze, and T-maze. Results: Our results demonstrate that the effects of developmental ethanol exposure on adolescent behavioral responses are highly dependent on strain. The low cell death strains, BXD60 and BXD71, showed minimal effect of ethanol exposure on all behavioral measures but did present sex differences. The parental -B6 and D2-strains and high cell death strains, BXD48a and BXD100, showed ethanol-induced effects on activity-related or anxiety-like behaviors. Interestingly, the high cell death strains were the only strains that showed a significant effect of postnatal ethanol exposure on hippocampal-dependent spatial learning and memory behaviors. Discussion: Overall, we identified effects of ethanol exposure, strain, and/or sex on multiple behavioral measures. Interestingly, the strains that showed the most effects of postnatal ethanol exposure on adolescent behavior were the BXD strains that show high ethanol-induced cell death in the neonatal hippocampus, consistent with our hypothesis. Additionally, we found evidence for interactions among strain and sex, demonstrating that these factors have a complex effect on alcohol responses and that both are important considerations.

Choline Supplementation Alters Hippocampal Cytokine Levels in Adolescence and Adulthood in an Animal Model of Fetal Alcohol Spectrum Disorders.

Alcohol (ethanol) exposure during pregnancy can adversely affect development, with long-lasting consequences that include neuroimmune, cognitive, and behavioral dysfunction. Alcohol-induced alterations in cytokine levels in the hippocampus may contribute to abnormal cognitive and behavioral outcomes in individuals with fetal alcohol spectrum disorders (FASD). Nutritional intervention with the essential nutrient choline can improve hippocampal-dependent behavioral impairments and may also influence neuroimmune function. Thus, we examined the effects of choline supplementation on hippocampal cytokine levels in adolescent and adult rats exposed to alcohol early in development. From postnatal day (PD) 4-9 (third trimester-equivalent), Sprague-Dawley rat pups received ethanol (5.25 g/kg/day) or sham intubations and were treated with choline chloride (100 mg/kg/day) or saline from PD 10-30; hippocampi were collected at PD 35 or PD 60. Age-specific ethanol-induced increases in interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and keratinocyte chemoattractant/human growth-regulated oncogene (KC/GRO) were identified in adulthood, but not adolescence, whereas persistent ethanol-induced increases of interleukin-6 (IL-6) levels were present at both ages. Interestingly, choline supplementation reduced age-related changes in interleukin-1 beta (IL-1ß) and interleukin-5 (IL-5) as well as mitigating the long-lasting increase in IFN-γ in ethanol-exposed adults. Moreover, choline influenced inflammatory tone by modulating ratios of pro- to -anti-inflammatory cytokines. These results suggest that ethanol-induced changes in hippocampal cytokine levels are more evident during adulthood than adolescence, and that choline can mitigate some effects of ethanol exposure on long-lasting inflammatory tone.

Effects of Genetics and Sex on Acute Gene Expression Changes in the Hippocampus Following Neonatal Ethanol Exposure in BXD Recombinant Inbred Mouse Strains.

Fetal alcohol spectrum disorders (FASD) are prevalent neurodevelopmental disorders. Genetics have been shown to have a role in the severity of alcohol's teratogenic effects on the developing brain. We previously identified recombinant inbred BXD mouse strains that show high (HCD) or low cell death (LCD) in the hippocampus following ethanol exposure. The present study aimed to identify gene networks that influence this susceptibility. On postnatal day 7 (3rd-trimester-equivalent), male and female neonates were treated with ethanol (5.0 g/kg) or saline, and hippocampi were collected 7hrs later. Using the Affymetrix microarray platform, ethanol-induced gene expression changes were identified in all strains with divergent expression sets found between sexes. Genes, such as Bcl2l11, Jun, and Tgfb3, showed significant strain-by-treatment interactions and were involved in many apoptosis pathways. Comparison of HCD versus LCD showed twice as many ethanol-induced genes changes in the HCD. Interestingly, these changes were regulated in the same direction suggesting (1) more perturbed effects in HCD compared to LCD and (2) limited gene expression changes that confer resistance to ethanol-induced cell death in LCD. These results demonstrate that genetic background and sex are important factors that affect differential cell death pathways after alcohol exposure during development that could have long-term consequences.

Choline Supplementation Modifies the Effects of Developmental Alcohol Exposure on Immune Responses in Adult Rats.

Prenatal alcohol exposure can disrupt the development of numerous systems, including the immune system. Indeed, alterations in cytokine levels may contribute to the neuropathological, behavioral, and cognitive problems, and other adverse outcomes observed in individuals with fetal alcohol spectrum disorders. Importantly, supplementation with the essential nutrient choline can improve performance in hippocampal-dependent behaviors; thus, the present study examined the effects of choline on plasma and hippocampal cytokines in adult rats exposed to ethanol in early development. From postnatal day (PD) 4-9 (third trimester equivalent), pups received ethanol (5.25 g/kg/day) or Sham intubations. Subjects were treated with choline chloride (100 mg/kg/day) or saline from PD10-30. On PD60, plasma and hippocampal tissue was collected before and after an immune challenge (lipopolysaccharide (LPS); 50 ug/kg). Prior to the immune challenge, ethanol-exposed subjects showed an overall increase in hippocampal pro-inflammatory cytokines, an effect mitigated by choline supplementation. In contrast, in the plasma, choline reduced LPS-related increases in pro-inflammatory markers, particularly in ethanol-exposed subjects. Thus, early choline supplementation may modify both brain and peripheral inflammation. These results suggest that early choline can mitigate some long-term effects of ethanol exposure on hippocampal inflammation, which may contribute to improved hippocampal function, and could also influence peripheral immune responses that may impact overall health.

Analytical Accuracy of RET Fusion Detection by Break-Apart Fluorescence In Situ Hybridization.

CONTEXT.­: RET gene fusions are oncogenic drivers in nonsmall cell lung cancer and nonmedullary thyroid cancer. Selpercatinib (RETEVMO), a targeted inhibitor of RET, was approved by the US Food and Drug Administration for the treatment of RET fusion-positive nonsmall cell lung cancer and nonmedullary thyroid cancer emphasizing the need for rapid and accurate diagnosis of RET fusions. Fluorescence in situ hybridization (FISH) has been used to detect gene rearrangements, but its performance detecting RET rearrangements is understudied. OBJECTIVE.­: To validate and describe the performance of Abbott Molecular RET break-apart FISH probes for detecting RET rearrangements. DESIGN.­: A training set with RET fusion-positive (13) and RET fusion-negative nonsmall cell lung cancer and nonmedullary thyroid cancer samples (12) was used to establish criteria for FISH scoring. The scoring criteria was then applied to a larger validation set of samples (96). RESULTS.­: A cutoff of 19% or more positive nuclei by FISH was established in the training set and determined by the mean ±3 SD. The validation set was tested using Abbott Molecular RET break-apart FISH compared with sequencing. With this cutoff, a sensitivity of 86% (12 of 14) and specificity of 99% (81 of 82) was achieved. Bootstrapping showed sensitivity could be optimized by using a greater than 13% cutoff with indeterminate samples of 13% to 18% abnormal nuclei requiring confirmation by an orthogonal method. Using this 3-tier scoring system sensitivity increased to 100% (14 of 14) and specificity was 96% (79 of 82). CONCLUSIONS.­: Abbott Molecular break-apart FISH probes can be used to detect RET fusions. Laboratories can optimize cutoffs and/or testing algorithms to maximize sensitivity and specificity to ensure appropriate patients receive effective, timely therapy.

Neonatal Alcohol Exposure in Mice Induces Select Differentiation- and Apoptosis-Related Chromatin Changes Both Independent of and Dependent on Sex.

Prenatal alcohol exposure (PAE) affects many aspects of physiology and behavior, including brain development. Specifically, ethanol can influence expression of genes important for brain growth, including chromatin modifiers. Ethanol can also increase apoptotic cell death in the brain and alter epigenetic profiles such as modifications to histones and DNA methylation. Although differential sex outcomes and disruptions to the function of multiple brain regions have been reported in fetal alcohol spectrum disorder (FASD), the majority of our knowledge on molecular epigenetic and apoptotic dysregulation in PAE is based on data from males and is sometimes limited to assessments of the whole brain or one brain region. Here, we examined histone modifications, DNA methylation, and expression of genes involved in differentiation and proliferation related-chromatin modifications and apoptosis in the cerebral cortex and cerebellum of C57BL/6J mice exposed to an acute alcohol challenge on postnatal day 7, with a focus on differential outcomes between sexes and brain regions. We found that neonatal alcohol exposure altered histone modifications, and impacted expression of a select few chromatin modifier and apoptotic genes in both the cortex and cerebellum. The results were observed primarily in a sex-independent manner, although some additional trends toward sexual dimorphisms were observed. Alcohol exposure induced trends toward increased bulk H3K4me3 levels, increased Kmt2e expression, and elevated levels of Casp6 mRNA and bulk γH2A.X. Additional trends indicated that ethanol may impact Kdm4a promoter DNA methylation levels and bulk levels of the histone variant H2A.Z, although further studies are needed. We comprehensively examined effects of ethanol exposure across different sexes and brain regions, and our results suggest that major impacts of ethanol on bulk chromatin modifications underlying differentiation and apoptosis may be broadly applicable across the rodent cortex and cerebellum in both sexes.

Genetic Influences on the Amount of Cell Death in the Neural Tube of BXD Mice Exposed to Acute Ethanol at Midgestation.

BACKGROUND: Fetal alcohol spectrum disorders (FASD) have a strong genetic component although the genes that underlie this are only beginning to be elucidated. In the present study, one of the most common phenotypes of FASD, cell death within the early developing neural tube, was examined across a genetic reference population in a reverse genetics paradigm with the goal of identifying genetic loci that could influence ethanol (EtOH)-induced apoptosis in the early developing neural tube. METHODS: BXD recombinant inbred mice as well as the parental strains were used to evaluate genetic differences in EtOH-induced cell death after exposure on embryonic day 9.5. Dams were given either 5.8 g/kg EtOH or isocaloric maltose-dextrin in 2 doses via intragastric gavage. Embryos were collected 7 hours after the initial exposure and cell death evaluated via TUNEL staining in the brainstem and forebrain. Genetic loci were evaluated using quantitative trait locus (QTL) analysis at GeneNetwork.org. RESULTS: Significant strain differences were observed in the levels of EtOH-induced cell death that were due to genetic effects and not confounding variables such as differences in developmental maturity or cell death kinetics. Comparisons between the 2 regions of the developing neural tube showed little genetic correlation with the QTL maps exhibiting no overlap. Significant QTLs were found on murine mid-chromosome 4 and mid-chromosome 14 only in the brainstem. Within these chromosomal loci, a number of interesting candidate genes were identified that could mediate this differential sensitivity including Nfia (nuclear factor I/A) and Otx2 (orthodenticle homeobox 2). CONCLUSIONS: These studies demonstrate that the levels of EtOH-induced cell death occur in strain- and region-dependent manners. Novel QTLs on mouse Chr4 and Chr14 were identified that modulate the differential sensitivity to EtOH-induced apoptosis in the embryonic brainstem. The genes underlying these QTLs could identify novel molecular pathways that are critical in this phenotype.

Healthcare Experiences of Underrepresented Lesbian and Bisexual Women: A Focus Group Qualitative Study.

Purpose: To understand the complex healthcare experiences of women identifying as lesbian or bisexual. who are also women of color, veterans, and/or 65 years of age and older. Methods: Inclusion criteria were age 25 or older, Los Angeles County resident, self-identification as a lesbian or bisexual woman, and as an African American, Latina, Asian-Pacific Islander, and/or a veteran. For the age 65 years and older group, participants were eligible regardless of their veteran status or race/ethnicity. Five focus groups were conducted (n=35) and the same questions were asked addressing their comfort interacting with healthcare providers, the provider knowing their sexual orientation, characteristics of a perfect provider, and barriers to care. Structured qualitative analyses were performed. Results: Participants identified concerns that providers often hold to heterosexual cultural norms. Participants varied on preferences for providers of the same race/ethnicity as themselves. Lesbians who are 65 years and older identified legal barriers as major concerns. All groups identified incorrect provider assumptions about sexual orientation and sexual practices as frequently compromising their care. Participants supported the idea of certification for providers skilled in lesbian, gay, bisexual, transgender, and queer (LGBTQ) health, but expressed skepticism that such programs would necessarily result in better care. Conclusion: Healthcare provider trainings need to address the specific concerns and experiences of underrepresented lesbian and bisexual women. Healthcare environments must be transformed to effectively address their needs. More research is needed on the separate healthcare experiences of specific marginalized populations related to their sexual orientation and/or gender identity.

The effect of alcohol on the differential expression of cluster of differentiation 14 gene, associated pathways, and genetic network.

Alcohol consumption affects human health in part by compromising the immune system. In this study, we examined the expression of the Cd14 (cluster of differentiation 14) gene, which is involved in the immune system through a proinflammatory cascade. Expression was evaluated in BXD mice treated with saline or acute 1.8 g/kg i.p. ethanol (12.5% v/v). Hippocampal gene expression data were generated to examine differential expression and to perform systems genetics analyses. The Cd14 gene expression showed significant changes among the BXD strains after ethanol treatment, and eQTL mapping revealed that Cd14 is a cis-regulated gene. We also identified eighteen ethanol-related phenotypes correlated with Cd14 expression related to either ethanol responses or ethanol consumption. Pathway analysis was performed to identify possible biological pathways involved in the response to ethanol and Cd14. We also constructed a genetic network for Cd14 using the top 20 correlated genes and present several genes possibly involved in Cd14 and ethanol responses based on differential gene expression. In conclusion, we found Cd14, along with several other genes and pathways, to be involved in ethanol responses in the hippocampus, such as increased susceptibility to lipopolysaccharides and neuroinflammation.

Analyses of differentially expressed genes after exposure to acute stress, acute ethanol, or a combination of both in mice.

Alcohol abuse is a complex disorder, which is confounded by other factors, including stress. In the present study, we examined gene expression in the hippocampus of BXD recombinant inbred mice after exposure to ethanol (NOE), stress (RSS), and the combination of both (RSE). Mice were given an intraperitoneal (i.p.) injection of 1.8 g/kg ethanol or saline, and subsets of both groups were exposed to acute restraint stress for 15 min or controls. Gene expression in the hippocampus was examined using microarray analysis. Genes that were significantly (p < 0.05, q < 0.1) differentially expressed were further evaluated. Bioinformatic analyses were predominantly performed using tools available at GeneNetwork.org, and included gene ontology, presence of cis-regulation or polymorphisms, phenotype correlations, and principal component analyses. Comparisons of differential gene expression between groups showed little overlap. Gene Ontology demonstrated distinct biological processes in each group with the combined exposure (RSE) being unique from either the ethanol (NOE) or stress (RSS) group, suggesting that the interaction between these variables is mediated through diverse molecular pathways. This supports the hypothesis that exposure to stress alters ethanol-induced gene expression changes and that exposure to alcohol alters stress-induced gene expression changes. Behavior was profiled in all groups following treatment, and many of the differentially expressed genes are correlated with behavioral variation within experimental groups. Interestingly, in each group several genes were correlated with the same phenotype, suggesting that these genes are the potential origins of significant genetic networks. The distinct sets of differentially expressed genes within each group provide the basis for identifying molecular networks that may aid in understanding the complex interactions between stress and ethanol, and potentially provide relevant therapeutic targets. Using Ptp4a1, a candidate gene underlying the quantitative trait locus for several of these phenotypes, and network analyses, we show that a large group of differentially expressed genes in the NOE group are highly interrelated, some of which have previously been linked to alcohol addiction or alcohol-related phenotypes.

A novel heat shock protein alpha 8 (Hspa8) molecular network mediating responses to stress- and ethanol-related behaviors.

Genetic differences mediate individual differences in susceptibility and responses to stress and ethanol, although, the specific molecular pathways that control these responses are not fully understood. Heat shock protein alpha 8 (Hspa8) is a molecular chaperone and member of the heat shock protein family that plays an integral role in the stress response and that has been implicated as an ethanol-responsive gene. Therefore, we assessed its role in mediating responses to stress and ethanol across varying genetic backgrounds. The hippocampus is an important mediator of these responses, and thus, was examined in the BXD family of mice in this study. We conducted bioinformatic analyses to dissect genetic factors modulating Hspa8 expression, identify downstream targets of Hspa8, and examined its role. Hspa8 is trans-regulated by a gene or genes on chromosome 14 and is part of a molecular network that regulates stress- and ethanol-related behaviors. To determine additional components of this network, we identified direct or indirect targets of Hspa8 and show that these genes, as predicted, participate in processes such as protein folding and organic substance metabolic processes. Two phenotypes that map to the Hspa8 locus are anxiety-related and numerous other anxiety- and/or ethanol-related behaviors significantly correlate with Hspa8 expression. To more directly assay this relationship, we examined differences in gene expression following exposure to stress or alcohol and showed treatment-related differential expression of Hspa8 and a subset of the members of its network. Our findings suggest that Hspa8 plays a vital role in genetic differences in responses to stress and ethanol and their interactions.

Identification of candidate genes that underlie the QTL on chromosome 1 that mediates genetic differences in stress-ethanol interactions.

Alcoholism, stress, and anxiety are strongly interacting heritable, polygenetic traits. In a previous study, we identified a quantitative trait locus (QTL) on murine chromosome (Chr) 1 between 23.0 and 31.5 Mb that modulates genetic differences in the effects of ethanol on anxiety-related phenotypes. The goal of the present study was to extend the analysis of this locus with a focus on identifying candidate genes using newly available data and tools. Anxiety-like behavior was evaluated with an elevated zero maze following saline or ethanol injections (1.8 g/kg) in C57BL/6J, DBA2J, and 72 BXD strains. We detected significant effects of strain and treatment and their interaction on anxiety-related behaviors, although surprisingly, sex was not a significant factor. The Chr1 QTL is specific to the ethanol-treated cohort. Candidate genes in this locus were evaluated using now standard bioinformatic criteria. Collagen 19a1 (Col19a1) and family sequence 135a (Fam135a) met most criteria but have lower expression levels and lacked biological verification and, therefore, were considered less likely candidates. In contrast, two other genes, the prenylated protein tyrosine phosphate family member Ptp4a1 (protein tyrosine phosphate 4a1) and the zinc finger protein Phf3 (plant homeoDomain finger protein 3) met each of our bioinformatic criteria and are thus strong candidates. These findings are also of translational relevance because both Ptp4a1 and Phf3 have been nominated as candidates genes for alcohol dependence in a human genome-wide association study. Our findings support the hypothesis that variants in one or both of these genes modulate heritable differences in the effects of ethanol on anxiety-related behaviors.

Prediction of in vivo clearance and associated variability of CYP2C19 substrates by genotypes in populations utilizing a pharmacogenetics-based mechanistic model.

It is important to examine the cytochrome P450 2C19 (CYP2C19) genetic contribution to drug disposition and responses of CYP2C19 substrates during drug development. Design of such clinical trials requires projection of genotype-dependent in vivo clearance and associated variabilities of the investigational drug, which is not generally available during early stages of drug development, but is essential for CYP2C19 substrates with multiple clearance pathways. This study evaluated the utility of pharmacogenetics-based mechanistic modeling in predicting such parameters. Hepatic CYP2C19 activity and variability within genotypes were derived from in vitro S-mephenytoin metabolic activity in genotyped human liver microsomes (N = 128). These data were then used in mechanistic models to predict genotype-dependent disposition of CYP2C19 substrates (i.e., S-mephenytoin, citalopram, pantoprazole, and voriconazole) by incorporating in vivo clearance or pharmacokinetics of wild-type subjects and parameters of other clearance pathways. Relative to the wild-type, the CYP2C19 abundance (coefficient of variation percentage) in CYP2C19*17/*17, *1/*17, *1/*1, *17/null, *1/null, and null/null microsomes was estimated as 1.85 (117%), 1.79 (155%), 1.00 (138%), 0.83 (80%), 0.38 (130%), and 0 (0%), respectively. The subsequent modeling and simulations predicted, within 2-fold of the observed, the means and variabilities of urinary S/R-mephenytoin ratio (36 of 37 genetic groups), the oral clearance of citalopram (9 of 9 genetic groups) and pantoprazole (6 of 6 genetic groups), and voriconazole oral clearance (4 of 4 genetic groups). Thus, relative CYP2C19 genotype-dependent hepatic activity and variability were quantified in vitro and used in a mechanistic model to predict pharmacokinetic variability, thus allowing the design of pharmacogenetics and drug-drug interaction trials for CYP2C19 substrates.

Contrasting effects of hyperoxia on GM-CSF gene transcription in alveolar epithelial cells and T cells.

Granulocyte/macrophage colony-stimulating factor (GM-CSF) is critically important for normal pulmonary innate immunity and for functional maturation of alveolar macrophages. Alveolar epithelial cells (AEC) are a major source of GM-CSF in the lung and express this growth factor constitutively, whereas most other cells, including T cells, express GM-CSF following inflammatory stimulation. AEC expression of GM-CSF is suppressed by oxidative stress, at least in part through induction of microRNA leading to increased mRNA turnover. In this report, we compare and contrast the effect of hyperoxia on transcriptional aspects of gene regulation of GM-CSF in lung epithelia and T cells of human and mouse origin. Similar to primary murine AEC, human H820 cells that express multiple characteristics of normal alveolar epithelial cells express GM-CSF constitutively, with decreased expression and increased mRNA turnover following exposure to hyperoxia. In contrast, hyperoxia induces augmented GM-CSF expression in human and murine activated T cells, in association with enhanced GM-CSF mRNA stability. Alveolar epithelial cells demonstrate constitutive transcription, with the proximal promoter in an open configuration in normoxia, without change in hyperoxia. Conversely, in both human and murine T cells, hyperoxia increased GM-CSF gene transcription. The proximal promoter was in a closed configuration in unstimulated T cells but became accessible upon activation and still more accessible in activated T cells exposed to hyperoxia. These fundamental differences in molecular regulation of GM-CSF expression highlight the distinctive niche of alveolar epithelial cell expression of GM-CSF and offer insights into the biology of GM-CSF in the setting of acute lung injury.

Immunohistochemical application of a highly sensitive and specific murine monoclonal antibody recognising the extracellular domain of the human hepatocyte growth factor receptor (MET).

AIMS: Development of novel targeted therapies directed against hepatocyte growth factor (HGF) or its receptor (MET) necessitates the availability of quality diagnostics to facilitate their safe and effective use. Limitations of some commercially available anti-MET antibodies have prompted development of the highly sensitive and specific clone A2H2-3. Here we report its analytical properties when applied by an automated immunohistochemistry method. METHODS AND RESULTS: Excellent antibody specificity was demonstrated by immunoblot, ELISA, and IHC evaluation of characterised cell lines including NIH3T3 overexpressing the related kinase MST1R (RON). Sensitivity was confirmed by measurements of MET in cell lines or characterised tissues. IHC correlated well with FISH and quantitative RT-PCR assessments of MET (P < 0.001). Good total agreement (89%) was observed with the anti-MET antibody clone SP44 using whole-tissue sections, but poor positive agreement (21-47%) was seen in tissue microarray cores. Multiple lots displayed appropriate reproducibility (R(2)  > 0.9). Prevalence of MET positivity by IHC was higher in non-squamous cell NSCLC, MET or EGFR amplified cases, and in tumours harbouring abnormalities in EGFR exon 19 or 21. CONCLUSIONS: The anti-MET antibody clone A2H2-3 displays excellent specificity and sensitivity. These properties make it suitable for clinical trial investigations and development as a potential companion diagnostic.

Pharmacogenomics of gemcitabine metabolism: functional analysis of genetic variants in cytidine deaminase and deoxycytidine kinase.

Gemcitabine (dFdC, 2',2'-difluorodeoxycytidine) is metabolized by cytidine deaminase (CDA) and deoxycytidine kinase (DCK), but the contribution of genetic variation in these enzymes to the variability in systemic exposure and response observed in cancer patients is unclear. Wild-type enzymes and variants of CDA (Lys27Gln and Ala70Thr) and DCK (Ile24Val, Ala119Gly, and Pro122Ser) were expressed in and purified from Escherichia coli, and enzyme kinetic parameters were estimated for cytarabine (Ara-C), dFdC, and its metabolite 2',2'-difluorodeoxyuridine (dFdU) as substrates. All three CDA proteins showed similar K(m) and V(max) for Ara-C and dFdC deamination, except for CDA70Thr, which had a 2.5-fold lower K(m) and 6-fold lower V(max) for Ara-C deamination. All four DCK proteins yielded comparable metabolic activity for Ara-C and dFdC monophosphorylation, except for DCK24Val, which demonstrated an approximately 2-fold increase (P < 0.05) in the intrinsic clearance of dFdC monophosphorylation due to a 40% decrease in K(m) (P < 0.05). DCK did not significantly contribute to dFdU monophosphorylation. In conclusion, the Lys27Gln substitution does not significantly modulate CDA activity toward dFdC, and therefore would not contribute to interindividual variability in response to gemcitabine. The higher in vitro catalytic efficiency of DCK24Val toward dFdC monophosphorylation may be relevant to dFdC clinical response. The substrate-dependent alterations in activities of CDA70Thr and DCK24Val in vitro were observed for the first time, and demonstrate that the in vivo consequences of these genetic variations should not be extrapolated from one substrate of these enzymes to another.