Oral Administration of Lactobacillus Inhibits the Permeability of Blood-Brain and Gut Barriers in a Parkinsonism Model.

It has recently been shown that the administration of probiotics can modulate the microbiota-gut-brain axis and may have favorable effects in models of Parkinson's disease. In this study, we used a hemiparkinsonism model induced by the neurotoxin 6-OHDA to evaluate the efficacy of the administration of a four-week administration of a mixture containing the microorganisms Lactobacillus fermentum LH01, Lactobacillus reuteri LH03, and Lactobacillus plantarum LH05. The hemiparkinsonism model induced an increase in rotations in the apomorphine test, along with a decrease in the latency time to fall in the rotarod test on days 14 and 21 after surgery, respectively. The administration of probiotics was sufficient to improve this condition. The model also showed a decrease in tyrosine hydroxylase immunoreactivity in the striatum and the number of labeled cells in the substantia nigra, both of which were counteracted by the administration of probiotics. The permeability of the blood-brain barrier was increased in the model, but this effect was reversed by the probiotics for both brain regions. The gut barrier was permeated with the model, and this effect was reversed and dropped to lower levels than the control group after the administration of probiotics. Finally, lipid peroxidation showed a pattern of differences similar to that of permeabilities. The inhibition of the permeability of the blood-brain and gut barriers mediated by the administration of probiotics will likely provide protection by downregulating oxidative stress, thus affecting the rotarod test performance.

Functional brain region-specific neural spheroids for modeling neurological diseases and therapeutics screening.

3D spheroids have emerged as powerful drug discovery tools given their high-throughput screening (HTS) compatibility. Here, we describe a method for generating functional neural spheroids by cell-aggregation of differentiated human induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes at cell type compositions mimicking specific regions of the human brain. Recordings of intracellular calcium oscillations were used as functional assays, and the utility of this spheroids system was shown through disease modeling, drug testing, and formation of assembloids to model neurocircuitry. As a proof of concept, we generated spheroids incorporating neurons with Alzheimer's disease-associated alleles, as well as opioid use disorder modeling spheroids induced by chronic treatment of a mu-opioid receptor agonist. We reversed baseline functional deficits in each pilot disease model with clinically approved treatments and showed that assembloid activity can be chemogenetically manipulated. Here, we lay the groundwork for brain region-specific neural spheroids as a robust functional assay platform for HTS studies.

ß-Caryophyllene decreases neuroinflammation and exerts neuroprotection of dopaminergic neurons in a model of hemiparkinsonism through inhibition of the NLRP3 inflammasome.

INTRODUCTION: Parkinson's disease represents a neurodegenerative condition characterized by the progressive loss of dopaminergic neurons within the Substantia Nigra pars compacta (SNpc), resulting in diminished dopamine levels in the striatum (STR) and chronic neuroinflammation. Recent investigations have proposed the neuroprotective potential of the endocannabinoid system in neurodegenerative disorders. ß-caryophyllene (BCP) is recognized for its antioxidant and anti-inflammatory properties, attributed to its activation of the type 2 cannabinoid receptor. This study aimed to assess the neuroprotective impact of BCP on dopaminergic neurons, with a particular focus on inhibiting the NLRP3 inflammasome. METHODS: A model of hemiparkinsonism, induced by 6-hydroxydopamine (6-OHDA), served as the experimental framework. Motor function was evaluated using the cylinder test, and inflammasome inhibition was determined by assessing the expression of NLRP3, caspase-1, and the pro-inflammatory cytokine IL-1ß in both the SNpc and STR through ELISA analysis. Furthermore, the evaluation of oxidative stress was facilitated by quantifying malondialdehyde (MDA) levels in the same regions. RESULTS: BCP treatment demonstrated significant improvements in motor dysfunction, as assessed by the cylinder test (p=0.0011) and exhibited a neuroprotective effect on dopaminergic neurons within the SNpc (p=0.0017), as well as nerve fibers in the STR (p=0.0399). In terms of its ability to inhibit the inflammasome, BCP led to decreased expression levels of NLRP3 (p=0.0401 in STR and p = 0.0139 in SNpc), caspase-1 (p=0.0004 in STR), and MDA (p=0.0085 in STR and p=0.0414 in SNpc). CONCLUSION: These results point to BCP's potential in mitigating the motor deficit, inhibiting NLRP3 inflammasome activation, and attenuating lipid peroxidation induced by 6-OHDA.

Studying the Inflammatory Responses to Amyloid Beta Oligomers in Brain-Specific Pericyte and Endothelial Co-culture from Human Stem Cells.

Background: Recently, the in vitro blood brain barrier (BBB) models derived from human pluripotent stem cells have been given extensive attention in therapeutics due to the implications it has with the health of the central nervous system. It is essential to create an accurate BBB model in vitro in order to better understand the properties of the BBB and how it can respond to inflammatory stimulation and be passed by targeted or non-targeted cell therapeutics, more specifically extracellular vesicles. Methods: Brain-specific pericytes (iPCs) were differentiated from iPSK3 cells using dual SMAD signaling inhibitors and Wnt activation plus fibroblast growth factor 2 (FGF-2). The derived cells were characterized by immunostaining, flow cytometry and RT-PCR. In parallel, blood vessels organoids were derived using Wnt activation, BMP4, FGF2, VEGF and SB431542. The organoids were replated and treated with retinoic acid to enhance the blood brain barrier (BBB) features in the differentiated brain endothelial cells (iECs). Co-culture was performed for the iPCs and iECs in transwell system and 3-D microfluidics channels. Results: The derived iPCs expressed common markers PDGFRb and NG2, as well as brain-specific genes FOXF2, ABCC9, KCNJ8, and ZIC1. The derived iECs expressed common endothelial cell markers CD31, VE-cadherin, as well as BBB-associated genes BRCP, GLUT-1, PGP, ABCC1, OCLN, SLC2A1. The co-culture of the two cell types responded to the stimulation of amyloid ß42 oligomers by the upregulation of expression of TNFa, IL6, NFKB, Casp3, SOD2 and TP53. The co-culture also showed the property of trans-endothelial electrical resistance. The proof-of-concept vascularization strategy was demonstrated in a 3-D microfluidics-based device. Conclusion: The derived iPCs and iECs have brain-specific properties and the co-culture of iPCs and iECs provides an in vitro BBB model that show inflammatory response. This study has significance in establishing micro-physiological systems for neurological disease modeling and drug screening.

Impacto de la COVID-19 en el control de la Tuberculosis en Paraguay. 2019-2020

La COVID-19 ha provocado una emergencia sanitaria mundial sin precedentes, afectando también negativamente a la prestación de atención a la tuberculosis (TB) en casi todos los países. Por tanto, esta investigación analiza el impacto de la COVID-19 en el control de la TB en Paraguay. Estudio descriptivo, retrospectivo, con enfoque mixto y de corte transversal. Resultados: siete de los 10 indicadores priorizados de la Estrategia "Fin de la TB" registraron resultados por debajo a lo notificado en los últimos tres años, siendo el 2020 el más afectado. Existe una brecha de 1.037 personas que no han sido diagnosticadas y tratadas por TB en Paraguay, de acuerdo a lo estimado por la Organización Mundial de la Salud (OMS) en 2020. Se analizaron 2.598 casos de TB en 2020, frente a 3.083 en el 2019, lo que representa una disminución del 15,7% en la notificación de casos; las notificaciones disminuyeron en un 24% en mujeres, 40,5% en niños y en un 19,6% de éxito de tratamiento. Se presentó una tasa de letalidad de 12%, la mayor en los últimos cuatro años. Las expertas encuestadas, opinan que la situación de la TB empeoró tras la llegada de la COVID-19 en el país, principalmente en la notificación de casos e investigación de contactos. La COVID-19 ha impactado negativamente en el control de la TB en Paraguay, esto representa un riesgo significativo en el control de la enfermedad y el avance a lo largo de los años se ve seriamente comprometido y amenaza con revertir los logros alcanzados.

COVID-19 has caused an unprecedented global health emergency, also negatively affecting the delivery of tuberculosis (TB) care in almost all countries. This research therefore analyses the impact of COVID-19 on TB control in Paraguay. Descriptive, retrospective, mixed-approach, cross-sectional study. Results: seven of the 10 prioritized indicators of the "End TB Strategy" registered results below what was reported in the last three years, with 2020 being the most affected. There is a gap of 1,037 people who have not been diagnosed and treated for TB in Paraguay, as estimated by the World Health Organisation (WHO) in 2020. There were 2,598 TB cases analysed in 2020, compared to 3,083 in 2019, representing a 15.7% decrease in case notification; notifications decreased by 24% in women, 40.5% in children and 19.6% treatment success. There was a case fatality rate of 12%, the highest in the last four years. The experts surveyed were of the opinion that the TB situation worsened after the advent of COVID-19 in the country, mainly in case notification and contact investigation. COVID-19 has had a negative impact on TB control in Paraguay, which represents a significant risk for the control of the disease, and progress over the years is seriously compromised and threatens to reverse the achievements made.

Intrinsic antiviral immunity of barrier cells revealed by an iPSC-derived blood-brain barrier cellular model.

Physiological blood-tissue barriers play a critical role in separating the circulation from immune-privileged sites and denying access to blood-borne viruses. The mechanism of virus restriction by these barriers is poorly understood. We utilize induced pluripotent stem cell (iPSC)-derived human brain microvascular endothelial cells (iBMECs) to study virus-blood-brain barrier (BBB) interactions. These iPSC-derived cells faithfully recapitulate a striking difference in in vivo neuroinvasion by two alphavirus isolates and are selectively permissive to neurotropic flaviviruses. A model of cocultured iBMECs and astrocytes exhibits high transendothelial electrical resistance and blocks non-neurotropic flaviviruses from getting across the barrier. We find that iBMECs constitutively express an interferon-induced gene, IFITM1, which preferentially restricts the replication of non-neurotropic flaviviruses. Barrier cells from blood-testis and blood-retinal barriers also constitutively express IFITMs that contribute to the viral resistance. Our application of a renewable human iPSC-based model for studying virus-BBB interactions reveals that intrinsic immunity at the barriers contributes to virus exclusion.

Estado actual, retos y soluciones operacionales a las Búsquedas activas de TB 2019-2020, Paraguay

RESUMEN Introducción: La búsqueda activa de casos de Tuberculosis (TB) pretende garantizar la detección temprana para reducir el riesgo de malos resultados de la enfermedad y las consecuencias sociales propias de la misma, así como cortar oportunamente la cadena de transmisión en la comunidad. Objetivo: Determinar el estado actual, retos y soluciones operacionales a las Búsquedas activas en las 18 Regiones Sanitarias del Paraguay. Métodos: Estudio descriptivo retrospectivo de corte transversal, fuente de datos: registros de búsqueda activa disponibles de las regiones sanitarias, sociedad civil y Programa Nacional de Control de Tuberculosis (PNCT) del Paraguay 2019-2020 y Sistema Experto del PNCT (SEPNCT) Para el análisis estadístico se utilizaron medidas de resumen como frecuencias absolutas, relativas y proporciones. Resultados: se analizaron las visitas de campo para búsquedas activas, en tres tipos de intervenciones, 5089 personas investigadas, 26.5% fueron identificadas como SR. La positividad general fue de 5%. Se necesita en promedio de 76 personas para encontrar un caso de TB. Las búsquedas activas que tuvieron mayor impacto fueron las realizadas por el nivel nacional con 49.3% de casos de TB aportados. Conclusiones: Las intervenciones basadas en la comunidad, como el establecimiento de redes de derivación y otras actividades que acercan la información y los servicios sobre la tuberculosis a las personas con síntomas, pueden contribuir a mejorar la notificación de casos de tuberculosis, cortar la cadena de transmisión y reducir la mortalidad por tuberculosis.

SUMMARY Introduction: The active search for cases of Tuberculosis (TB) aims to ensure early detection to reduce the risk of poor outcomes of the disease and the social consequences of the disease, as well as to cut the chain of transmission in the community in a timely manner. Objective . To determine the current status, challenges and operational solutions to the Active Searches in the 18 Health Regions of Paraguay. Methods: Retrospective descriptive cross-sectional study, data source: active search records available from the health regions, civil society and National Tuberculosis Control Program (PNCT) of Paraguay 2019-2020 and Expert System of the PNCT (SEPNCT) For the statistical analysis summary measures such as absolute and relative frequencies and proportions were used. Results: field visits were analyzed for active searches, in three types of interventions, 5089 people investigated, 26.5% were identified as RH. The overall positivity was 5%. It takes an average of 76 persons to find a TB case. The active searches that had the greatest impact were those conducted by the national level with 49.3% of TB cases contributed. Conclusions: Community-based interventions, such as the establishment of referral networks and other activities that bring TB information and services closer to people with symptoms, can help improve TB case notification, cut the chain of transmission, and reduce TB mortality.

iPS Cell Differentiation into Brain Microvascular Endothelial Cells.

The blood-brain barrier is a tissue structure that modulates the selective entry of molecules into the brain compartment. This barrier offers protection to the brain microenvironment from toxins or any fluctuations in the composition of the blood plasma via a layer of endothelial cells connected by tight junctions and supported by pericytes and astrocytes. Disruption of the barrier can be either a cause or a consequence of central nervous system pathogenesis. Therefore, research based on understanding the structure, function, and the mechanisms of breaching the blood-brain barrier is of primary interest for diverse disciplines including drug discovery, brain pathology, and infectious disease. The following protocol describes a detailed differentiation method that uses defined serum components during stem cell culture to deliver cellular cues in order to drive the cells towards brain endothelial cell lineage. This method can be used to obtain reproducible and scalable cultures of brain microvascular endothelial cells with barrier characteristics and functionality. These endothelial cells can also be stored long term or shipped frozen.

Inhibition of zika virus infection by fused tricyclic derivatives of 1,2,4,5-tetrahydroimidazo[1,5-a]quinolin-3(3aH)-one.

Zika virus (ZIKV) infection represents a significant threat to the global health system, and the search for efficient antivirals to ZIKV remains necessary and urgent. In this study, we extended the exploration of our previously discovered scaffold of 1H-pyrrolo[1,2-c]imidazol-1-one and revealed that two trans isomers of compounds 2 and 7 and one mixture with major trans isomer of compound 3 as novel tetrahydroquinoline-fused imidazolone derivatives are active against ZIKV infection but they are not virucidal. Western Blot and ELISA analyses of ZIKV NS5 and NS1 further demonstrate that compounds of (±)-2, (±)-3 and (±)-7 act as effective agents against ZIKV infection. We show that the N10's basicity is not the basic requirement for these compounds' antiviral activity in the current work. Importantly, tuning of some pharmacophores including substituents at arene can generate promising candidates for anti-ZIKV agents.

Renewing Traditions: A Sensory and Chemical Characterisation of Mexican Pigmented Corn Beers.

This study was undertaken to explore how the use of pigmented corn as brewing ingredient influences the sensory profile of craft beers, by using both sensory and chemical analyses. Six pigmented corn and barley beers were brewed and then analysed to obtain their sensory characteristics, volatile composition and non-volatile (alcohol, bitterness, anthocyanins and polyphenol content) composition. ANOVAs, Principal Component Analysis (PCA) and Multiple Factor Analysis (MFA) were used to visualise these data for exploring the differences between beers based on the type of malt and to characterise corn beers considering the relationships between their sensory characteristics and their chemical parameters. The sensory attributes such as fermented fruits, cooked vegetables, tortillas, bread, dried fruits and dried chili characterised beers made 100% with pigmented corn. Over 100 volatiles were identified by head space-solid phase micro-extraction coupled with gas chromatography-mass spectrometry (HS-SPME/GC-MS). Among them, phenols and terpenes were the groups of volatiles that better characterised beers containing corn. The content of anthocyanins in corn beers provide the 'amber-red-cooper' colours in beers and may prevent the development of off-aromas and tastes. The use of pigmented corn seems to be a good option to renew the traditional 'Sendechó' while preserving some of its sensory attributes.

Zika Virus Infection Induces DNA Damage Response in Human Neural Progenitors That Enhances Viral Replication.

Zika virus (ZIKV) infection attenuates the growth of human neural progenitor cells (hNPCs). As these hNPCs generate the cortical neurons during early brain development, the ZIKV-mediated growth retardation potentially contributes to the neurodevelopmental defects of the congenital Zika syndrome. Here, we investigate the mechanism by which ZIKV manipulates the cell cycle in hNPCs and the functional consequence of cell cycle perturbation on the replication of ZIKV and related flaviviruses. We demonstrate that ZIKV, but not dengue virus (DENV), induces DNA double-strand breaks (DSBs), triggering the DNA damage response through the ATM/Chk2 signaling pathway while suppressing the ATR/Chk1 signaling pathway. Furthermore, ZIKV infection impedes the progression of cells through S phase, thereby preventing the completion of host DNA replication. Recapitulation of the S-phase arrest state with inhibitors led to an increase in ZIKV replication, but not of West Nile virus or DENV. Our data identify ZIKV's ability to induce DSBs and suppress host DNA replication, which results in a cellular environment favorable for its replication.IMPORTANCE Clinically, Zika virus (ZIKV) infection can lead to developmental defects in the cortex of the fetal brain. How ZIKV triggers this event in developing neural cells is not well understood at a molecular level and likely requires many contributing factors. ZIKV efficiently infects human neural progenitor cells (hNPCs) and leads to growth arrest of these cells, which are critical for brain development. Here, we demonstrate that infection with ZIKV, but not dengue virus, disrupts the cell cycle of hNPCs by halting DNA replication during S phase and inducing DNA damage. We further show that ZIKV infection activates the ATM/Chk2 checkpoint but prevents the activation of another checkpoint, the ATR/Chk1 pathway. These results unravel an intriguing mechanism by which an RNA virus interrupts host DNA replication. Finally, by mimicking virus-induced S-phase arrest, we show that ZIKV manipulates the cell cycle to benefit viral replication.

[Factors associated with tuberculosis mortality in Paraguay, 2015-2016Fatores associados à mortalidade por tuberculose no Paraguai, 2015-2016]. / Factores asociados a la mortalidad por tuberculosis en Paraguay, 2015-2016.

OBJECTIVE: To describe the socio-demographic and clinical-epidemiological characteristics and to determine the factors associated with the mortality of people diagnosed with tuberculosis (TB) in Paraguay. METHODS: Operational research with a retrospective cohort design of cases diagnosed with TB in Paraguay between 2015-2016. The database of the National Tuberculosis Control Program was used. Chi-square and relative risk (RR) tests with a 95% confidence interval (95% CI) were used to determine the factors associated with mortality; in addition, a robust Poisson multiple regression model was adjusted. A significance level of 5% was used. RESULTS: Five hundred and forty-one cases of TB were studied, of which 11.5% died. The factors increasing the risk of death were male sex (RR 1.26; 95% CI 1.1-1.50), infection with human immunodeficiency virus (RR 4.78; 95% CI 4.04-5.65) and chronic obstructive pulmonary disease (RR 1.70; 95% CI 1.19-2.42). Being deprived of one's liberty was a protective factor (RR 0.37; 95% CI 0.24-0.61). CONCLUSIONS: The highest risk of death is presented by men and people with TB/HIV coinfection and the lowest risk is presented by people deprived of liberty. There is a need to improve diagnosis and follow-up of TB cases, with effective implementation of directly observed treatment (DOTS) and timely management of associated diseases such as HIV and chronic obstructive pulmonary disease.

OBJETIVO: Descrever as características sociodemográficas e clínico-epidemiológicas e determinar os fatores associados à mortalidade de pessoas diagnosticadas com tuberculose (TB) no Paraguai. MÉTODOS: Pesquisa operacional com desenho de coorte retrospectivo de casos diagnosticados com TB no Paraguai entre 2015-2016. Foi utilizada a base de dados do Programa Nacional de Controle da Tuberculose. Testes de qui-quadrado e risco relativo (RR) com intervalo de confiança de 95% (IC95%) foram utilizados para determinar os fatores associados à mortalidade; além disso, um robusto modelo de regressão múltipla de Poisson foi ajustado. Foi utilizado nível de significância de 5%. RESULTADOS: Foram estudados quinhentos e quarenta e um casos de TB, dos quais 11,5% morreram. Os fatores que aumentaram o risco de morte foram sexo masculino (RR 1,26; IC95% 1,1-1,50), infecção por vírus da imunodeficiência humana (RR 4,78; IC95% 4,04-5,65) e doença pulmonar obstrutiva crônica (RR 1,70; 95 % CI 1,19-2,42). Ser privado de liberdade foi um fator protetor (RR 0,37; IC 95% 0,24-0,61). CONCLUSÕES: O maior risco de morte é apresentado por homens e pessoas com co-infecção TB / HIV e o menor risco é apresentado por pessoas privadas de liberdade. É necessário melhorar o diagnóstico e o acompanhamento dos casos de TB, com a implementação efetiva do tratamento diretamente observado (DOTS) e o gerenciamento oportuno de doenças associadas, como o HIV e a doença pulmonar obstrutiva crônica.

Association between perceived social support and induced abortion: A study in maternal health centers in Lima, Peru.

OBJECTIVES: This study aimed to assess the association between perceived social support and induced abortion among young women in Lima, Peru. In addition, prevalence and incidence of induced abortion was estimated. METHODS/PRINCIPAL FINDINGS: A cross-sectional study enrolling women aged 18-25 years from maternal health centers in Southern Lima, Peru, was conducted. Induced abortion was defined as the difference between the total number of pregnancies ended in abortion and the number of spontaneous abortions; whereas perceived social support was assessed using the DUKE-UNC scale. Prevalence and incidence of induced abortion (per 100 person-years risk) was estimated, and the association of interest was evaluated using Poisson regression models with robust variance. A total of 298 women were enrolled, mean age 21.7 (± 2.2) years. Low levels of social support were found in 43.6% (95%CI 38.0%-49.3%), and 17.4% (95%CI: 13.1%- 21.8%) women reported at least one induced abortion. The incidence of induced abortion was 2.37 (95%CI: 1.81-3.11) per 100 person-years risk. The multivariable model showed evidence of the association between low perceived social support and induced abortion (RR = 1.94; 95%CI: 1.14-3.30) after controlling for confounders. CONCLUSIONS: There was evidence of an association between low perceived social support and induced abortion among women aged 18 to 25 years. Incidence of induced abortion was similar or even greater than rates of countries where abortion is legal. Strategies to increase social support and reduce induced abortion rates are needed.

Adenoviruses in fecal samples from asymptomatic rhesus macaques, United States.

Adenoviruses can cause infectious diarrheal disease or respiratory infections in humans; 2 recent reports have indicated probable human infection with simian adenoviruses (SAdVs). To assess the possibility of animal-to-human transmission of SAdVs, we tested fecal samples from asymptomatic rhesus macaques housed in 5 primate facilities in the United States and cultured 23 SAdV isolates. Of these, 9 were purified and completely sequenced; 3 SAdV samples from the American Type Culture Collection (SAdV-6, SAdV-18, and SAdV-20) were also completely sequenced. The sequence of SAdV-18 was closely related to that of human adenovirus F across the whole genome, and the new isolates were found to harbor 2 fiber genes similar to those of human adenovirus (HAdV) strains HAdV-40 and HAdV-41, which can cause infectious diarrhea. The high prevalence of adenoviruses in fecal samples from asymptomatic rhesus macaques and the similarity of the isolates to human strains indicates the possibility of animal-to-human transmission of SAdVs.

Construction of gene transfer vectors based on simian adenovirus 7.

The complete nucleotide sequence of an isolate of simian adenovirus 7 (SAdV-7) was determined. The genome organization of this isolate was found to be similar to that of other primate adenoviruses with two principal notable points: severe truncation of the E1A and E1B 19K proteins and an E3 region encoding only the 12.5K homologue. The viral gene products of SAdV-7 are most closely related to simian adenovirus 1 (SAdV-1), and like SAdV-1, are related to the human adenovirus species HAdV-F, such as the enteric adenoviruses HAdV-40 and HAdV-41 and the recently defined HAdV-G (HAdV-52). Two kinds of gene transfer vectors were made: a replication-competent SAdV-7-based vector with no genomic deletion, and a standard replication-incompetent vector deleted for E1. Importantly, the E1-deleted vector could be propagated to high titre by trans-complementation in human HEK 293 cells.

Application of a BRAF pyrosequencing assay for mutation detection and copy number analysis in malignant melanoma.

Mutations in the BRAF gene are found in the majority of cutaneous malignant melanomas and subsets of other tumors. These mutations lead to constitutive activation of BRAF with increased downstream ERK (extracellular signal-regulated kinase) signaling; therefore, the development of RAF kinase inhibitors for targeted therapy is being actively pursued. A methodology that allows sensitive, cost-effective, high-throughput analysis of BRAF mutations will be needed to triage patients for specific molecular-based therapies. Pyrosequencing is a high-throughput, sequencing-by-synthesis method that is particularly useful for analysis of single nucleotide polymorphisms or hotspot mutations. Mutational analysis of BRAF is highly amenable to pyrosequencing because the majority of mutations in this gene localize to codons 600 and 601 and consist of single or dinucleotide substitutions. In this study, DNAs from a panel of melanocyte cell lines, melanoma cell lines, and melanoma tumors were used to validate a pyrosequencing assay to detect BRAF mutations. The assay demonstrates high accuracy and precision for detecting common and variant exon 15 BRAF mutations. Further, comparison of pyrosequencing data with 100K single nucleotide polymorphism microarray data allows characterization of BRAF amplification events that may accompany BRAF mutation. Pyro-sequencing serves as an excellent platform for BRAF genotyping of tumors from patients entering clinical trial.

microRNAs exhibit high frequency genomic alterations in human cancer.

MicroRNAs (miRNAs) are endogenous noncoding RNAs, which negatively regulate gene expression. To determine genomewide miRNA DNA copy number abnormalities in cancer, 283 known human miRNA genes were analyzed by high-resolution array-based comparative genomic hybridization in 227 human ovarian cancer, breast cancer, and melanoma specimens. A high proportion of genomic loci containing miRNA genes exhibited DNA copy number alterations in ovarian cancer (37.1%), breast cancer (72.8%), and melanoma (85.9%), where copy number alterations observed in >15% tumors were considered significant for each miRNA gene. We identified 41 miRNA genes with gene copy number changes that were shared among the three cancer types (26 with gains and 15 with losses) as well as miRNA genes with copy number changes that were unique to each tumor type. Importantly, we show that miRNA copy changes correlate with miRNA expression. Finally, we identified high frequency copy number abnormalities of Dicer1, Argonaute2, and other miRNA-associated genes in breast and ovarian cancer as well as melanoma. These findings support the notion that copy number alterations of miRNAs and their regulatory genes are highly prevalent in cancer and may account partly for the frequent miRNA gene deregulation reported in several tumor types.

Transcriptional coactivator Drosophila eyes absent homologue 2 is up-regulated in epithelial ovarian cancer and promotes tumor growth.

Epithelial ovarian cancer is the most frequent cause of gynecologic malignancy-related mortality in women. To identify genes up-regulated in ovarian cancer, PCR-select cDNA subtraction was done and Drosophila Eyes Absent Homologue 2 (EYA2) was isolated as a promising candidate. The transcriptional coactivator eya controls essential cellular functions during organogenesis of Drosophila. EYA2 mRNA was found to be up-regulated in ovarian cancer by real-time reverse transcription-PCR, whereas its protein product was detected in 93.6% of ovarian cancer specimens by immunohistochemistry (n = 140). EYA2 was amplified in 14.8% of ovarian carcinomas, as detected by array-based comparative genomic hybridization (n = 88). Most importantly, EYA2 overexpression was significantly associated with short overall survival in advanced ovarian cancer (n = 99, P = 0.0361). EYA2 was found to function as transcriptional activator in ovarian cancer cells by Gal4 assay and to promote tumor growth in vivo in xenograft models. Therefore, this study suggests an important role of EYA2 in ovarian cancer and its potential application as a therapeutic target.