Functional analysis of recurrent CDC20 promoter variants in human melanoma.

Small nucleotide variants in non-coding regions of the genome can alter transcriptional regulation, leading to changes in gene expression which can activate oncogenic gene regulatory networks. Melanoma is heavily burdened by non-coding variants, representing over 99% of total genetic variation, including the well-characterized TERT promoter mutation. However, the compendium of regulatory non-coding variants is likely still functionally under-characterized. We developed a pipeline to identify hotspots, i.e. recurrently mutated regions, in melanoma containing putatively functional non-coding somatic variants that are located within predicted melanoma-specific regulatory regions. We identified hundreds of statistically significant hotspots, including the hotspot containing the TERT promoter variants, and focused on a hotspot in the promoter of CDC20. We found that variants in the promoter of CDC20, which putatively disrupt an ETS motif, lead to lower transcriptional activity in reporter assays. Using CRISPR/Cas9, we generated an indel in the CDC20 promoter in human A375 melanoma cell lines and observed decreased expression of CDC20, changes in migration capabilities, increased growth of xenografts, and an altered transcriptional state previously associated with a more proliferative and less migratory state. Overall, our analysis prioritized several recurrent functional non-coding variants that, through downregulation of CDC20, led to perturbation of key melanoma phenotypes.

Bidialectal and monodialectal differences in morphosyntactic processing of AAE and MAE: Evidence from ERPs and acceptability judgments.

INTRODUCTION: African American English (AAE) has never been examined through neurophysiological methods in investigations of dual-language variety processing. This study examines whether contrastive and non-contrastive morphosyntactic features in sentences with and without AAE constructions elicit differing neural and/or behavioral responses in bidialectal speakers of AAE and Mainstream American English (MAE), compared to monodialectal MAE speakers. We compared electroencephalographic (EEG) and behavioral (grammatical acceptability judgment) data to determine whether two dialects are processed similarly to distinct languages, as seen in studies of bilingual codeswitching where the P600 event related potential (ERP) has been elicited when processing a switch between language varieties. METHODS: Bidialectal AAE-MAE speakers (n = 15) and monodialectal MAE speakers (n = 12) listened to sentences in four conditions, while EEG was recorded to evaluate time-locked brain responses to grammatical differences between sentence types. The maintained verb form in the present progressive tense sentences (e.g., The black cat lap/s the milk) was the morphosyntactic feature of interest for comparing P600 responses as an indicator of error detection. Following each trial, responses and reaction times to a grammatical acceptability judgment task were collected and compared. RESULTS: Findings indicate distinct neurophysiological profiles between bidialectal and monodialectal speakers. Monodialectal speakers demonstrated a P600 response within 500-800ms following presentation of an AAE morphosyntax feature, indicating error detection; this response was not seen in the bidialectal group. Control sentences with non-contrasting grammar revealed no differences in ERP responses between groups. Behaviorally, bidialectal speakers showed greater acceptance of known dialectal variation and error (non-contrastive) sentence types compared to the monodialectal group. CONCLUSIONS: ERP and behavioral responses are presented as preliminary evidence of dual-language representation in bidialectal speakers. Increased consideration of AAE language processing would enhance equity in the study of language at large, improving the work of clinicians, researchers, educators and policymakers alike.

Acute responses of stevia and d-tagatose intake on metabolic parameters and appetite/satiety in insulin resistance.

OBJECTIVE: To examine the effects of d-tagatose or stevia preloads on carbohydrate metabolism markers after an oral glucose load, as well as subjective and objective appetite in women with insulin resistance (IR). RESEARCH DESIGN AND METHODS: Randomized controlled crossover study. Women with IR without T2DM (n = 33; aged 23.4 ± 3.8; BMI 28.1 ± 3.4 kg × m-2) underwent three oral glucose loads (3 h each) on three different days. Ten min before oral glucose load, volunteers consumed a preload of 60 mL water (control), 60 mL water with stevia (15.3 mg), or d-tagatose (5000 mg). Serum glucose and C-peptide were evaluated at -10, 30-, 60-, 90-, 120-, and 180-min. Subjective appetite was determined with a visual analog scale. Food intake was measured at ad libitum buffet after 180 min. RESULTS: C-peptide iAUC was significantly higher for stevia (median (IQR): 1033 (711-1293) ng × min × L-1) vs. d-tagatose (794 (366-1134) ng × min × L-1; P = 0.001) or control (730 (516-1078) ng × min × L-1; P = 0.012). At 30- and 60-min serum glucose was higher for stevia vs other conditions (P < 0.01). Volunteers reported greater satiety for stevia and d-tagatose vs. control at 60 min and greater desire to eat for stevia vs. control at 120- min (all P < 0.05). Objective appetite did not vary by condition (P = 0.06). CONCLUSIONS: Our findings suggest that these NNS are not inert. Stevia intake produced an acute response on C-peptide release while increased serum glucose at earlier times. It is possible that NNS affects subjective but not objective appetite. This trial is registered at clinicaltrials.gov as NCT04327245. CLINICAL TRIAL REGISTRY: NCT04327245.

Transcriptional profile and chromatin accessibility in zebrafish melanocytes and melanoma tumors.

Transcriptional and epigenetic characterization of melanocytes and melanoma cells isolated from their in vivo context promises to unveil key differences between these developmentally related normal and cancer cell populations. We therefore engineered an enhanced Danio rerio (zebrafish) melanoma model with fluorescently labeled melanocytes to allow for isolation of normal (wild type) and premalignant (BRAFV600E-mutant) populations for comparison to fully transformed BRAFV600E-mutant, p53 loss-of-function melanoma cells. Using fluorescence-activated cell sorting to isolate these populations, we performed high-quality RNA- and ATAC-seq on sorted zebrafish melanocytes vs. melanoma cells, which we provide as a resource here. Melanocytes had consistent transcriptional and accessibility profiles, as did melanoma cells. Comparing melanocytes and melanoma, we note 4128 differentially expressed genes and 56,936 differentially accessible regions with overall gene expression profiles analogous to human melanocytes and the pigmentation melanoma subtype. Combining the RNA- and ATAC-seq data surprisingly revealed that increased chromatin accessibility did not always correspond with increased gene expression, suggesting that though there is widespread dysregulation in chromatin accessibility in melanoma, there is a potentially more refined gene expression program driving cancerous melanoma. These data serve as a resource to identify candidate regulators of the normal vs. diseased states in a genetically controlled in vivo context.

Functional in vivo characterization of sox10 enhancers in neural crest and melanoma development.

The role of a neural crest developmental transcriptional program, which critically involves Sox10 upregulation, is a key conserved aspect of melanoma initiation in both humans and zebrafish, yet transcriptional regulation of sox10 expression is incompletely understood. Here we used ATAC-Seq analysis of multiple zebrafish melanoma tumors to identify recurrently open chromatin domains as putative melanoma-specific sox10 enhancers. Screening in vivo with EGFP reporter constructs revealed 9 of 11 putative sox10 enhancers with embryonic activity in zebrafish. Focusing on the most active enhancer region in melanoma, we identified a region 23 kilobases upstream of sox10, termed peak5, that drives EGFP reporter expression in a subset of neural crest cells, Kolmer-Agduhr neurons, and early melanoma patches and tumors with high specificity. A ~200 base pair region, conserved in Cyprinidae, within peak5 is required for transgenic reporter activity in neural crest and melanoma. This region contains dimeric SoxE/Sox10 dimeric binding sites essential for peak5 neural crest and melanoma activity. We show that deletion of the endogenous peak5 conserved genomic locus decreases embryonic sox10 expression and disrupts adult stripe patterning in our melanoma model background. Our work demonstrates the power of linking developmental and cancer models to better understand neural crest identity in melanoma.

Comparison of Reproducibility, Accuracy, Sensitivity, and Specificity of miRNA Quantification Platforms.

Given the increasing interest in their use as disease biomarkers, the establishment of reproducible, accurate, sensitive, and specific platforms for microRNA (miRNA) quantification in biofluids is of high priority. We compare four platforms for these characteristics: small RNA sequencing (RNA-seq), FirePlex, EdgeSeq, and nCounter. For a pool of synthetic miRNAs, coefficients of variation for technical replicates are lower for EdgeSeq (6.9%) and RNA-seq (8.2%) than for FirePlex (22.4%); nCounter replicates are not performed. Receiver operating characteristic analysis for distinguishing present versus absent miRNAs shows small RNA-seq (area under curve 0.99) is superior to EdgeSeq (0.97), nCounter (0.94), and FirePlex (0.81). Expected differences in expression of placenta-associated miRNAs in plasma from pregnant and non-pregnant women are observed with RNA-seq and EdgeSeq, but not FirePlex or nCounter. These results indicate that differences in performance among miRNA profiling platforms impact ability to detect biological differences among samples and thus their relative utility for research and clinical use.

Author Correction: Transposable elements drive widespread expression of oncogenes in human cancers.

In the version of this article initially published, grant PF-17-201-01-TBG from the American Cancer Society to author Erica C. Pehrsson was not included in the Acknowledgements. The error has been corrected in the HTML and PDF versions of the article.

Transposable elements drive widespread expression of oncogenes in human cancers.

Transposable elements (TEs) are an abundant and rich genetic resource of regulatory sequences1-3. Cryptic regulatory elements within TEs can be epigenetically reactivated in cancer to influence oncogenesis in a process termed onco-exaptation4. However, the prevalence and impact of TE onco-exaptation events across cancer types are poorly characterized. Here, we analyzed 7,769 tumors and 625 normal datasets from 15 cancer types, identifying 129 TE cryptic promoter-activation events involving 106 oncogenes across 3,864 tumors. Furthermore, we interrogated the AluJb-LIN28B candidate: the genetic deletion of the TE eliminated oncogene expression, while dynamic DNA methylation modulated promoter activity, illustrating the necessity and sufficiency of a TE for oncogene activation. Collectively, our results characterize the global profile of TE onco-exaptation and highlight this prevalent phenomenon as an important mechanism for promiscuous oncogene activation and ultimately tumorigenesis.

Large Differences in Small RNA Composition Between Human Biofluids.

Extracellular microRNAs (miRNAs) and other small RNAs are implicated in cellular communication and may be useful as disease biomarkers. We systematically compared small RNAs in 12 human biofluid types using RNA sequencing (RNA-seq). miRNAs and tRNA-derived RNAs (tDRs) accounted for the majority of mapped reads in all biofluids, but the ratio of miRNA to tDR reads varied from 72 in plasma to 0.004 in bile. miRNA levels were highly correlated across all biofluids, but levels of some miRNAs differed markedly between biofluids. tDR populations differed extensively between biofluids. Y RNA fragments were seen in all biofluids and accounted for >10% of reads in blood plasma, serum, and cerebrospinal fluid (CSF). Reads mapping exclusively to Piwi-interacting RNAs (piRNAs) were very rare, except in seminal plasma. These results demonstrate extensive differences in small RNAs between human biofluids and provide a useful resource for investigating extracellular RNA biology and developing biomarkers.

Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling.

RNA-seq is increasingly used for quantitative profiling of small RNAs (for example, microRNAs, piRNAs and snoRNAs) in diverse sample types, including isolated cells, tissues and cell-free biofluids. The accuracy and reproducibility of the currently used small RNA-seq library preparation methods have not been systematically tested. Here we report results obtained by a consortium of nine labs that independently sequenced reference, 'ground truth' samples of synthetic small RNAs and human plasma-derived RNA. We assessed three commercially available library preparation methods that use adapters of defined sequence and six methods using adapters with degenerate bases. Both protocol- and sequence-specific biases were identified, including biases that reduced the ability of small RNA-seq to accurately measure adenosine-to-inosine editing in microRNAs. We found that these biases were mitigated by library preparation methods that incorporate adapters with degenerate bases. MicroRNA relative quantification between samples using small RNA-seq was accurate and reproducible across laboratories and methods.

A New Improved and Extended Version of the Multicell Bacterial Simulator gro.

gro is a cell programming language developed in Klavins Lab for simulating colony growth and cell-cell communication. It is used as a synthetic biology prototyping tool for simulating multicellular biocircuits and microbial consortia. In this work, we present several extensions made to gro that improve the performance of the simulator, make it easier to use, and provide new functionalities. The new version of gro is between 1 and 2 orders of magnitude faster than the original version. It is able to grow microbial colonies with up to 105 cells in less than 10 min. A new library, CellEngine, accelerates the resolution of spatial physical interactions between growing and dividing cells by implementing a new shoving algorithm. A genetic library, CellPro, based on Probabilistic Timed Automata, simulates gene expression dynamics using simplified and easy to compute digital proteins. We also propose a more convenient language specification layer, ProSpec, based on the idea that proteins drive cell behavior. CellNutrient, another library, implements Monod-based growth and nutrient uptake functionalities. The intercellular signaling management was improved and extended in a library called CellSignals. Finally, bacterial conjugation, another local cell-cell communication process, was added to the simulator. To show the versatility and potential outreach of this version of gro, we provide studies and novel examples ranging from synthetic biology to evolutionary microbiology. We believe that the upgrades implemented for gro have made it into a powerful and fast prototyping tool capable of simulating a large variety of systems and synthetic biology designs.

Laser heating tunability by off-resonant irradiation of gold nanoparticles.

Temperature changes in the vicinity of a single absorptive nanostructure caused by local heating have strong implications in technologies such as integrated electronics or biomedicine. Herein, the temperature changes in the vicinity of a single optically trapped spherical Au nanoparticle encapsulated in a thermo-responsive poly(N-isopropylacrylamide) shell (Au@pNIPAM) are studied in detail. Individual beads are trapped in a counter-propagating optical tweezers setup at various laser powers, which allows the overall particle size to be tuned through the phase transition of the thermo-responsive shell. The experimentally obtained sizes measured at different irradiation powers are compared with average size values obtained by dynamic light scattering (DLS) from an ensemble of beads at different temperatures. The size range and the tendency to shrink upon increasing the laser power in the optical trap or by increasing the temperature for DLS agree with reasonable accuracy for both approaches. Discrepancies are evaluated by means of simple models accounting for variations in the thermal conductivity of the polymer, the viscosity of the aqueous solution and the absorption cross section of the coated Au nanoparticle. These results show that these parameters must be taken into account when considering local laser heating experiments in aqueous solution at the nanoscale. Analysis of the stability of the Au@pNIPAM particles in the trap is also theoretically carried out for different particle sizes.

La vacuna polio oral en lactantes no interfiere con la detección de enterovirus en sangre / Oral polio vaccine in infants does not interfere in detection of enterovirus in blood

Introduction: There is not known if a viraemia post-oral polio vaccine (OPV) is detectable by modern molecular techniques. Such viraemia could affect the performance of the real time-polymerase chain reaction (PCR) for non polio enterovirus (EV) detection, technique of growing clinical use for the study of febrile infants. Objective: To determine viraemia post-first dose of OPV in healthy infants, by molecular techniques. Patients and Methods: 50 infants less than three months without previous VPO were randomized in 5 groups: a control group with pre-vaccination blood sample (BS), group 1 BS at day 2, group 2 BS at day 4, group 3, BS at day 6 and group 4, BS at day 8 post-vaccination. Conventional and specific PCR for poliovirus and real time PCR for non polio EV were performed in BS and in OPV samples. Results: No genetic material of poliovirus was detected in any infant, while in 9 of them (18%) non polio EV was identified. Real time PCR for EV did not amplify poliovirus from OPV samples. Discussion: Results suggest that no post VPO viraemia detectable by molecular methods exists. Considering that real time PCR for EV does not allow to identify polio virus, no false positives of the test are expected as a result of a recent VPO vaccination. We documented presence of non polio EV in blood of healthy asymptomatic infants.

Introducción: No existen estudios que indiquen si la vacuna polio oral (VPO) produce viremia detectable mediante métodos moleculares. Una eventual viremia podría afectar el rendimiento de la RPC tiempo real para detectar enterovirus (EV) no polio, examen de creciente uso clínico en lactantes pequeños con fiebre sin foco. Objetivo: Determinar viremia post VPO en lactantes sanos, por métodos moleculares. Métodos: 50 menores de 3 meses, al momento de recibir su primera VPO se distribuyeron en forma aleatoria en 5 grupos: control, muestra de sangre pre-vacunación; grupo 1, muestra al 2° día; grupo 2, al 4° día; grupo 3, al 6° día y grupo 4, al 8° día post-vacunación. Se realizó RPC convencional específica para virus polio y RPC tiempo real para EV no polio en las muestras de sangre y en muestras de VPO. Resultados: No se identificó presencia de material genético de virus polio en lactante alguno, mientras que en 9 (18%) se identificó presencia de EV no polio. La RPC tiempo real para EV no polio no amplificó material genético a partir de las muestras de VPO. Discusión: Los resultados sugieren que no existe viremia post-VPO detectable por métodos moleculares. Considerando que la RPC tiempo real de EV no polio de uso clínico no permite identificar la presencia de virus polio, estos hallazgos indican que no existirán falsos positivos de este examen como resultado de una vacunación VPO reciente. Adicionalmente se documentó presencia de EV no polio en sangre de lactantes asintomáticos.

Synthetic approaches to study transcriptional networks and noise in mammalian systems.

Synthetic biology aims to build new functional organisms and to rationally re-design existing ones by applying the engineering principle of modularity. Apart from building new life forms to perform technical applications, the approach of synthetic biology is useful to dissect complex biological phenomena into simple and easy to understand synthetic modules. Synthetic gene networks have been successfully implemented in prokaryotes and lower eukaryotes, with recent approaches moving ahead towards the mammalian environment. However, synthetic circuits in higher eukaryotes present a more challenging scenario, since its reliability is compromised because of the strong stochastic nature of transcription. Here, the authors review recent approaches that take advantage of the noisy response of synthetic regulatory circuits to learn key features of the complex machinery that orchestrates transcription in higher eukaryotes. Understanding the causes and consequences of biological noise will allow us to design more reliable mammalian synthetic circuits with revolutionary medical applications.

[Oral polio vaccine in infants does not interfere in detection of enterovirus in blood]. / La vacuna polio oral en lactantes no interfiere con la detección de enterovirus en sangre.

INTRODUCTION: There is not known if a viraemia post-oral polio vaccine (OPV) is detectable by modern molecular techniques. Such viraemia could affect the performance of the real time-polymerase chain reaction (PCR) for non polio enterovirus (EV) detection, technique of growing clinical use for the study of febrile infants. OBJECTIVE: To determine viraemia post-first dose of OPV in healthy infants, by molecular techniques. PATIENTS AND METHODS: 50 infants less than three months without previous VPO were randomized in 5 groups: a control group with pre-vaccination blood sample (BS), group 1 BS at day 2, group 2 BS at day 4, group 3, BS at day 6 and group 4, BS at day 8 post-vaccination. Conventional and specific PCR for poliovirus and real time PCR for non polio EV were performed in BS and in OPV samples. RESULTS: No genetic material of poliovirus was detected in any infant, while in 9 of them (18%) non polio EV was identified. Real time PCR for EV did not amplify poliovirus from OPV samples. DISCUSSION: Results suggest that no post VPO viraemia detectable by molecular methods exists. Considering that real time PCR for EV does not allow to identify polio virus, no false positives of the test are expected as a result of a recent VPO vaccination. We documented presence of non polio EV in blood of healthy asymptomatic infants.

Common features of sexual dimorphism in the cranial airways of different human populations.

Sexual dimorphism in the human craniofacial system is an important feature of intraspecific variation in recent and fossil humans. Although several studies have reported different morphological patterns of sexual dimorphism in different populations, this study searches for common morphological aspects related to functional anatomy of the respiratory apparatus. 3D geometric morphometrics were used to test the hypothesis that due to higher daily energy expenditure and associated greater respiratory air consumption as well as differences in body composition, males should have absolutely and relatively greater air passages in the bony cranial airways than females. We measured 25 3D landmarks in five populations (N = 212) of adult humans from different geographic regions. Male average cranial airways were larger in centroid sizes than female ones. Males tended to show relatively taller piriform apertures and, more consistently, relatively taller internal nasal cavities and choanae than females. Multivariate regressions and residual analysis further indicated that after standardizing to the same size, males still show relatively larger airway passages than females. Because the dimensions of the choanae are limiting factors for air transmission towards the noncranial part of the respiratory system, the identified sex-specific differences in cranial airways, possibly shared among human populations, may be linked with sex-specific differences in body size, composition, and energetics. These findings may be important to understanding trends in hominin facial evolution.

Development and characterization of a highly specific and sensitive SYBR green reverse transcriptase PCR assay for detection of the 2009 pandemic H1N1 influenza virus on the basis of sequence signatures.

The emergence and rapid spread of the 2009 H1N1 pandemic influenza virus showed that many diagnostic tests were unsuitable for detecting the novel virus isolates. In most countries the probe-based TaqMan assay developed by the U.S. Centers for Disease Control and Prevention was used for diagnostic purposes. The substantial sequence data that became available during the course of the pandemic created the opportunity to utilize bioinformatics tools to evaluate the unique sequence properties of this virus for the development of diagnostic tests. We used a comprehensive computational approach to examine conserved 2009 H1N1 sequence signatures that are at least 20 nucleotides long and contain at least two mismatches compared to any other known H1N1 genome. We found that the hemagglutinin (HA) and neuraminidase (NA) genes contained sequence signatures that are highly conserved among 2009 H1N1 isolates. Based on the NA gene signatures, we used Visual-OMP to design primers with optimal hybridization affinity and we used ThermoBLAST to minimize amplification artifacts. This procedure resulted in a highly sensitive and discriminatory 2009 H1N1 detection assay. Importantly, we found that the primer set can be used reliably in both a conventional TaqMan and a SYBR green reverse transcriptase (RT)-PCR assay with no loss of specificity or sensitivity. We validated the diagnostic accuracy of the NA SYBR green assay with 125 clinical specimens obtained between May and August 2009 in Chile, and we showed diagnostic efficacy comparable to the CDC assay. Our approach highlights the use of systematic computational approaches to develop robust diagnostic tests during a viral pandemic.

Andes virus antigens are shed in urine of patients with acute hantavirus cardiopulmonary syndrome.

Hantavirus cardiopulmonary syndrome (HCPS) is a highly pathogenic emerging disease (40% case fatality rate) caused by New World hantaviruses. Hantavirus infections are transmitted to humans mainly by inhalation of virus-contaminated aerosol particles of rodent excreta and secretions. At present, there are no antiviral drugs or immunotherapeutic agents available for the treatment of hantaviral infection, and the survival rates for infected patients hinge largely on early virus recognition and hospital admission and aggressive pulmonary and hemodynamic support. In this study, we show that Andes virus (ANDV) interacts with human apolipoprotein H (ApoH) and that ApoH-coated magnetic beads or ApoH-coated enzyme-linked immunosorbent assay plates can be used to capture and concentrate the virus from complex biological mixtures, such as serum and urine, allowing it to be detected by both immunological and molecular approaches. In addition, we report that ANDV-antigens and infectious virus are shed in urine of HCPS patients.

Prospective evaluation of household contacts of persons with hantavirus cardiopulmonary syndrome in chile.

BACKGROUND: Andes virus (ANDV) infection, which has a case fatality rate of 37% in Chile, often occurs in household clusters and may be transmitted from person to person. METHODS: To determine the incidence and risk factors for additional household cases, we conducted a prospective study among recent household contacts of persons with hantavirus cardiopulmonary syndrome (HCPS) in Chile, including testing of serum for anti-hantavirus antibodies and blood cells for ANDV RNA by reverse-transcription polymerase chain reaction (RT-PCR). RESULTS: We enrolled 76 index case patients and 476 household contacts, of whom 16 (3.4%) developed HCPS; 32.6% of 92 cases occurred in household clusters. The risk of HCPS was 17.6% among sex partners of index case patients, versus 1.2% among other household contacts (P<.001). Person-to-person transmission was definite in 3, probable in 9, and possible in 2 of the 16 additional household case patients. We detected ANDV RNA by RT-PCR in peripheral blood cells 5-15 days before the onset of symptoms or the appearance of anti-hantavirus antibodies. CONCLUSIONS: In recent household contacts of persons with HCPS in Chile, the risk of HCPS was greatest among sex partners. Among the household contacts who developed HCPS, viremia preceded the onset of symptoms and the appearance of anti-hantavirus antibodies by up to 2 weeks.