Nanogram-scale boron isotope analysis through micro-distillation and Nu Plasma 3 MC-ICP-MS.

The determination of boron isotopes (δ11B) represents a powerful tool for a variety of applications such as the reconstruction of past ocean pH and atmospheric pCO2 from the analysis of marine biogenic carbonates. In recent years, MC-ICP-MS has gained popularity over other techniques thanks to its superior sample throughput and high ionization efficiency. This study evaluates, for the first time, the performance of the Nu Instruments Plasma 3 MC-ICP-MS for measuring δ11B using different sample introduction systems and detector configurations. The main goal is to provide a detailed methodology for nanogram-scale boron isotope analysis through a straightforward approach that can be easily adopted. Boron (B) purification from the carbonate matrix was performed through micro-distillation, using a temperature of 95 °C and a minimum heating duration of 15 h, allowing the full recovery of B from up to 3 mg of carbonate mass. We attained blank values (on average 14 ± 6 pg, 1 SD, n = 27) comparable to the lowest micro-distillation blanks reported in the literature. Three sample introduction systems were tested, and the 30 µL min-1 nebuliser system outperformed the 50 and 170 µL min-1 systems in terms of signal intensity per mass of B. Two detector configurations were used based on the total boron signal intensity achieved: (1) FC11/FC12, with two Faraday cups fitted to 1011 Ω and 1012 Ω amplifier resistors to detect 11B and 10B ion beams, respectively, and (2) FC12/IC, with which we investigated, for the first time, the feasibility of combining an ion counter for detecting 10B, and a Faraday cup fitted to a 1012 Ω amplifier for 11B. The FC12/IC configuration provided accurate results compared to the use of two Faraday cups for total boron signals lower than 0.35 V (∼12 ng of B in the analysed solution). The proposed analytical procedure was validated through the analysis of several reference materials with varying boron amounts, including clam JCt-1, coral JCp-1, NIST RM 8301 Foram and Coral solutions, and boric acid ERM-AE121. Furthermore, the long-term reproducibility was assessed with two in-house standards (coral CLD-1 and foraminifera GINF-1), providing values of 25.68 ± 0.23 ‰ (2SD, n = 53; with 14-36 ng of B) and 14.90 ± 0.16 ‰ (2SD, n = 12; with 11-16 ng of B), respectively.

Efectividad de técnicas analgésicas preventivas con bloqueos regionales en el control del dolor postoperatorio tardío del cáncer de mama / Effectiveness of preventive analgesic techniques with regional blocks in the control of late postoperative breast cancer pain

RESUMEN Introducción: el cáncer de mama es una proliferación maligna de las células epiteliales que revisten los conductos o lobulillos mamarios. Objetivo: caracterizar la efectividad de técnicas analgésicas preventivas con bloqueos regionales en el control del dolor postoperatorio tardío del cáncer de mama, en el Hospital Provincial Docente Clínico Quirúrgico "León Cuervo Rubio", durante el período 2017 a 2019. Métodos: se realizó una investigación observacional, analítica, longitudinal y prospectiva en coordinación con el servicio de Anestesiología y Cirugía del Hospital Provincial Docente Clínico Quirúrgico "León Cuervo Rubio". El universo de estudio fue de 260 pacientes con diagnóstico positivo de cáncer de mama y la muestra escogida fue de 176 pacientes, la misma cumplió con los criterios de inclusión. Resultados: el mayor número de mujeres afectadas estuvo enmarcado entre la quinta y séptima década de la vida para un 65,6 % del total incluido en el estudio, en los dos grupos predominó la ausencia de dolor a dolor ligero; se observó que las complicaciones fueron escasas en los dos grupos, aunque con un ligero incremento en el grupo A con predominio de las náuseas y vómitos en 23 pacientes (26,14 %). Conclusiones: se evidenció una estabilidad hemodinámica en ambos grupos de estudio, donde la variación de la intensidad del dolor es mínima y la complicación postoperatorio más frecuente es las náuseas y los vómitos.

ABSTRACT Introduction: breast cancer is a malignant proliferation of epithelial cells lining the breast ducts or lobules. Objective: to characterize the effectiveness of preventive analgesic techniques with regional blocks in the control of late postoperative pain of breast cancer at Leon Cuervo Rubio Provincial Surgical Clinical Teaching Hospital during the period 2017 to 2019. Methods: an observational, analytical, longitudinal and prospective research was conducted in coordination with the service of Anesthesiology and Surgery at Leon Cuervo Rubio Provincial Surgical Clinical Teaching Hospital. The study target group comprised 260 patients with positive diagnosis of breast cancer and the sample chosen was 176 patients, it met the inclusion criteria. Results: the greatest number of affected women was between the fifth and seventh decade of life, representing 65,6 % of the total included in the study, in both groups there was a predominance of absence of pain to slight pain; it was observed that complications were scarce in both groups, although with a slight increase in group A with a predominance of nausea and vomiting in 23 patients (26,14 %). Conclusions: hemodynamic stability was evidenced in both study groups, where the variation in pain intensity was minimal and the most frequent postoperative complication was nausea and vomiting.

Eficacia del tratamiento alternativo con oleozón tópico en pacientes con onicomicosis / Effectiveness of alternative treatment with topical oleozon in patients with onychomycosis

RESUMEN Introducción: las onicomicosis son infecciones fúngicas de la lámina ungueal y tejidos adyacentes. Objetivo: determinar la eficacia del tratamiento alternativo con oleozón tópico en pacientes con onicomicosis de los consultorios 24 y 25 del Policlínico Universitario "Luis Augusto Turcios Lima" de la provincia de Pinar del Río, de 2017-2018. MétodoS: se realizó un estudio observacional, descriptivo y de corte transversal a los pacientes diagnosticados con onicomicosis en los consultorios 24 y 25 del Policlínico "Luis Augusto Turcios Lima" durante el 2017-2018, el universo estuvo conformado por 90 pacientes con el diagnostico de esta enfermedad, se trabajó con la totalidad de ellos; se estratificó en tres grupos, grupo A (ketoconazol tópico más fluconazol tableta), grupo B (oleozón tópico) y grupo C (fluconazol tableta más oleozón tópico). Resultados: predominó el grupo etáreo de 60-69 (31,1 %) y el sexo masculino (65,6 %), predominaron los pacientes que presentaron cambios de coloración en las uñas (32 %), en el grupo A el 56,7 % de los pacientes presentaron mejoría entre tres y seis meses, el grupo B el 93,3 % en el mismo periodo que el grupo A y en el grupo C el 100 % se curaron en el mismo periodo que los demás grupos. Conclusiones: predominó el grupo de 60-69 años de edad, el sexo masculino fue el más afectado. El signo más frecuente fue el cambio de coloración y el síntoma el dolor; el tratamiento combinado fue el más efectivo.

ABSTRACT Introduction: onychomycoses are fungal infections of the nail plate and adjacent tissues. Objective: to determine the efficacy of alternative treatment with topical oleozon in patients with onychomycosis of the 24th and 25th Doctor's Offices belonging to Luis Augusto Turcios Lima University Polyclinic in Pinar del Rio province during 2017-2018. Methods: an observational, descriptive and cross-sectional study was conducted on patients diagnosed with onychomycosis in the 24th and 25th Doctor's Offices belonging to Luis Augusto Turcios Lima University Polyclinic in Pinar del Rio province during 2017-2018. The target group comprised 90 patients diagnosed with this disease, working with all of them, which were stratified into three groups, group-A (topical ketoconazole plus fluconazole tablet), group-B (topical oleozon) and group-C (fluconazole tablet plus topical oleozon). Results: the age group 60-69 (31,1 %) and male sex (65,6 %) predominated, patients with nail discoloration changes predominated (32 %), in group-A 56,7 % of patients showed improvement between 3 and 6 months, group-B 93,3 % in the same period as group-A and in group-C 100 % were cured in the same period as the other groups. Conclusions: the age group from 60-69 predominated, male sex was the most affected. The most frequent sign was discoloration change and the symptom was pain; combined treatment was the most effective.

Ethical challenges for a new generation of early-phase pediatric gene therapy trials.

After decades of setbacks, gene therapy (GT) is experiencing major breakthroughs. Five GTs have received US regulatory approval since 2017, and over 900 others are currently in development. Many of these GTs target rare pediatric diseases that are severely life-limiting, given a lack of effective treatments. As these GTs enter early-phase clinical trials, specific ethical challenges remain unresolved in three domains: evaluating risks and potential benefits, selecting participants fairly, and engaging with patient communities. Drawing on our experience as clinical investigators, basic scientists, and bioethicists involved in a first-in-human GT trial for an ultrarare pediatric disease, we analyze these ethical challenges and offer points to consider for future GT trials.

Giant axonal neuropathy: cross-sectional analysis of a large natural history cohort.

Giant axonal neuropathy (GAN) is an ultra-rare autosomal recessive, progressive neurodegenerative disease with early childhood onset that presents as a prominent sensorimotor neuropathy and commonly progresses to affect both the PNS and CNS. The disease is caused by biallelic mutations in the GAN gene located on 16q23.2, leading to loss of functional gigaxonin, a substrate specific ubiquitin ligase adapter protein necessary for the regulation of intermediate filament turnover. Here, we report on cross-sectional data from the first study visit of a prospectively collected natural history study of 45 individuals, age range 3-21 years with genetically confirmed GAN to describe and cross-correlate baseline clinical and functional cohort characteristics. We review causative variants distributed throughout the GAN gene in this cohort and identify a recurrent founder mutation in individuals with GAN of Mexican descent as well as cases of recurrent uniparental isodisomy. Through cross-correlational analysis of measures of strength, motor function and electrophysiological markers of disease severity, we identified the Motor Function Measure 32 to have the strongest correlation across measures and age in individuals with GAN. We analysed the Motor Function Measure 32 scores as they correspond to age and ambulatory status. Importantly, we identified and characterized a subcohort of individuals with a milder form of GAN and with a presentation similar to Charcot-Marie-Tooth disease. Such a clinical presentation is distinct from the classic presentation of GAN, and we demonstrate how the two groups diverge in performance on the Motor Function Measure 32 and other functional motor scales. We further present data on the first systematic clinical analysis of autonomic impairment in GAN as performed on a subset of the natural history cohort. Our cohort of individuals with genetically confirmed GAN is the largest reported to date and highlights the clinical heterogeneity and the unique phenotypic and functional characteristics of GAN in relation to disease state. The present work is designed to serve as a foundation for a prospective natural history study and functions in concert with the ongoing gene therapy trial for children with GAN.

Cytoplasmic aggregation of uranium in human dopaminergic cells after continuous exposure to soluble uranyl at non-cytotoxic concentrations.

Uranium exposure can lead to neurobehavioral alterations in particular of the monoaminergic system, even at non-cytotoxic concentrations. However, the mechanisms of uranium neurotoxicity after non-cytotoxic exposure are still poorly understood. In particular, imaging uranium in neurons at low intracellular concentration is still very challenging. We investigated uranium intracellular localization by means of synchrotron X-ray fluorescence imaging with high spatial resolution (< 300 nm) and high analytical sensitivity (< 1 µg.g-1 per 300 nm pixel). Neuron-like SH-SY5Y human cells differentiated into a dopaminergic phenotype were continuously exposed, for seven days, to a non-cytotoxic concentration (10 µM) of soluble natural uranyl. Cytoplasmic submicron uranium aggregates were observed accounting on average for 62 % of the intracellular uranium content. In some aggregates, uranium and iron were co-localized suggesting common metabolic pathways between uranium and iron storage. Uranium aggregates contained no calcium or phosphorous indicating that detoxification mechanisms in neuron-like cells are different from those described in bone or kidney cells. Uranium intracellular distribution was compared to fluorescently labeled organelles (lysosomes, early and late endosomes) and to fetuin-A, a high affinity uranium-binding protein. A strict correlation could not be evidenced between uranium and the labeled organelles, or with vesicles containing fetuin-A. Our results indicate a new mechanism of uranium cytoplasmic aggregation after non-cytotoxic uranyl exposure that could be involved in neuronal defense through uranium sequestration into less reactive species. The remaining soluble fraction of uranium would be responsible for protein binding and for the resulting neurotoxic effects.

Incidencia del microcarcinoma papilar en enfermos operados de cáncer de tiroides / Incidence of papillary microcarcinoma in patients operated on thyroid cancer

RESUMEN Introducción: aunque el microcarcinoma papilar de tiroides ha sufrido un aumento en la incidencia en las últimas décadas, todavía es una controversia la extensión de la resección de la glándula y el tratamiento oncológico. Objetivo: determinar la incidencia del microcarcinoma papilar de tiroides en enfermos operados de cáncer de tiroides en el Hospital "León Cuervo Rubio" de Pinar del Río, en el período entre enero de 2018 a marzo de 2020. Métodos: se realizó un estudio descriptivo y transversal de 22 enfermos operados de microcarcinoma de tiroides, a los cuales se les practicó una tiroidectomía total. Se tomaron los datos de las historias clínicas, informes operatorios y las biopsias. Se utilizaron los métodos de observación y analítico. Las variables analizadas fueron: tamaño del tumor, resultados de la BAAF, diagnóstico definitivo, variedad histológica, enfermedades de tiroides asociadas y extensión de la enfermedad Resultados: los 22 enfermos operados de microcarcinoma de tiroides representan el 42,30 % del total de caso operados por cáncer. Predomina el sexo femenino y edad promedio 49,9 años. El 31,81 % de los enfermos con biopsia corresponden al grupo Bethesda I, II Y III; sin embargo, eran portadores de microcarcinomas. Un 18 % de los enfermos eran portadores de dos variedades histológicas de carcinoma. Conclusiones: el microcarcinoma papilar de tiroides aumenta su incidencia sobre todo en enfermos mayores de 45 años, con extensión más allá de la glándula lo cual agrava el pronóstico del enfermo, y requiere de la atención médica multidisciplinaria para determinar factores de riesgo pronóstico. De esta forma mejorar la conducta a seguir y la calidad de vida del paciente.

ABSTRACT Introduction: although papillary thyroid microcarcinoma has suffered an increase in incidence in the last decades, the extension of gland resection and oncological treatment is still a controversy. Objective: to determine the incidence of papillary thyroid microcarcinoma in patients who have undergone surgery for thyroid cancer at Leon Cuervo Rubio Hospital in Pinar del Río, 2018-2020. Methods: a descriptive and cross-sectional study of 22 patients operated for thyroid microcarcinoma was carried out, who underwent a total thyroidectomy, collecting data from the clinical histories, surgery reports and biopsies. Observation and analysis approaches were used. Variables to be analyzed: size of the tumor, BAAF results, definitive diagnosis, histological variety, associated thyroid diseases and extension of the disease Results: the 22 patients operated on thyroid microcarcinoma represented 42.30% of the total number of cases operated for cancer reasons. Female sex predominated and average age is 49.9 years; 31.81% of the patients with biopsy correspond to the Bethesda I, II and III group, however they were carriers of microcarcinoma; 18% of the patients were carriers of two histological varieties of carcinoma. Conclusions: papillary thyroid microcarcinoma continues the increase of its incidence, especially in patients over 45 years old, with extension beyond the gland, which aggravates the prognosis of the patient suffering from it and requires multidisciplinary medical care to determine prognostic risk factors and to improve the behavior to be followed and the quality of life of the patient.

O Ideal Profissional na Formação Médica / The Professional Ideal in Medical Formation

Resumo Introdução: No curso de Medicina, os estudantes recebem não somente conhecimento e habilidades técnicas em sala de aula e nos campos de prática, mas também aprendem valores, atitudes e comportamentos profissionais. Nesse processo, ocorre a construção social do médico, em que será desenvolvido o ideal profissional. Esta pesquisa teve como objetivo compreender o processo de formação da identidade profissional a partir dos seguintes aspectos: a identificação dos ideais de profissão e de profissionais sobre as qualidades pessoais e profissionais dos docentes do curso médico, a imagem de profissional e os fatores contribuintes para o ingresso na profissão. Métodos: Para isso, utilizou-se uma metodologia qualitativa de caráter exploratório, realizada por meio de entrevista semiestruturada com 20 discentes do primeiro ao sétimo semestre do curso de Medicina de uma escola médica de João Pessoa. As informações coletadas foram estudadas com base na análise do discurso. As categorias utilizadas para análise foram: o "bom médico", o "bom professor" e a idealização do profissional "bem-sucedido". Resultados: Os sujeitos da pesquisa tinham em média 22,2 anos de idade e 13 eram do sexo feminino e sete do sexo masculino. Mais da metade dos alunos se autodeclarou de cor branca e 45% informaram renda familiar maior que 20 salários mínimos. Quando se exploraram as categorias, percebeu-se que a imagem profissional mescla a ideia de status, por meio do reconhecimento da sociedade e da estabilidade financeira, e a de intenção de cuidar das pessoas. Conclusão: Observou-se que o "bom médico" deve ser um profissional "humano"; "estudioso", "atualizado" e "resolutivo"; "profissional comprometido" e "responsável". Além disso, os estudantes esperam desenvolver esses aspectos durante a graduação, mas muitas vezes se deparam com o que "não querem ser". Outro elemento importante foi a percepção do conflito entre as ideias de "técnico competente" e "médico humano". Essas características são transferidas para o "bom professor", sendo reconhecido o educador que detém mais características do ideal profissional.

Abstract Introduction: In medical school, students receive not only technical knowledge and skills in the classroom and fields of practice, but also learn values, attitudes, and professional behaviors. In this process, the social construction of the physician takes place, in which the professional ideal will be developed. The aim of this research was to understand the process of professional identity formation based on: the identification of the ideals of the profession and professionals on the personal and professional qualities of the medical course teachers; professional image; and the factors contributing to the entry into the profession. Methods: For this purpose, a qualitative exploratory methodology was used through a semi-structured interview with 20 students from the first to the seventh semesters of the medical course of a medical school in João Pessoa. The collected information was studied from the discourse analysis. The categories used for analysis were: the "good doctor"; the "good teacher"; and the idealization of the "successful" professional. Results: The subjects were 22.2 years of age; 13 were females and 7 were males. More than half of the students self-declared their ethnicity as white and 45% reported a family income greater than twenty minimum wages. Exploring the categories, it was observed that the professional image merges the idea of status, through the recognition of society and financial stability, and the intention to take care of people. Conclusion: It was observed that the "good doctor" must be a "humane" professional; "studious," "updated," and "resolutive"; "committed professional" and "responsible". In addition, the students expect to develop these aspects during their undergraduate education, but often encounter what they "do not want to be." Another important element was the perception of the conflict between the ideas of a "competent technician" and a "humane doctor". These characteristics are transferred to the "good teacher", thus recognizing the educator that holds the most characteristics of the professional ideal.

Isotopic variations of copper at the protein fraction level in neuronal human cells exposed in vitro to uranium.

The study of isotopic variations of endogenous and toxic metals in fluids and tissues is a recent research topic with an outstanding potential in biomedical and toxicological investigations. Most of the analyses have been performed so far in bulk samples, which can make the interpretation of results entangled, since different sources of stress or the alteration of different metabolic processes can lead to similar variations in the isotopic compositions of the elements in bulk samples. The downscaling of the isotopic analysis of elements at the sub-cellular level, is considered as a more promising alternative. Here we present for the first time the accurate determination of Cu isotopic ratios in four main protein fractions from lysates of neuron-like human cells exposed in vitro to 10 µM of natural uranium for seven days. These protein fractions were isolated by Size Exclusion Chromatography and analysed by Multi-Collector Inductively Coupled Plasma Mass Spectrometry to determine the Cu isotopic variations in each protein fraction with regard to the original cell lysate. Values obtained, expressed as δ65Cu, were -0.03 ± 0.14 ‰ (Uc, k = 2), -0.55 ± 0.20 ‰ (Uc, k = 2), -0.32 ± 0.21 ‰ (Uc, k = 2) and +0.84 ± 0.21 ‰ (Uc, k = 2) for the four fractions, satisfying the mass balance. The results obtained in this preliminary study pave the way for dedicated analytical developments to identify new specific disease biomarkers, to gain insight into stress-induced altered metabolic processes, as well as to decipher metabolic pathways of toxic elements.

Deciphering the uranium target proteins in human dopaminergic SH-SY5Y cells.

Uranium (U) is the heaviest naturally occurring element ubiquitously present in the Earth's crust. Human exposure to low levels of U is, therefore, unavoidable. Recently, several studies have clearly pointed out that the brain is a sensitive target for U, but the mechanisms leading to the observed neurological alterations are not fully known. To deepen our knowledge of the biochemical disturbances resulting from U(VI) toxicity in neuronal cells, two complementary strategies were set up to identify the proteins that selectively bind U(VI) in human dopaminergic SH-SY5Y cells. The first strategy relies on the selective capture of proteins capable of binding U(VI), using immobilized metal affinity chromatography, and starting from lysates of cells grown in a U(VI)-free medium. The second strategy is based on the separation of U-enriched protein fractions by size-exclusion chromatography, starting from lysates of U(VI)-exposed cells. High-resolution mass spectrometry helped us to highlight 269 common proteins identified as the urano-proteome. They were further analyzed to characterize their cellular localization and biological functions. Four canonical pathways, related to the protein ubiquitination system, gluconeogenesis, glycolysis, and the actin cytoskeleton proteins, were particularly emphasized due to their high content of U(VI)-bound proteins. A semi-quantification was performed to concentrate on the ten most abundant proteins, whose physico-chemical characteristics were studied in particular depth. The selective interaction of U(VI) with these proteins is an initial element of proof of the possible metabolic effects of U(VI) on neuronal cells at the molecular level.

Impact of uranium uptake on isotopic fractionation and endogenous element homeostasis in human neuron-like cells.

The impact of natural uranium (U) on differentiated human neuron-like cells exposed to 1, 10, 125, and 250 µM of U for seven days was assessed. In particular, the effect of the U uptake on the homeostatic modulation of several endogenous elements (Mg, P, Mn, Fe, Zn, and Cu), the U isotopic fractionation upon its incorporation by the cells and the evolution of the intracellular Cu and Zn isotopic signatures were studied. The intracellular accumulation of U was accompanied by a preferential uptake of 235U for cells exposed to 1 and 10 µM of U, whereas no significant isotopic fractionation was observed between the extra- and the intracellular media for higher exposure U concentrations. The U uptake was also found to modulate the homeostasis of Cu, Fe, and Mn for cells exposed to 125 and 250 µM of U, but the intracellular Cu isotopic signature was not modified. The intracellular Zn isotopic signature was not modified either. The activation of the non-specific U uptake pathway might be related to this homeostatic modulation. All together, these results show that isotopic and quantitative analyses of toxic and endogenous elements are powerful tools to help deciphering the toxicity mechanisms of heavy metals.

Uranium exposure of human dopaminergic cells results in low cytotoxicity, accumulation within sub-cytoplasmic regions, and down regulation of MAO-B.

Natural uranium is an ubiquitous element present in the environment and human exposure to low levels of uranium is unavoidable. Although the main target of acute uranium toxicity is the kidney, some concerns have been recently raised about neurological effects of chronic exposure to low levels of uranium. Only very few studies have addressed the molecular mechanisms of uranium neurotoxicity, indicating that the cholinergic and dopaminergic systems could be altered. The main objective of this study was to investigate the mechanisms of natural uranium toxicity, after 7-day continuous exposure, on terminally differentiated human SH-SY5Y cells exhibiting a dopaminergic phenotype. Cell viability was first assessed showing that uranium cytotoxicity only occurred at high exposure concentrations (> 125 µM), far from the expected values for uranium in the blood even after occupational exposure. SH-SY5Y differentiated cells were then continuously exposed to 1, 10, 125 or 250 µM of natural uranium for 7 days and uranium quantitative subcellular distribution was investigated by means of micro-PIXE (Particle Induced X-ray Emission). The subcellular element imaging revealed that uranium was located in defined perinuclear regions of the cytoplasm, suggesting its accumulation in organelles. Uranium was not detected in the nucleus of the differentiated cells. Quantitative analysis evidenced a very low intracellular uranium content at non-cytotoxic levels of exposure (1 and 10 µM). At higher levels of exposure (125 and 250 µM), when cytotoxic effects begin, a larger and disproportional intracellular accumulation of uranium was observed. Finally the expression of dopamine-related genes was quantified using real time qRT-PCR. The expression of monoamine oxidase B (MAO-B) gene was statistically significantly decreased after exposure to uranium while other dopamine-related genes were not modified. The down regulation of MAO-B was confirmed at the protein level. This original result suggests that the inhibition of dopamine catabolism, but also of other MAO-B substrates, could constitute selective effects of uranium neurotoxicity.

A new procedure for high precision isotope ratio determinations of U, Cu and Zn at nanogram levels in cultured human cells: What are the limiting factors?

The monitoring of isotopic fractionations in in vitro cultured human cell samples is a very promising and under-exploited tool to help identify the metabolic processes leading to disease-induced isotopic fractionations or decipher metabolic pathways of toxic metals in these samples. One of the limitations is that the analytes are often present at small amounts, ranging from tens to hundreds of ng, thus making challenging low-uncertainty isotope ratio determinations. Here we present a new procedure for U, Cu and Zn purification and isotope ratio determinations in cultured human neuron-like cells exposed to natural U. A thorough study of the influence of the limiting factors impacting the uncertainty of δ238U, δ66Zn and δ65Cu is also carried out. These factors include the signal intensity, which determines the within-day measurement reproducibility, the procedural blank correction and the matrix effects, which determine the accuracy of the mass bias correction models. Given the small Cu and U amounts in the cell samples, 15-30 and 20ng respectively, a highly efficient sample introduction system was employed in order to improve the analyte transport to the plasma and, hence, the signal intensity. With this device, the procedural blanks became the main uncertainty source of δ238U and δ65Cu values, accounting over 65% of the overall uncertainty. The matrix effects gave rise to inaccuracies in the mass bias correction models for samples finally dissolved in the minimal volumes required for the analysis, 100-150µL, leading to biases for U and Cu. We will show how these biases can be cancelled out by dissolving the samples in volumes of at least 300µL for Cu and 450µL for U. Using our procedure, expanded uncertainties (k = 2) of around 0.35‰ for δ238U and 0.15‰ for δ66Zn and δ65Cu could be obtained. The analytical approach presented in this work is also applicable to other biological microsamples and can be extended to other elements and applications.

Crystal structure of the Entamoeba histolytica RNA lariat debranching enzyme EhDbr1 reveals a catalytic Zn2+ /Mn2+ heterobinucleation.

The RNA lariat debranching enzyme, Dbr1, is a metallophosphoesterase that cleaves 2'-5' phosphodiester bonds within intronic lariats. Previous reports have indicated that Dbr1 enzymatic activity is supported by diverse metal ions including Ni2+ , Mn2+ , Mg2+ , Fe2+ , and Zn2+ . While in initial structures of the Entamoeba histolytica Dbr1 only one of the two catalytic metal-binding sites were observed to be occupied (with a Mn2+ ion), recent structures determined a Zn2+ /Fe2+ heterobinucleation. We solved a high-resolution X-ray crystal structure (1.8 Å) of the E. histolytica Dbr1 and determined a Zn2+ /Mn2+ occupancy. ICP-AES corroborate this finding, and in vitro debranching assays with fluorescently labeled branched substrates confirm activity.

Evidence of isotopic fractionation of natural uranium in cultured human cells.

The study of the isotopic fractionation of endogen elements and toxic heavy metals in living organisms for biomedical applications, and for metabolic and toxicological studies, is a cutting-edge research topic. This paper shows that human neuroblastoma cells incorporated small amounts of uranium (U) after exposure to 10 µM natural U, with preferential uptake of the 235U isotope with regard to 238U. Efforts were made to develop and then validate a procedure for highly accurate n(238U)/n(235U) determinations in microsamples of cells. We found that intracellular U is enriched in 235U by 0.38 ± 0.13‰ (2σ, n = 7) relative to the exposure solutions. These in vitro experiments provide clues for the identification of biological processes responsible for uranium isotopic fractionation and link them to potential U incorporation pathways into neuronal cells. Suggested incorporation processes are a kinetically controlled process, such as facilitated transmembrane diffusion, and the uptake through a high-affinity uranium transport protein involving the modification of the uranyl (UO22+) coordination sphere. These findings open perspectives on the use of isotopic fractionation of metals in cellular models, offering a probe to track uptake/transport pathways and to help decipher associated cellular metabolic processes.

International system of units traceable results of Hg mass concentration at saturation in air from a newly developed measurement procedure.

Data most commonly used at present to calibrate measurements of mercury vapor concentrations in air come from a relationship known as the "Dumarey equation". It uses a fitting relationship to experimental results obtained nearly 30 years ago. The way these results relate to the international system of units (SI) is not known. This has caused difficulties for the specification and enforcement of limit values for mercury concentrations in air and in emissions to air as part of national or international legislation. Furthermore, there is a significant discrepancy (around 7% at room temperature) between the Dumarey data and data calculated from results of mercury vapor pressure measurements in the presence of only liquid mercury. As an attempt to solve some of these problems, a new measurement procedure is described for SI traceable results of gaseous Hg concentrations at saturation in milliliter samples of air. The aim was to propose a scheme as immune as possible to analytical biases. It was based on isotope dilution (ID) in the liquid phase with the (202)Hg enriched certified reference material ERM-AE640 and measurements of the mercury isotope ratios in ID blends, subsequent to a cold vapor generation step, by inductively coupled plasma mass spectrometry. The process developed involved a combination of interconnected valves and syringes operated by computer controlled pumps and ensured continuity under closed circuit conditions from the air sampling stage onward. Quantitative trapping of the gaseous mercury in the liquid phase was achieved with 11.5 µM KMnO4 in 2% HNO3. Mass concentrations at saturation found from five measurements under room temperature conditions were significantly higher (5.8% on average) than data calculated from the Dumarey equation, but in agreement (-1.2% lower on average) with data based on mercury vapor pressure measurement results. Relative expanded combined uncertainties were estimated following a model based approach. They ranged from 2.2% to 2.8% (k = 2). The volume of air samples was traceable to the kilogram via weighing of water for the calibration of the sampling syringe. Procedural blanks represented on average less than 0.1% of the mass of Hg present in 7.4 cm(3) of air, and correcting for these blanks was not an important source of uncertainty.

Autotransfecting short interfering RNA through facile covalent polymer escorts.

Short interfering ribonucleic acids (siRNAs) are important agents for RNA interference (RNAi) that have proven useful in gene function studies and therapeutic applications. However, the efficacy of exogenous siRNAs for gene knockdown remains hampered by their susceptibility to cellular nucleases and impermeability to cell membranes. We report here new covalent polymer-escort siRNA constructs that address both of these constraints simultaneously. By simple postsynthetic click conjugation of polymers to the passenger strand of an siRNA duplex followed by annealing with the complementary guide strand, we obtained siRNA in which one strand includes terminal polymer escorts. The polymer escorts both confer protection against nucleases and facilitate cellular internalization of the siRNA. These autotransfecting polymer-escort siRNAs are viable in RNAi and effective in knocking down reporter and endogenous genes.

Star polymers with a cationic core prepared by ATRP for cellular nucleic acids delivery.

Poly(ethylene glycol) (PEG)-based star polymers with a cationic core were prepared by atom transfer radical polymerization (ATRP) for in vitro nucleic acid (NA) delivery. The star polymers were synthesized by ATRP of 2-(dimethylamino)ethyl methacrylate (DMAEMA) and ethylene glycol dimethacrylate (EGDMA). Star polymers were characterized by gel permeation chromatography, zeta potential, and dynamic light scattering. These star polymers were combined with either plasmid DNA (pDNA) or short interfering RNA (siRNA) duplexes to form polyplexes for intracellular delivery. These polyplexes with either siRNA or pDNA were highly effective in NA delivery, particularly at relatively low star polymer weight or molar ratios, highlighting the importance of NA release in efficient delivery systems.

Backbone-branched DNA building blocks for facile angular control in nanostructures.

Nanotechnology based on the highly specific pairing of nucleobases in DNA has been used to generate a wide variety of well-defined two- and three-dimensional assemblies, both static and dynamic. However, control over the junction angles to achieve them has been limited. To achieve higher order assemblies, the strands of the DNA duplex are typically made to deviate at junctions with configurations based on crossovers or non-DNA moieties. Such strand crossovers tend to be intrinsically unstructured with the overall structural rigidity determined by the architecture of the nanoassembly, rather than the junction itself. Specific approaches to define nanoassembly junction angles are based either on the cooperative twist- and strain-promoted tuning of DNA persistence length leading to bent DNA rods for fairly large nano-objects, or de novo synthesis of individual junction inserts that are typically non-DNA and based on small organic molecules or metal-coordinating ligand moieties. Here, we describe a general strategy for direct control of junction angles in DNA nanostructures that are completely tunable about the DNA helix. This approach is used to define angular vertices through readily accessible backbone-branched DNAs (bbDNAs). We demonstrate how such bbDNAs can be used as a new building block in DNA nanoconstruction to obtain well-defined nanostructures. Angular control through readily accessible bbDNA building block provides a general and versatile approach for incorporating well-defined junctions in nanoconstructs and expands the toolkit toward achieving strain free, highly size- and shape-tunable DNA based architectures.