Nanobody-mediated targeting of zinc phthalocyanine with polymer micelles as nanocarriers.

Photodynamic therapy (PDT) is a suitable alternative to currently employed cancer treatments. However, the hydrophobicity of most photosensitizers (e.g., zinc phthalocyanine (ZnPC)) leads to their aggregation in blood. Moreover, non-specific accumulation in skin and low clearance rate of ZnPC leads to long-lasting skin photosensitization, forcing patients with a short life expectancy to remain indoors. Consequently, the clinical implementation of these photosensitizers is limited. Here, benzyl-poly(ε-caprolactone)-b-poly(ethylene glycol) micelles encapsulating ZnPC (ZnPC-M) were investigated to increase the solubility of ZnPC and its specificity towards cancers cells. Asymmetric flow field-flow fractionation was used to characterize micelles with different ZnPC-to-polymer ratios and their stability in human plasma. The ZnPC-M with the lowest payload (0.2 and 0.4% ZnPC w/w) were the most stable in plasma, exhibiting minimal ZnPC transfer to lipoproteins, and induced the highest phototoxicity in three cancer cell lines. Nanobodies (Nbs) with binding specificity towards hepatocyte growth factor receptor (MET) or epidermal growth factor receptor (EGFR) were conjugated to ZnPC-M to facilitate cell targeting and internalization. MET- and EGFR-targeting micelles enhanced the association and the phototoxicity in cells expressing the target receptor. Altogether, these results indicate that ZnPC-M decorated with Nbs targeting overexpressed proteins on cancer cells may provide a better alternative to currently approved formulations.

Effect of Polyplex Size on Penetration into Tumor Spheroids.

Ovarian cancer is one of the most lethal gynecological cancers in the world. In recent years, nucleic acid (NA)-based formulations have been shown to be promising treatments for ovarian cancer, including tumor nodules. However, gene therapy is not that far advanced in clinical reality due to unfavorable physicochemical properties of the NAs, such as high molecular weight, poor cellular uptake, rapid degradation by nucleases, etc. One of the strategies used to overcome these drawbacks is the complexation of anionic NAs via electrostatic interactions with cationic polymers, resulting in the formation of so-called polyplexes. In this work, the role of the size of pDNA and siRNA polyplexes on their penetration into ovarian-cancer-based tumor spheroids was investigated. For this, a methoxypoly(ethylene glycol) poly(2-(dimethylamino)ethyl methacrylate) (mPEG-pDMAEMA) diblock copolymer was synthesized as a polymeric carrier for NA binding and condensation with either plasmid DNA (pDNA) or short interfering RNA (siRNA). When prepared in HEPES buffer (10 mM, pH 7.4) at a nitrogen/phosphate (N/P) charge ratio of 5 and pDNA polyplexes were formed with a size of 162 ± 11 nm, while siRNA-based polyplexes displayed a size of 25 ± 2 nm. The polyplexes had a slightly positive zeta potential of +7-8 mV in the same buffer. SiRNA and pDNA polyplexes were tracked in vitro into tumor spheroids, resembling in vivo avascular ovarian tumor nodules. For this purpose, reproducible spheroids were obtained by coculturing ovarian carcinoma cells with primary mouse embryonic fibroblasts in different ratios (5:2, 1:1, and 2:5). Penetration studies revealed that after 24 h of incubation, siRNA polyplexes were able to penetrate deeper into the homospheroids (composed of only cancer cells) and heterospheroids (cancer cells cocultured with fibroblasts) compared to pDNA polyplexes which were mainly located in the rim. The penetration of the polyplexes was slowed when increasing the fraction of fibroblasts present in the spheroids. Furthermore, in the presence of serum siRNA polyplexes encoding for luciferase showed a high cellular uptake in 2D cells resulting in ∼50% silencing of luciferase expression. Taken together, these findings show that self-assembled small siRNA polyplexes have good potential as a platform to test ovarian tumor nodulus penetration..

Mechanistic Study on the Degradation of Hydrolysable Core-Crosslinked Polymeric Micelles.

Core-crosslinked polymeric micelles (CCPMs) are an attractive class of nanocarriers for drug delivery. Two crosslinking approaches to form CCPMs exist: either via a low-molecular-weight crosslinking agent to connect homogeneous polymer chains with reactive handles or via cross-reactive handles on polymers to link them to each other (complementary polymers). Previously, CCPMs based on methoxy poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide-lactate] (mPEG-b-PHPMAmLacn) modified with thioesters were crosslinked via native chemical ligation (NCL, a reaction between a cysteine residue and thioester resulting in an amide bond) using a bifunctional cysteine containing crosslinker. These CCPMs are degradable under physiological conditions due to hydrolysis of the ester groups present in the crosslinks. The rapid onset of degradation observed previously, as measured by the light scattering intensity, questions the effectiveness of crosslinking via a bifunctional agent. Particularly due to the possibility of intrachain crosslinks that can occur using such a small crosslinker, we investigated the degradation mechanism of CCPMs generated via both approaches using various analytical techniques. CCPMs based on complementary polymers degraded slower at pH 7.4 and 37 °C than CCPMs with a crosslinker (the half-life of the light scattering intensity was approximately 170 h versus 80 h, respectively). Through comparative analysis of the degradation profiles of the two different CCPMs, we conclude that partially ineffective intrachain crosslinks are likely formed using the small crosslinker, which contributed to more rapid CCPM degradation. Overall, this study shows that the type of crosslinking approach can significantly affect degradation kinetics, and this should be taken into consideration when developing new degradable CCPM platforms.

The Impact of Nanobody Density on the Targeting Efficiency of PEGylated Liposomes.

Nanoparticles (NPs) are commonly modified with tumor-targeting moieties that recognize proteins overexpressed on the extracellular membrane to increase their specific interaction with target cells. Nanobodies (Nbs), the variable domain of heavy chain-only antibodies, are a robust targeting ligand due to their small size, superior stability, and strong binding affinity. For the clinical translation of targeted Nb-NPs, it is essential to understand how the number of Nbs per NP impacts the receptor recognition on cells. To study this, Nbs targeting the hepatocyte growth factor receptor (MET-Nbs) were conjugated to PEGylated liposomes at a density from 20 to 800 per liposome and their targeting efficiency was evaluated in vitro. MET-targeted liposomes (MET-TLs) associated more profoundly with MET-expressing cells than non-targeted liposomes (NTLs). MET-TLs with approximately 150-300 Nbs per liposome exhibited the highest association and specificity towards MET-expressing cells and retained their targeting capacity when pre-incubated with proteins from different sources. Furthermore, a MET-Nb density above 300 Nbs per liposome increased the interaction of MET-TLs with phagocytic cells by 2-fold in ex vivo human blood compared to NTLs. Overall, this study demonstrates that adjusting the MET-Nb density can increase the specificity of NPs towards their intended cellular target and reduce NP interaction with phagocytic cells.

Attritional evaluation of lipophilic and hydrophilic metallated phthalocyanines for oncological photodynamic therapy.

BACKGROUND AND AIM: Oncological photodynamic therapy (PDT) relies on photosensitizers (PSs) to photo-oxidatively destroy tumor cells. Currently approved PSs yield satisfactory results in superficial and easy-to-access tumors but are less suited for solid cancers in internal organs such as the biliary system and the pancreas. For these malignancies, second-generation PSs such as metallated phthalocyanines are more appropriate. Presently it is not known which of the commonly employed metallated phtahlocyanines, namely aluminum phthalocyanine (AlPC) and zinc phthalocyanine (ZnPC) as well as their tetrasulfonated derivatives AlPCS4 and ZnPCS4, is most cytotoxic to tumor cells. This study therefore employed an attritional approach to ascertain the best metallated phthalocyanine for oncological PDT in a head-to-head comparative analysis and standardized experimental design. METHODS: ZnPC and AlPC were encapsulated in PEGylated liposomes. Analyses were performed in cultured A431 cells as a template for tumor cells with a dysfunctional P53 tumor suppressor gene and EGFR overexpression. First, dark toxicity was assessed as a function of PS concentration using the WST-1 and sulforhodamine B assay. Second, time-dependent uptake and intracellular distribution were determined by flow cytometry and confocal microscopy, respectively, using the intrinsic fluorescence of the PSs. Third, the LC50 values were established for each PS at 671 nm and a radiant exposure of 15 J/cm2 following 1-h PS exposure. Finally, the mode of cell death as a function of post-PDT time and cell cycle arrest at 24 h after PDT were analyzed. RESULTS: In the absence of illumination, AlPC and ZnPC were not toxic to cells up to a 1.5-µM PS concentration and exposure for up to 72 h. Dark toxicity was noted for AlPCS4 at 5 µM and ZnPCS4 at 2.5 µM. Uptake of all PSs was observed as early as 1 min after PS addition to cells and increased in amplitude during a 2-h incubation period. After 60 min, the entire non-nuclear space of the cell was photosensitized, with PS accumulation in multiple subcellular structures, especially in case of AlPC and AlPCS4. PDT of cells photosensitized with ZnPC, AlPC, and AlPCS4 yielded LC50 values of 0.13 µM, 0.04 µM, and 0.81 µM, respectively, 24 h post-PDT (based on sulforhodamine B assay). ZnPCS4 did not induce notable phototoxicity, which was echoed in the mode of cell death and cell cycle arrest data. At 4 h post-PDT, the mode of cell death comprised mainly apoptosis for ZnPC and AlPC, the extent of which was gradually exacerbated in AlPC-photosensitized cells during 8 h. ZnPC-treated cells seemed to recover at 8 h post-PDT compared to 4 h post-PDT, which had been observed before in another cell line. AlPCS4 induced considerable necrosis in addition to apoptosis, whereby most of the cell death had already manifested at 2 h after PDT. During the course of 8 h, necrotic cell death transitioned into mainly late apoptotic cell death. Cell death signaling coincided with a reduction in cells in the G0/G1 phase (ZnPC, AlPC, AlPCS4) and cell cycle arrest in the S-phase (ZnPC, AlPC, AlPCS4) and G2 phase (ZnPC and AlPC). Cell cycle arrest was most profound in cells that had been photosensitized with AlPC and subjected to PDT. CONCLUSIONS: Liposomal AlPC is the most potent PS for oncological PDT, whereas ZnPCS4 was photodynamically inert in A431 cells. AlPC did not induce dark toxicity at PS concentrations of up to 1.5 µM, i.e., > 37 times the LC50 value, which is favorable in terms of clinical phototoxicity issues. AlPC photosensitized multiple intracellular loci, which was associated with extensive, irreversible cell death signaling that is expected to benefit treatment efficacy and possibly immunological long-term tumor control, granted that sufficient AlPC will reach the tumor in vivo. Given the differential pharmacokinetics, intracellular distribution, and cell death dynamics, liposomal AlPC may be combined with AlPCS4 in a PS cocktail to further improve PDT efficacy.

Avaliação fisiológica de neonatos prematuros submetidos ao banho de imersão em banheira e banho humanizado / Physiological evaluation of premature neonates submitted to bath and humanized bath / Evaluación fisiológica de neonatos prematuros presentados al baño y baño humanizado

Objetivos: descrever e comparar os sinais fisiológicos apresentados pelos bebês prematuros durante o banho de imersão em banheira e banho humanizado. Método: estudo quantitativo, transversal e observacional, realizado através da filmagem e análise dos banhos. Participaram do estudo todos os prematuros hemodinamicamente estáveis. A coleta de dados ocorreu de junho de 2018 até junho de 2019. A análise de dados ocorreu por estatística descritiva. Resultados: o banho de imersão em banheira apresentou uma grande perda na temperatura corporal de 50% (15) dos prematuros. O banho humanizado não apresentou perda de significativa de temperatura em nenhum neonato. Conclusão: a técnica humanizada promove uma melhor assistência ao recém-nascido prematuro, gerando menos alterações fisiológicas

Objectives: were to describe and compare the physiological signs presented by premature babies during bath immersion in a bath and humanized bath. Method: quantitative, cross-sectional and observational study, carried out through the filming and analysis of the baths. All hemodynamically stable preterm infants participated in the study. Data collection took place from June 2018 to June 2019. Data analysis was performed using descriptive statistics. Results: the bath immersion bath showed a great loss in body temperature of 50% (15) of premature infants. The humanized bath did not show any significant loss of temperature in any neonate. Conclusion: the humanized technique promotes better care for the premature newborn, generating less physiological changes

Objetivos: fueron describir y comparar los signos fisiológicos presentados por los bebés prematuros durante la inmersión en un baño y un baño humanizado. Método: estudio cuantitativo, transversal y observacional, realizado a través de la filmación y análisis de los baños. Todos los recién nacidos prematuros hemodinámicamente estables participaron en el estudio. La recopilación de datos se realizó entre junio de 2018 y junio de 2019. El análisis de datos se realizó mediante estadísticas descriptivas. Resultados: el baño de inmersión mostró una gran pérdida de temperatura corporal del 50% (15) de los bebés prematuros. El baño humanizado no mostró ninguna pérdida significativa de temperatura en ningún recién nacido. Conclusión: la técnica humanizada promueve un mejor cuidado del recién nacido prematuro, generando menos cambios fisiológicos

Publisher Correction: Pulmonary venous circulating tumor cell dissemination before tumor resection and disease relapse.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Ensino inclusivo em um curso de educação física: análise das adaptações pedagógicas para o estudante cego na educação superior / Inclusive teaching in a physical education graduation: analysis of pedagogical adaptations for blind student in higher education / Educación inclusiva en un curso de educación física: analisis de adaptaciones pedagógicas para el estudiante ciego en lá educación superior

O objetivo desta pesquisa foi analisar as estratégias de aces- sibilidade pedagógica para atender as necessidades educacionais de um estudante cego, matriculado na disciplina de anatomia humana de um curso de Educação Física em uma universidade pública, por meio da pesquisa-ação. O estudo foi realizado no primeiro semestre de 2018. Verificou-se a necessidade de adaptações na transmissão do conteúdo, da construção de peças anatômicas com materiais de baixo custo em diferentes texturas e a utilização de peças anatômicas sintéticas. Os re- sultados indicam que as adaptações pedagógicas e instrumentais utili - zadas foram essenciais ao processo de ensino-aprendizagem do aluno cego, aliado à sua utilização em conjunto com toda a turma. O estudante atingiu o domínio do conteúdo da disciplina, identificado por meio das avaliações realizadas.

This study aimed to analyze strategies of pedagogical accessibility to meet the educational needs of blind student enrolled in Human Anatomy subject in a Physical Education Graduation of a Public University, Brazil, through an action research. The study was carried out at first semester of 2018. There was need to construction anatomical pieces of low cost with different textures, and use of synthetics anatomical parts. The results indicate that the pedagogical and instrumental adaptations used were essentials to teaching-learning process of the blind student, besides it's used by whole class. The student appropriated the subject identified through the assessment.

El objetivo fue analizar las estrategias de accesibilidad pedagógica para satisfacer las necesidades educativas de un estudiante ciego matriculado en la disciplina de anatomía humana de un curso de Educación Física en una universidad pública, a través da la investigación- acción. El estudio se realizó en el primer semestre de 2018. Los resultados indican que las adaptaciones pedagógicas e instrumentales utilizadas fueron esenciales para el proceso de enseñanza y aprendizaje del estudiante ciego, aliado a su uso junto con toda la clase. El estudiante se apropió del contenido de la disciplina identificada a través de las evaluaciones realizadas.

EGFR-Targeted Nanobody Functionalized Polymeric Micelles Loaded with mTHPC for Selective Photodynamic Therapy.

meta-Tetra(hydroxyphenyl)chlorin (mTHPC) is one of the most potent second-generation photosensitizers, clinically used for photodynamic therapy (PDT) of head and neck squamous cell carcinomas. However, improvements are still required concerning its present formulation (i.e., Foscan, a solution of mTHPC in ethanol/propylene glycol (40:60 w/w)), as mTHPC has the tendency to aggregate in aqueous media, e.g., biological fluids, and it has limited tumor specificity. In the present study, polymeric micelles with three different diameters (17, 24, and 45 nm) based on benzyl-poly(ε-caprolactone)-b-poly(ethylene glycol) (PCLn-PEG; n = 9, 15, or 23) were prepared with mTHPC loadings ranging from 0.5 to 10 wt % using a film-hydration method as advanced nanoformulations for this photosensitizer. To favor the uptake of the micelles by cancer cells that overexpress the epidermal growth factor receptor (EGFR), the micelles were decorated with an EGFR-targeted nanobody (named EGa1) through maleimide-thiol chemistry. The enhanced binding of the EGFR-targeted micelles at 4 °C to EGFR-overexpressing A431 cells, compared to low-EGFR-expressing HeLa cells, confirmed the specificity of the micelles. In addition, an enhanced uptake of mTHPC-loaded micelles by A431 cells was observed when these were decorated with the EGa1 nanobody, compared to nontargeted micelles. Both binding and uptake of targeted micelles were blocked by an excess of free EGa1 nanobody, demonstrating that these processes occur through EGFR. In line with this, mTHPC loaded in EGa1-conjugated PCL23-PEG (EGa1-P23) micelles demonstrated 4 times higher photocytotoxicity on A431 cells, compared to micelles lacking the nanobody. Importantly, EGa1-P23 micelles also showed selective PDT against A431 cells compared to the low-EGFR-expressing HeLa cells. Finally, an in vivo pharmacokinetic study shows that after intravenous injection, mTHPC incorporated in the P23 micelles displayed prolonged blood circulation kinetics, compared to free mTHPC, independently of the presence of EGa1. Thus, these results make these micelles a promising nanomedicine formulation for selective therapy.

Tuning liposome composition to modulate corona formation in human serum and cellular uptake.

Nano-sized objects such as liposomes are modified by adsorption of biomolecules in biological fluids. The resulting corona critically changes nanoparticle behavior at cellular level. A better control of corona composition could allow to modulate uptake by cells. Within this context, in this work, liposomes of different charge were prepared by mixing negatively charged and zwitterionic lipids to different ratios. The series obtained was used as a model system with tailored surface properties to modulate corona composition and determine the effects on liposome interactions with cells. Uptake efficiency and uptake kinetics of the different liposomes were determined by flow cytometry and fluorescence imaging. Particular care was taken in optimizing the methods to isolate the corona forming in human serum to prevent liposome agglomeration and to exclude residual free proteins, which could confuse the results. Thanks to the optimized methods, mass spectrometry of replicate corona isolations showed excellent reproducibility and this allowed semi-quantitative analysis to determine for each formulation the most abundant proteins in the corona. The results showed that by changing the fraction of zwitterionic and charged lipids in the bilayer, the amount and identity of the most abundant proteins adsorbed from serum differed. Interestingly, the formulations also showed very different uptake kinetics. Similar approaches can be used to tune lipid composition in a systematic way in order to obtain formulations with the desired corona and cell uptake behavior. STATEMENT OF SIGNIFICANCE: Liposomes and other nano-sized objects when introduced in biological fluids are known to adsorb biomolecules forming the so-called nanoparticle corona. This layer strongly affects the subsequent interactions of liposomes with cells. Here, by tuning lipid composition in a systematic way, a series of liposomes with tailored surface properties has been prepared to modulate the corona forming in human serum. Liposomes with very different cellular uptake kinetics have been obtained and their corona was identified in order to determine the most enriched proteins on the different formulations. By combining corona composition and uptake kinetics candidate corona proteins associated with reduced or increased uptake by cells can be identified and the liposome formulation can be tuned to obtain the desired uptake behavior.

Pulmonary venous circulating tumor cell dissemination before tumor resection and disease relapse.

Approximately 50% of patients with early-stage non-small-cell lung cancer (NSCLC) who undergo surgery with curative intent will relapse within 5 years1,2. Detection of circulating tumor cells (CTCs) at the time of surgery may represent a tool to identify patients at higher risk of recurrence for whom more frequent monitoring is advised. Here we asked whether CellSearch-detected pulmonary venous CTCs (PV-CTCs) at surgical resection of early-stage NSCLC represent subclones responsible for subsequent disease relapse. PV-CTCs were detected in 48% of 100 patients enrolled into the TRACERx study3, were associated with lung-cancer-specific relapse and remained an independent predictor of relapse in multivariate analysis adjusted for tumor stage. In a case study, genomic profiling of single PV-CTCs collected at surgery revealed higher mutation overlap with metastasis detected 10 months later (91%) than with the primary tumor (79%), suggesting that early-disseminating PV-CTCs were responsible for disease relapse. Together, PV-CTC enumeration and genomic profiling highlight the potential of PV-CTCs as early predictors of NSCLC recurrence after surgery. However, the limited sensitivity of PV-CTCs in predicting relapse suggests that further studies using a larger, independent cohort are warranted to confirm and better define the potential clinical utility of PV-CTCs in early-stage NSCLC.

Riscos ocupacionais no processo de trabalho do agente comunitário de saúde: revisão integrativa / Occupational risks in the work process of the community health agent: integrated review

Objective:To identify, in the scientific literature, publications on the main occupational risks in the work of the Community Health Agent (ACS). Method:Qualitative research of the type integrative literature review, having as guiding question: What are the risks that the ACS are exposed in their work activity? Data collection took place in May 2019, in the Latin American and Caribbean Literature in Health Sciences (LILACS) and Scientific Electronic Library Online (SciELO) databases, through the Virtual Health Library (VHL) portal, the main collection of the Web of Science, through the Portal of Periodicals of Capes. Results:We identified 43 publications that, after filtering according to inclusion and exclusion criteria, rusulted in a sample of 12 articles. Four types of risk were identified, presented in the categories of analysis categories, namely: Physical risks; Chemical risks; Biological risks; Psychosocial risks. Conclusions:Given the importance of the ACS to the Family Health Strategy and public health, it is important to reflect on the working conditions in which these professionals are and its hoped that managers and the ACS and other professionals are expected to mobilize to structure mechanisms of support and defenseof this category, minimizing and eliminating occupational risks.

Objetivo:Identificar, na literatura científica, publicações sobre os principais riscos ocupacionais no trabalho do Agente Comunitário de Saúde (ACS). Método:Pesquisa qualitativa do tipo revisão integrativa de literatura, tendo como questão norteadora: Quais os riscos que os ACS estão expostos na sua atividade laboral? A coleta de dados ocorreu em maio de 2019, nas bases de dados Literatura Latino-Americana e do Caribe em Ciências da Saúde (LILACS) e Scientific Electronic Library Online (SciELO), por meio do portal da Biblioteca Virtual em Saúde (BVS), a coleção principal da Web of Science, através do Portal de Periódicos da Capes. Resultados:Foram identificadas 43 publicações que, após a filtragem segundo critérios de inclusão e exclusão, culminou em uma amostra de 12 artigos. Foram identificados quatro tipos de riscos, apresentados no formato de categorias de análise, a saber: Riscos físicos; Riscos químicos; Riscos biológicos; Riscos psicossociais. Conclusões:Diante da tamanha relevância do ACS para a Estratégia Saúde da Família e a saúde pública, cabe a reflexão sobre as condições laborais nas quais estes profissionais se encontram e espera-se que gestores, os próprios ACS e demais profissionais se mobilizem para estruturar mecanismos de apoio e defesa desta categoria, minimizando e sanando os riscos ocupacionais.

Myocardial infarction before and after the age of 45: Possible role of platelet receptor polymorphisms.

INTRODUCTION: We examined the potential role of polymorphisms of the platelet genes GP1BA (rs2243093, rs6065 and VNTR), ITGB3 (rs5918), ITGA2 (rs938043469) and P2RY12 (rs2046934, rs6801273 and rs6798347) as risk factors for myocardial infarction (MI). METHODS: The study population was divided into three groups: controls (n=235), MI at age ≤45 years (MI ≤45, n=44), and MI at age >45 years (MI >45, n=78). The control group was further divided into two subgroups (control ≤45 and >45), and subgroups including only men were also considered for statistical analysis. Polymorphisms were detected by polymerase chain reaction and restriction fragment length polymorphism analysis. RESULTS: Regarding non-genetic risk factors, the control group differed statistically from the MI ≤45 group (p<00.5) in terms of smoking, hypertension, diabetes and obesity, and from the MI >45 group (p<0.05) in terms of hypertension, diabetes, obesity, family history of thrombosis and high cholesterol. For the studied ITGA2 polymorphism, a statistical difference was found when MI >45 was compared with the control group, with a higher risk of MI in the TT genotype (OR 2.852; 95% CI: 1.092-7.451; p=0.032). In the GP1BA rs6065 polymorphism, a statistically significant difference was found between control ≤45 only men and MI ≤45 only men, with a higher risk in the CT genotype (OR 5.568; 95% CI: 1.421-21.822; p=0.016), despite the low numbers included. The other polymorphisms studied did not show any statistically significant correlations. CONCLUSION: There is a statistically significant association between the TT genotype of the ITGA2 rs938043469 polymorphism and increased risk for MI >45.

Molecular analysis of single circulating tumour cells following long-term storage of clinical samples.

The CellSearch® semiautomated CTC enrichment and staining system has been established as the 'gold standard' for CTC enumeration with CellSearch® CTC counts recognized by the FDA as prognostic for a number of cancers. We and others have gone on to show that molecular analysis of CellSearch® CTCs isolated shortly after CellSearch® enrichment provides another valuable layer of information that has potential clinical utility including predicting response to treatment. Although CellSearch® CTCs can be readily isolated after enrichment, the process of analysing a single CellSearch® patient sample, which may contain many CTCs, is both time-consuming and costly. Here, we describe a simple process that will allow storage of all CellSearch® -enriched cells in glycerol at -20 °C for up to 2 years without any measurable loss in the ability to retrieve single cells or in the genome integrity of the isolated cells. To establish the suitability of long-term glycerol storage for single-cell molecular analysis, we isolated individual CellSearch® -enriched cells by DEPArray™ either shortly after CellSearch® enrichment or following storage of matched enriched cells in glycerol at -20 °C. All isolated cells were subjected to whole-genome amplification (WGA), and the efficacy of single-cell WGA was evaluated by multiplex PCR to generate a Genome Integrity Index (GII). The GII results from 409 single cells obtained from both 'spike-in' controls and clinical samples showed no statistical difference between values obtained pre- and postglycerol storage and that there is no further loss in integrity when DEPArray™-isolated cells are then stored at -80 °C for up to 2 years. In summary, we have established simple yet effective 'stop-off' points along the CTC workflow enabling CTC banking and facilitating selection of suitable samples for intensive analysis once patient outcomes are known.

Gold nanorods induce early embryonic developmental delay and lethality in zebrafish (Danio rerio).

Due to their unique electronic and optical features, gold nanoparticles (AuNP) have received a great deal of attention for application in different fields such as catalysis, electronics, and biomedicine. The large-volume manufacturing predicted for future decades and the inevitable release of these substances into the environment necessitated an assessment of potential adverse human and ecological risks due to exposure to AuNP. Accordingly, this study aimed to examine the acute and developmental toxicity attributed to a commercial suspension of Au nanorods stabilized with cetyltrimethylammonium bromide (CTAB-AuNR) using early embryonic stages of zebrafish (Danio rerio), a well-established model in ecotoxicology. Zebrafish embryos were exposed to CTAB-AuNR (0-150 µg/L) to determine for developmental assessment until 96 hr post fertilization (hpf) and lethality. Uptake of CTAB-AuNR by embryos and nanoparticles potential to induce DNA damage was also measured at 48 and 96 hpf. Analysis of the concentration-response curves with cumulative mortality at 96 hpf revealed a median lethal concentration (LC50,96h) of 110.2 µg/L. At sublethal concentrations, CTAB-AuNR suspensions were found to produce developmental abnormalities such as tail deformities, pericardial edema, decreased body length, and delayed eye, head, and tail elongation development. Further, less than 1% of the initial concentration of CTAB-AuNR present in the exposure media was internalized by zebrafish embryos prior to (48 hpf) and after hatching (96 hpf). In addition, no marked DNA damage was detected in embryos after exposure to CTAB-AuNR. Overall, CTAB-AuNR suspensions produced lethal and sublethal effects on zebrafish embryos with possible repercussions in fitness of adult stages. However, these results foresee a low risk for fish since the observed effects occurred at concentrations above the levels expected to find in the aquatic environment.

Molecular analysis of circulating tumor cells identifies distinct copy-number profiles in patients with chemosensitive and chemorefractory small-cell lung cancer.

In most patients with small-cell lung cancer (SCLC)-a metastatic, aggressive disease-the condition is initially chemosensitive but then relapses with acquired chemoresistance. In a minority of patients, however, relapse occurs within 3 months of initial treatment; in these cases, disease is defined as chemorefractory. The molecular mechanisms that differentiate chemosensitive from chemorefractory disease are currently unknown. To identify genetic features that distinguish chemosensitive from chemorefractory disease, we examined copy-number aberrations (CNAs) in circulating tumor cells (CTCs) from pretreatment SCLC blood samples. After analysis of 88 CTCs isolated from 13 patients (training set), we generated a CNA-based classifier that we validated in 18 additional patients (testing set, 112 CTC samples) and in six SCLC patient-derived CTC explant tumors. The classifier correctly assigned 83.3% of the cases as chemorefractory or chemosensitive. Furthermore, a significant difference was observed in progression-free survival (PFS) (Kaplan-Meier P value = 0.0166) between patients designated as chemorefractory or chemosensitive by using the baseline CNA classifier. Notably, CTC CNA profiles obtained at relapse from five patients with initially chemosensitive disease did not switch to a chemorefractory CNA profile, which suggests that the genetic basis for initial chemoresistance differs from that underlying acquired chemoresistance.

Influence of genetic variations in platelet glycoproteins and eNOS in the development of arterial ischaemia of lower limbs in type 2 diabetes mellitus patients.

Endothelial and platelet dysfunction increase the atherothrombotic risk in diabetes mellitus patients. Therefore, arterial ischaemia of lower limbs is an important complication in diabetes mellitus. In the present work, type 2 diabetic patients were classified by a podiatrist into presence or absence of arterial ischaemia of lower limbs. Several polymorphisms in platelet glycoproteins and eNOS genes were evaluated. Our results suggest that the -5CC genotype in Kozak sequence of GPIbα may be associated with a higher risk of developing arterial ischaemia of lower limbs in type 2 diabetes mellitus patients.

TERT promoter mutations in pancreatic endocrine tumours are rare and mainly found in tumours from patients with hereditary syndromes.

One of the hallmarks of cancer is its unlimited replicative potential that needs a compensatory mechanism for the consequential telomere erosion. Telomerase promoter (TERTp) mutations were recently reported as a novel mechanism for telomerase re-activation/expression in order to maintain telomere length. Pancreatic endocrine tumors (PETs) were so far recognized to rely mainly on the alternative lengthening of telomeres (ALT) mechanism. It was our objective to study if TERTp mutations were present in pancreatic endocrine tumors (PET) and could represent an alternative mechanism to ALT. TERTp mutations were detected in 7% of the cases studied and were mainly associated to patients harbouring hereditary syndromes. In vitro, using PET-derived cell lines and by luciferase reporter assay, these mutations confer a 2 to 4-fold increase in telomerase transcription activity. These novel alterations are able to recruit ETS transcription factor members, in particular GABP-α and ETV1, to the newly generated binding sites. We report for the first time TERTp mutations in PETs and PET-derived cell lines. Additionally, our data indicate that these mutations serve as an alternative mechanism and in an exclusive manner to ALT, in particular in patients with hereditary syndromes.

Clinical evaluation of a novel microfluidic device for epitope-independent enrichment of circulating tumour cells in patients with small cell lung cancer.

Circulating tumour cells (CTCs) have potential utility as minimally-invasive biomarkers to aid cancer treatment decision making. However, many current CTC technologies enrich CTCs using specific surface epitopes that do not necessarily reflect CTC heterogeneity. Here we evaluated the epitope-independent Parsortix system which enriches CTCs based on size and rigidity using both healthy normal volunteer blood samples spiked with tumour cells and blood samples from patients with small cell lung cancer (SCLC). Blood samples were maintained unfractionated at room temperature for up to 4 days followed by plasma removal for circulating free DNA (cfDNA) isolation and direct application of the remaining cell component to the Parsortix system. For tumour cells expressing the EpCAM cell surface marker the numbers of spiked cells retained using the Parsortix system and by EpCAM-positive selection using CellSearch® were not significantly different, whereas only the Parsortix system showed strong enrichment of cells with undetectable EpCAM expression. In a pilot clinical study we banked both enriched CTCs as well as plasma from SCLC patient blood samples. Upon retrieval of the banked Parsortix cellular samples we could detect cytokeratin positive CTCs in all 12 SCLC patients tested. Interestingly, processing parallel samples from the same patients by EpCAM enrichment using CellSearch® revealed only 83% (10/12) with cytokeratin positive CTCs indicating the Parsortix system is enriching for EpCAM negative SCLC CTCs. Our combined results indicate the Parsortix system is a valuable tool for combined cfDNA isolation and CTC enrichment that enables CTC analysis to be extended beyond dependence on surface epitopes.

Allelic and genotypic frequencies of platelet glycoprotein polymorphisms in a Portuguese population.

INTRODUCTION AND OBJECTIVES: We studied genotypic and allelic frequencies of polymorphisms that can affect platelet function, namely the Kozak, VNTR and HPA-2 polymorphisms of glycoprotein Ibα, the Pl(A) polymorphism of glycoprotein IIIa and the C807T polymorphism of glycoprotein Ia, in a Portuguese population composed of 227 donors. METHODS: PCR-RFLP was used to assess the Kozak, HPA-2, Pl(A) and C807T polymorphisms. The VNTR polymorphism was discriminated by different weight bands on electrophoresis. RESULTS: All genotypic frequencies were in Hardy-Weinberg equilibrium and do not differ from other Caucasian populations. Genotypic frequencies were 68.3%, 26.9% and 4.8% for Pl(A1/A1), Pl(A1/A2) and Pl(A2/A2) genotypes of the Pl(A) polymorphism, 79.3%, 20.3% and 0.4% for TT, TC and CC genotypes of the Kozak polymorphism, 81.1%, 18.9% and 0.0% for aa, ab and bb genotypes of the HPA-2 polymorphism, 15.4%, 0.9%, 70.5%, 11.5%, 1.3% and 0.4% for BC, BD, CC, CD, DD and CE genotypes of the VNTR polymorphism, and 39.7%, 50.2% and 10.1% for CC, CT and TT genotypes of the C807T polymorphism. CONCLUSIONS: The Portuguese population has now been characterized in terms of major platelet glycoprotein polymorphisms, which will be an important tool for further studies to assess the role of platelet glycoproteins in individual predisposition to prothrombotic conditions and response to antithrombotic therapy.