Assessment of RAS-RAF-MAPK Pathway Mutation Status in Healthy Skin, Benign Nevi, and Cutaneous Melanomas: Pilot Study Using Droplet Digital PCR.

In the present study, we employed the ddPCR and IHC techniques to assess the prevalence and roles of RAS and RAF mutations in a small batch of melanoma (n = 22), benign moles (n = 15), and normal skin samples (n = 15). Mutational screening revealed the coexistence of BRAF and NRAS mutations in melanomas and nevi and the occurrence of NRAS G12/G13 variants in healthy skin. All investigated nevi had driver mutations in the BRAF or NRAS genes and elevated p16 protein expression, indicating cell cycle arrest despite an increased mutational burden. BRAF V600 mutations were identified in 54% of melanomas, and NRAS G12/G13 mutations in 50%. The BRAF mutations were associated with the Breslow index (BI) (p = 0.029) and TIL infiltration (p = 0.027), whereas the NRAS mutations correlated with the BI (p = 0.01) and the mitotic index (p = 0.04). Here, we demonstrate that the "young" ddPCR technology is as effective as a CE-IVD marked real-time PCR method for detecting BRAF V600 hotspot mutations in tumor biopsies and recommend it for extended use in clinical settings. Moreover, ddPCR was able to detect low-frequency hotspot mutations, such as NRAS G12/G13, in our tissue specimens, which makes it a promising tool for investigating the mutational landscape of sun-damaged skin, benign nevi, and melanomas in more extensive clinical studies.

Assessment of Immune Cell Populations in Tumor Tissue and Peripheral Blood Samples from Head and Neck Squamous Cell Carcinoma Patients.

Head and neck squamous cell carcinoma (HNSCC) is a common type of cancer worldwide. Strong connections have been revealed between immune cells and the pathogenesis of HNSCC. Important differences regarding the levels of immune cell subpopulations in both peripheral circulation and tumor microenvironment were emphasized, with some of them having prognostic significance. In our study, we performed an analysis of immune changes in the tumor tissue and the peripheral blood of untreated HNSCC patients, investigating the proportions of different immune cell populations in these two compartments. The local infiltrating lymphocytes were mainly cytotoxic T cells (CD8+). We have also revealed an increased level of B lymphocytes (CD19+) in the tumor microenvironment. In peripheral blood, the most important lymphocyte subtype was represented by the helper T lymphocytes (CD4+). We also found an increased proportion of circulating NK cells (CD56+). Our results showed significant differences between all investigated lymphocyte subtypes in the peripheral blood and the tumor tissue of untreated HNSCC patients, suggesting that the local and systemic expressions of antitumor immune responses are different and that investigation of immune cell proportions in peripheral circulation has different cues that do not reflect the immune infiltrate pattern within the tumor microenvironment. Further studies are necessary to unveil the complex interplay involving local and systemic events in the immune system's fight against cancer.

Reliability of Repeated Trials Protocols for Body Composition Assessment by Air Displacement Plethysmography.

Air displacement plethysmography (ADP) is fast, accurate, and reliable. Nevertheless, in about 3% of the cases, standard ADP tests provide rogue results. To spot these outliers and improve precision, repeated trials protocols have been devised, but few works have addressed their reliability. This study was conducted to evaluate the test-retest reliabilities of two known protocols and a new one, proposed here. Ninety-two healthy adults (46 men and 46 women) completed six consecutive ADP tests. To evaluate the reliability of single measurements, we used the results of the first two tests; for multiple measures protocols, we computed the test result from trials 1-3 and the retest result from trials 4-6. Bland-Altman analysis revealed that the bias and the width of the 95% interval of agreement were smaller for multiple trials than for single ones. For percent body fat (%BF)/fat-free mass, the technical error of measurement was 1% BF/0.68 kg for single trials and 0.62% BF/0.46 kg for the new protocol of multiple trials, which proved to be the most reliable. The minimal detectable change (MDC) was 2.77% BF/1.87 kg for single trials and 1.72% BF/1.26 kg for the new protocol.

Safety and efficacy assessment of aerogels for biomedical applications.

The unique physicochemical properties of aerogels have made them an attractive class of materials for biomedical applications such as drug delivery, regenerative medicine, and wound healing. Their low density, high porosity, and ability to regulate the pore structure makes aerogels ideal nano/micro-structures for loading of drugs and active biomolecules. As a result of this, the number of in vitro and in vivo studies on the therapeutic efficacy of these porous materials has increased substantially in recent years and continues to be an area of great interest. However, data about their in vivo performance and safety is limited. Studies have shown that polymer-based, silica-based and some hybrid aerogels are generally regarded as safe but given that studies on the acute, subacute, and chronic toxicity for the majority of aerogel types is missing, more work is still needed. This review presents a comprehensive summary of different biomedical applications of aerogels proposed to date as well as new and innovative applications of aerogels in other areas such as decontamination. We have also reviewed their biological effect on cells and living organisms with a focus on therapeutic efficacy and overall safety (in vivo and in vitro).

Reliability of body composition assessment using A-mode ultrasound in a heterogeneous sample.

BACKGROUND/OBJECTIVES: Several studies have addressed the validity of ultrasound (US) for body composition assessment, but few have evaluated its reliability. This study aimed to determine the reliability of percent body fat (%BF) estimates using A-mode US in a heterogeneous sample. SUBJECTS/METHODS: A group of 144 healthy adults (81 men and 63 women), 30.4 (10.1) years (mean (SD)), BMI 24.6 (4.7) kg/m2, completed 6 consecutive measurements of the subcutaneous fat layer thickness at 8 anatomical sites. The measurements were done, alternatively, by two testers, using a BodyMetrix™ instrument. To compute %BF, 4 formulas from the BodyView™ software were applied: 7-sites Jackson and Pollock, 3-sites Jackson and Pollock, 3-sites Pollock, and 1-point biceps. RESULTS: The formula with the most anatomic sites provided the best reliability quantified by the following measures: intraclass correlation coefficient (ICC) = 0.979 for Tester 1 (T1) and 0.985 for T2, technical error of measurement (TEM) = 1.07% BF for T1 and 0.89% BF for T2, and minimal detectable change (MDC) = 2.95% BF for T1, and 2.47% BF for T2. The intertester bias was -0.5% BF, whereas the intertester ICC was 0.972. The intertester MDC was 3.43% BF for the entire sample, 3.24% BF for men, and 3.65% BF for women. CONCLUSIONS: A-mode US is highly reliable for %BF assessments, but it is more precise for men than for women. Examiner performance is a source of variability that needs to be mitigated to further improve the precision of this technique.

Critical assessment and integration of separate lines of evidence for risk assessment of chemical mixtures.

Humans are exposed to multiple chemicals on a daily basis instead of to just a single chemical, yet the majority of existing toxicity data comes from single-chemical exposure. Multiple factors must be considered such as the route, concentration, duration, and the timing of exposure when determining toxicity to the organism. The need for adequate model systems (in vivo, in vitro, in silico and mathematical) is paramount for better understanding of chemical mixture toxicity. Currently, shortcomings plague each model system as investigators struggle to find the appropriate balance of rigor, reproducibility and appropriateness in mixture toxicity studies. Significant questions exist when comparing single-to mixture-chemical toxicity concerning additivity, synergism, potentiation, or antagonism. Dose/concentration relevance is a major consideration and should be subthreshold for better accuracy in toxicity assessment. Previous work was limited by the technology and methodology of the time, but recent advances have resulted in significant progress in the study of mixture toxicology. Novel technologies have added insight to data obtained from in vivo studies for predictive toxicity testing. These include new in vitro models: omics-related tools, organs-on-a-chip and 3D cell culture, and in silico methods. Taken together, all these modern methodologies improve the understanding of the multiple toxicity pathways associated with adverse outcomes (e.g., adverse outcome pathways), thus allowing investigators to better predict risks linked to exposure to chemical mixtures. As technology and knowledge advance, our ability to harness and integrate separate streams of evidence regarding outcomes associated with chemical mixture exposure improves. As many national and international organizations are currently stressing, studies on chemical mixture toxicity are of primary importance.

Toxicological and efficacy assessment of post-transition metal (Indium) phthalocyanine for photodynamic therapy in neuroblastoma.

Metallo-phthalocyanines due to their photophysical characteristics as high yield of triplet state and long lifetimes, appear to be good candidates for photodynamic therapy (PDT). Complexes with diamagnetic metals such as Zn2+, Al3+ Ga3+ and In3+meet such requirements and are recognized as potential PDT agents. Clinically, Photofrin® PDT in neuroblastoma therapy proved in pediatric subjects diagnosed with progressive/recurrent malignant brain tumors increased progression free survival and overall survival outcome. Our study focuses on the dark toxicity testing of a Chloro-Indium-phthalocyanine photosensitizer (In-Pc) upon SH-SY5Y neuroblastoma cell line and its experimental in vitro PDT. Upon testing, In-Pc has shown a relatively high singlet oxygen quantum yield within the cells subjected to PDT (0.553), and 50 µg/mL IC50. Classical toxicological and efficacy assessment were completed with dynamic cellular impedance measurement methodology. Using this technology we have shown that long time incubation of neuroblastoma cell lines in In-Pc (over 5 days) does not significantly hinder cell proliferation when concentration are ≤ 10 µg/mL. When irradiating neuroblastoma cells loaded with non-toxic concentration of In-Pc, 50% of cells entered apoptosis. Transmission electron microscopy has confirmed apoptotic characteristics of cells. Investigating the proliferative capacity of the in vitro treated cells we have shown that cells that "escape" the irradiation protocol, present a reduced proliferative capacity. In conclusion, In-Pc represents another photosensitizer that can display sound PDT properties enhancing neuroblastoma therapy armentarium.

Computer simulations of in vitro morphogenesis.

One of the most important challenges of contemporary biology is understanding how cells assemble into tissues. The complexity of morphogenesis calls for computational tools able to identify the dominant mechanisms involved in shaping tissues. This narrative review presents individual-based computational models that proved useful in simulating phenomena of interest in tissue engineering (TE), a research field that aims to create tissue replacements in the laboratory. First, we briefly describe morphogenetic mechanisms. Then, we present several computational models of cellular and subcellular resolution, along with applications that illustrate their potential to address problems of TE. Finally, we analyze experiments that may be used to validate computational models of tissue constructs made of cohesive cells. Our analysis shows that the models available in the literature are not exploited to their full potential. We argue that, upon validation, a computational model can be used to optimize cell culture conditions and to design new experiments.

Computational modeling of epithelial-mesenchymal transformations.

An epithelial-mesenchymal transformation (EMT) involves alterations in cell-cell and cell-matrix adhesion, the detachment of epithelial cells from their neighbors, the degradation of the basal lamina and acquisition of mesenchymal phenotype. Here we present Monte Carlo simulations for a specific EMT in early heart development: the formation of cardiac cushions. Cell rearrangements are described in accordance with Steinberg's differential adhesion hypothesis, which states that cells possess a type-dependent adhesion apparatus and are sufficiently motile to give rise to the tissue conformation with the largest number of strong bonds. We also implement epithelial and mesenchymal cell proliferation, cell type change and extracellular matrix production by mesenchymal cells. Our results show that an EMT is promoted more efficiently by an increase in cell-substrate adhesion than by a decrease in cell-cell adhesion. In addition to cushion tissue formation, the model also accounts for the phenomena of matrix invasion and mesenchymal condensation. We conclude that in order to maintain epithelial integrity during EMT the number of epithelial cells must increase at a controlled rate. Our model predictions are in qualitative agreement with available experimental data.