Abstract Objective To investigate methodological aspects in body fat (BF) measurements in 7-to-10-year-old children. Study design Systematic review of the literature. Methods The studies were chosen ...from the PubMed and Scielo databases according to a protocol that defined: inclusion criteria; a search and quality-assessment strategy; and information extraction. Results 27 studies published from 2004 to 2014 were included. The literature describes skinfold measurements and dual energy X-ray absorptiometry (DEXA) as being the reference methods most widely used in the assessment of the ability of methods to identify BF. The most commonly-used statistical analyses were the Pearson correlation coefficient, and sensitivity and specificity performance analyses. The comparison between the tested methods and the references showed that body mass index (BMI) and waist circumference (WC) are strongly correlated to BF as calculated by bioelectrical impedance or skinfolds, and that there is a moderate positive correlation with percent body fat as calculated by DEXA, air-displacement plethysmography (ADP) or isotope dilution. There was a moderate positive correlation between weight-to-height ratio (WtHR) and BF, as estimated by ADP and skinfolds. Performance studies suggest that BMI and WC are very specific but less sensitive methods. Conclusions The results of this systematic review show favourable evidence for the use of anthropometric indicators – above all BMI and WC– in the measurement of BF, when more accurate techniques such as DEXA and ADP are not feasible. They also demonstrate features that make them advantageous for epidemiological studies in a child population, since they are easy and safe to obtain and well tolerated by the children.
Currently the study of Regulated Cell Death (RCD) processes is limited to the use of lysed cell populations for Western blot analysis of each separate RCD process. We have previously shown that ...intracellular antigen flow cytometric analysis of RIP3, Caspase-3 and cell viability dye allowed the determination of levels of apoptosis (Caspase-3
+ ve
/RIP3
− ve
), necroptosis (RIP3
Hi + ve
/Caspase-3
− ve
) and RIP1-dependent apoptosis (Caspase-3
+ ve
/RIP3
+ ve
) in a single Jurkat cell population. The addition of more intracellular markers allows the determination of the incidence of parthanatos (PARP), DNA Damage Response (DDR, H2AX), H2AX hyper-activation of PARP (H2AX/PARP) autophagy (LC3B) and ER stress (PERK), thus allowing the identification of 124 sub-populations both within live and dead cell populations. Shikonin simultaneously induced Jurkat cell apoptosis and necroptosis the degree of which can be shown flow cytometrically together with the effects of blockade of these forms of cell death by zVAD and necrostatin-1 have on specific RCD populations including necroptosis, early and late apoptosis and RIP1-dependent apoptosis phenotypes in live and dead cells. Necrostatin-1 and zVAD was shown to modulate levels of shikonin induced DDR, hyper-action of PARP and parthanatos in the four forms of RCD processes analysed. LC3B was up-regulated by combined treatment of zVAD with chloroquine which also revealed that DNA damage was reduced in live cells but enhanced in dead cells indicating the role of autophagy in maintaining cell health. This approach to RCD research should be a great advance to understanding the mechanisms of drugs and their effects upon RCD populations.
Summary
Background
Gap‐junctional intercellular communication is crucial for epidermal cellular homeostasis. Inability to establish melanocyte–keratinocyte contact and loss of the intercellular ...junction’s integrity may contribute to melanoma development. Connexins, laminins and desmocollins have been implicated in the control of melanoma growth, where their reduced expression has been reported in metastatic lesions.
Objectives
The aim of this study was to investigate connexin 31·1 (GJB5) expression and identify any association with BRAF mutational status, prognosis of patients with melanoma and mitogen‐activated protein kinase (MAPK) inhibitor (MAPKi) treatment.
Methods
GJB5 expression was measured at RNA and protein level in melanoma clinical samples and established cell lines treated (or not) with BRAF and MEK inhibitors (MEKi), as well as in cell lines which developed MAPKi resistance. Findings were further validated and confirmed by analysis of independent datasets.
Results
Our analysis reveals significant downregulation of GJB5 expression in metastatic melanoma lesions compared with primary ones and in BRAF‐mutated vs. BRAF‐wildtype (BRAFWT) melanomas. Likewise, GJB5 expression is significantly lower in BRAFV600E compared with BRAFWT cell lines and increases on MAPKi treatment. MAPKi‐resistant melanoma cells display a similar expression pattern compared with BRAFWT cells, with increased GJB5 expression associated with morphological changes. Enhancement of BRAFV600E expression in BRAFWT melanoma cells significantly upregulates miR‐335‐5p expression with consequent downregulation of GJB5, one of its targets. Furthermore, overexpression of miR‐335‐5p in two BRAFWT cell lines confirms specific GJB5 protein downregulation. Reverse transcriptase quantitative polymerase chain reaction analysis also revealed upregulation of miR‐335 in BRAFV600E melanoma cells, which is significantly downregulated in cells resistant to MEKi. Our data were further validated using the TCGA_SKCM dataset, where BRAF mutations associate with increased miR‐335 expression and inversely correlate with GJB5 expression. In clinical samples, GJB5 underexpression is also associated with patient overall worse survival, especially at early stages.
Conclusions
We identified a significant association between metastases/BRAF mutation and low GJB5 expression in melanoma. Our results identify a novel mechanism of gap‐junctional protein regulation, suggesting a prognostic role for GJB5 in cutaneous melanoma.
What is already known about this topic?
GJB5 expression has never been studied in melanoma.
Although there is very limited knowledge about connexins in melanoma, these types of gap‐junction proteins have recently been linked with late stages of tumorigenesis and metastasis and are considered as tumour suppressors.
What does this study add?
This study establishes a significant association between BRAFV600E, metastases and GJB5 downregulation in melanoma.
GJB5 underexpression also correlates with worse patient overall survival. Therefore, the data support a prognostic role for GJB5 in cutaneous melanoma.
What is the translational message?
This study highlights the importance of monitoring the integrity of connexin and junctional proteins during melanomagenesis, providing novel therapeutic target options for melanoma treatment, as well as a novel prognostic biomarker to predict melanoma progression.
Linked Comment: J.E. Fromme and P. Zigrino. Br J Dermatol 2022; 186:13–14.
Plain language summary available online
•Photon correlation spectroscopy allows to detect differences between tears of contact lens wearers of different materials.•Average hydrodynamic diameter of tear particles of IV-FDA hydrogel wearers ...is 50% higher compared to non-wearers.•Average hydrodynamic diameter of tear particles of silicone-hydrogel wearers is 40% higher compared to non-wearers.•Average hydrodynamic diameter of tear particles of IV-FDA hydrogel wearers is 30% higher compared to II-FDA hydrogel wearers.•Average hydrodynamic diameter of tear particles of silicone-hydrogel wearers is 20% higher compared to II-FDA hydrogel wearers.
The purpose was to evaluate if there are differences between tears of contact lens (CL) wearers of different materials detectable by measuring the hydrodynamic diameter of tear components through photon correlation spectroscopy (PCS).
Tears of 59 CL wearers and tears of 39 non-wearers were collected by glass capillary. Wearers were divided into groups depending on the CL material: (i) hydrogels of II FDA group (H-II, 15 subjects), (ii) hydrogels of IV FDA group (H-IV, 13 subjects), (iii) silicone hydrogels (SH, 31 subjects). PCS analyses were performed at 25 °C on samples diluted with deionized water with tear concentration (10 ± 2)% V/W to obtain, for each subject, the average hydrodynamic diameter (dH,avg) of tear components by analyzing intensity fluctuations in time of scattered light.
Means of dH,avg calculated on each group were found, on increasing order, to be 256 nm (std dev 18 nm) for non-wearers, 297 nm (std dev 45 nm) for H-II, 360 nm (std dev 76 nm) for SH, and 391 nm (std dev 85 nm) for H-IV with statistical differences between each group of wearers compared to non-wearers and between groups of wearers except between SH and H-IV.
PCS reveals the differences between tears of CL wearers of different materials, not only between tears of wearers and non-wearers.
RAS mutations occur frequently in human cancer and activated RAS signalling contributes to tumour development and progression. Apart from its oncogenic effects on cell growth, active RAS has ...tumour-suppressive functions via its ability to induce cellular senescence and apoptosis. RAS is known to induce p53-dependent cell cycle arrest, yet its effect on p53-dependent apoptosis remains unclear. We report here that apoptosis-stimulating protein of p53 (ASPP) 1 and 2, two activators of p53, preferentially bind active RAS via their N-terminal RAS-association domains (RAD). Additionally, ASPP2 colocalises with and contributes to RAS cellular membrane localisation and potentiates RAS signalling. In cancer cells, ASPP1 and ASPP2 cooperate with oncogenic RAS to enhance the transcription and apoptotic function of p53. Thus, loss of ASPP1 and ASPP2 in human cancer cells may contribute to the full transforming property of RAS oncogene.
Novel prognostic biomarkers and therapeutic strategies are urgently required for malignant melanoma. Ecto-5-prime-nucleotidase (NT5E; CD73) overexpression has been reported in several human cancers. ...The mechanism(s) underlying deregulated expression and the clinical consequences of changes in expression are not known.
We used RT-PCR, qPCR, methylation-specific PCR and pyrosequencing to analyse expression and regulation of NT5E in malignant melanoma cell lines and primary and metastatic melanomas.
NT5E is subject to epigenetic regulation in melanoma. NT5E mRNA is downregulated by methylation-dependent transcriptional silencing in the melanoma cell lines SKMel2, SKMel23, WM35, Mel501, Mel505 and C81-61 and expression is reactivated by azacytidine. In contrast, the CpG island is unmethylated and the gene expressed in cultured normal melanocytes. In clinical cases of melanoma, methylation in the NT5E CpG island occurs in both primary and metastatic melanomas and correlates with transcriptional downregulation of NT5E mRNA. Relapse with metastatic disease, particularly to the visceral sites and brain, is more common in primary melanomas lacking NT5E methylation. Primary melanomas with methylation in NT5E show limited metastatic potential or more commonly metastasise predominantly to nodal sites rather than viscera and brain (P=0.01).
Deregulation of NT5E expression in melanoma occurs via epigenetic changes in the NT5E CpG island. Confirmation of our results in larger clinical series would support the candidacy of NT5E as a clinical biomarker in melanoma, which could be applied in both primary and relapsed disease. Inhibition of NT5E may have therapeutic potential in melanoma, particularly in patients with more aggressive disease metastatic to viscera or the brain.
The mode of action of Ecteinascidin-743 (ET-743), a marine tetrahydroisoquinoline alkaloid isolated from
Ecteinascidia turbinata, which has shown very potent antitumour activity in preclinical ...systems and encouraging results in Phase I clinical trials was investigated at a cellular level. Both SW620 and LoVo human intestinal carcinoma cell lines exposed for 1 h to ET-743 progress through S phase more slowly than control cells and then accumulate in the G
2M phase. The sensitivity to ET-743 of G
1 synchronised cells was much higher than that of cells synchronised in S phase and even higher than that of cells synchronised in G
2M. ET-743 concentrations up to four times higher than the IC
50 value caused no detectable DNA breaks or DNA–protein cross-links as assessed by alkaline elution techniques. ET-743 induced a significant increase in p53 levels in cell lines expressing wild-type (wt) (p53). However, the p53 status does not appear to be related to the ET-743 cytotoxic activity as demonstrated by comparing the drug sensitivity in p53 (−/−) or (+/+) mouse embryo fibroblasts and in A2780 ovarian cancer cells or the A2780/CX3 sub-line transfected with a dominant-negative mutant
TP53. The cytotoxic potency of ET-743 was comparatively evaluated in CHO cell lines proficient or deficient in nucleotide excision repair (NER), and it was found that ET-743 was approximately 7–8 times less active in
ERCC3/XPB and
ERCC1-deficient cells than control cells. The findings that G
1 phase cells are hypersensitive and that NER-deficient cells are resistant to ET-743 indicate that the mode of action of ET-743 is unique and different from that of other DNA-interacting drugs.
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