Regulation of Focal Adhesion Dynamics and Cell Migration by PLC/PI3K-Mediated Metabolism of PtdIns (4,5) P2 in a Breast Cancer Cell Line

Background:Focal adhesions (FAs) are highly dynamic complex structures that assembled and disassembled on an ongoing basis. The balance between the two processes mediates various aspects of cell behavior, ranging from cell adhesion to cell migration. Assembly and disassembly processes of FAs are regulated by a variety of cellular signaling proteins and adaptors. We previously demonstrated that local levels of Phosphatidylinositol 4,5‐bisphosphate (PtdIns(4,5)P2) in MDA-MB-231 cells increases during FA assembly and declines during disassembly. In this study we aimed to investigate whether PtdIns(4,5)P2 regulates FA turnover.Methods:MDA-MB-231 cells were co-transfected with a labeling vinculin (or zyxin) and the PLC𝛅1-PH biosensor to visualize FA localization and PtdIns(4,5)P2 in the cell membrane. We also used pharmacological inhibitors to determine the mechanism underlying the changes of PtdIns(4,5)P2 level during FA turnover and cell migration. Immunostaining, immunoprecipitation, and Western blotting were used to examine the localization and interaction between phospholipase C (PLC)/phosphatidylinositol 3-kinase (PI3K) FA proteins.Results:We showed that inhibition of PLC, PI3K significantly reduced the decline of PtdIns(4,5)P2 levels within FA disassembly and the slowdown rate of FA turnover and cell migration. We also showed that the inhibition of enzymes implicated in the downstream pathway of PtdIns(4,5)P2, such as diacylglycerol kinase (DAGK) and protein kinase C (PKC) significantly reduced FA turnover time and the speed of cell migration. Additionally, we demonstrated that PLC but not PI3K interact with FAs. In conclusion,DiscussionThis study suggests that dynamical changes of PtdIns(4,5)P2 might regulate FA turnover and facilitate cell migration.Key Words: Cell Migration, Focal adhesion turnover, MDA-MB-231 Breast Cancer Cell Line, Ptdins(4,5)P2, PLC, PI3K     

Genetic diversity of SSR and ISSR markers in wild populations of Brachypodium distachyon and its close relatives B. stacei and B. hybridum (Poaceae)

The genetic diversity of seven wild populations of the grass Brachypodium distachyon (2n = 10), 4 of B. stacei (2n = 20) and 13 of B. hybridum (2n = 30) from the Mediterranean and southern areas of the Iberian Peninsula was studied via the analysis of microsatellite (SSR) and inter-microsatellite (ISSR) markers. The 11 SSR markers analysed provided a total of 156 polymorphic fragments. The B. hybridum populations returned more fragments (98) than the B. stacei populations (87), and more than twice the number recorded for the B. distachyon populations (44). Some fragments were specific to the B. distachyon (3.85 %), B. stacei (27.56 %) or B. hybridum populations (18.58 %). The analysis of 16 ISSR markers returned similar results: the B. hybridum populations returned more polymorphic fragments than the B. distachyon or B. stacei populations. The analysis of molecular variance, with distances between individuals, populations and species estimated on the basis of the presence/absence of the SSR and ISSR fragments, showed most of the variation (67 %) to occur among populations. This was followed by differences among individuals within populations (24 %), and finally among species (9 %). Grouping based on UPGMA and principal coordinate analysis showed a clear separation of three groups corresponding to the populations of the same species. Principal component analysis, involving chromosome number, low-molecular weight-glutenin subunits and the most influential climatic and geographic factors, was also performed. This revealed an obvious separation among the populations of B. distachyon and B. hybridum. The index of Mediterraneity and altitude explained 70.56 % of the total variation associated with the first axis. A trend was seen towards a greater presence of B. distachyon forms in areas of the Peninsular interior and higher altitude, with the B. hybridum forms more common in regions of greater summer rainfall and a lower index of Mediterraneity.     

Prepubertal goat oocytes from large follicles result in similar blastocyst production and embryo ploidy than those from adult goats

Developmental competence of oocytes from prepubertal females is lower than those from adult females. Oocyte development competence is positively related to follicular diameter. Most of the follicles of prepubertal goat ovaries are smaller than 3 mm. The aim of this study was to compare oocytes of two follicle sizes (< 3 mm and ≥ 3 mm) from prepubertal goats with oocytes from adult goats in relation to their in vitro production and quality of blastocysts. Oocytes from prepubertal goats were obtained from slaughterhouse ovaries and selected according to the follicle diameter whereas oocytes from adult goats were recovered in vivo by LOPU technique without prior selection of follicle size. COCs were IVM for 27 h, IVF at the conventional conditions with fresh semen and presumptive zygotes were cultured in SOF medium for 8 days. Blastocysts obtained were vitrified and after warming their blastocoele re-expansion and the ploidy by FISH technique were assessed. We found significant differences between blastocysts yield of oocytes recovered from follicles smaller than 3 mm of prepubertal goats compared to those from adult goats (5.45% vs 20. 83%, respectively) however, these differences disappear if oocytes were recovered form large follicles (18.07%). A total of 28 blastocysts were analysed and 96.43% showed mixoploidy. Age did not affect the number of embryos with abnormal ploidy or blastocyst re-expansion after warming. Furthermore, the percentage of diploid blastomeres per embryo was similar in the 3 groups studied, adult, prepubertal from follicles ≥ 3 mm and < 3 mm (68.6%, 80.8% and 73.6%, respectively). In conclusion, IVP of blastocysts coming from follicles larger than 3 mm of goats 45 days old were not different to the blastocysts produced from adult goats, both in terms of quantity and quality.     

Cell Volume Changes Regulate Slick (Slo2.1), but Not Slack (Slo2.2) K+ Channels

Slick (Slo2.1) and Slack (Slo2.2) channels belong to the family of high-conductance K⁺ channels and have been found widely distributed in the CNS. Both channels are activated by Na⁺ and Cl⁻ and, in addition, Slick channels are regulated by ATP. Therefore, the roles of these channels in regulation of cell excitability as well as ion transport processes, like regulation of cell volume, have been hypothesized. It is the aim of this work to evaluate the sensitivity of Slick and Slack channels to small, fast changes in cell volume and to explore mechanisms, which may explain this type of regulation. For this purpose Slick and Slack channels were co-expressed with aquaporin 1 in Xenopus laevis oocytes and cell volume changes of around 5% were induced by exposure to hypotonic or hypertonic media. Whole-cell currents were measured by two electrode voltage clamp. Our results show that Slick channels are dramatically stimulated (196% of control) by cell swelling and inhibited (57% of control) by a decrease in cell volume. In contrast, Slack channels are totally insensitive to similar cell volume changes. The mechanism underlining the strong volume sensitivity of Slick channels needs to be further explored, however we were able to show that it does not depend on an intact actin cytoskeleton, ATP release or vesicle fusion. In conclusion, Slick channels, in contrast to the similar Slack channels, are the only high-conductance K⁺ channels strongly sensitive to small changes in cell volume.     

Intérêt de la scintigraphie rénale au DMSA dans les pyélonéphrites aiguës de l’enfant

IntroductionAcute pyelonephritis is responsible for significant morbidity during the acute phase and distant irreversible kidney damage. However, diagnosis is often difficult in children. The aim of our study was to evaluate in children hospitalized for suspected acute pyelonephritis prevalence of acute lesions revealed by scintigraphy with ^99mTc labeled dimercaptosuccinic acid (DMSA) and the relationship between renal parenchymal lesions and different clinical factors and biological risks.MethodsThis is a prospective study collecting 40 children admitted with suspected infection of the high urinary tract. All have benefited from DMSA renal scintigraphy.ResultsThe average age of our patients was 3.25 years. The DMSA scan showed signs of acute pyelonephritis in 32.5% of cases. Taking this examination as a reference, fever was the most sensitive clinical signs (76.9%), but the specificity was only 48.1%. CRP (≥ 20 mg/mL) was the most sensitive (92.3%) but with a specificity of 40.7% organic signs. A high CRP was associated with acute pyelonephritis significantly (P = 0.0033). Sixty-three percent of refluxing kidneys were associated with acute pyelonephritis versus 20.8% of non-refluxing kidneys, with a significant difference (P = 0.007).ConclusionIn case of urinary tract infection in children, a complete clinical and laboratory table may be sufficient to retain the diagnosis of acute pyelonephritis. However, this possibility is rare. In the opposite case, a DMSA scan should be performed to confirm the diagnosis.     

A Comparative Study to Visualize PtdIns(4,5) P2 and PtdIns(3,4,5) P3 in MDA-MB-231 Breast Cancer Cell Line

Background:Phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5) P3) and Phosphatidylinositol 4,5-trisphosphate (PtdIns(4,5) P2] form an insignificant amount of phospholipids but play important roles in controlling membrane-bound signalling. Little attention has been given to visualize and monitor changes or differences in the local generation of PtdIns(4,5) P2 and PtdIns(3,4,5) P3 in the cell membranes of MDA-MB-231 breast cancer cell lines.Methods:PLCδ1-PH-GFP and Btk-PH-GFP were used as biosensors to detected PtdIns(4,5) P2 and PtdIns(3,4,5)P3 respectively. These biosensors and antibodies were transfected, immuostained and then visualized by confocal microscopy on different cell surfaces.Results:Our results showed that PLCδ1-PH-GFP/mCherry was localized at the cell membrane, while Btk-PH-GFP/mCherry was sometimes localized at the cell membrane but there was also a large amount of fluorescence present in the cytosol and nucleus. Our results also showed that the cells that expressed low levels of Btk-PH-GFP the fluorescence was predominantly localised to the cell membrane. While the cells that expressed high levels of Btk-PH-GFP the fluorescence was localization in the cytosol and cell membrane. Our results demonstrated that both anti-PtdIns(4,5)P2 and anti-PtdIns(3,4,5)P3 antibodies were localized everywhere in cell.Conclusion:Our results suggest that PLCδ1-PH-GFP and Btk-PH-GFP/mCherry have more specificity, reliability, suitability and accuracy than antibodies in binding with and detecting PtdIns(4,5)P2 and PtdIns(3,4,5)P3 and in studying the molecular dynamics of phospholipids in live and fixed cells.Key Words: Antibodies, Biosensors, MDA-MB-231, Phosphatidylinositol     

Identification of Proteins Sensitive to Thermal Stress in Human Neuroblastoma and Glioma Cell Lines

Heat-shock is an acute insult to the mammalian proteome. The sudden elevation in temperature has far-reaching effects on protein metabolism, leads to a rapid inhibition of most protein synthesis, and the induction of protein chaperones. Using heat-shock in cells of neuronal (SH-SY5Y) and glial (CCF-STTG1) lineage, in conjunction with detergent extraction and sedimentation followed by LC-MS/MS proteomic approaches, we sought to identify human proteins that lose solubility upon heat-shock. The two cell lines showed largely overlapping profiles of proteins detected by LC-MS/MS. We identified 58 proteins in detergent insoluble fractions as losing solubility in after heat shock; 10 were common between the 2 cell lines. A subset of the proteins identified by LC-MS/MS was validated by immunoblotting of similarly prepared fractions. Ultimately, we were able to definitively identify 3 proteins as putatively metastable neural proteins; FEN1, CDK1, and TDP-43. We also determined that after heat-shock these cells accumulate insoluble polyubiquitin chains largely linked via lysine 48 (K-48) residues. Collectively, this study identifies human neural proteins that lose solubility upon heat-shock. These proteins may represent components of the human proteome that are vulnerable to misfolding in settings of proteostasis stress.     

Interleukin IL-1B gene polymorphism in Tunisian patients with chronic hepatitis B infection: Association with replication levels

Approaches based on association studies have proven useful in identifying genetic predictors for many diseases, including susceptibility to chronic hepatitis B. In this study we were interested by the IL-1B genetic variants that have been involved in the immune response and we analyzed their role in the susceptibility to develop chronic hepatitis B in the Tunisian population. IL - 1B is a potent proinflammatory cytokine that plays an important role in inflammation of the liver. Polymorphic gene IL-1 ( − 511, +3954) was analyzed in a total of 476 individuals: 236 patients with chronic hepatitis B from different cities of Tunisia recruited in Pasteur Institute between January 2017 and December 2018 and 240 controls. Genomic DNA was obtained using the standard salting-out method and genotyping was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism. For − 511C>T polymorphism a significant association was found between patients and controls when comparing the genotypic (P = 0.007; χ² = 9.74 and odds ratio [OR] = 0.60; confidence interval [CI] = 0.41–0.89) and allelic (P = 0.001; χ² = 10.60) frequencies. When the viral load was taken into account a highly significant difference was found (P = 9 × 10⁻⁴; χ² = 10.89). For +3954C>T polymorphism a significant association was found between patients and controls when comparing genotypic (P = 0.0058; χ² = 7.60 and OR = 1.67; CI = 1.14–2.46) and allelic (P = 0.0029; χ² = 8.81) frequencies. T allele can be used as a strong marker for hepatitis B virus disease for both polymorphisms.     

Emphysema Predicts Hospitalisation and Incident Airflow Obstruction among Older Smokers: A Prospective Cohort Study

Background

Emphysema on CT is common in older smokers. We hypothesised that emphysema on CT predicts acute episodes of care for chronic lower respiratory disease among older smokers.

Materials and Methods

Participants in a lung cancer screening study age ≥60 years were recruited into a prospective cohort study in 2001–02. Two radiologists independently visually assessed the severity of emphysema as absent, mild, moderate or severe. Percent emphysema was defined as the proportion of voxels ≤ −910 Hounsfield Units. Participants completed a median of 5 visits over a median of 6 years of follow-up. The primary outcome was hospitalization, emergency room or urgent office visit for chronic lower respiratory disease. Spirometry was performed following ATS/ERS guidelines. Airflow obstruction was defined as FEV1/FVC ratio <0.70 and FEV1<80% predicted.

Results

Of 521 participants, 4% had moderate or severe emphysema, which was associated with acute episodes of care (rate ratio 1.89; 95% CI: 1.01–3.52) adjusting for age, sex and race/ethnicity, as was percent emphysema, with similar associations for hospitalisation. Emphysema on visual assessment also predicted incident airflow obstruction (HR 5.14; 95% CI 2.19–21.1).

Conclusion

Visually assessed emphysema and percent emphysema on CT predicted acute episodes of care for chronic lower respiratory disease, with the former predicting incident airflow obstruction among older smokers.