Differential expression of 16 genes coding for cell cycle – and apoptosis-related proteins in vitamin D-induced differentiation of HL-60 cells

Objective: The aim of the study was to analyze expression of several genes related to cell cycle regulation and apoptosis in realization of 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3)-induced differentiation of HL-60 cells. Methods: The cultured HL-60 cells were treated with 1,25(OH)2D3. Quantitative real-time PCR was used for analyzing the changes in expression of 16 genes (Bcl-2, Bcl-xL, Mcl-1, Bik, caspase 6, caspase 7, cytochrome c, TNFR1, Myc, TGF-beta, JNK1, p38MAPK, p21, p27, Cdk2, cyclin E) at early phases of cell differentiation of HL-60 cells induced by 1,25(OH)2D3. Results: Among investigated genes, Bik and Myc gene expression was down-regulated at 48 h time points. JNK1 gene was markedly up-regulated and caspase-6 and cyclin E genes were down-regulated at 18 h time point. Conclusion: These findings suggest that there are no distinct apoptotic signals at early phases of cell differentiation. It is speculated that changes in the expression of the genes involved in vitamin D-induced apoptosis of HL-60 cells could be better visualized after the terminal stages of cell differentiation.Цель: проанализировать экспрессию ряда генов, участвующих в регуляции клеточного цикла и апоптоза в дифференцировке клеток HL-60 промиелоцитарного лейкоза человека, индуцированной 1альфа,25-дигидроксивитамином 3(1,25(OH 2 3). Методы: культивируемые клетки HL-60 инкуировали с 1,25(OH 2 D3. Количественную ПЦР в режиме реального времени использовали для анализа изменений экспрессии 16 генов (Bcl-2, Bc l-xL , l 1, k, каспаза 6, каспаза 7, цитохром с, TNFR1, Myc, TGF- beta, K 1, p38 MAPK, p21, p , Cdk2, иклин Е ) при индукции апоптоза и дифференцировки в клетках HL-60 под дествием 1,25(OH 2 D3. Результаты: среди исследованных генов отмечено снижение экспресси Bik и Myc через 48 ч. Экспрессия гена JNK1 повышена, а генов каспазы-6 и циклина Е снижена в точке 18 ч. Выводы: эти данные свидетельствуют об отсутствии четких сигналов экспрессии апоптоз-ассоциированных генов на ранних фазах клеточной дифференцировки. Предполагается, что изменения в экспрессии генов, участвующих в реализации апоптоза клеток HL-60, индуцированного витаминомD, могут быть выражены лишь на терминальных стадиях дифференцировки этих клеток

Proteomic Profiling of Saliva and Tears in Radiated Head and Neck Cancer Patients as Compared to Primary Sjögren’s Syndrome Patients

Patients with head and neck cancer (HNC) and patients with primary Sjögren’s syndrome (pSS) may exhibit similar symptoms of dry mouth and dry eyes, as a result of radiotherapy (RT) or a consequence of disease progression. To identify the proteins that may serve as promising disease biomarkers, we analysed saliva and tears from 29 radiated HNC patients and 21 healthy controls, and saliva from 14 pSS patients by mass spectrometry-based proteomics. The study revealed several upregulated, and in some instances overlapping, proteins in the two patient groups. Histone H1.4 and neutrophil collagenase were upregulated in whole saliva of both patient groups, while caspase-14, histone H4, and protein S100-A9 were upregulated in HNC saliva only. In HCN tear fluid, the most highly upregulated protein was mucin-like protein 1. These overexpressed proteins in saliva and tears play central roles in inflammation, host cell injury, activation of reactive oxygen species, and tissue repair. In conclusion, the similarities and differences in overexpressed proteins detected in saliva from HNC and pSS patients may contribute to the overall understanding of the different pathophysiological mechanisms inducing dry mouth. Thus, the recurring proteins identified could possibly serve as future promising biomarkerspublishedVersio

The Hippo signaling pathway is required for salivary gland development and its dysregulation is associated with Sjogren's-like disease

Sjogren's syndrome (SS) is a complex autoimmune disease that primarily affects salivary and lacrimal glands and is associated with high morbidity. Although the prevailing dogma is that immune system pathology drives SS, increasing evidence points to structural defects, including defective E-cadherin adhesion, to be involved in its etiology. We have shown that E-cadherin plays pivotal roles in the development of the mouse salivary submandibular gland (SMG) by organizing apical-basal polarity in acinar and ductal progenitors and by signaling survival for differentiating duct cells. Recently, E-cadherin junctions have been shown to interact with effectors of the Hippo signaling pathway, a core pathway regulating organ size, cell proliferation and differentiation. We now show that Hippo signaling is required for SMG branching morphogenesis and is involved in the pathophysiology of SS. During SMG development, a Hippo pathway effector, TAZ, becomes increasingly phosphorylated and associated with E-cadherin and α-catenin, consistent with the activation of Hippo signaling. Inhibition of Lats2, an upstream kinase that promotes TAZ phosphorylation, results in dysmorphogenesis of the SMG and impaired duct formation. SMGs from NOD mice, a mouse model for SS, phenocopy the Lats2-inhibited SMGs and exhibit a reduction in E-cadherin junctional components, including TAZ. Importantly, labial specimens from human SS patients display mislocalization of TAZ from junctional regions to the nucleus, coincident with accumulation of extracellular matrix components, fibronectin and CTGF, known downstream targets of TAZ. Our studies show that Hippo signaling plays a crucial role in SMG branching morphogenesis and provide evidence that defects in this pathway are associated with SS in humans

Protocol for derivation of organoids and patient-derived orthotopic xenografts from glioma patient tumors

Tumor organoids and patient-derived orthotopic xenografts (PDOXs) are some of the most valuable pre-clinical tools in cancer research. In this protocol, we describe efficient derivation of organoids and PDOX models from glioma patient tumors. We provide detailed steps for organoid culture, intracranial implantation, and detection of tumors in the brain. We further present technical adjustments for standardized functional assays and drug testing.publishedVersio

Localization of AQP5 during development of the mouse submandibular salivary gland

Aquaporin 5 (AQP5) is known to be central for salivary fluid secretion. A study of the temporal-spatial distribution of AQP5 during submandibular gland (SMG) development and in adult tissues might offer further clues to its unknown role during development. In the present work, SMGs from embryonic day (E) 14.5–18.5 and postnatal days (P) 0, 2, 5, 25, and 60 were immunostained for AQP5 and analyzed using light microscopy. Additional confocal and transmission electron microscopy were performed on P60 glands. Our results show that AQP5 expression first occurs in a scattered pattern in the late canalicular stage and becomes more prominent and organized in the terminal tubuli/pro-acinar cells towards birth. Additional apical membrane staining in the entire intralobular duct is found just prior to birth. During postnatal development, AQP5 is expressed in both the luminal and lateral membrane of pro-acinar/acinar cells. AQP5 is also detected in the basal membrane of acinar cells at P25 and P60. In the intercalated ducts at P60, the male glands show apical staining in the entire segment, while only the proximal region is positive in the female glands. These results demonstrate an evolving distribution of AQP5 during pre- and postnatal development in the mouse SMGs

The use of non-conventional CPTe data in determination of 3-D electrical resistivity distribution

The spatial distribution of the electrical resistivity data provides useful information for investigating and modeling the fluid transport processes. 3D electrical resistivity distribution provides information about water flow and changes in electrical resistivity of the pore fluid.Therefore, to assist in understanding and modeling of the fluid transport process, 3D spatial distribution of the electrical resistivity data with the corresponded 3D geological section were mapped and interpreted in the test site located in western Germany. A process of deriving electrical resistivity values from the mechanical and radioactive parameters of cone penetration tests (CPT) and geological information of boreholes was presented. A reliable method which gives accurate resistivity values in cases of near surface sediments was introduced. Then a field test was executed where the calculated resistivity values were compared with the measured CPTe resistivity data. The CPTe (cone penetration test with electrical extension) data were also used in correlating to the ERT (electrical resistivity tomography) data. Consequently, obtained dense CPT surveys give us the possibility to determine a high resolution resistivity distribution of the investigated area. (C) 2010 Elsevier B.V. All rights reserved

SLOPE STABILITY ASSESSMENT IN THE OPENCAST QUARRY IN HAMMAM DALAA (M'SILA, ALGERIA)

The aim of this study is to identify the main factors that affect the site's stability by using the adequate solutions, which help to solve the problems of slope stability at the quarry of Chouf Amar, Hammam Dalaa, Algeria