Loss-of-function RNAi screens in breast cancer cells identify AURKB,PLK1, PIK3R1, MAPK12, PRKD2, and PTK6 as sensitizing targets of rapamycin activity

The use of molecularly targeted drugs as single agents has shown limited utility in many tumor types, largely due to the complex and redundant nature of oncogenic signaling networks. Targeting of the PI3K/AKT/mTOR pathway through inhibition of mTOR in combination with aromatase inhibitors has seen success in particular sub-types of breast cancer and there is a need to identify additional synergistic combinations to maximize the clinical potential of mTOR inhibitors. We have used loss-of-function RNAi screens of the mTOR inhibitor rapamycin to identify sensitizers of mTOR inhibition. RNAi screens conducted in combination with rapamycin in multiple breast cancer cell lines identified six genes, AURKB, PLK1, PIK3R1, MAPK12, PRKD2, and PTK6 that when silenced, each enhanced the sensitivity of multiple breast cancer lines to rapamycin. Using selective pharmacological agents we confirmed that inhibition of AURKB or PLK1 synergizes with rapamycin. Compound-associated gene expression data suggested histone deacetylation (HDAC) inhibition as a strategy for reducing the expression of several of the rapamycin-sensitizing genes, and we tested and validated this using the HDAC inhibitor entinostat in vitro and in vivo. Our findings indicate new approaches for enhancing the efficacy of rapamycin including the use of combining its application with HDAC inhibition.     

A novel model of human implantation: 3D endometrium-like culture system to study attachment of human trophoblast (Jar) cell spheroids

There is an urgent need to develop optimized experimental models to examine human implantation. These studies aimed to (i) establish a human endometrium-like three-dimensional (3D) culture system, and (ii) examine the attachment of trophoblast-like Jar spheroids to the culture. In the present work, 3D endometrial cultures were constructed with fibrin-agarose as matrix scaffold, and using epithelial and stromal cells from both human primary cultures and established cell lines. An attachment assay between trophoblast cells and the 3D culture was developed. Epithelial cells (cytokeratin(+)) concentrated on top of the matrix forming a monolayer, and stromal cells (vimentin(+)) resided within the matrix, resembling the normal endometrial structure. The capability of primary epithelial cells to form glands spontaneously was observed. Human trophoblast cells (Jar cells) were hCG(+) by immunostaining, allowed to form spheroids, and confirmed to secrete hCG into the medium. Time-dependent experiments demonstrated a high rate of attachment of Jar spheroids to the epithelium, and adhesion was strongly related to the various cell types present in the 3D culture. An architecturally and functionally competent 3D endometrial culture system was established, that coupled with Jar spheroids mimicking trophoblast cells, provides a unique in vitro model for the study of certain aspects of human implantation.     

Dendritic processes of osteocytes are mechanotransducers that induce the opening of hemichannels

Osteocytes with long dendritic processes are known to sense mechanical loading, which is essential for bone remodeling. There has been a long-standing debate with regard to which part(s) of osteocyte, the cell body versus the dendritic process, acts as a mechanical sensor. To address this question experimentally, we used a transwell filter system that differentiates the cell body from the dendritic processes. Mechanical loading was applied to either the cell body or the dendrites, and the osteocyte’s response was observed through connexin 43 hemichannel opening. The hemichannels located on the cell body were induced to open when mechanical loading was applied to either the dendritic processes or the cell body. However, no significant hemichannel activity in the dendrites was detected when either part of the cell was mechanically stimulated. Disruption of the glycocalyx by hyaluronidase on the dendrite side alone is sufficient to diminish a dendrite’s ability to induce the opening of hemichannels on the cell body, while hyaluronidase has no such effect when applied to the cell body. Importantly, hyaluronidase treatment to the dendrite side resulted in formation of poor integrin attachments with the reduced ability of the dendrites to form integrin attachments on the underside of the transwell filter. Together, our study suggests that the glycocalyx of the osteocyte dendritic process is required for forming strong integrin attachments. These integrin attachments probably serve as the mechanotransducers that transmit the mechanical signals to the cell body leading to the opening of hemichannels, which permits rapid exchange of factors important for bone remodeling.It is widely accepted that osteocytes are the principal mechanosensory cells of the bone tissue and are implicated to be involved in bone homeostasis (1). Osteocytes are embedded inside the bone tissue and are surrounded by fluid filled spaces known as lacunae (2). Long dendritic processes of osteocytes form a network connecting the neighboring osteocytes and the cells on the bone surface such as osteoblasts and osteoclasts (3). Osteocytic dendrites are surrounded by canalicular wall, thus forming a lacuno-canalicular network (4). Mechanical signals sensed by osteocytes are converted to chemical signals, and the lacuno-canalicular network plays a critical role in conveying these signals to osteoblasts, osteoclasts, and bone-lining cells (5). Gap junctions are present at the tip of osteocytic dendritic processes and play an important role in communicating cellular signals (6). Gap junctions are formed by proteins known as connexins, and hexameric forms of connexins are called connexons (7). Two such connexons present on adjacent cells dock onto each other to form a gap junction channel. When connexons are present on an unapposed cell membrane they are known as hemichannels (8). Hemichannels are involved in communicating with the extracellular environment and are implicated in the exchange of molecules that are involved in bone remodeling, such as prostaglandin E2 (PGE₂) (9). Our previous studies have shown that hemichannels formed by connexin 43 (Cx43) are abundantly expressed in osteocytes and are mechanosensitive (10). We also have shown that their opening is adaptively regulated by different magnitudes of mechanical stimulation (11).Mechanical loading of bone causes fluid flow in the lacuno-canalicular network that is proposed to result in drag forces that act directly on the tethering filaments that attach the osteocyte cell process membrane to the canalicular wall (12, 13). The flow-induced drag forces on the tethering filaments are then transmitted via linker molecules to the central actin filament bundle of the process. This is proposed to lead to a large amplification of whole tissue strains at the cellular level and result in cytoskeletal reorganization and cellular signaling (12, 14). These strains are concentrated around integrin attachments along the dendritic process, attachments that are absent from the pericellular space surrounding the cell body in its lacuna (15, 16). Mechanical stimulation of osteocytes through fluid flow leads to increased dendritic processes, and osteocytes are shown to arrange in the direction of the flow (17).Extracellular matrix proteins are implicated in bringing about changes in the cytoskeletal organization (18). The glycocalyx present in the endothelial extracellular matrix has been shown to act as a transducer of mechanical signals in endothelial cells and is involved in changes in the actin cytoskeletal organization (19, 20). In osteocytes, it is shown that the glycocalyx degradation results in reduced PGE₂ release after fluid flow shear stress (21). These studies imply that the glycocalyx could be involved in causing changes in the cell membrane leading to actin cytoskeletal reorganization and thereby regulating the release of molecules, such as PGE₂. In addition, it has been shown that osteocyte morphology and alignment also affect mechanosensory nature of these cells (22). As these studies implicate the unique role of cell morphology in regulating the mechanosensing by osteocytes, it is imperative to experimentally differentiate which part(s) of these cells, dendrites or the cell body, is involved in mechanotransduction. By taking advantage of a transwell filter system, we show here that osteocyte dendritic processes sense mechanical loading, which is likely to be transmitted through the glycocalyx, leading to the opening of hemichannels on the cell body. This study provides direct evidence suggesting the specific role of dendrites and the glycocalyx in transducing mechanical signals and in the regulation of the opening of hemichannels.     

Pineal parenchymal tumor of intermediate differentiation: imaging spectrum of an unusual tumor in 11 cases

Introduction  

Pineal parenchymal tumor of intermediate differentiation (PPTID) was recognized in the 2007 World Health Organization (WHO) classification as a new pineal parenchymal neoplasm, intermediate in malignancy (WHO grade II or III) between pineocytoma (grade I) and pineoblastoma (grade IV). The imaging spectrum of this new tumor has not been previously delineated. We describe the imaging spectrum in 11 pathologically proven PPTIDs and identify findings that may suggest the preoperative diagnosis of this newly recognized entity.     

Incidence of pulmonary hypertension in patients with chronic myeloproliferative disorders

STUDY OBJECTIVE: To assess the incidence of pulmonary hypertension (PH) in patients with chronic myeloproliferative disorders (CMPD). METHOD: Twenty-seven patients with a diagnosis of CMPD were included in the study. Patients were excluded if they had a secondary cause of PH. Diagnosis of PH was established if right ventricular systolic pressure (RVSP) by transthoracic echocardiography (TTE) was >35 mmHg. RESULTS: Diagnosis of PH was established in 14 out of 27 patients. Two patients were excluded from analysis because of poor ejection fraction on TTE, resulting in a final diagnosis of PH in 12 of 25 (48%) patients. Of these 25 patients, seven of nine with essential thrombocytosis (ET), five of 14 with polycythemia vera (PV), and 0 out of two with chronic myeloid leukemia (CML) had PH. All patients were asymptomatic at the time of their most recent visit. There was no relationship between PH and age at diagnosis, duration of disease, platelet count and hematocrit at diagnosis or during follow-up, both for the entire cohort or for specific diagnosis of ET or PV. CONCLUSION: Pulmonary hypertension appears to be common in patients with CMPD. Further studies are needed to evaluate the impact of treatment on PH and long-term survival in these patients.     

Synthesis, antibacterial and antimycobacterial activities of some new 4-aryl/heteroaryl-2,6-dimethyl-3,5-bis-N-(aryl)-carbamoyl-1,4-dihydropyridines

A novel class of 4-aryl/heteroaryl-2,6-dimethyl-3,5-bis-N-(phenyl/substituted phenyl)-carbamoyl-1,4-dihydropyridines has been synthesized by simple, economical and eco-friendly, modified Hantzsch condensation reaction making use of N-arylacetoacetamides, aryl or heteroaryl aldehydes and ammonium acetate. The newly synthesized compounds were characterized by their spectral (IR, ¹H NMR, Mass), elemental analyses data and evaluated for in vitro antitubercular activity against Mycobacterium tuberculosis H₃₇Rv ATCC 27294 and antibacterial activity against different Gram +ve and Gram −ve bacteria. The preliminary screening results revealed that some of the compounds possess promising antimicrobial activity. Amongst the new series of compounds, 6m containing pyrrolyl and 4-methylphenyl groups and 6r possessing 2-pyridyl and 2-methylphenyl groups were found to exhibit a significant antitubercular activity (MIC = 12.5-25 μg/mL) in comparison with the first line drug pyrazinamide.     

Gene regulation of extracellular matrix remodeling in human bone marrow stem cell-seeded tissue-engineered grafts

Tissue-engineered heart valves are prone to early structural deterioration. We hypothesize that cell-scaffold interaction and mechanical deformation results in upregulation of genes related to osteogenic/chondrogenic differentiation and thus changes extracellular matrix (ECM) composition in human bone marrow mesenchymal stem cell (hBMSC)-derived tissue-engineered grafts. hBMSC were expanded and seeded onto poly-glycolic acid/poly-lactic acid scaffold for 14 days. Seeded tissue-engineered constructs (TEC) were subjected to cyclic flexure for 24 h, whereas control TEC was maintained in roller bottles for the same duration. hBMSC, TEC, and mechanically deformed TEC were subjected to gene-array and histological analysis. Expression levels of RNA and/or protein markers related to chondrogenesis (Sox9, MGP, RunX2, Col II, Col X, and Col XI) and osteogenesis (ALPL, BMP2, EDN1, RunX1, and Col I) were increased in TEC compared to unseeded hBMSC. Histological sections of TEC stained positive for Saffranin O, alkaline phosphatase activity, and calcium deposits. The expression levels of the above gene and protein markers further increased in deformed TEC compared to static TEC. Cell-scaffold interactions and mechanical stress results in gene expression suggestive of endochondral-ossification that impact upon ECM composition and may predispose them to eventual calcification.     

Vitamin B12 deficiency in a large cohort of healthcare professionals across the network of an eyecare organization in India

Purpose:To evaluate Vitamin B12 levels in healthcare professionals at a tertiary eyecare centre in India.Methods:This was a cross-sectional study conducted among healthcare professionals working at a tertiary eyecare centre in India. The sample included 2,374 employees. Chemiluminescent immunoassay method (reference range, 211–911 pg/ml) was used to assess serum vitamin B12 levels. Effect of age and gender was analyzed in vitamin B12 normal and vitamin B12 deficient groups. To evaluate risk factors, questions related to vitamin B12 deficiency were asked to the study participants in a survey.Results:The mean age of employees was 29.2 ± 0.7 years. Around 26% of them were vitamin B12 deficient. The proportion of males in the vitamin B12 deficient group (61.2%) was significantly higher (P < 0.0001) than that of the vitamin B12 normal group (44.9%). There was no effect of age on vitamin B12 levels in both vitamin B12 normal and vitamin B12 deficient groups. Mean vitamin B12 levels in males (289.1 ± 22.2 pg/ml) was significantly lower (P < 0.0001) than that of females (338.7 ± 30.0 pg/ml).Conclusion:This is the first such study on eyecare professionals. One-fourth of the eyecare professionals were vitamin B12 deficient. The proportion of males was higher in the vitamin B12 deficiency group. Males had lower vitamin B12 levels than females. Annual blood tests for vitamin B12 are recommended for timely diagnosis and management of vitamin B12 deficiency, particularly in males.