Cancer-associated mesothelial cells are regulated by the anti-Müllerian hormone axis.

Cancer-associated mesothelial cells (CAMCs) in the tumor microenvironment are thought to promote growth and immune evasion. We find that, in mouse and human ovarian tumors, cancer cells express anti-Müllerian hormone (AMH) while CAMCs express its receptor AMHR2, suggesting a paracrine axis. Factors secreted by cancer cells induce AMHR2 expression during their reprogramming into CAMCs in mouse and human in vitro models. Overexpression of AMHR2 in the Met5a mesothelial cell line is sufficient to induce expression of immunosuppressive cytokines and growth factors that stimulate ovarian cancer cell growth in an AMH-dependent way. Finally, syngeneic cancer cells implanted in transgenic mice with Amhr2-/- CAMCs grow significantly slower than in wild-type hosts. The cytokine profile of Amhr2-/- tumor-bearing mice is altered and their tumors express less immune checkpoint markers programmed-cell-death 1 (PD1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4). Taken together, these data suggest that the AMH/AMHR2 axis plays a critical role in regulating the pro-tumoral function of CAMCs in ovarian cancer.

Target highlights in CASP13: Experimental target structures through the eyes of their authors.

The functional and biological significance of selected CASP13 targets are described by the authors of the structures. The structural biologists discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP13 experiment.

A gene expression signature distinguishes innate response and resistance to proteasome inhibitors in multiple myeloma.

Extensive interindividual variation in response to chemotherapy is a major stumbling block in achieving desirable efficacy in the treatment of cancers, including multiple myeloma (MM). In this study, our goal was to develop a gene expression signature that predicts response specific to proteasome inhibitor (PI) treatment in MM. Using a well-characterized panel of human myeloma cell lines (HMCLs) representing the biological and genetic heterogeneity of MM, we created an in vitro chemosensitivity profile in response to treatment with the four PIs bortezomib, carfilzomib, ixazomib and oprozomib as single agents. Gene expression profiling was performed using next-generation high-throughput RNA-sequencing. Applying machine learning-based computational approaches including the supervised ensemble learning methods Random forest and Random survival forest, we identified a 42-gene expression signature that could not only distinguish good and poor PI response in the HMCL panel, but could also be successfully applied to four different clinical data sets on MM patients undergoing PI-based chemotherapy to distinguish between extraordinary (good and poor) outcomes. Our results demonstrate the use of in vitro modeling and machine learning-based approaches to establish predictive biomarkers of response and resistance to drugs that may serve to better direct myeloma patient treatment options.

Single-cell analysis of targeted transcriptome predicts drug sensitivity of single cells within human myeloma tumors.

Multiple myeloma (MM) is characterized by significant genetic diversity at subclonal levels that have a defining role in the heterogeneity of tumor progression, clinical aggressiveness and drug sensitivity. Although genome profiling studies have demonstrated heterogeneity in subclonal architecture that may ultimately lead to relapse, a gene expression-based prediction program that can identify, distinguish and quantify drug response in sub-populations within a bulk population of myeloma cells is lacking. In this study, we performed targeted transcriptome analysis on 528 pre-treatment single cells from 11 myeloma cell lines and 418 single cells from 8 drug-naïve MM patients, followed by intensive bioinformatics and statistical analysis for prediction of proteasome inhibitor sensitivity in individual cells. Using our previously reported drug response gene expression profile signature at the single-cell level, we developed an R Statistical analysis package available at https://github.com/bvnlabSCATTome, SCATTome (single-cell analysis of targeted transcriptome), that restructures the data obtained from Fluidigm single-cell quantitative real-time-PCR analysis run, filters missing data, performs scaling of filtered data, builds classification models and predicts drug response of individual cells based on targeted transcriptome using an assortment of machine learning methods. Application of SCATT should contribute to clinically relevant analysis of intratumor heterogeneity, and better inform drug choices based on subclonal cellular responses.

MicroRNAs as mediators and communicators between cancer cells and the tumor microenvironment.

Cancer cells grow in an environment comprised of multiple components that support tumor growth and contribute to therapy resistance. Major cell types in the tumor microenvironment are fibroblasts, endothelial cells and infiltrating immune cells all of which communicate with cancer cells. One way that these cell types promote cancer progression is by altering the expression of microRNAs (miRNAs), small noncoding RNAs that negatively regulate protein expression, either in the cancer cells or in the associated normal cells. Changes in miRNA expression can be brought about by direct interaction between the stromal cells and cancer cells, by paracrine factors secreted by any of the cell types or even through direct communication between cells through secreted miRNAs. Understanding the role of miRNAs in the complex interactions between the tumor and cells in its microenvironment is necessary if we are to understand tumor progression and devise new treatments.

Microenvironment-induced downregulation of miR-193b drives ovarian cancer metastasis.

The cross-talk between ovarian cancer (OvCa) cells and the metastatic microenvironment is an essential determinant of successful colonization. MicroRNAs (miRNAs) have several critical roles during metastasis; however, the role of microenvironmental cues in the regulation of miRNAs in metastasizing cancer cells has not been studied. Using a three-dimensional culture model that mimics the human omentum, one of the principal sites of OvCa metastasis, we identified and characterized the microenvironment-induced downregulation of a tumor suppressor miRNA, miR-193b, in metastasizing OvCa cells. The direct interaction of the OvCa cells with mesothelial cells, which cover the surface of the omentum, caused a DNA methyltransferase 1-mediated decrease in the expression of miR-193b in the cancer cells. The reduction in miR-193b enabled the metastasizing cancer cells to invade and proliferate into human omental pieces ex vivo and into the omentum of a mouse xenograft model of OvCa metastasis. The functional effects of miR-193b were mediated, in large part, by the concomitant increased expression of its target, urokinase-type plasminogen activator, a known tumor-associated protease. These findings link paracrine signals from the microenvironment to the regulation of a key miRNA in cancer cells. Targeting miR-193b, which is essential for metastatic colonization of cancer cells could prove effective in the treatment of OvCa metastasis.

A novel packing arrangement of AcrB in the lipid bilayer membrane.

The central component AcrB of the Escherichia coli drug efflux complex AcrA-AcrB-TolC has been extensively investigated by X-ray crystallography of detergent-protein 3-D crystals. In these crystals, AcrB packs as trimers - the functional unit. We visualized the AcrB-AcrB interaction in its native environment by examining E. coli lipid reconstituted 2-D crystals, which were overwhelmingly formed by asymmetric trimers stabilized by strongly-interacting monomers from adjacent trimers. Most interestingly, we observed lattices formed by an arrangement of AcrB monomers distinct from that in traditional trimers. This hitherto unobserved packing, might play a role in the biogenesis of trimeric AcrB.

Ritonavir inhibits HIF-1α-mediated VEGF expression in retinal pigment epithelial cells in vitro.

PURPOSE: Retinal hypoxia-mediated activation of the hypoxia-inducible factor (HIF pathway) leading to angiogenesis is a major signaling mechanism underlying a number of sight-threatening diseases. Inhibiting this signaling mechanism with an already approved therapeutic molecule may have promising anti-angiogenic role with fewer side effects. Hence, the primary objective of this study was to examine the expression of HIF-1α and VEGF in human retinal pigment epithelial cells treated with ritonavir under hypoxic and normoxic conditions. METHODS: ARPE-19 and D407 cells were cultured in normoxic or hypoxic conditions, alone or in the presence of ritonavir. Quantitative real-time polymerase chain reaction, immunoblot analysis, sandwich ELISA, endothelial cell proliferation, and cytotoxicity were performed. RESULTS: A 12-h hypoxic exposure resulted in elevated mRNA expression levels of both HIF-1α and VEGF in ARPE-19 and D407 cells. Hence, this time point was selected for subsequent experiments. Presence of ritonavir in the culture medium strongly inhibited VEGF expression in a concentration-dependent manner under hypoxic conditions. Immunoblot analysis demonstrated a substantially reduced protein expression of HIF-1α in the presence of ritonavir. Further, hypoxic exposure-induced VEGF secretion was also inhibited by ritonavir, as demonstrated using ELISA. Finally, ritonavir significantly diminished the proliferation of choroid-retinal endothelial (RF/6A) cells demonstrating potential anti-angiogenic activity. Cytotoxicity studies showed that ritonavir is non-toxic to RPE cells. CONCLUSIONS: This study demonstrates for the first time that ritonavir can inhibit HIF-1α and VEGF in ARPE-19 and D407 cells. Such inhibition may form a platform for application of ritonavir in the treatment of various ocular diseases.

Exon 8-9 mutations of DNA polymerase ß in ovarian carcinoma patients from Haldia, India.

BACKGROUND: Ovarian cancer is the number one killer among all the gynecological cancers. We undertook association study to identify potential alterations in the genomic DNA of a DNA repair gene, DNA polymerase beta (polß), involved in base excision repair (BER), in ovarian carcinomas of patients from Haldia, India. Mutations, splice variants have been reported earlier in different tumors other than ovarian tumors. AIM: In this study we explored the possibility of association of any mutation of pol beta (Exon 8) with prognosis in 152 ovarian cancer samples. RESULTS: Alteration in the exon 8 region (Exon 8:468, AgC; 15.1%) was noted among fifty seven polymorphism positive samples. Alteration in the intervening sequence 8 (IVS8, -25, AgC; 3.9%) was also noted. All alterations are heterozygous in nature. CONCLUSIONS: We found no significant association among the samples from serous type, stage IV, and the polß mutations (P ≤ 0.01). Only a slight tendency of association was evident between IVS8, -25, A to C; and stage III. Further analysis with a larger number of samples is needed.

Association of two polymorphisms of DNA polymerase beta in exon-9 and exon-11 with ovarian carcinoma in India.

BACKGROUND: DNA polymerase beta (polß ) is a key enzyme in the base excision repair pathway. It is 39 kDa protein, with two subunits, one large subunit of 31 kDa having catalytic activity between exon V to exon XIV, and an 8 kDa smaller subunit having single strand DNA binding activity. Exons V to VII have double strand DNA binding activity, whereas exons VIII to XI account for the nucleotidyl transferase activity and exons XII to XIV the dNTP selection activity. AIM: To examine the association between polß polymorphisms and the risk of ovarian cancer, the present case control study was performed using 152 cancer samples and non-metastatic normal samples from the same patients. In this study, mutational analysis of polß genomic DNA was undertaken using primers from exons IX to XIV - the portion having catalytic activity. RESULTS: We detected alteration in DNA polymerase beta by SSCP. Two specific heterozygous point mutations of polß were identified in Exon 9:486, A->C (polymorphism 1; 11.18%) and in Exon 11:676, A->C (polymorphism 2; 9.86%). The correlation study involving polymorphism 1 and 4 types of tissue showed a significant correlation between mucinous type with a Pearson correlation value of 4.03 (p=0.04). The association among polymorphism 2 with serous type and stage IV together have shown Pearson χ2 value of 3.28 with likelihood ratio of 4.4 (p=0.07) with OR =2.08 (0.3- 14.55). This indicates that there is a tendency of correlation among polymorphism 2, serous type and stage IV, indicating a risk factor for ovarian cancer. CONCLUSION: Hence, the results indicate that there is a tendency for polß polymorphisms being a risk factor for ovarian carcinogenesis in India.

Effect of high temperature and the presence of stone-wales defects on the mechanical behavior of a single wall carbon nanotube under tension and compression.

On the basis of molecular dynamics simulation, a zigzag (7, 0) SWCNT is examined under axial tension and compression to observe its mechanical behavior and fracture pattern in the absence and presence of Stone-Wales (SW) defects coupled with the variation of temperature. A defect-free tube is subjected to tensile or compressive stresses at different temperatures. Then SW defects are introduced into it and stress is applied with the increase of temperature. The defective and defect-free tubes show different fracture patterns with increasing temperature showing significant changes in their mechanical properties. Compression of the tube also reveals interesting changes in its buckling behavior with variation of temperature and inclusion of defects.

Functional characterization of folate transport proteins in Staten's Seruminstitut rabbit corneal epithelial cell line.

PURPOSE: The overall objective of this study was to investigate and characterize the expression of folate transport proteins in Staten's Seruminstitut rabbit corneal (SIRC) epithelial cell line. METHODS: [(3)H]Folic acid uptake was studied with respect to time, pH, temperature, sodium, and chloride ion dependency. Inhibition studies were conducted with structural analogs, vitamins, and metabolic inhibitors. [(3)H]Folic acid uptake was also determined with varying concentrations of cold folic acid. Uptake kinetics was studied in the presence of various modulators of intracellular regulatory pathways, protein kinases A and C (PKA and PKC), protein tyrosine kinase (PTK), and calcium-calmodulin modulators. Ex vivo corneal permeability studies were carried out with [(3)H]folic acid in the presence and absence of 1 mM cold folic acid. RESULTS: Linear increase in [(3)H]folic acid uptake was observed over 30 min. The process followed saturation kinetics with apparent K(m) of 14.2 ± 0.2 nM, V(max) of (1.5 ± 0.1)*10(-5) micro.moles/min/mg protein, and K(d) of (2.1 ± 0.2)*10(-6) min(-1). The uptake process was found to be dependent on pH, sodium ions, chloride ions, temperature, and energy. Uptake was inhibited in the presence of structural analogs (cold folic acid, methyltetrahydro folate, and methotrexate), but structurally unrelated vitamins did not show any effect. Membrane transport inhibitors SITS, DIDS, probenecid and endocytic inhibitor, colchicine significantly inhibited the [(3)H]folic acid uptake indicating the involvement of receptor/transporter mediated process. PKA, PTK, and Ca(2+)/calmodulin pathways significantly regulate the process. RT-PCR and Western blot analysis confirmed the presence of folate receptor-α (FR-alpha) and proton-coupled folate transporter (PCFT). Permeability of [(3)H]folic acid across the rabbit cornea was (1.48 ± 0.13)*10(-05) cm/sec, and in the presence of cold folic acid it was (1.08 ± 0.10)*10(-05) cm/sec. CONCLUSIONS: This work demonstrated the functional and molecular presence of FR-alpha and PCFT in SIRC epithelial cell line.

Ligand-independent activation of c-Met by fibronectin and α(5)ß(1)-integrin regulates ovarian cancer invasion and metastasis.

The role of the fibronectin receptor, α(5)ß(1)-integrin, as an adhesion receptor and in angiogenesis is well established. However, its role in cancer cell invasion and metastasis is less clear. We describe a novel mechanism by which fibronectin regulates ovarian cancer cell signaling and promotes metastasis. Fibronectin binding to α(5)ß(1)-integrin led to a direct association of α(5)-integrin with the receptor tyrosine kinase, c-Met, activating it in a hepatocyte growth factor/scatter factor (HGF/SF) independent manner. Subsequently, c-Met associated with Src, and activated Src and focal adhesion kinase (FAK). Inhibition of α(5)ß(1)-integrin decreased the phosphorylation of c-Met, FAK and Src, both in vitro and in vivo. Independent activation of c-Met by its native ligand, HGF/SF, or overexpression of a constitutively active FAK in HeyA8 cells could overcome the effect of α(5)ß(1)-integrin inhibition on tumor cell invasion, indicating that α(5)ß(1)-integrin is upstream of c-Met, Src and FAK. Inhibition of α(5)ß(1)-integrin on cancer cells in two xenograft models of ovarian cancer metastasis resulted in a significant decrease of tumor burden, which was independent of the effect of α(5)ß(1)-integrin on angiogenesis. These data suggest that fibronectin promotes ovarian cancer invasion and metastasis through an α(5)ß(1)-integrin/c-Met/FAK/Src-dependent signaling pathway, transducing signals through c-Met in an HGF/SF-independent manner.

Viral obesity: fact or fiction?

The aetiology of obesity is multifactorial. An understanding of the contributions of various causal factors is essential for the proper management of obesity. Although it is primarily thought of as a condition brought on by lifestyle choices, recent evidence shows there is a link between obesity and viral infections. Numerous animal models have documented an increased body weight and a number of physiologic changes, including increased insulin sensitivity, increased glucose uptake and decreased leptin secretion that contribute to an increase in body fat in adenovirus-36 infection. Other viral agents associated with increasing obesity in animals included canine distemper virus, rous-associated virus 7, scrapie, Borna disease virus, SMAM-1 and other adenoviruses. This review attempted to determine if viral infection is a possible cause of obesity. Also, this paper discussed mechanisms by which viruses might produce obesity. Based on the evidence presented in this paper, it can be concluded that a link between obesity and viral infections cannot be ruled out. Further epidemiologic studies are needed to establish a causal link between the two, and determine if these results can be used in future management and prevention of obesity.

Three-photon-induced four-photon absorption and nonlinear refraction in ZnO quantum dots.

Three-photon-induced four-photon absorption via excited-state absorption and self-defocusing nonlinear refraction are reported for the first time, to our knowledge, in ZnO quantum dots with average sizes of 2.0+/-0.1 nm with 1064 nm radiation from a Q-switched Nd:YAG laser at a peak intensity of 2.5 GW/cm(2). By employing the three-level two-step model, the experimental results can be explained quite satisfactorily.

The multifunctional histone-like protein Lsr2 protects mycobacteria against reactive oxygen intermediates.

Mycobacterium tuberculosis has evolved a number of strategies to survive within the hostile environment of host phagocytes. Reactive nitrogen and oxygen intermediates (RNI and ROI) are among the most effective antimycobacterial molecules generated by the host during infection. Lsr2 is a M. tuberculosis protein with histone-like features, including the ability to regulate a variety of transcriptional responses in mycobacteria. Here we demonstrate that Lsr2 protects mycobacteria against ROI in vitro and during macrophage infection. Furthermore, using macrophages derived from NOS(-/-) and Phox(-/-) mice, we demonstrate that Lsr2 is important in protecting against ROI but not RNI. The protection provided by Lsr2 protein is not the result of its ability to either bind iron or scavenge hydroxyl radicals. Instead, electron microscopy and DNA-binding studies suggest that Lsr2 shields DNA from reactive intermediates by binding bacterial DNA and physically protecting it. Thus, Lsr2 appears to be a unique protein with both histone-like properties and protective features that may be central to M. tuberculosis pathogenesis. In addition, evidence indicates that lsr2 is an essential gene in M. tuberculosis. Because of its essentiality, Lsr2 may represent an excellent candidate as a drug target.

EDXRF analysis of municipal solid waste using (109)Cd source.

Elemental compositions of municipal solid waste (MSW) samples have been analyzed using the non-destructive energy dispersive X-ray fluorescence (EDXRF) technique. The samples were collected from three different dumping sites of urban and suburban areas of the city of Kolkata, West Bengal, India. The EDXRF spectrometer consisted of a (109)Cd radioactive source and a Si (Li) detector. To check the reliability of the system, NIST Standard Reference Material-1648 UPM had been analyzed and it was found that within the experimental errors, our results agree quite well with the certified and non-certified values. The elemental compositions of all the three MSW samples were subsequently estimated using the same procedure. The matrix effects were estimated following the emission-transmission method. It was observed that except Fe, all the elements from Ti to Pb show concentration levels higher by a factor of 2-7 than the ecological screening values where as in the case of Fe, this factor varies from 100 to 200.