Detection of MET mRNA in gastric cancer in situ. Comparison with immunohistochemistry and sandwich immunoassays

Determination of predictive biomarkers by immunohistochemistry (IHC) relies on antibodies with high selectivity. RNA in situ hybridization (RNA ISH) may be used to confirm IHC and may potentially replace it if suitable antibodies are not available or are insufficiently selective to discriminate closely related protein isoforms. We validated RNA ISH as specificity control for IHC and as a potential alternative method for selecting patients for treatment with MET inhibitors. MET, the HGF receptor, is encoded by the MET proto-oncogene that may be activated by mutation or amplification. MET expression and activity were tested in a panel of control cell lines. MET could be detected in formalin fixed paraffin, embedded (FFPE) samples by IHC and RNA ISH, and this was confirmed by sandwich immunoassays of fresh frozen samples. Gastric cancer cell lines with high MET expression and phosphorylation of tyrosine-1349 respond to the MET inhibitor, BAY-853474. High expression and phosphorylation of MET is a predictive biomarker for response to MET inhibitors. We then analyzed MET expression and activity in a matched set of FFPE vs. fresh frozen tumor samples consisting of 20 cases of gastric cancer. Two of 20 clinical samples investigated exhibited high MET expression with RNA ISH and IHC. Both cases were shown by sandwich immunoassays to exhibits strong functional activity. Expression levels and functional activity in these two cases were in a range that predicted response to treatment. Our findings indicate that owing to its high selectivity, RNA ISH can be used to confirm findings obtained by IHC and potentially may replace IHC for certain targets if no suitable antibodies are available. RNA ISH is a valid platform for testing predictive biomarkers for patient selection.     

Heterologous expression and purification of active L‐asparaginase I of Saccharomyces cerevisiae in Escherichia coli host

l ‐asparaginase (ASNase) is a biopharmaceutical widely used to treat child leukemia. However, it presents some side effects, and in order to provide an alternative biopharmaceutical, in this work, the genes encoding ASNase from Saccharomyces cerevisiae (Sc_ASNaseI and Sc_ASNaseII) were cloned in the prokaryotic expression system Escherichia coli. In the 93 different expression conditions tested, the Sc_ASNaseII protein was always obtained as an insoluble and inactive form. However, the Sc_ASNaseI (His)₆‐tagged recombinant protein was produced in large amounts in the soluble fraction of the protein extract. Affinity chromatography was performed on a Fast Protein Liquid Chromatography (FPLC) system using Ni²⁺‐charged, HiTrap Immobilized Metal ion Affinity Chromatography (IMAC) FF in order to purify active Sc_ASNaseI recombinant protein. The results suggest that the strategy for the expression and purification of this potential new biopharmaceutical protein with lower side effects was efficient since high amounts of soluble Sc_ASNaseI with high specific activity (110.1 ± 0.3 IU mg^?1) were obtained. In addition, the use of FPLC‐IMAC proved to be an efficient tool in the purification of this enzyme, since a good recovery (40.50 ± 0.01%) was achieved with a purification factor of 17‐fold. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:416–424, 2017     

Treating metastatic prostate cancer with microRNA-145

Prostate cancer (PCa) is an incurable disease at the metastatic stage. Although there are different options for treatment, the results are limited. MicroRNAs (miRNAs) are a group of small, noncoding, regulatory RNAs with important roles in regulating gene expression. miR-145 is reported to be a key tumor suppressor miRNA (tsmiR) that controls important oncogenes, such as MYC and RAS. In this study, in vitro studies were performed to show the control of MYC and RAS by miR-145. Flow cytometry was used to analyze cell proliferation and apoptosis. The efficacy of miR-145 in treating metastatic PCa was tested in nude mice using a model of bone metastasis promoted by intraventricular injection of PC-3MLuc-C6 cells. Tumor growth was evaluated by an in vivo bioluminescence system. After the full establishment of metastases on day 21, six animals were treated with three intravenous doses of miR-145 (on days 21, 24 and 27), and six were injected with scramble miRNA as controls. Compared to the controls, tumor growth was significantly reduced in animals receiving miR-145, most importantly on day 7 after the third and last dose of miRNA. After discontinuing the treatment, tumor growth resumed, becoming similar to the group of non-treated animals. A decrease in MYC and RAS expression was observed in all cell lines after treatment with miR-145, although statistical significance was achieved only in experiments with LNCaP and PC3 cell lines, with a decrease in 56% (p?=?0.012) and 31% (p?=?0.013) of RAS expression, respectively. Our results suggest that miR-145 is a potential molecule to be tested for treatment of metastatic, castration-resistant PCa.     

Cell surface nanoanatomy of Leishmania major as revealed by fracture-flip. A surface meshwork of 44 nm fusiform filaments identifies infective developmental stage promastigotes

Fracture-flip (Anderson-Forsman and Pinto da Silva, J. Cell Sci. 90, 531-541; 1988) was used to reveal the nanoanatomy of the surface of Leishmania major promastigotes. Over the cell surface of infective metacyclic promastigotes we identify a meshwork of 44 nm long, fusiform filaments. These filaments are not seen in noninfective stages of the parasite. Replica-staining immunocytochemistry with monoclonal antibody against infective metacyclic lipophosphoglycan shows a uniform distribution of protein A-colloidal gold complexes over the cell surface. Thin sections show that acquisition of the high molecular weight lipophosphoglycan is reflected in a thicker glycocalyx. Conventional freeze-fracture shows that in infective metacyclic promastigotes there is a reversal of the partition of intramembrane particles--an additional morphological marker for the infective developmental stage. We hypothesize that the fusiform filaments represent metacyclic developmental lipophosphoglycan.     

Drivers of beta diversity of macroinvertebrate communities in tropical forest streams

1. There has recently been increasing interest in patterns of beta diversity but we still lack a comprehensive understanding of these patterns in various regions (e.g. the tropics), ecosystems (e.g. streams) and organism groups (e.g. invertebrates). 2. Our aim was to investigate the patterns of beta diversity of stream macroinvertebrates in relation to key environmental (i.e. stream size, pH and habitat degradation) and geographical variables (i.e. latitude, longitude, altitude) in a tropical region. We surveyed a total of 8–10 riffle sites in each of 34 streams (altogether 337 riffle sites were sampled) in Peninsular Malaysia to examine variation in macroinvertebrate community composition at within‐stream and among‐stream scales. 3. Based on test of homogeneity of dispersion, we found that the streams studied differed significantly in within‐stream variation in community composition (i.e. among‐site variation of within stream beta diversity). The patterns were similar based on Bray–Curtis coefficient on abundance data, Sorensen coefficient on presence–absence data and Simpson coefficient on presence–absence data. We also found that within‐stream beta diversity was significantly related to stream size, pH and latitude, with each of these variables individually accounting for around 20% of the variation in beta diversity in simple regressions, while the total variation explained by the three significant variables amounted to around 50% in multiple regressions. By contrast, habitat degradation, longitude and altitude were not significantly related to beta diversity. We also found that the factor drainage basin accounted for much of the variation in beta diversity in general linear models, suppressing the effects of environmental variables. 4. We concluded that within‐stream beta diversity is mainly related to a combination of the identity of a drainage basin and stream environmental factors. Our findings provide important background for stream environmental assessment and conservation planning by emphasising that (i) macroinvertebrate communities within streams are not homogeneous, but show considerable beta diversity, (ii) streams differ in their degree of within‐stream beta diversity, (iii) stream size and water pH should be considered in applied contexts related to within‐stream beta diversity and (iv) historical effects may be different in different drainage basins and may affect present‐day patterns of within‐stream beta diversity.     

Splenectomy Improves Hemostatic and Liver Functions in Hepatosplenic Schistosomiasis Mansoni

Background

Schistosomiasis mansoni is a chronic liver disease, in which some patients (5–10%) progress to the most severe form, hepatosplenic schistosomiasis. This form is associated with portal hypertension and splenomegaly, and often episodes of gastrointestinal bleeding, even with liver function preserved. Splenectomy is a validated procedure to reduce portal hypertension following digestive bleeding. Here, we evaluate beneficial effects of splenectomy on blood coagulation factors and liver function tests in hepatosplenic schistosomiasis mansoni compared to non-operated patients.

Methodology/Principal Findings

Forty-five patients who had undergone splenectomy surgery were assessed by laboratory analyses and ultrasound examination and compared to a non-operated group (n = 55). Blood samples were obtained for liver function tests, platelet count and prothrombin time. Coagulation factors (II, VII, VIII, IX and X), protein C and antithrombin IIa, plasminogen activator inhibitor-1 were measured by routine photometric, chromogenic or enzyme-linked immunosorbent assays, while hyperfibrinolysis was defined by plasminogen activator inhibitor-1 levels. Both groups had similar age, gender and pattern of periportal fibrosis. Splenectomized patients showed significant reductions in portal vein diameter, alkaline phosphatase and bilirubin levels compared to non-operated patients, while for coagulation factors there were significant improvement in prothrombin, partial thromboplastin times and higher levels of factor VII, VIII, IX, X, protein C and plasminogen activator inhibitor-1.

Conclusion/Significance

This study shows that the decrease of flow pressure in portal circulation after splenectomy restores the capacity of hepatocyte synthesis, especially on the factor VII and protein C levels, and these findings suggest that portal hypertension in patients with hepatosplenic schistosomiasis influences liver functioning and the blood coagulation status.