Overexpression of biologically active VEGF121 fusion proteins in Escherichia coli

Vascular endothelial growth factor-A (VEGF) exists as five different isoforms, which exert their growth stimulatory effects through interaction with the FLK and KDR receptors. The VEGF(121) isoform has been employed as a highly selective carrier of therapeutic agents to target tumor endothelial cells resulting in inhibition of tumor growth and metastasis. VEGF(121) and VEGF(121)/rGel fusion toxin containing hexa-histidine tags were expressed in Escherichia coli AD494 (DE3) pLysS. Media containing glycerol as a primary carbon source increased the specific expression levels of soluble VEGF(121) and VEGF(121)/rGel (mg/L/OD10) by more than two-fold over LB media when grown in a batchtype cultivation in a bioreactor. High cell densities over OD 40 were achieved using a fed-batch method and employing feeding medium containing glycerol and yeast extract. The overall production of the target proteins was improved 18-fold for VEGF(121) (59.2mg/L) and 27-fold for VEGF(121)/rGel (42.5mg/L), respectively, compared to the conventional flask cultivation method (3.3 and 1.6mg/L for VEGF(121) and VEGF(121)/rGel, respectively). The purified VEGF(121) and VEGF(121)/rGel fusion proteins were biologically active as assessed by phosphorylation of KDR receptors and cytotoxicity against KDR expressing cells.     

Immunotherapy of myeloid leukaemia

The treatment of myeloid leukaemia has progressed in recent years with the advent of donor leukocyte infusions (DLI), haemopoietic stem cell transplants (HSCTs) and targeted therapies. However, relapse has a high associated morbidity rate and a method for removing diseased cells in first remission, when a minimal residual disease state is achieved and tumour load is low, has the potential to extend remission times and prevent relapse especially when used in combination with conventional treatments. Acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) are heterogeneous diseases which lack one common molecular target while chronic myeloid leukaemia (CML) patients have experienced prolonged remissions through the use of targeted therapies which remove BCR-ABL⁺ cells effectively in early chronic phase. However, escape mutants have arisen and this therapy has little effectivity in the late chronic phase. Here we review the immune therapies which are close to or in clinical trials for the myeloid leukaemias and describe their potential advantages and disadvantages.     

Quantification of mycorrhizal water uptake via high-resolution on-line water content sensors

The benefits of mycorrhizas for host plants are well known for a large number of species. However, experimental evaluations of the hyphal contribution to the total water uptake and the assessment of the bulk flow velocity in the hyphae are so far contradictory. Barley (Hordeum vulgaris L. Scarlet) with the inoculum Glomus intraradices was grown in a split plant-hyphal chamber with a 5 mm air gap. During the preparation of the chambers with a loamy-silt soil, water content sensors were inserted in each of the plant and the hyphal compartments. These sensors allow non-destructive measurements with high resolution. In total, 8 drying periods with a length of several days were applied with repeated watering following each drying period. A clear decline in water content in the hyphal compartment during each drying period supports the ability of hyphae to transfer water into the plant compartment. The difference between the decline in the hyphal compartment with and without arbuscular mycorrhyzal fungi is significant at the p?<?0.000001 level. The direct and indirect hyphal contribution to the total water uptake was estimated to be about 20%. The application of capacitance sensors for water content determination with a special geometry adapted to the plant-hyphal chambers allows the evaluation of the hyphal water flow with high accuracy.     

A novel multiplexed immunoassay identifies CEA,IL-8 and prolactin as prospective markers for Dukes’ stages A-D colorectal cancers

Background

Current methods widely deployed for colorectal cancers (CRC) screening lack the necessary sensitivity and specificity required for population-based early disease detection. Cancer-specific protein biomarkers are thought to be produced either by the tumor itself or other tissues in response to the presence of cancers or associated conditions. Equally, known examples of cancer protein biomarkers (e.g., PSA, CA125, CA19-9, CEA, AFP) are frequently found in plasma at very low concentration (pg/mL-ng/mL). New sensitive and specific assays are therefore urgently required to detect the disease at an early stage when prognosis is good following surgical resection. This study was designed to meet the longstanding unmet clinical need for earlier CRC detection by measuring plasma candidate biomarkers of cancer onset and progression in a clinical stage-specific manner. EDTA plasma samples (1 μL) obtained from 75 patients with Dukes’ staged CRC or unaffected controls (age and sex matched with stringent inclusion/exclusion criteria) were assayed for expression of 92 human proteins employing the Proseek® Multiplex Oncology I proximity extension assay. An identical set of plasma samples were analyzed utilizing the Bio-Plex Pro™ human cytokine 27-plex immunoassay.

Results

Similar quantitative expression patterns for 13 plasma antigens common to both platforms endorsed the potential efficacy of Proseek as an immune-based multiplex assay for proteomic biomarker research. Proseek found that expression of Carcinoembryonic Antigen (CEA), IL-8 and prolactin are significantly correlated with CRC stage.

Conclusions

CEA, IL-8 and prolactin expression were found to identify between control (unaffected), non-malignant (Dukes’ A + B) and malignant (Dukes’ C + D) stages.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9081-x) contains supplementary material, which is available to authorized users.     

Proteomics Reveals Cell‐Surface Urokinase Plasminogen Activator Receptor Expression Impacts Most Hallmarks of Cancer

While metastasis is the primary cause of colorectal cancer (CRC) mortality, the molecular mechanisms underpinning it remains elusive. Metastasis is propagated through driver oncogene/suppressor gene mutations, accompanied by passenger mutations and underlying genomic instability. To understand cancer biology, a unifying framework called the “hallmarks of cancer” (HoCs) has been developed, which organizes cell biological alterations under ten key hallmarks. Underlying these HoCs, genome instability generates mutational diversity that is amplified by inflammation. Recognizing how critical cancer cell‐surface proteins influence, these HoCs have been proposed to accelerate precision medicine therapeutic development. A moderate decrease (43%↓) in HCT116 cell surface urokinase plasminogen activator receptor (uPAR) expression mitigates against many HoCs driven by these cell's KRAS and PIK3CA mutational signature. Comprehensive proteomics (whole cell lysis with two membrane protein enrichments) coupled with ingenuity pathway analysis (IPA) demonstrates that uPAR negates essential pathways across the HoC spectrum, particularly those associated with metastasis, resisting cell death, and sustaining proliferation, and parallels Cancer Hallmarks Analytics Tool analysis. Decreasing uPAR predominantly alters metastasis‐related and uPAR‐interactome protein expression (e.g., EGFR, caveolin, vitronectin, integrin β4). Collectively, it is demonstrated that uPAR is a lynchpin protein capable of regulating several HoC pathways in a classical CRC mutational background.     

A functional assay for microRNA target identification and validation

MicroRNAs (miRNA) are a class of small RNA molecules that regulate numerous critical cellular processes and bind to partially complementary sequences resulting in down-regulation of their target genes. Due to the incomplete homology of the miRNA to its target site identification of miRNA target genes is difficult and currently based on computational algorithms predicting large numbers of potential targets for a given miRNA. To enable the identification of biologically relevant miRNA targets, we describe a novel functional assay based on a 3'-UTR-enriched library and a positive/negative selection strategy. As proof of principle we have used mir-130a and its validated target MAFB to test this strategy. Identification of MAFB and five additional targets and their subsequent confirmation as mir-130a targets by western blot analysis and knockdown experiments validates this strategy for the functional identification of miRNA targets.     

Correlations between Integrin ανβ6 Expression and Clinico-Pathological Features in Stage B and Stage C Rectal Cancer

Integrin ανβ6 is highly expressed in a range of human cancers and frequently correlates with patient survival. This study examines correlations between ανβ6 expression and patient clinico-pathological features in Stage B and Stage C rectal cancer, including overall survival. Expression of ανβ6 was measured in 362 Stage B or C rectal cancer tissue samples at the tumour central region, invasive tumour front and adjacent non-neoplastic mucosa using immunohistochemistry. Distribution of ανβ6 was found to be significantly higher at the invasive front compared to central regions of the tumour (p<0.001) or adjacent non-neoplastic mucosa (p<0.001) suggesting ανβ6 plays a role in tumour cell invasion. However, integrin ανβ6 expression was not associated with clinico-pathological features or overall survival indicating it is not an independent prognostic marker differentiating Stage B or C rectal cancer. Previous ανβ6 studies have suggested the expression of ανβ6 is involved in the earlier stages (i.e. Stages A/B) of tumour progression rather than the later stages (i.e. Stages C/D). However, our study has revealed that in rectal cancer ανβ6 expression does not increase between Stages B and C, but may occur earlier, namely before or during Stage B cancer.     

Characterisation of the actions of group I metabotropic glutamate receptor subtype selective ligands on excitatory amino acid release and sodium-dependent re-uptake in rat cerebrocortical minislices

In this study we have tested the effects of a wide range of metabotropic glutamate receptor ligands on (i) depolarisation-evoked efflux of pre-accumulated d-[3H]aspartic acid (d-[3H]asp) from rapidly superfused rat cerebrocortical minislices, and (ii) Na+-dependent uptake of d-[3H]asp into cerebrocortical tissue. Transient elevations in extracellular K+ produced concentration-dependent increases in d-[3H]asp efflux. A submaximally effective concentration (50 mm) was used in all subsequent experiments. The broad-spectrum mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD; EC50 17.8 microm], the group I mGlu-selective agonist (S)-3,5-dihydroxyphenylglycine [(S)-3,5-DHPG; EC50 0.5 microm] and the mGlu5 receptor subtype-selective agonist (RS)-2-chloro-5-hydroxyphenylglycine [(RS)-CHPG; EC50 7.3 microm] all concentration-dependently potentiated high K+-evoked d-[3H]asp efflux in the absence of effects on basal outflow of radiolabel. At concentrations selective for mGlu1 receptors, the antagonists (RS)-1-aminoindan-1,5-dicarboxylic acid [(RS)-AIDA; 10-300 microm]; (+)-2-methyl-4-carboxyphenylglycine [LY367385; 1-100 microm] and 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylate ethyl ester [CPCCOEt, 1-30 microm] all failed to inhibit responses to (S)-3,5-DHPG. However, the broad-spectrum mGlu receptor antagonist (S)-alpha-methyl-4-carboxyphenylglycine [(S)-MCPG; IC50 88.5 microm] together with the recently described mGlu5-selective antagonists, 2-methyl-6-(phenylethynyl)-pyridine (MPEP; IC50 0.6 microm), 6-methyl-2-(phenyl-azo)-3-pyridinol (SIB-1757; IC50 4.4 microm) and (E)-2-methyl-6-(2-phenylethenyl)pyridine (SIB-1893; IC50 3.1 microm), at mGlu5-selective concentrations, all powerfully and concentration-dependently inhibited (S)-3,5-DHPG-evoked responses. Two selective excitatory amino acid (EAA) uptake inhibitors, l-trans-2,4-pyrrolidine dicarboxylate (l-trans-2,4-PDC; IC50 229 microm) and dl-threo-beta-benzyloxyaspartate (dl-TBOA; IC50 665 microm) both inhibited the Na+-dependent uptake of d-[3H]asp into cerebrocortical minislices. Importantly, none of the mGlu ligands utilized in the present study significantly inhibited d-[3H]asp uptake at concentrations shown to potentiate K+-evoked efflux. These data demonstrate for the first time that mGlu5 ligands modulate extracellular EAA concentrations by a direct effect on mGlu5-type autoreceptors on EAA nerve terminals as they evoke clear changes in EAA release in the absence of any effects on EAA uptake. Selective mGlu5 receptor antagonists that show high potency and good central bioavailability may provide novel classes of neuroprotective agents for the treatment of brain disorders associated with abnormal EAAergic neurotransmission.