Chronic immune therapy induces a progressive increase in intratumoral T suppressor activity and a concurrent loss of tumor-specific CD8+ T effectors in her-2/neu transgenic mice bearing advanced spontaneous tumors

A single intratumoral injection of IL-12 and GM-CSF-encapsulated microspheres induces the complete regression of advanced spontaneous tumors in her-2/neu transgenic mice. However, tumor regression in this model is transient and long-term cure is not achieved due to recurrence. Posttherapy molecular analysis of immune activation/suppression markers within the tumor microenvironment demonstrated a dramatic up-regulation of IFN-gamma and a concomitant down-regulation of Forkhead/winged-helix protein 3 (Foxp3), TGFbeta, and IL-10 expression. Therapy-induced reversion of immune suppression was transient since all three markers of suppression recovered rapidly and surpassed pretherapy levels by day 7 after treatment, resulting in tumor resurgence. Repeated treatment enhanced short-term tumor regression, but did not augment long-term survival. Serial long-term analysis demonstrated that although chronic stimulation enhanced the IFN-gamma response, this was countered by a parallel increase in Foxp3, TGFbeta, and IL-10 expression. Analysis of tumor-infiltrating T lymphocyte populations showed that the expression of Foxp3 and IL-10 was associated with CD4(+)CD25(+) T cells. Repeated treatment resulted in a progressive increase in tumor-infiltrating CD4(+)CD25(+)Foxp3(+) T suppressor cells establishing their role in long-term neutralization of antitumor activity. Analysis of tumor-infiltrating CD8(+) T cells demonstrated that although treatment enhanced IFN-gamma production, antitumor cytotoxicity was diminished. Monitoring of CD8(+) T cells that specifically recognized a dominant MHC class I her-2/neu peptide showed a dramatic increase in tetramer-specific CD8(+) T cells after the first treatment; however, continuous therapy resulted in the loss of this population. These results demonstrate that both enhanced suppressor activity and deletion of tumor-specific T cells are responsible for the progressive loss of efficacy that is associated with chronic immune therapy.     

A computer-based model for the regulation of mitogen activated protein kinase (MAPK) activation

Computer simulations and mathematical modeling of biological processes are becoming increasingly popular, and yet the complexity of the biochemical systems or the differences between experimental setups make it very difficult to establish a standard formula for these modeling projects. Before we can start using computer-based models for predictions or targeted experiment designs, it is very important to establish a reliable model on which those predictions can be based and experimentally tested. Here we attempt to present a computer model for the mitogen-activated protein kinase (MAPK) signaling cascade which is consistent with previously published experimental results. In this study we have focused our attention to a generic MAPK ERK (extracellular signal-regulated kinase) pathway activated by epidermal growth factor (EGF) in an attempt to understand how receptors may achieve different activation kinetics of the MAPK signaling. We successfully show that the level of receptor expression is one key determinant in this regulation, and that the binding affinity of the active receptor to adaptor proteins can have a small but albeit direct effect on the downstream activation.     

Cutting edge: the toll pathway is required for resistance to gram-positive bacterial infections in Drosophila

In Drosophila, the response against various microorganisms involves different recognition and signaling pathways, as well as distinct antimicrobial effectors. On the one hand, the immune deficiency pathway regulates the expression of antimicrobial peptides that are active against Gram-negative bacteria. On the other hand, the Toll pathway is involved in the defense against filamentous fungi and controls the expression of antifungal peptide genes. The gene coding for the only known peptide with high activity against Gram-positive bacteria, Defensin, is regulated by both pathways. So far, survival experiments to Gram-positive bacteria have been performed with Micrococcus luteus and have failed to reveal the involvement of one or the other pathway in host defense against such infections. In this study, we report that the Toll pathway, but not that of immune deficiency, is required for resistance to other Gram-positive bacteria and that this response does not involve Defensin.     

Whitemere, a lake that defies some conventions about nutrients

1. Whitemere, a kettle-hole lake in north-west England (z_m,14 m, area 22.5 ha) has extremely high maximal concentrations (around 1 mg L⁻¹) of total phosphorus (TP) and soluble reactive phosphorus (SRP), and comparatively low maximal concentrations of dissolved inorganic nitrogen (<0.5 mg N L⁻¹). Bioassays indicate that its phytoplankton is nitrogen limited, and it has surface blue-green algal blooms in summer. Palaeoecological investigations have shown that high populations of cyanophytes occurred more than 6000 years ago, even before human settlement of the catchment.
2. The reasons for this combination of features are not immediately clear. This paper attempts to distinguish between two hypotheses: that the high phosphorus concentrations arise from input of phosphate-rich groundwater; and that the high phosphorus concentrations arise from concentration and recycling mechanisms within the lake.
3. The lake is entirely fed by ground water, direct rainfall and surface sheet flow. The concentrations of phosphorus in the ground water are much lower than in the lake water. The lake is stratified and has high phosphorus concentrations in the anaerobic hypolimnion in summer as a result of substantial release from the sediment. Vertical turbulence in the relatively weakly stratified water column may transfer substantial amounts to the epilimnion.There is also substantial phosphorus release from the sediments under the aerobic epilimnion.
4. There is little outflow of water and phosphorus. Most of the phosphorus is returned to the sediment under winter isothermal conditions, to be released again the following summer. The lake has probably maintained these mechanisms for millennia. Recent eutrophication is likely to have been driven by nitrogen inputs from greater intensification of agriculture in the catchment.     

Chinese hamster ovary (CHO) host cell engineering to increase sialylation of recombinant therapeutic proteins by modulating sialyltransferase expression

N‐Glycans of human proteins possess both α2,6‐ and α2,3‐linked terminal sialic acid (SA). Recombinant glycoproteins produced in Chinese hamster overy (CHO) only have α2,3‐linkage due to the absence of α2,6‐sialyltransferase (St6gal1) expression. The Chinese hamster ST6GAL1 was successfully overexpressed using a plasmid expression vector in three recombinant immunoglobulin G (IgG)‐producing CHO cell lines. The stably transfected cell lines were enriched for ST6GAL1 overexpression using FITC‐Sambucus nigra (SNA) lectin that preferentially binds α2,6‐linked SA. The presence of α2,6‐linked SA was confirmed using a novel LTQ Linear Ion Trap Mass Spectrometry (LTQ MS) method including MSn fragmentation in the enriched ST6GAL1 Clone 27. Furthermore, the total SA (mol/mol) in IgG produced by the enriched ST6GAL1 Clone 27 increased by 2‐fold compared to the control. For host cell engineering, the CHOZN^® GS host cell line was transfected and enriched for ST6GAL1 overexpression. Single‐cell clones were derived from the enriched population and selected based on FITC‐SNA staining and St6gal1 expression. Two clones (“ST6GAL1 OE Clone 31 and 32”) were confirmed for the presence of α2,6‐linked SA in total host cell protein extracts. ST6GAL1 OE Clone 32 was subsequently used to express SAFC human IgG1. The recombinant IgG expressed in this host cell line was confirmed to have α2,6‐linked SA and increased total SA content. In conclusion, overexpression of St6gal1 is sufficient to produce recombinant proteins with increased sialylation and more human‐like glycoprofiles without combinatorial engineering of other sialylation pathway genes. This work represents our ongoing effort of glycoengineering in CHO host cell lines for the development of “bio‐better” protein therapeutics and cell culture vaccine production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:334–346, 2015     

“Constructing” the Cell Cycle in 3D

The cycle of duplication and division, known as the cell cycle, is the essential mechanism by which all living organisms reproduce. This activity allows students to develop an understanding of the main events that occur during the typical eukaryotic cell cycle mostly in the process of mitotic phase that divides the duplicated genetic material creating two genetically identical daughter cells.     

Crosslinked aggregates of Rhizopus oryzae lipase as industrial biocatalysts: Preparation, optimization, characterization, and application for enantioselective resolution reactions

Lipase from Rhizopus oryzae (ROL) was immobilized as crosslinked enzyme aggregate (CLEA) via precipitation with ammonium sulfate and simultaneous crosslinking with glutaraldehyde. The optimum conditions of the immobilization process were determined. Lipase CLEAs showed a twofold increase in activity when Tween 80‐pretreated lipase was used for CLEA preparation. CLEAs were shown to have several advantages compared to free lipase. CLEAs were more stable at 50°C and 60°C as well as for a wide range of pH. After incubation at 50°C, CLEA showed 74% of initial activity whereas free enzyme was totally inactivated. Reduction of Schiff bases has been performed for the first time in the CLEA preparation process significantly improving the chemically modified CLEAs' reusability, thus providing an enzyme with high potential for recycling even under aqueous reaction conditions where enzyme leakage is, in general, one of the major problems. The CLEA retained 91% activity after 10 cycles in aqueous medium. The immobilized enzyme was used for kinetic resolution reactions. Results showed that immobilization had an enhancing effect on the conversion (c) as well as on the enantiomeric ratio (E). ROL CLEA displayed five times higher enantioselectivity for the hydrolysis of (R,S)‐1‐phenylethyl acetate and likewise 1.5 times higher enantioselectivity for the transesterification of racemic (R–S)‐1‐phenylethanol with vinylacetate. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 937–945, 2012 This article was published online on June 26, 2012. An edit was subsequently requested. This notice is included in the online and print versions to indicate that both have been corrected [27 June 2012].     

Alterations in promoter methylation status of tumor suppressor HIC1, SFRP2, and DAPK1 genes in prostate carcinomas

Hypermethylated genomic DNA is a common feature in tumoral tissues, although the prevalence of this modification remains poorly understood. We aimed to determine the frequency of five tumor suppressor (TS) genes in prostate cancer and the correlation between promoter hypermethylation of these genes and low and high grade of prostate carcinomas. A total of 30 prostate tumor specimens were investigated for promoter methylation status of TS hypermethylated in cancer 1 (HIC1), death-associated protein kinase 1 (DAPK1), secreted frizzled-related protein 2 (SFRP2), cyclin-dependent kinase inhibitor 2A (p16), and O-6-methylguanine-DNA methyltransferase (MGMT) genes by using bisulfite modifying method. A high frequency of promoter hypermethylation was found in HIC1 (70.9%), SFRP2 (58.3%), and DAPK1 (33.3%) genes in tumor samples that were examined. The current data show high frequency of hypermethylation changes in HIC1, SFRP2, and DAPK1 genes in prostate carcinomas of high Gleason Score (GS).     

Design,synthesis and docking study of novel coumarin ligands as potential selective acetylcholinesterase inhibitors

New coumaryl-thiazole derivatives with the acetamide moiety as a linker between the alkyl chains and/or the heterocycle nucleus were synthesized and in vitro tested as acetylcholinesterase (AChE) inhibitors. 2-(diethylamino)-N-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)acetamide (6c, IC₅₀ value of 43?nM) was the best AChE inhibitor with a selectivity index of 4151.16 over BuChE. Kinetic study of AChE inhibition revealed that 6c was a mixed-type inhibitor. Moreover, the result of H4IIE hepatoma cell toxicity assay for 6c showed negligible cell death. Molecular docking studies were also carried out to clarify the inhibition mode of the more active compounds. Best pose of compound 6c is positioned into the active site with the coumarin ring wedged between the residues of the CAS and catalytic triad of AChE. In addition, the coumarin ring is anchored into the gorge of the enzyme by H-bond with Tyr130.