Tuning of CHO secretional machinery improve activity of secreted therapeutic sulfatase 150-fold.

Rare diseases are, despite their name, collectively common and millions of people are affected daily of conditions where treatment often is unavailable. Sulfatases are a large family of activating enzymes related to several of these diseases. Heritable genetic variations in sulfatases may lead to impaired activity and a reduced macromolecular breakdown within the lysosome, with several severe and lethal conditions as a consequence. While therapeutic options are scarce, treatment for some sulfatase deficiencies by recombinant enzyme replacement are available. The recombinant production of such sulfatases suffers greatly from both low product activity and yield, further limiting accessibility for patient groups. To mitigate the low product activity, we have investigated cellular properties through computational evaluation of cultures with varying media conditions and comparison of two CHO clones with different levels of one active sulfatase variant. Transcriptome analysis identified 18 genes in secretory pathways correlating with increased sulfatase production. Experimental validation by upregulation of a set of three key genes improved the specific enzymatic activity at varying degree up to 150-fold in another sulfatase variant, broadcasting general production benefits. We also identified a correlation between product mRNA levels and sulfatase activity that generated an increase in sulfatase activity when expressed with a weaker promoter. Furthermore, we suggest that our proposed workflow for resolving bottlenecks in cellular machineries, to be useful for improvements of cell factories for other biologics as well.

Comparative treatment planning study for mediastinal Hodgkin's lymphoma: impact on normal tissue dose using deep inspiration breath hold proton and photon therapy.

BACKGROUND: Late effects induced by radiotherapy (RT) are of great concern for mediastinal Hodgkin's lymphoma (HL) patients and it is therefore important to reduce normal tissue dose. The aim of this study was to investigate the impact on the normal tissue dose and target coverage, using various combinations of intensity modulated proton therapy (IMPT), volumetric modulated arc therapy (VMAT) and 3-dimensional conformal RT (3D-CRT), planned in both deep inspiration breath hold (DIBH) and free breathing (FB). MATERIAL AND METHODS: Eighteen patients were enrolled in this study and planned with involved site RT. Two computed tomography images were acquired for each patient, one during DIBH and one during FB. Six treatment plans were created for each patient; 3D-CRT in FB, 3D-CRT in DIBH, VMAT in FB, VMAT in DIBH, IMPT in FB and IMPT in DIBH. Dosimetric impact on the heart, left anterior descending (LAD) coronary artery, lungs, female breasts, target coverage, and also conformity index and integral dose (ID), was compared between the different treatment techniques. RESULTS: The use of DIBH significantly reduced the lung dose for all three treatment techniques, however, no significant difference in the dose to the female breasts was observed. Regarding the heart and LAD doses, large individual variations were observed. For VMAT, the mean heart and LAD doses were significantly reduced using DIBH, but no significant difference was observed for 3D-CRT and IMPT. Both IMPT and VMAT resulted in improved target coverage and more conform dose distributions compared to 3D-CRT. IMPT generally showed the lowest organs at risk (OAR) doses and significantly reduced the ID compared to both 3D-CRT and VMAT. CONCLUSIONS: The majority of patients benefited from treatment in DIBH, however, the impact on the normal tissue dose was highly individual and therefore comparative treatment planning is encouraged. The lowest OAR doses were generally observed for IMPT in combination with DIBH.

Treatment planning for spinal radiosurgery : A competitive multiplatform benchmark challenge.

PURPOSE: To investigate the quality of treatment plans of spinal radiosurgery derived from different planning and delivery systems. The comparisons include robotic delivery and intensity modulated arc therapy (IMAT) approaches. Multiple centers with equal systems were used to reduce a bias based on individual's planning abilities. The study used a series of three complex spine lesions to maximize the difference in plan quality among the various approaches. METHODS: Internationally recognized experts in the field of treatment planning and spinal radiosurgery from 12 centers with various treatment planning systems participated. For a complex spinal lesion, the results were compared against a previously published benchmark plan derived for CyberKnife radiosurgery (CKRS) using circular cones only. For two additional cases, one with multiple small lesions infiltrating three vertebrae and a single vertebra lesion treated with integrated boost, the results were compared against a benchmark plan generated using a best practice guideline for CKRS. All plans were rated based on a previously established ranking system. RESULTS: All 12 centers could reach equality (n = 4) or outperform (n = 8) the benchmark plan. For the multiple lesions and the single vertebra lesion plan only 5 and 3 of the 12 centers, respectively, reached equality or outperformed the best practice benchmark plan. However, the absolute differences in target and critical structure dosimetry were small and strongly planner-dependent rather than system-dependent. Overall, gantry-based IMAT with simple planning techniques (two coplanar arcs) produced faster treatments and significantly outperformed static gantry intensity modulated radiation therapy (IMRT) and multileaf collimator (MLC) or non-MLC CKRS treatment plan quality regardless of the system (mean rank out of 4 was 1.2 vs. 3.1, p = 0.002). CONCLUSIONS: High plan quality for complex spinal radiosurgery was achieved among all systems and all participating centers in this planning challenge. This study concludes that simple IMAT techniques can generate significantly better plan quality compared to previous established CKRS benchmarks.

Inverse treatment planning for spinal robotic radiosurgery: an international multi-institutional benchmark trial.

Stereotactic radiosurgery (SRS) is the accurate, conformal delivery of high-dose radiation to well-defined targets while minimizing normal structure doses via steep dose gradients. While inverse treatment planning (ITP) with computerized optimization algorithms are routine, many aspects of the planning process remain user-dependent. We performed an international, multi-institutional benchmark trial to study planning variability and to analyze preferable ITP practice for spinal robotic radiosurgery. 10 SRS treatment plans were generated for a complex-shaped spinal metastasis with 21 Gy in 3 fractions and tight constraints for spinal cord (V14Gy < 2 cc, V18Gy < 0.1 cc) and target (coverage > 95%). The resulting plans were rated on a scale from 1 to 4 (excellent-poor) in five categories (constraint compliance, optimization goals, low-dose regions, ITP complexity, and clinical acceptability) by a blinded review panel. Additionally, the plans were mathemati-cally rated based on plan indices (critical structure and target doses, conformity, monitor units, normal tissue complication probability, and treatment time) and compared to the human rankings. The treatment plans and the reviewers' rankings varied substantially among the participating centers. The average mean overall rank was 2.4 (1.2-4.0) and 8/10 plans were rated excellent in at least one category by at least one reviewer. The mathematical rankings agreed with the mean overall human rankings in 9/10 cases pointing toward the possibility for sole mathematical plan quality comparison. The final rankings revealed that a plan with a well-balanced trade-off among all planning objectives was preferred for treatment by most par-ticipants, reviewers, and the mathematical ranking system. Furthermore, this plan was generated with simple planning techniques. Our multi-institutional planning study found wide variability in ITP approaches for spinal robotic radiosurgery. The participants', reviewers', and mathematical match on preferable treatment plans and ITP techniques indicate that agreement on treatment planning and plan quality can be reached for spinal robotic radiosurgery.

A truncated analogue of CCL2 mediates anti-fibrotic effects on murine fibroblasts independently of CCR2.

The truncated [1+9-76] CCL2 analogue, also known as 7ND, has been described in numerous reports as an anti-inflammatory and anti-fibrotic agent in a wide spectrum of animal models, e.g. models of cardiovascular disease, graft versus host disease and bleomycin-induced pulmonary fibrosis. 7ND has been reported to function as a competitive inhibitor of CCL2 signaling via CCR2 in human in vitro systems. In contrast, the mechanistic basis of 7ND action in animal models has not been previously reported. Here we have studied how 7ND interacts with CCL2 and CCR2 of murine origin. Surprisingly, 7ND was shown to be a weak inhibitor of murine CCL2/CCR2 signaling and displaced murine CCL2 (JE) from the receptor with a K(i)>1 µM. Using surface plasmon resonance, we found that 7ND binds murine CCL2 with a K(d) of 670 nM, which may indicate that 7ND inhibits murine CCL2/CCR2 signaling by a dominant negative mechanism rather than by competitive binding to the CCR2 receptor. In addition we observed that sub-nanomolar levels of 7ND mediate anti-fibrotic effects in CCR2 negative fibroblasts cultured from fibrotic lung of bleomycin-induced mice. Basal levels of extracellular matrix proteins were reduced (collagen type 1 and fibronectin) as well as expression levels of α-smooth muscle actin and CCL2. Our conclusion from these data is that the previously reported effects of 7ND in murine disease models most probably are mediated via mechanisms independent of CCR2.

Structural development of mercaptophenol self-assembled monolayers and the overlying mineral phase during templated CaCO3 crystallization from a transient amorphous film.

Formation of biomineral structures is increasingly attributed to directed growth of a mineral phase from an amorphous precursor on an organic matrix. While many in vitro studies have used calcite formation on organothiol self-assembled monolayers (SAMs) as a model system to investigate this process, they have generally focused on the stability of amorphous calcium carbonate (ACC) or maximizing control over the order of the final mineral phase. Little is known about the early stages of mineral formation, particularly the structural evolution of the SAM and mineral. Here we use near-edge X-ray absorption spectroscopy (NEXAFS), photoemission spectroscopy (PES), X-ray diffraction (XRD), and scanning electron microscopy (SEM) to address this gap in knowledge by examining the changes in order and bonding of mercaptophenol (MP) SAMs on Au(111) during the initial stages of mineral formation as well as the mechanism of ACC to calcite transformation during template-directed crystallization. We demonstrate that formation of ACC on the MP SAMs brings about a profound change in the morphology of the monolayers: although the as-prepared MP SAMs are composed of monomers with well-defined orientations, precipitation of the amorphous mineral phase results in substantial structural disorder within the monolayers. Significantly, a preferential face of nucleation is observed for crystallization of calcite from ACC on the SAM surfaces despite this static disorder.

Baculovirus-mediated gene transfer and recombinant protein expression do not interfere with insulin dependent phosphorylation of PKB/Akt in human SHSY-5Y and C3A cells.

BACKGROUND: Recombinant adenovirus vectors and transfection agents comprising cationic lipids are widely used as gene delivery vehicles for functional expression in cultured cells. Consequently, these tools are utilized to investigate the effects of functional over-expression of proteins on insulin mediated events. However, we have previously reported that cationic lipid reagents cause a state of insulin unresponsiveness in cell cultures. In addition, we have found that cultured cells often do not respond to insulin stimulation following adenovirus treatment. Infection with adenovirus compromises vital functions of the host cell leading to the activation of protein kinases central to insulin signalling, such as protein kinase B/Akt. Therefore, we investigated the effect of adenovirus infection on insulin unresponsiveness by means of Akt activation in cultured cells. Moreover, we investigated the use of baculovirus as a heterologous viral gene delivery vehicle to circumvent these phenomena. Since the finding that baculovirus can efficiently transduce mammalian cells, the applications of this viral system in gene delivery has greatly expanded and one advantage is the virtual absence of cytotoxicity in mammalian cells. RESULTS: We show that infection of human neuroblastoma SHSY-5Y and liver C3A cells with recombinant adenovirus results in the activation of Akt in a dose dependent manner. In addition, this activation makes treated cells unresponsive to insulin stimulation as determined by an apparent lack of differential phosphorylation of Akt on serine-473. Our data further indicate that the use of recombinant baculovirus does not increase the phosphorylation of Akt in SHSY-5Y and C3A cells. Moreover, following infection with baculovirus, SHSY-5Y and C3A cells respond to insulin by means of phosphorylation of Akt on serine-473 in the same manner as uninfected cells. CONCLUSION: Widely-used adenovirus vectors for gene delivery cause a state of insulin unresponsiveness in human SHSY-5Y and C3A cells in culture due to the activation of central protein kinases of the insulin signalling pathway. This phenomenon can be avoided when studying insulin signalling by using recombinant baculovirus as a heterologous viral expression system. In addition, our data may contribute to an understanding of the molecular mechanisms underlying baculovirus infection of human cells.

Effect of ring substitution position on the structural conformation of mercaptobenzoic acid self-assembled monolayers on Au(111).

Near-edge X-ray absorption fine structure (NEXAFS) spectroscopy, photoemission spectroscopy (PES), and contact angle measurements have been used to examine the structure and bonding of self-assembled monolayers (SAMs) prepared on Au(111) from the positional isomers of mercaptobenzoic acid (MBA). The isomer of MBA and solvent chosen in SAM preparation has considerable bearing upon film morphology. Carbon K-edge NEXAFS measurements indicate that the monomers of 2-, 3-, and 4-MBA have well-defined orientations within their respective SAMs. Monomers of 3- and 4-MBA assume an upright orientation on the Au substrates in monolayers prepared using an acetic acid in ethanol solvent. The aryl ring and carboxyl group of these molecules are tilted from the surface normal by a colatitudal angle of approximately 30 degrees . Preparation of 4-MBA SAMs using pure ethanol solvent, a more traditional means of synthesis, had no appreciable effect upon the monomer orientation. Nonetheless, S(2p) PES measurements illustrate that it results in extensive bilayer formation via carboxyl group hydrogen-bonding between 4-MBA monomers. In 2-MBA monolayers prepared using acetic acid/ethanol solvent, the monomers adopt a more prostrate orientation on the Au substrates, in which the aryl ring and carboxyl group of the molecules are tilted approximately 50 degrees from the surface normal. This configuration is consistent with an interaction between both the mercaptan sulfur and carboxyl group of 2-MBA with the underlying substrate. S(2p) and C(1s) PES experiments provide supporting evidence for a bidentate interaction between 2-MBA and Au(111).

Production of a truncated soluble human semicarbazide-sensitive amine oxidase mediated by a GST-fusion protein secreted from HEK293 cells.

Elevated levels of semicarbazide-sensitive amine oxidase (SSAO) activity have been observed in several human conditions such as congestive heart failure, diabetes mellitus, and inflammation. The reactive aldehydes and hydrogen peroxide produced by SSAO have been suggested to contribute to the progression of vascular complications associated with these conditions. In addition, SSAO activity has been shown to be involved in the leukocyte extravasation process at sites of inflammation. To facilitate characterization and development of specific and selective inhibitors of SSAO, we have developed a method for production of recombinant human SSAO. The extracellular region (residues 29-763) of human SSAO was expressed in HEK293 cells in fusion with a mutated Schistosoma japonicum glutathione S-transferase (GST) and secreted to the culture medium. The mutGST-SSAO fusion protein was purified in a single step by glutathione-affinity chromatography followed by site-specific cleavage using a GST-3C protease fusion protein to remove the mutGST fusion partner. A second glutathione-affinity chromatography step was then used to capture both the mutGST fusion partner and the GST-3C protease, resulting in milligram quantities of pure, enzymatically active, and soluble recombinant human SSAO.

Structure of human semicarbazide-sensitive amine oxidase/vascular adhesion protein-1.

Semicarbazide-sensitive amine oxidase (SSAO) belongs to a ubiquitous family of copper-containing amine oxidases (CuAOs). SSAO is also known as vascular adhesion protein-1 (VAP-1) and has been identified as one of the adhesion molecules involved in the leukocyte-extravasation process. The structure of a truncated soluble form of human SSAO has been solved and refined to 2.5 A. As expected, SSAO is a homodimer with a fold typical of the CuAO family. The topaquinone (TPQ) cofactor and a copper ion characteristic of CuAOs are present in the active site, with the TPQ in the active ;off-copper' conformation. The structure reveals that a leucine residue (Leu469) located adjacent to the active site could function as a gate controlling its accessibility. An RGD motif is displayed on the surface, where it could be involved in integrin binding and possibly play a role in the shedding of SSAO from the membrane. Carbohydrate moieties are observed at five of six potential N-glycosylation sites. Carbohydrates attached to Asn232 flank the active-site entrance and might influence substrate specificity. The structure of an adduct of SSAO and the irreversible inhibitor 2-hydrazinopyridine has been solved and refined to 2.9 A resolution. Together, these structures will aid efforts to identify natural substrates, provide valuable information for the design of specific inhibitors and direct further studies.

Crystallization of a truncated soluble human semicarbazide-sensitive amine oxidase.

Human semicarbazide-sensitive amine oxidase (SSAO) is a homodimeric copper-containing monoamine oxidase that occurs in both a membrane-bound and a soluble form. SSAO is also known as vascular adhesion protein-1 (VAP-1). A truncated soluble form of human SSAO (comprising residues 29-763) was expressed in human embryonic kidney 293 cells and purified to homogeneity. Tetragonal crystals were obtained and a data set extending to 2.5 A was collected. The crystals are merohedrally twinned and the estimation of the twinning fraction was complicated by pseudo-symmetry and the anisotropic character of the crystals. Using a recently developed method for twinning detection that is insensitive to phenomena such as anisotropy or pseudo-symmetry [Padilla & Yeates (2003), Acta Cryst. D59, 1124-1130], the twinning fraction was estimated to be 0.3. The structure was eventually solved by molecular replacement in space group P4(3).

High-level production and optimization of monodispersity of 11beta-hydroxysteroid dehydrogenase type 1.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is an intraluminally oriented, endoplasmic reticulum (ER)-bound enzyme catalyzing the interconversion between inactive cortisone and hormonally active cortisol. Heterologous production of 11beta-HSD1, devoid of its N-terminal transmembrane segment, is possible but yields only small amounts of soluble protein. Here we show that the soluble portion of recombinant 11beta-HSD1 produced in E. coli is found mainly as multimeric aggregates in the absence of detergent, and to a large extent associated with the endogenous chaperonin GroEL and other E. coli proteins. By co-overexpressing GroEL/ES and adding an 11beta-HSD1 inhibitor during protein synthesis, we have increased the accumulation of soluble 11beta-HSD1 by more than one order of magnitude. Using monodispersity as a screening criterion, we have also optimized the purification process by evaluating various solubilizing systems for the chromatographic steps, finally obtaining stable monodisperse preparations of both human and guinea pig 11beta-HSD1. By analytical ultracentrifugation, we could demonstrate that 11beta-HSD1 mainly exists as a dimer in the solubilized state. Moreover, active site titration of human 11beta-HSD1 revealed that at least 75% of the protein in a typical preparation represents active enzyme. Equilibrium unfolding experiments indicate that addition of inhibitor and the cofactor NADP(H) can stabilize the conformational stability of this enzyme in an additive manner. The outlined procedure may provide a general method for preparing similar proteins to oligomeric homogeneity and with retained biological activity.

Overexpression of semicarbazide-sensitive amine oxidase in smooth muscle cells leads to an abnormal structure of the aortic elastic laminas.

Elevated semicarbazide-sensitive amine oxidase (SSAO) activity has been observed in several human conditions, eg, diabetes, and it has been speculated that SSAO contributes to the development of vasculopathies associated with this disease. To investigate in vivo consequences of elevated expression of SSAO in vascular tissues, we have developed a transgenic model for overexpression of human SSAO in mice. A smooth muscle-specific promoter, smooth muscle alpha-actin promoter 8 (SMP8) was used. Transgenic expression of human SSAO in tissues with a high content of smooth muscle cells was confirmed by Northern blot analysis. Enzymatic analysis of homogenates from transgenic tissues showed elevated levels of SSAO activity compared to non-transgenic littermates. Furthermore, when plasma SSAO activity was analyzed, much higher activity was detected compared to plasma from control mice, indicating that plasma SSAO may originate from smooth muscle cells. Histopathological evaluation of aorta and renal artery from transgenic mice revealed an abnormal structure of the elastin tissue. Instead of the regularly folded elastic laminae normally found in tunica media of sacrificed mice, the elastic laminae were straight and unfolded with irregularly arranged elastic fibers, forming tangled webs, between the intercalating elastic laminae. These alterations of the elastin structures suggest that overexpression of SSAO has led to a reduced elasticity of the arteries. Moreover, the mean femoral arterial pressure of the SMP8 SSAO transgenic mice was significantly lower in comparison to non-transgenic littermates. This suggests that the transgenic mice have a defect in their ability to regulate blood pressure.