No difference in between-country variability in use of newly approved orphan and non- orphan medicinal products - a pilot study

Background

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease, which rapidly leads to chronic respiratory failure requiring mechanical ventilation. Currently, forced vital capacity (FVC) < 50% is considered as physiologic marker for admitting patients to Noninvasive Positive Pressure Ventilation (NPPV) intervention, although it has been recently shown the median survival of patients with baseline FVC < 75% much shorter than median survival of patients with baseline FVC > 75%, independently by any treatment.

Aim

To assess the role of NPPV in improving outcome of ALS, a retrospective analysis was performed to investigate 1 year survival of ALS patients with FVC < 75% and nocturnal respiratory insufficiency, treated with NPPV, compared to a well-matched population of ALS patients, who refused or was intolerant to NPPV.

Methods

We investigated seventy-two consecutive ALS patients who underwent pulmonary function test. Forty-four presented a FVC > 75% and served as control group. Twenty-eight patients presented a FVC < 75% and showed, at polysomnography analysis, nocturnal respiratory insufficiency, requiring NPPV; sixteen were treated with NPPV, while twelve refused or were intolerant.

Results

Increased survival rate at 1 year in patients with FVC < 75% treated with NPPV, as compared to those who refused or could not tolerate NPPV (p = 0.02), was observed. The median rate of decline in FVC% was slower in NPPV patients than in patients who did not use NPPV (95% CI: 0.72 to 1.85; p < 0.0001).

Conclusion

This report demonstrates that early treatment with NPPV prolongs survival and reduces decline of FVC% in ALS.     

Combinatorial Domain Hunting: An effective approach for the identification of soluble protein domains adaptable to high-throughput applications

Exploitation of potential new targets for drug and vaccine development has an absolute requirement for multimilligram quantities of soluble protein. While recombinant expression of full-length proteins is frequently problematic, high-yield soluble expression of functional subconstructs is an effective alternative, so long as appropriate termini can be identified. Bioinformatics localizes domains, but doesn't predict boundaries with sufficient accuracy, so that subconstructs are typically found by trial and error. Combinatorial Domain Hunting (CDH) is a technology for discovering soluble, highly expressed constructs of target proteins. CDH combines unbiased, finely sampled gene-fragment libraries, with a screening protocol that provides "holistic" readout of solubility and yield for thousands of protein fragments. CDH is free of the "passenger solubilization" and out-of-frame translational start artifacts of fusion-protein systems, and hits are ready for scale-up expression. As a proof of principle, we applied CDH to p85alpha, successfully identifying soluble and highly expressed constructs encapsulating all the known globular domains, and immediately suitable for downstream applications.     

IGFBP7 induces apoptosis of acute myeloid leukemia cells and synergizes with chemotherapy in suppression of leukemia cell survival

Despite high remission rates after chemotherapy, only 30–40% of acute myeloid leukemia (AML) patients survive 5 years after diagnosis. This extremely poor prognosis of AML is mainly caused by treatment failure due to chemotherapy resistance. Chemotherapy resistance can be caused by various features including activation of alternative signaling pathways, evasion of cell death or activation of receptor tyrosine kinases such as the insulin growth factor-1 receptor (IGF-1R). Here we have studied the role of the insulin-like growth factor-binding protein-7 (IGFBP7), a tumor suppressor and part of the IGF-1R axis, in AML. We report that IGFBP7 sensitizes AML cells to chemotherapy-induced cell death. Moreover, overexpression of IGFBP7 as well as addition of recombinant human IGFBP7 is able to reduce the survival of AML cells by the induction of a G2 cell cycle arrest and apoptosis. This effect is mainly independent from IGF-1R activation, activated Akt and activated Erk. Importantly, AML patients with high IGFBP7 expression have a better outcome than patients with low IGFBP7 expression, indicating a positive role for IGFBP7 in treatment and outcome of AML. Together, this suggests that the combination of IGFBP7 and chemotherapy might potentially overcome conventional AML drug resistance and thus might improve AML patient survival.Only 30–40% of acute myeloid leukemia (AML) patients survive 5 years after diagnosis.¹ This extremely poor prognosis is mainly caused by treatment failure due to chemotherapy resistance. This resistance is often a multifactorial phenomenon that can include enhanced expression or activation of receptor tyrosine kinases such as the insulin growth factor-1 receptor (IGF-1R).^{2, 3} The IGF-1R stimulates proliferation, protects cells from apoptosis and has been implicated in the development and maintenance of various cancers.^{4, 5} Several oncogenes require an intact IGF-1R pathway for their transforming activity⁶ and moreover, disruption or inhibition of IGF-1R activity has been shown to inhibit the growth and motility of a wide range of cancer cells in vitro and in mouse models.^{4, 5} IGF-1Rs are membrane receptors and binding of their ligand, the insulin-like growth factor-1 (IGF-1), results in receptor phosphorylation and activation of MAPK and PI3K/Akt signaling.⁴ Importantly, IGF-1, normally produced by the liver and bone marrow stromal cells, can stimulate the proliferation of cancer cells in vitro and genetic manipulations that reduce IGF-1 signaling can lead to decreased tumor growth.^{7, 8}In hematological malignancies, a role for IGF-1 signaling has been demonstrated in multiple myeloma (MM) where it stimulates growth and potently mediates survival.⁹ Several anti-IGF-1R strategies have been shown to inhibit MM growth.^{10, 11} In AML, expression of the IGF-1R and IGF-1 was detected in AML cell lines and primary AML blasts and stimulation with IGF-1 can promote the growth of AML cells.^{12, 13, 14} In addition, neutralizing IGF-1R antibodies and the tyrosine kinase inhibitors (TKIs) NVP-AEW541 and NVP-ADW742, have been shown to inhibit proliferation and to induce apoptosis.^{15, 16}In addition to its mitogenic and anti-apoptotic roles, directly influencing tumor development, IGF-1R appears to be a critical determinant of response to numerous anti-cancer therapies, including TKIs and chemotherapy.^{2, 3, 17, 18, 19, 20, 21, 22} In AML, activated IGF-1R signaling has been linked to cytarabine resistance, a drug included in every AML treatment schedule.¹⁷ Notably, in several cancer cell lines, a small subpopulation of drug-tolerant cancer cells exists that maintains their viability, after treatment with a lethal drug dose, via engagement of the IGF-1R.¹⁸The activity of the IGF-1R is tightly controlled at multiple levels, including their processing, endocytosis, trafficking and availability of its ligands.⁴ Ligand bioavailability is partly controlled by the family of secreted insulin-like growth factor-binding protein (IGFBP1 to IGFBP6), which can bind to IGFs therewith regulating the interaction of these ligands to their receptors. However, as IGFBPs are able to induce IGF-dependent and IGF-independent effects, the results of several studies on their role in cancer cell survival appeared to be controversial and complex.^{23, 24} In addition to IGFBPs, various IGFBP-related proteins have been identified.^{23, 25} One of these is the IGFB-related protein 1, also known as insulin-like growth factor-binding protein-7 (IGFBP7). IGFBP7 has 30% homology to IGFBP1 to IGFBP6 in its N-terminal domain and functions predominantly as a tumor suppressor.^{23, 24, 25, 26} In contrast to IGFBP1 to IGFBP6, which bind to the IGFs,²³ IGFBP7 is a secreted protein that can directly bind to the IGF-1R and thereby inhibits its activity.²⁷ The abundance of IGFBP7 is inversely correlated with tumor progression in hepatocellular carcinoma.²⁸ Importantly, decreased expression of IGFBP7 has been associated with therapy resistance^{29, 30} and increasing IGFBP7 levels can inhibit melanoma and breast cancer growth.^{31, 32} IGFBP7 was originally identified as being involved in Raf-mediated apoptosis and senescence³³ and also has been shown to induce senescence in mesenchymal stromal cells.³⁴We established that IGFBP7 induces a cell cycle block and apoptosis in AML cells and cooperates with chemotherapy in the induction of leukemia cell death. AML patients with low IGFBP7 expression have a worse outcome than patients with high IGFBP7 expression, indicating that AML patients might benefit from a combination therapy consisting of chemotherapy and IGFBP7. Our results define IGFBP7 as a focus to enhance chemotherapy efficacy and improve AML patient survival.     

Amylose conformational transitions in binary DMSO/water mixtures

The effects on amylose conformation of percentage water in dimethyl sulfoxide (DMSO)/water mixtures were measured by following changes in specific optical rotation, limiting viscosity number, and ¹³C-NMR chemical shifts. The temperature dependence of specific optical rotation showed differences in amylose conformation at four chosen ratios of dimethyl sulfoxide/water. An amylose conformational change was also deduced from ¹³C-NMR chemical shift data. Changes in limiting viscosity of amylose in different proportions of DMSO/water, and the effect of tetramethylurea on the specific rotation of amylose, indicate that intramolecular hydrogen bonding decreases with increased water content. 66.6% DMSO appears to be a crossover concentration, below which the helical conformation is progressively lost as water is added. When water content is over 60%, transition to a conformation which allows iodine complexation to take place is complete. A transition of amylose conformation from helix to loose helix to random coil with increasing water content was deduced from the experimental results.     

Development of multiple strain competitive index assays for <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> using pIMC; a new site-specific integrative vector

Background  

The foodborne, gram-positive pathogen, Listeria monocytogenes, is capable of causing lethal infections in compromised individuals. In the post genomic era of L. monocytogenes research, techniques are required to identify and validate genes involved in the pathogenicity and environmental biology of the organism. The aim here was to develop a widely applicable method to tag L. monocytogenes strains, with a particular emphasis on the development of multiple strain competitive index assays.     

Molecular Simulation of Adsorption of Guest Molecules in Zeolitic Materials: A Comparative Study of Intermolecular Potentials

Abstract

Grand Canonical Monte Carlo simulation, together with an appropriate guest-host forcefield is shown to provide reasonably accurate predictions of adsorption properties of guest molecules in a variety of zeolitic materials. The use of a simple guest-host Kiselev-type potential permits the calculations to capture the essence of the behavior of simple guest-host systems such as rare gases or methane molecules in neutral AlPO₄-5. However, a full scale potential is needed in the more complex cases of large anisotropic molecules adsorbed in cationic zeolites (such as xylene isomers in faujasite). The guest-host potential model developed by Nicholson and coworkers is shown to allow an excellent transferability of the potential parameters from one guest/host system to another.     

An h.p.l.c. method for determining chain-length distribution in some glycogens.

Human, oyster, Streptococcus mitis, and phyto-glycogen samples were debranched using Pseudomonas amylodermosa isoamylase (EC 3.2.1.68). The distribution of chain lengths was studied by high-performance liquid chromatography on reversed-phase columns, with water as eluent. Quantitative data was obtained over the degree of polymerisation range three to eighteen (d.p. 3-18), and oligosaccharides up to d.p. 26 were detected. No single column was found suitable for the resolution of the complete range of oligosaccharides, two columns being necessary for the quantitative analysis. The resulting "fingerprints" of chain lengths are characteristic of the glycogen source and should be useful for both comparison purposes among glycogens and for monitoring procedures of glycogen isolation.