Induction of secretory phospholipase A2 in reactive astrocytes in response to transient focal cerebral ischemia in the rat brain

Although mRNA expression of group IIA secretory phospholipase A2 (sPLA2-IIA) has been implicated in responses to injury in the CNS, information on protein expression remains unclear. In this study, we investigated temporal and spatial expression of sPLA2-IIA mRNA and immunoreactivity in transient focal cerebral ischemia induced in rats by occlusion of the middle cerebral artery. Northern blot analysis showed a biphasic increase in sPLA2-IIA mRNA expression following 60-min of ischemia-reperfusion: an early phase at 30 min and a second increase at a late phase ranging from 12 h to 14 days. In situ hybridization localized the early-phase increase in sPLA2-IIA mRNA to the affected ischemic cortex and the late-phase increase to the penumbral area. Besides sPLA2-IIA mRNA, glial fibrillary acidic protein (GFAP) and cyclo-oxygenase-2 mRNAs, but not cytosolic PLA2, also showed an increase in the penumbral area at 3 days after ischemia-reperfusion. Immunohistochemistry of sPLA2-IIA indicated positive cells in the penumbral area similar to the GFAP-positive astrocytes but different from the isolectin B4-positive microglial cells. Confocal microscopy further confirmed immunoreactivity of sPLA2-IIA in reactive astrocytes but not in microglial cells. Taken together, these results demonstrate for the first time an up-regulation of the inflammatory sPLA2-IIA in reactive astrocytes in response to cerebral ischemia-reperfusion.     

Assessing the performance of the photo‐acoustic infrared gas monitor for measuring CO2, N2O,and CH4 fluxes in two major cereal rotations

Rapid, precise, and globally comparable methods for monitoring greenhouse gas (GHG) fluxes are required for accurate GHG inventories from different cropping systems and management practices. Manual gas sampling followed by gas chromatography (GC) is widely used for measuring GHG fluxes in agricultural fields, but is laborious and time‐consuming. The photo‐acoustic infrared gas monitoring system (PAS) with on‐line gas sampling is an attractive option, although it has not been evaluated for measuring GHG fluxes in cereals in general and rice in particular. We compared N₂O, CO_2, and CH₄ fluxes measured by GC and PAS from agricultural fields under the rice–wheat and maize–wheat systems during the wheat (winter), and maize/rice (monsoon) seasons in Haryana, India. All the PAS readings were corrected for baseline drifts over time and PAS‐CH₄ (PCH₄) readings in flooded rice were corrected for water vapor interferences. The PCH₄ readings in ambient air increased by 2.3 ppm for every 1000 mg cm^?3 increase in water vapor. The daily CO₂, N₂O, and CH₄ fluxes measured by GC and PAS from the same chamber were not different in 93–98% of all the measurements made but the PAS exhibited greater precision for estimates of CO₂ and N₂O fluxes in wheat and maize, and lower precision for CH₄ flux in rice, than GC. The seasonal GC‐ and PAS‐N₂O (PN₂O) fluxes in wheat and maize were not different but the PAS‐CO₂ (PCO₂) flux in wheat was 14–39% higher than that of GC. In flooded rice, the seasonal PCH₄ and PN₂O fluxes across N levels were higher than those of GC‐CH₄ and GC‐N₂O fluxes by about 2‐ and 4fold, respectively. The PAS (i) proved to be a suitable alternative to GC for N₂O and CO₂ flux measurements in wheat, and (ii) showed potential for obtaining accurate measurements of CH₄ fluxes in flooded rice after making correction for changes in humidity.     

Rv0802c acetyltransferase from Mycobacterium tuberculosis H37Rv

Rv0802c acetyltransferase is a mycobacterial RNase E-associated protein. 6His and FLAG-tagged acetyltransferase was cloned from Mycobacterium tuberculosis H37Rv, expressed in Escherichia coli and partially purified. It is a 25 kDa protein showing a modest sequence homology with other acetyltransferases. The R-X-X-G-X-G sequence for acetyl-coenzyme A recognition and binding can be found in the molecule.     

Juvenile animal studies for the development of paediatric medicines: a description and conclusions from a European medicines agency workshop on juvenile animal testing for nonclinical assessors

A workshop organised by the European Medicines Agency involved assessors and experts present in a Nonclinical Working Group evaluating juvenile animal studies for Paediatric Investigation Plans in collaboration with the Paediatric Committee and the Safety Working Party of the Committee for Human Medicinal Products. The objective of the workshop was to analyse which juvenile animal studies proposals were received and agreed by the Paediatric Committee, to check consistency and how to apply the existing European guideline on juvenile animal studies. A comparison of main organ system development in man vs. animal species was presented to guide the review and to support species selection and protocol design. An analysis of juvenile animal studies included in finalised PIP's was also presented. Out of 109 paediatric investigation plans finalised between November 2008 and March 2009, 43 included one or more juvenile animal studies. In most cases the preferred species was the rat; one species only was requested to be studied (20/22), but in a minority two species were required (2/22). When deciding on the characteristics of the juvenile animal studies, such as age of animals at study start, the age of the children targeted by the medicine was considered. It is expected that the increasing experience gained by Applicants and Regulators will allow further refining the criteria for these juvenile animal studies. Further research on this topic is highly encouraged in the European Regulatory framework. Birth Defects Res (Part B) 89:467–473, 2010. © 2010 Wiley‐Liss, Inc.     

Serum 27-nor-5β-cholestane-3,7,12,24,25 pentol glucuronide discovered by metabolomics as potential diagnostic biomarker for epithelium ovarian cancer

The aim of this study was to use a two steps strategy metabolomics to screen/identify and validate novel metabolic biomarker(s) for epithelial ovarian cancer (EOC). In the screening step, serum samples from 27 healthy women, 28 benign ovarian tumors, and 29 EOCs were analyzed by using LC-MS based nontargeted metabolomics. The three groups were separated with OSC filtered PLS-DA model, and six metabolites (27-nor-5β-cholestane-3,7,12,24,25 pentol glucuronide (CPG), phenylalanine, glycocholic acid, propionylcarnitine, Phe-Phe and Lyso PC (18:2)) were considered as potential biomarker candidates. In the validation step, the six metabolites were analyzed in targeted metabolomics by LC-selective ion monitoring mass spectrometry in another 685 serum samples with various clinical backgrounds. As a result, CPG was evaluated to be a potential biomarker and its content was elevated in EOC tissues compared with benign ovarian tumor tissues (p = 0.0005). Besides, CPG levels were found to be up-regulated in early stage EOC and in the three types of EOC histological types. Other variables such as nonovarian diseases, medicine consumption, gynecological inflammations, and menopausal state did not interfere in using CPG as diagnosis marker. CPG was found to be complementary to CA125. Our findings suggest that CPG can be considered a statistical relevant biomarker of EOC, ready for early stage detection.     

Alpha repeat proteins (αRep) as expression and crystallization helpers

We have previously described a highly diverse library of artificial repeat proteins based on thermostable HEAT-like repeats, named αRep. αReps binding specifically to proteins difficult to crystallize have been selected and in several examples, they made possible the crystallization of these proteins. To further simplify the production and crystallization experiments we have explored the production of chimeric proteins corresponding to covalent association between the targets and their specific binders strengthened by a linker. Although chimeric proteins with expression partners are classically used to enhance expression, these fusions cannot usually be used for crystallization. With specific expression partners like a cognate αRep this is no longer true, and chimeric proteins can be expressed purified and crystallized. αRep selection by phage display suppose that at least a small amount of the target protein should be produced to be used as a bait for selection and this might, in some cases, be difficult. We have therefore transferred the αRep library in a new construction adapted to selection by protein complementation assay (PCA). This new procedure allows to select specific binders by direct interaction with the target in the cytoplasm of the bacteria and consequently does not require preliminary purification of target protein. αRep binders selected by PCA or by phage display can be used to enhance expression, stability, solubility and crystallogenesis of proteins that are otherwise difficult to express, purify and/or crystallize.     

Mitochondrial DNA of ancient Cumanians: culturally Asian steppe nomadic immigrants with substantially more western Eurasian mitochondrial DNA lineages

The Cumanians were originally Asian pastoral nomads who in the 13th century migrated to Hungary. We have examined mitochondrial DNA from members of the earliest Cumanian population in Hungary from two archeologically well-documented excavations and from 74 modern Hungarians from different rural locations in Hungary. Haplogroups were defined based on HVS I sequences and examinations of haplogroup-associated polymorphic sites of the protein coding region and of HVS II. To exclude contamination, some ancient DNA samples were cloned. A database was created from previously published mtDNA HVS I sequences (representing 2,615 individuals from different Asian and European populations) and 74 modem Hungarian sequences from the present study. This database was used to determine the relationships between the ancient Cumanians, modern Hungarians, and Eurasian populations and to estimate the genetic distances between these populations. We attempted to deduce the genetic trace of the migration of Cumanians. This study is the first ancient DNA characterization of an eastern pastoral nomad population that migrated into Europe. The results indicate that, while still possessing a Central Asian steppe culture, the Cumanians received a large admixture of maternal genes from more westerly populations before arriving in Hungary. A similar dilution of genetic, but not cultural, factors may have accompanied the settlement of other Asian nomads in Europe.     

Effect of Peroxisome Proliferator-Activated Receptor-γ Coactivator-1 Alpha Variants on Spontaneous Clearance and Fibrosis Progression during Hepatitis C Virus Infection in Moroccan Patients

Virologica Sinica - Hepatitis C virus (HCV) is still one of the main causes of liver disease worldwide. Metabolic disorders, including non-alcoholic fatty liver disease (NAFLD), induced by HCV have...     

Patient and observer reported outcome measures to evaluate health-related quality of life in inherited metabolic diseases: a scoping review

Background

Health-related Quality of Life (HrQoL) is a multidimensional measure, which has gained clinical and social relevance. Implementation of a patient-centred approach to both clinical research and care settings, has increased the recognition of patient and/or observer reported outcome measures (PROMs or ObsROMs) as informative and reliable tools for HrQoL assessment. Inherited Metabolic Diseases (IMDs) are a group of heterogeneous conditions with phenotypes ranging from mild to severe and mostly lacking effective therapies. Consequently, HrQoL evaluation is particularly relevant.

Objectives

We aimed to: (1) identify patient and/or caregiver-reported HrQoL instruments used among IMDs; (2) identify the main results of the application of each HrQoL tool and (3) evaluate the main limitations of HrQoL instruments and study design/methodology in IMDs.

Methods

A scoping review was conducted using methods outlined by Arksey and O’Malley. Additionally, we critically analysed each article to identify the HrQoL study drawbacks.

Results

Of the 1954 studies identified, 131 addressed HrQoL of IMDs patients using PROMs and/or ObsROMs, both in observational or interventional studies. In total, we identified 32 HrQoL instruments destined to self- or proxy-completion; only 2% were disease-specific. Multiple tools (both generic and disease-specific) proved to be responsive to changes in HrQoL; the SF-36 and PedsQL questionnaires were the most frequently used in the adult and pediatric populations, respectively. Furthermore, proxy data often demonstrated to be a reliable approach complementing self-reported HrQoL scores. Nevertheless, numerous limitations were identified especially due to the rarity of these conditions.

Conclusions

HrQoL is still not frequently assessed in IMDs. However, our results show successful examples of the use of patient-reported HrQoL instruments in this field. The importance of HrQoL measurement for clinical research and therapy development, incites to further research in HrQoL PROMs’ and ObsROMs’ creation and validation in IMDs.

     

Structural and spectropotentiometric analysis of Blastochloris viridis heterodimer mutant reaction center

Heterodimer mutant reaction centers (RCs) of Blastochloris viridis were crystallized using microfluidic technology. In this mutant, a leucine residue replaced the histidine residue which had acted as a fifth ligand to the bacteriochlorophyll (BChl) of the primary electron donor dimer M site (HisM200). With the loss of the histidine-coordinated Mg, one bacteriochlorophyll of the special pair was converted into a bacteriopheophytin (BPhe), and the primary donor became a heterodimer supermolecule. The crystals had dimensions 400 × 100 × 100 μm, belonged to space group P4₃2₁2, and were isomorphous to the ones reported earlier for the wild type (WT) strain. The structure was solved to a 2.5 Å resolution limit. Electron-density maps confirmed the replacement of the histidine residue and the absence of Mg. Structural changes in the heterodimer mutant RC relative to the WT included the absence of the water molecule that is typically positioned between the M side of the primary donor and the accessory BChl, a slight shift in the position of amino acids surrounding the site of the mutation, and the rotation of the M194 phenylalanine. The cytochrome subunit was anchored similarly as in the WT and had no detectable changes in its overall position. The highly conserved tyrosine L162, located between the primary donor and the highest potential heme C₃₈₀, revealed only a minor deviation of its hydroxyl group. Concomitantly to modification of the BChl molecule, the redox potential of the heterodimer primary donor increased relative to that of the WT organism (772 mV vs. 517 mV). The availability of this heterodimer mutant and its crystal structure provides opportunities for investigating changes in light-induced electron transfer that reflect differences in redox cascades.