Molecular patterns behind immunological and metabolic alterations in lysinuric protein intolerance

Lysinuric protein intolerance (LPI) is a recessively inherited disorder characterised by reduced plasma and increased urinary levels of cationic amino acids (CAAs), protein malnutrition, growth failure and hyperlipidemia. Some patients develop severe immunological, renal and pulmonary complications. All Finnish patients share the same LPIFin mutation in the SLC7A7 gene that encodes CAA transporter y+LAT1. The aim of this study was to examine molecular factors contributing to the various symptoms, systemic metabolic and lipid profiles, and innate immune responses in LPI. The transcriptomes, metabolomes and lipidomes were analysed in whole-blood cells and plasma using RNA microarrays and gas or liquid chromatography-mass spectrometry techniques, respectively. Toll-like receptor (TLR) signalling in monocyte-derived macrophages exposed to pathogens was scrutinised using qRT-PCR and the Luminex technology. Altered levels of transcripts participating in amino acid transport, immune responses, apoptosis and pathways of hepatic and renal metabolism were identified in the LPI whole-blood cells. The patients had increased non-essential amino acid, triacylglycerol and fatty acid levels, and decreased plasma levels of phosphatidylcholines and practically all essential amino acids. In addition, elevated plasma levels of eight metabolites, long-chain triacylglycerols, two chemoattractant chemokines and nitric oxide correlated with the reduced glomerular function in the patients with kidney disease. Accordingly, it can be hypothesised that the patients have increased autophagy, inflammation, oxidative stress and apoptosis, leading to hepatic steatosis, uremic toxicity and altered intestinal microbe metabolism. Furthermore, the LPI macrophages showed disruption in the TLR2/1, TLR4 and TLR9 pathways, suggesting innate immune dysfunctions with an excessive response to bacterial infections but a deficient viral DNA response.

L'école de la chasse aux chiens courants / par Le Verrier de la Conterie ; Illustré de huit estampes de Bidinger, de bandeaux d'après Routar et des 27 figures techniques des éditions anciennes

Collection : Les maîtres de la vénerie ; ̊ 5

Flore complète de la France et de la Suisse (comprenant aussi toutes les espèces de Belgique) pour trouver facilement les noms des plantes sans mots techniques : 5338 figures représentant les caractères de toutes les espèces... (5e éd. revue et corrigée) / par Gaston Bonnier,... et G. de Layens,...

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Development of techniques for testing the effect of the E1 region on adenovirus integration /

Adenoviral vectors are currently the most widely used gene therapeutic vectors, but their inability to integrate into host chromosomal DNA shortened their transgene expression and limited their use in clinical trials. In this project, we initially planned to develop a technique to test the effect of the early region 1 (E1) on adenovirus integration by comparing the integration efficiencies between an E1-deleted adenoviral vector (SubE1) and an Elcontaining vector (SubE3). However, we did not harvest any SubE3 virus, even if we repeated the transfection and successfully rescued the SubE1 virus (2/4 transfections generated viruses) and positive control virus (6/6). The failure of rescuing SubE3 could be caused by the instability of the genomic plasmid pFG173, as it had frequent intemal deletions when we were purifying It. Therefore, we developed techniques to test the effect of E1 on homologous recombination (HR) since literature suggested that adenovirus integration is initiated by HR. We attempted to silence the E1 in 293 cells by transfecting E1A/B-specific small interfering RNA (siRNA). However, no silenced phenotype was observed, even if we varied the concentrations of E1A/B siRNA (from 30 nM to 270 nM) and checked the silencing effects at different time points (48, 72, 96 h). One possible explanation would be that the E1A/B siRNA sequences are not potent enough to Induce the silenced phenotype. For evaluating HR efficiencies, an HR assay system based on bacterial transfonmatJon was designed. We constmcted two plasmids ( designated as pUC19-dl1 and pUC19-dl2) containing different defective lacZa cassettes (forming white colonies after transformation) that can generate a functional lacZa cassette (forming blue colonies) through HR after transfecting into 293 cells. The HR efficiencies would be expressed as the percentages of the blue colonies among all the colonies. Unfortunately, after transfonnation of plasmid isolated from 293 cells, no colony was found, even at a transformation efficiency of 1.8x10^ colonies/pg pUC19, suggesting the sensitivity of this system was low. To enhance the sensitivity, PCR was used. We designed a set of primers that can only amplify the recombinant plasmid fomied through HR. Therefore, the HR efficiencies among different treatments can be evaluated by the amplification results, and this system could be used to test the effect of E1 region on adenovirus integration. In addition, to our knowledge there was no previous studies using PCR/ Realtime PCR to evaluate HR efficiency, so this system also provides a PCR-based method to carry out the HR assays.

Arsenic speciation by ion-exchange chromatography with on- line determination by hydride generation: electronic modification of the D.C. plasma by photon counting

A method using L-cysteine for the determination of arsenous acid (As(III)), arsenic acid (As(V)), monomethylarsonic acid (MMAA), and dimethylarsinic acid (DMAA) by hydride generation was demonstrated. The instrument used was a d.c. plasma atomic emission spectrometer (OCP-AES). Complete recovery was reported for As(III), As(V), and DMAA while 86% recovery was reported for MMAA. Detection limits were determined, as arsenic for the species listed previously, to be 1.2, 0.8, 1.1, and 1.0 ngemL-l, respectively. Precision values, at 50 ngemL-1 arsenic concentration, were f.80/0, 2.50/0, 2.6% and 2.6% relative standard deviation, respectively. The L-cysteine reagent was compared directly with the conventional hydride generation technique which uses a potassium iodide-hydrochloric acid medium. Recoveries using L-cysteine when compared with the conventional method provided the following results: similar recoveries were obtained for As(III), slightly better recoveries were obtained for As(V) and MMAA, and significantly better recoveries for DMAA. In addition, tall and sharp peak shapes were observed for all four species when using L-cysteine. The arsenic speciation method involved separation by ion exchange .. high perfonnance liquid chromatography (HPLC) with on-line hydride generation using the L.. cysteine reagent and measurement byOCP-AES. Total analysis time per sample was 12 min while the time between the start of subsequent runs was approximately 20 min. A binary . gradient elution program, which incorporated the following two eluents: 0.01 and 0.5 mM tri.. sodium citrate both containing 5% methanol (v/v) and both at a pH of approximately 9, was used during the separation by HPLC. Recoveries of the four species which were measured as peak area, and were normalized against As(III), were 880/0, 290/0, and 40% for DMAA, MMAA and As(V), respectively. Resolution factors between adjacent analyte peaks of As(III) and DMAA was 1.1; DMAA and MMAA was 1.3; and MMAA and As(V) was 8.6. During the arsenic speciation study, signals from the d.c. plasma optical system were measured using a new photon-signal integrating device. The_new photon integrator developed and built in this laboratory was based on a previously published design which was further modified to reflect current available hardware. This photon integrator was interfaced to a personal computer through an AID convertor. The .photon integrator has adjustable threshold settings and an adjustable post-gain device.

Investigation of the composition of linear alkylbenzenes with emphasis on the identification and quantitation of some trace compounds using GS/MS system in both electron impact and chemical ionization modes

Linear alkylbenzenes, LAB, formed by the Alel3 or HF catalyzed alkylation of benzene are common raw materials for surfactant manufacture. Normally they are sulphonated using S03 or oleum to give the corresponding linear alkylbenzene sulphonates In >95 % yield. As concern has grown about the environmental impact of surfactants,' questions have been raised about the trace levels of unreacted raw materials, linear alkylbenzenes and minor impurities present in them. With the advent of modem analytical instruments and techniques, namely GCIMS, the opportunity has arisen to identify the exact nature of these impurities and to determine the actual levels of them present in the commercial linear ,alkylbenzenes. The object of the proposed study was to separate, identify and quantify major and minor components (1-10%) in commercial linear alkylbenzenes. The focus of this study was on the structure elucidation and determination of impurities and on the qualitative determination of them in all analyzed linear alkylbenzene samples. A gas chromatography/mass spectrometry, (GCIMS) study was performed o~ five samples from the same manufacturer (different production dates) and then it was followed by the analyses of ten commercial linear alkylbenzenes from four different suppliers. All the major components, namely linear alkylbenzene isomers, followed the same elution pattern with the 2-phenyl isomer eluting last. The individual isomers were identified by interpretation of their electron impact and chemical ionization mass spectra. The percent isomer distribution was found to be different from sample to sample. Average molecular weights were calculated using two methods, GC and GCIMS, and compared with the results reported on the Certificate of Analyses (C.O.A.) provided by the manufacturers of commercial linear alkylbenzenes. The GC results in most cases agreed with the reported values, whereas GC/MS results were significantly lower, between 0.41 and 3.29 amu. The minor components, impurities such as branched alkylbenzenes and dialkyltetralins eluted according to their molecular weights. Their fragmentation patterns were studied using electron impact ionization mode and their molecular weight ions confirmed by a 'soft ionization technique', chemical ionization. The level of impurities present i~ the analyzed commercial linear alkylbenzenes was expressed as the percent of the total sample weight, as well as, in mg/g. The percent of impurities was observed to vary between 4.5 % and 16.8 % with the highest being in sample "I". Quantitation (mg/g) of impurities such as branched alkylbenzenes and dialkyltetralins was done using cis/trans-l,4,6,7-tetramethyltetralin as an internal standard. Samples were analyzed using .GC/MS system operating under full scan and single ion monitoring data acquisition modes. The latter data acquisition mode, which offers higher sensitivity, was used to analyze all samples under investigation for presence of linear dialkyltetralins. Dialkyltetralins were reported quantitatively, whereas branched alkylbenzenes were reported semi-qualitatively. The GC/MS method that was developed during the course of this study allowed identification of some other trace impurities present in commercial LABs. Compounds such as non-linear dialkyltetralins, dialkylindanes, diphenylalkanes and alkylnaphthalenes were identified but their detailed structure elucidation and the quantitation was beyond the scope of this study. However, further investigation of these compounds will be the subject of a future study.

Investigations into the extraction and the determination of polycyclic aromatic hydrocarbons (PAHs) from water by solid-phase extraction and capillary gas chromatography/ mass spectrometry

Factors affecting the detennination of PAHs by capillary GC/MS were studied. The effect of the initial column temperature and the injection solvent on the peak areas and heights of sixteen PAHs, considered as priority pollutants, USillg crosslinked methyl silicone (DB!) and 5% diphenyl, 94% dimethyl, 1% vinyl polysiloxane (DBS) columns was examined. The possibility of using high boiling point alcohols especially butanol, pentanol, cyclopentanol, and hexanol as injection solvents was investigated. Studies were carried out to optimize the initial column temperature for each of the alcohols. It was found that the optimum initial column temperature is dependent on the solvent employed. The peak areas and heights of the PAHs are enhanced when the initial column temperature is 10-20 c above the boiling point of the solvent using DB5 column, and the same or 10 C above the boiling point of the solvent using DB1 column. Comparing the peak signals of the PAHs using the alcohols, p-xylene, n-octane, and nonane as injection solvents, hexanol gave the greatest peak areas and heights of the PAHs particularly the late-eluted peaks. The detection limits were at low pg levels, ranging from 6.0 pg for fluorene t9 83.6 pg for benzo(a)pyrene. The effect of the initial column temperature on the peak shape and the separation efficiency of the PARs was also studied using DB1 and DB5 columns. Fronting or splitting of the peaks was obseIVed at very low initial column temperature. When high initial column temperature was used, tailing of the peaks appeared. Great difference between DB! and.DB5 columns in the range of the initial column temperature in which symmetrical.peaks of PAHs can be obtained is observed. Wider ranges were shown using DB5 column. Resolution of the closely-eluted PAHs was also affected by the initial column temperature depending on the stationary phase employed. In the case of DB5, only the earlyeluted PAHs were affected; whereas, with DB1, all PAHs were affected. An analytical procedure utilizing solid phase extraction with bonded phase silica (C8) cartridges combined with GC/MS was developed to analyze PAHs in water as an alternative method to those based on the extraction with organic solvent. This simple procedure involved passing a 50 ml of spiked water sample through C8 bonded phase silica cartridges at 10 ml/min, dried by passing a gentle flow of nitrogen at 20 ml/min for 30 sec, and eluting the trapped PAHs with 500 Jll of p-xylene at 0.3 ml/min. The recoveries of PAHs were greater than 80%, with less than 10% relative standard deviations of nine determinations. No major contaminants were present that could interfere with the recognition of PAHs. It was also found that these bonded phase silica cartridges can be re-used for the extraction of PAHs from water.