The interferon-induced gene ISG12 is regulated by various cytokines as the gene 6-16 in human cell lines

The gene for ISG12 (originally designated p27) was isolated as an oestrogen-induced gene. The authors undertook a comprehensive study using quantitative RT-PCR, in which we delineate the regulation of ISG12 by seven different cytokines including interferons and poly(I). poly(C) in seven human cell lines of different origin. In all cell lines ISG12 is strongly induced by IFN-alpha and only slightly by IFNgamma. Poly(I).poly(C) induces ISG12 in a cell line-dependent manner, whereas none of the other cytokines tested elicited a response. Comparing the induction pattern of ISG12 to that of 6-16 a high degree of similarity was found. The induction levels varied, however, between cell lines.     

Fractionation, solid-phase immobilization and chemical degradation of long pectin oligogalacturonides. Initial steps towards sequencing of oligosaccharides

This work presents the optimized separation of pectin oligomers, their analysis by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), their subsequent immobilization to supports, and our initial steps towards solid-support assisted sequencing. The ambient pressure strong anion-exchange resin Source 15Q combined with ammonium formate buffer (AF) was used for the separation of unsaturated and saturated pectic oligogalacturonides (OGAs) derived from enzymatic digestion of pectin. Routinely, multi-milligram quantities of defined sizes OGAs with DPs from 5 to 19 were produced in excellent purity (>95%). Elution of OGAs followed by direct analysis of the peak fractions by MALDI-TOF MS. Purified OGAs (DP 5-7) were chemoselectively immobilized onto aminooxy-terminated polyethylene glycol polyacrylamide (PEGA) supports. Solid-phase anchoring took place at the reducing end of the oligosaccharide and resulted in the formation of an oxime linkage. The very high coupling yields confirmed the general suitability of aminooxy-PEGA resins for the immobilization of OGAs of different lengths. The OGA-functionalized PEGA supports were subsequently treated with aq TFA at 40 or 60 degrees C, and the chemical degradation products released from the support were analyzed by ESIMS. In all cases, the original OGA was degraded into smaller oligomers of various sizes down to the monomer. This work illustrates some of the basic principles underlying a strategy ultimately aimed at solid-support assisted sequencing of oligosaccharides.     

Towards on-site pathogen detection using antibody-based sensors

In this paper, the recent progress within biosensors for plant pathogen detection will be reviewed. Bio-recognition layers on sensors can be designed in various ways, however the most popular approach is to immobilise antibodies for specific capture of analytes. Focus will be put on antibody surface-immobilisation strategies as well as the use of antibodies in the widely used sensors, quartz crystal microbalance, surface plasmon resonance and cantilevers. We will describe the available data on antibody-based plant pathogen detection and furthermore use examples from detection of the pathogens Salmonella, Listeria monocytogenes, Streptococcus mutans, Bacillus cereus, Bacillus anthracis, Campylobacter and Escherichia coli. We will touch upon optimal assay design and further discuss the strengths and limitations of current sensor technologies for detection of viruses, bacteria and fungi.     

Optimization and clinical validation of a pathogen detection microarray

DNA microarrays used as 'genomic sensors' have great potential in clinical diagnostics. Biases inherent in random PCR-amplification, cross-hybridization effects, and inadequate microarray analysis, however, limit detection sensitivity and specificity. Here, we have studied the relationships between viral amplification efficiency, hybridization signal, and target-probe annealing specificity using a customized microarray platform. Novel features of this platform include the development of a robust algorithm that accurately predicts PCR bias during DNA amplification and can be used to improve PCR primer design, as well as a powerful statistical concept for inferring pathogen identity from probe recognition signatures. Compared to real-time PCR, the microarray platform identified pathogens with 94% accuracy (76% sensitivity and 100% specificity) in a panel of 36 patient specimens. Our findings show that microarrays can be used for the robust and accurate diagnosis of pathogens, and further substantiate the use of microarray technology in clinical diagnostics.     

FLCN,a novel autophagy component,interacts with GABARAP and is regulated by ULK1 phosphorylation

Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant condition caused by mutations in the FLCN gene and characterized by benign hair follicle tumors, pneumothorax, and renal cancer. Folliculin (FLCN), the protein product of the FLCN gene, is a poorly characterized tumor suppressor protein, currently linked to multiple cellular pathways. Autophagy maintains cellular homeostasis by removing damaged organelles and macromolecules. Although the autophagy kinase ULK1 drives autophagy, the underlying mechanisms are still being unraveled and few ULK1 substrates have been identified to date. Here, we identify that loss of FLCN moderately impairs basal autophagic flux, while re-expression of FLCN rescues autophagy. We reveal that the FLCN complex is regulated by ULK1 and elucidate 3 novel phosphorylation sites (Ser406, Ser537, and Ser542) within FLCN, which are induced by ULK1 overexpression. In addition, our findings demonstrate that FLCN interacts with a second integral component of the autophagy machinery, GABA(A) receptor-associated protein (GABARAP). The FLCN-GABARAP association is modulated by the presence of either folliculin-interacting protein (FNIP)-1 or FNIP2 and further regulated by ULK1. As observed by elevation of GABARAP, sequestome 1 (SQSTM1) and microtubule-associated protein 1 light chain 3 (MAP1LC3B) in chromophobe and clear cell tumors from a BHD patient, we found that autophagy is impaired in BHD-associated renal tumors. Consequently, this work reveals a novel facet of autophagy regulation by ULK1 and substantially contributes to our understanding of FLCN function by linking it directly to autophagy through GABARAP and ULK1.     

Simultaneous quantitative determination of the HIV protease inhibitors indinavir,amprenavir, ritonavir,lopinavir, saquinavir,nelfinavir and the nelfinavir active metabolite M8 in plasma by liquid chromatography

A simple HPLC method that quantitates all six currently available protease inhibitors and the nelfinavir active metabolite M8 in one assay is presented. A 500-microliter plasma sample was treated by liquid-liquid extraction with a mixture of heptane and ethyl acetate. After evaporation, the residue was redissolved in sodium dihydrogenphosphate and acetonitrile and washed twice with heptane. Chromatography was performed with an analytical C(18) column. Ultraviolet detection at 210 and 239 nm was used. The present method is associated with high accuracy and low imprecision in the concentration range of 25-5000 ng/ml of all six protease inhibitors and M8. This makes it suitable for monitoring purposes.     

Perceived wellbeing of patients one year post stroke in general practice - <Emphasis Type="Italic">recommendations for quality aftercare</Emphasis>

Background  

Annually, 41,000 people in the Netherlands have strokes. This has multiple physical and psychosocial consequences. Most patients return home after discharge from hospital. Quality aftercare by general practitioners is important to support patients at home. The purpose of this study is to examine the wellbeing of patients who returned home immediately after discharge from hospital, one year post stroke, in comparison with the general Dutch population of the same age and to determine factors that could influence wellbeing.     

Recombinant chymosin used for exact and complete removal of a prochymosin derived fusion tag releasing intact native target protein

Fusion tags add desirable properties to recombinant proteins, but they are not necessarily acceptable in the final products. Ideally, fusion tags should be removed releasing the intact native protein with no trace of the tag. Unique endoproteinases with the ability to cleave outside their own recognition sequence can potentially cleave at the boundary of any native protein. Chymosin was recently shown to cleave a pro‐chymosin derived fusion tag releasing native target proteins. In our hands, however, not all proteins are chymosin‐resistant under the acidic cleavage conditions (pH 4.5) used in this system. Here, we have modified the pro‐chymosin fusion tag and demonstrated that chymosin can remove this tag at more neutral pH (pH 6.2); conditions, that are less prone to compromise the integrity of target proteins. Chymosin was successfully used to produce intact native target protein both at the level of small and large‐scale preparations. Using short peptide substrates, we further examined the influence of P1′ amino acid (the N‐terminus of the native target protein) and found that chymosin accepts many different, although not all, amino acids. We conclude that chymosin has several appealing characteristics for the exact removal of fusion tags. It is readily available in highly purified recombinant versions approved by the FDA for preparation of food for human consumption. We suggest that one should consider extending the use of chymosin to the preparation of pharmaceutical proteins.