Increased urinary excretion of free N-acetylneuraminic acid in thirteen patients with Salla disease

Thirteen severely retarded patients with Salla disease, a new type of lysosomal storage disorder, have been studied biochemically. All patients excreted approximately ten times more free sialic acid than normal individuals. The isolated sialic acid was characterized by paper chromatography, thin-layer chromatography, optical rotation, 13C and 1H nuclear magnetic resonance spectroscopy, and mass spectrometry of its permethylated derivative. The results clearly indicated that the excreted sialic acid was identical to N-acetylneuraminic acid. The main sialylated trisaccharide present in the urine of the patients was identified as 3'-sialyllactose by sugar and methylation analysis. The excreted amounts were found to be within normal range.     

Assessment of Blood Contamination in Biological Fluids Using MALDI-TOF MS

Biological fluid sample collection often includes the risk of blood contamination that may alter the proteomic profile of biological fluid. In proteomics studies, exclusion of contaminated samples is usually based on visual inspection and counting of red blood cells in the sample; analysis of specific blood derived proteins is less used. To fill the gap, we developed a fast and sensitive method for ascertainment of blood contamination in crude biological fluids, based on specific blood-derived protein, hemoglobin detection by MALDI-TOF MS. The MALDI-TOF MS based method allows detection of trace hemoglobin with the detection limit of 0.12 nM. UV-spectrometry, which was used as reference method, was found to be less sensitive. The main advantages of the presented method are that it is fast, effective, sensitive, requires very small sample amount and can be applied for detection of blood contamination in various biological fluids collected for proteomics studies. Method applicability was tested on human cerebrospinal and follicular fluid, which proteomes generally do not contain hemoglobin, however, which possess high risk for blood contamination. Present method successfully detected the blood contamination in 12 % of cerebrospinal fluid and 24 % of follicular fluid samples. High percentage of contaminated samples accentuates the need for initial inspection of proteomic samples to avoid incorrect results from blood proteome overlap.     

Characterization of a New Chronic Lymphocytic Leukemia Cell Line for Mechanistic In Vitro and In Vivo Studies Relevant to Disease

Studies of chronic lymphocytic leukemia (CLL) have yielded substantial progress, however a lack of immortalized cell lines representative of the primary disease has hampered a full understanding of disease pathogenesis and development of new treatments. Here we describe a novel CLL cell line (OSU-CLL) generated by EBV transformation, which displays a similar cytogenetic and immunophenotype observed in the patient’s CLL (CD5 positive with trisomy 12 and 19). A companion cell line was also generated from the same patient (OSU-NB). This cell line lacked typical CLL characteristics, and is likely derived from the patient’s normal B cells. In vitro migration assays demonstrated that OSU-CLL exhibits migratory properties similar to primary CLL cells whereas OSU-NB has significantly reduced ability to migrate spontaneously or towards chemokine. Microarray analysis demonstrated distinct gene expression patterns in the two cell lines, including genes on chromosomes 12 and 19, which is consistent with the cytogenetic profile in this cell line. Finally, OSU-CLL was readily transplantable into NOG mice, producing uniform engraftment by three weeks with leukemic cells detectable in the peripheral blood spleen and bone marrow. These studies describe a new CLL cell line that extends currently available models to study gene function in this disease.     

Complex Patterns of Chromosome 11 Aberrations in Myeloid Malignancies Target CBL,MLL, DDB1 and LMO2

Exome sequencing of primary tumors identifies complex somatic mutation patterns. Assignment of relevance of individual somatic mutations is difficult and poses the next challenge for interpretation of next generation sequencing data. Here we present an approach how exome sequencing in combination with SNP microarray data may identify targets of chromosomal aberrations in myeloid malignancies. The rationale of this approach is that hotspots of chromosomal aberrations might also harbor point mutations in the target genes of deletions, gains or uniparental disomies (UPDs). Chromosome 11 is a frequent target of lesions in myeloid malignancies. Therefore, we studied chromosome 11 in a total of 813 samples from 773 individual patients with different myeloid malignancies by SNP microarrays and complemented the data with exome sequencing in selected cases exhibiting chromosome 11 defects. We found gains, losses and UPDs of chromosome 11 in 52 of the 813 samples (6.4%). Chromosome 11q UPDs frequently associated with mutations of CBL. In one patient the 11qUPD amplified somatic mutations in both CBL and the DNA repair gene DDB1. A duplication within MLL exon 3 was detected in another patient with 11qUPD. We identified several common deleted regions (CDR) on chromosome 11. One of the CDRs associated with de novo acute myeloid leukemia (P=0.013). One patient with a deletion at the LMO2 locus harbored an additional point mutation on the other allele indicating that LMO2 might be a tumor suppressor frequently targeted by 11p deletions. Our chromosome-centered analysis indicates that chromosome 11 contains a number of tumor suppressor genes and that the role of this chromosome in myeloid malignancies is more complex than previously recognized.     

Neighboring Deschampsia flexuosa and Trientalis europaea harbor contrasting root fungal endophytic communities

Fungal endophytic communities and potential host preference of root-inhabiting fungi of boreal forest understory plants are poorly known. The objective of this study was to find out whether two neighboring plant species, Deschampsia flexuosa (Poaceae) and Trientalis europaea (Primulaceae), share similar root fungal endophytic communities and whether the communities differ between two sites. The study was carried out by analysis of pure culture isolates and root fungal colonization percentages. A total of 84 isolates from D. flexuosa and 27 isolates from T. europaea were obtained. The roots of D. flexuosa harbored 16 different isolate types based on macromorphological characteristics, whereas only 4 isolate types were found in T. europaea. The root colonization by dark septate and hyaline septate hyphae correlated with isolate numbers being higher in D. flexuosa compared to T. europaea. The different isolate types were further identified on the basis of internal transcribed spacer sequence and phylogenetic analysis. An isolate type identified as dark septate endophyte Phialocephala fortinii colonized 50 % of the T. europaea and 21 % of the D. flexuosa specimens. In addition, Meliniomyces variabilis, Phialocephala sphaeroides, and Umbelopsis isabellina were found colonizing the grass, D. flexuosa, for the first time and Mycena sp. was confirmed as an endophyte of D. flexuosa. Site-specific differences were observed in the abundance and diversity of endophytic fungi in the roots of both study plants, but the differences were not as predominant as those between plant species. It is concluded that D. flexuosa harbors both higher amount and more diverse community of endophytic fungi in its roots compared to T. europaea.     

The first detection of influenza in the Finnish pig population: a retrospective study

Background

Swine influenza is an infectious acute respiratory disease of pigs caused by influenza A virus. We investigated the time of entry of swine influenza into the Finnish pig population. We also describe the molecular detection of two types of influenza A (H1N1) viruses in porcine samples submitted in 2009 and 2010.This retrospective study was based on three categories of samples: blood samples collected for disease monitoring from pigs at major slaughterhouses from 2007 to 2009; blood samples from pigs in farms with a special health status taken in 2008 and 2009; and diagnostic blood samples from pigs in farms with clinical signs of respiratory disease in 2008 and 2009.The blood samples were tested for influenza A antibodies with an antibody ELISA. Positive samples were further analyzed for H1N1, H3N2, and H1N2 antibodies with a hemagglutination inhibition test.Diagnostic samples for virus detection were subjected to influenza A M-gene-specific real-time RT-PCR and to pandemic influenza A H1N1-specific real-time RT-PCR. Positive samples were further analyzed with RT-PCRs designed for this purpose, and the PCR products were sequenced and sequences analyzed phylogenetically.

Results

In the blood samples from pigs in special health class farms producing replacement animals and in diagnostic blood samples, the first serologically positive samples originated from the period July–August 2008. In samples collected for disease monitoring, < 0.1%, 0% and 16% were positive for antibodies against influenza A H1N1 in the HI test in 2007, 2008, and 2009, respectively.Swine influenza A virus of avian-like H1N1 was first detected in diagnostic samples in February 2009. In 2009 and 2010, the avian-like H1N1 virus was detected on 12 and two farms, respectively. The pandemic H1N1 virus (A(H1N1)pdm09) was detected on one pig farm in 2009 and on two farms in 2010.

Conclusions

Based on our study, swine influenza of avian-like H1N1 virus was introduced into the Finnish pig population in 2008 and A(H1N1)pdm09 virus in 2009. The source of avian-like H1N1 infection could not be determined. Cases of pandemic H1N1 in pigs coincided with the period when the A(H1N1)pdm09 virus was spread in humans in Finland.