Quantitative analysis of LacCer/CDw17 in human myelogenous leukaemic cells

LacCer/CDw17 is the most abundant GSL in neutrophils. The cell-surface and intracellular presence of LacCer was determined quantitatively using anti-CDw17 mAbs in a flow cytometry assay. The quantified alterations in the level of CDw17 antigen expression are consistent with alterations in LacCer content, determined chemically. Our results show that CDw17 antigen expression defines successive stages in the maturation of the myeloid cell. The assessment of cell-surface and intracellular CDw17 expression may be useful in evaluating neutrophil physiological status.     

Protein homeostasis,regulation of energy production and activation of DNA damage-repair pathways are involved in the heat stress response of Pseudogymnoascus spp.

Proteome changes can be used as an instrument to measure the effects of climate change, predict the possible future state of an ecosystem and the direction in which is headed. In this study, proteomic and gene ontology functional enrichment analysis of six Pseudogymnoascus spp. isolated from various global biogeographical regions were carried out to determine their response to heat stress. In total, 2122 proteins were identified with high confidence. Comparative quantitative analysis showed that changes in proteome profiles varied greatly between isolates from different biogeographical regions. Although the identities of the proteins that changed varied between the different regions, the functions they governed were similar. Gene ontology analysis showed enrichment of proteins involved in multiple protective mechanisms, including the modulation of protein homeostasis, regulation of energy production and activation of DNA damage and repair pathways. Our proteomic analysis did not show any clear relationship between protein changes and the strains' biogeographical origins.     

A new dicrocoeliid from the bank vole Clethrionomys glareolus (Rodentia: Microtidae) from Poland

A new dicrocoeliid trematode, Brachylecithum glareoli n. sp., is described from the biliary ducts of the bank vole, Clethrionomys glareolus, in southwest Poland. This is the first dicrocoeliid species described in rodents from Poland. It is characterized mainly by the maximum body width at the level of the vitellaria; large, longitudinally oval testes; round, or transversely oval, ovary that is smaller than the testes; vitellaria located in the midbody; cirrus sac dorsally overlapping ventral sucker, but never reaching beyond half of its length; and large, distinctly elongated eggs.     

Single nucleotide polymorphism detection by polymerase chain reaction-restriction fragment length polymorphism

Accurate analysis of DNA sequence variation in not only humans and animals but also other organisms has played a significant role in expanding our knowledge about genetic variety and diversity in a number of different biological areas. The search for an understanding of the causes of genetic variants and mutations has resulted in the development of a simple laboratory technique, known as the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, for the detection of single nucleotide polymorphisms (SNPs). PCR-RFLP allows rapid detection of point mutations after the genomic sequences are amplified by PCR. The mutation is discriminated by digestion with specific restriction endonucleases and is identified by gel electrophoresis after staining with ethidium bromide (EtBr). This convenient and simple method is inexpensive and accurate for SNP genotyping and especially useful in small basic research studies of complex genetic diseases. The whole protocol takes only a day to carry out.     

Cost-efficient multiplex PCR for routine genotyping of up to nine classical HLA loci in a single analytical run of multiple samples by next generation sequencing

Background

HLA genotyping by next generation sequencing (NGS) requires three basic steps, PCR, NGS, and allele assignment. Compared to the conventional methods, such as PCR-sequence specific oligonucleotide primers (SSOP) and -sequence based typing (SBT), PCR-NGS is extremely labor intensive and time consuming. In order to simplify and accelerate the NGS-based HLA genotyping method for multiple DNA samples, we developed and evaluated four multiplex PCR methods for genotyping up to nine classical HLA loci including HLA-A, HLA-B, HLA-C, HLA-DRB1/3/4/5, HLA-DQB1, and HLA-DPB1.

Results

We developed multiplex PCR methods using newly and previously designed middle ranged PCR primer sets for genotyping different combinations of HLA loci, (1) HLA-DRB1/3/4/5, (2) HLA-DQB1 (3.8 kb to 5.3 kb), (3) HLA-A, HLA-B, HLA-C, and (4) HLA-DPB1 (4.6 kb to 7.2 kb). The primer sets were designed to genotype polymorphic exons to the field 3 level or 6-digit typing. When we evaluated the PCR method for genotyping all nine HLA loci (9LOCI) using 46 Japanese reference subjects who represented a distribution of more than 99.5% of the HLA alleles at each of the nine HLA loci, all of the 276 alleles genotyped, except for HLA-DRB3/4/5 alleles, were consistent with known alleles assigned by the conventional methods together with relevant locus balance and no excessive allelic imbalance. One multiplex PCR method (9LOCI) was able to provide precise genotyping data even when only 1 ng of genomic DNA was used for the PCR as a sample template.

Conclusions

In this study, we have demonstrated that the multiplex PCR approach for NGS-based HLA genotyping could serve as an alternative routine HLA genotyping method, possibly replacing the conventional methods by providing an accelerated yet robust amplification step. The method also could provide significant merits for clinical applications with its ability to amplify lower quantity of samples and the cost-saving factors.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1514-4) contains supplementary material, which is available to authorized users.     

Vitreous humour as a potential DNA source for postmortem human identification

PURPOSE: The aim of this study was the assessment of vitreous humor as a potential DNA for forensic human postmortem identification. MATERIAL AND METHODS: Vitreous humor samples were collected using two alternative approaches from 25 corpses of either sex during autopsies. DNA was extracted by standard organic method. Recovered DNA was quantitiated fluorometrically. AmpFlSTR SGM Plus kit and ABI 310 Genetic Analyzer (Applera) were used to obtain genetic profiles. RESULTS: Different DNA yields were quantitated in vitreous body depending on cause of death and sampling approach. CONCLUSION: Vitreous humor is a potential DNA for forensic human postmortem identification depending on a sampling method used.