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.     

The NADPH- and iron-dependent lipid peroxidation in human placental microsomes

In pregnant females, placenta is the most important source of lipid hydroperoxides and other reactive oxygen species (ROS). The increased production of lipid peroxides is often linked to preeclampsia. In our study, we revealed that NADPH- and iron-dependent lipid peroxidation in human placental microsomes (HPM) occurred. In the presence of Fe²⁺ ion, HPM produced small amounts of thiobarbituric acid-reactive substances (TBARS) – a final product of lipid peroxidation. NADPH caused a strong increase of iron stimulated TBARS formation. TBARS formation was inhibited by superoxide dismutase, butylated hydroxytoluene and α-tocopherol but not by mannitol or catalase. TBARS and superoxide radical production was inhibited in similar manner by cytochrome P450 inhibitors. The results obtained led us to the following conclusions: (1) microsomal lipid peroxidation next to mitochondrial lipid peroxidation may by an important source of lipid hydroperoxides in blood during pregnancy and (2) superoxide radical released by microsomal cytochrome P450 is an important factor in NADPH- and iron-dependent lipid peroxidation in HPM.     

Non-omnia moriantur—toxicity of mancozeb on dead wood microarthropod fauna

The effect of Dithane M-45 (dithiocarbamate fungicide; active substance: mancozeb) was studied on microarthropod fauna inhabiting dead wood. Although the exposure was almost never 100% lethal for the majority of observed taxa, almost all (Mesostigmata, Oribatida, some Uropodina, Actinedida, Collembola and Diplopoda) showed very high correlation between concentration of the fungicide and mortality (r > 0.86). Only Stigmaeidae showed low correlation (r = 0.293). For the majority of taxa LC₅₀ values were close to the concentrations used during agrochemical activities in woods. Only Trachytes aegrota showed full susceptibility to the fungicide within the range of recommended field concentrations used in forestry (characterised by the low LC₉₅ value). Tolerance of mesostigmatid and oribatid mites was found to differ between juveniles and adults, but not consistently. Related Uropodina species varied in susceptibility to the fungicide.