High Resolution Size Analysis of Fetal DNA in the Urine of Pregnant Women by Paired-End Massively Parallel Sequencing

Background

Fetal DNA in maternal urine, if present, would be a valuable source of fetal genetic material for noninvasive prenatal diagnosis. However, the existence of fetal DNA in maternal urine has remained controversial. The issue is due to the lack of appropriate technology to robustly detect the potentially highly degraded fetal DNA in maternal urine.

Methodology

We have used massively parallel paired-end sequencing to investigate cell-free DNA molecules in maternal urine. Catheterized urine samples were collected from seven pregnant women during the third trimester of pregnancies. We detected fetal DNA by identifying sequenced reads that contained fetal-specific alleles of the single nucleotide polymorphisms. The sizes of individual urinary DNA fragments were deduced from the alignment positions of the paired reads. We measured the fractional fetal DNA concentration as well as the size distributions of fetal and maternal DNA in maternal urine.

Principal Findings

Cell-free fetal DNA was detected in five of the seven maternal urine samples, with the fractional fetal DNA concentrations ranged from 1.92% to 4.73%. Fetal DNA became undetectable in maternal urine after delivery. The total urinary cell-free DNA molecules were less intact when compared with plasma DNA. Urinary fetal DNA fragments were very short, and the most dominant fetal sequences were between 29 bp and 45 bp in length.

Conclusions

With the use of massively parallel sequencing, we have confirmed the existence of transrenal fetal DNA in maternal urine, and have shown that urinary fetal DNA was heavily degraded.     

Bayesian Phylogeography of Crimean-Congo Hemorrhagic Fever Virus in Europe

Crimean-Congo hemorrhagic fever (CCHF) is a zoonosis mainly transmitted by ticks that causes severe hemorrhagic fever and has a mortality rate of 5-60%. The first outbreak of CCHF occurred in the Crimean peninsula in 1944-45 and it has recently emerged in the Balkans and eastern Mediterranean. In order to reconstruct the origin and pathway of the worldwide dispersion of the virus at global and regional (eastern European) level, we investigated the phylogeography of the infection by analysing 121 publicly available CCHFV S gene sequences including two recently characterised Albanian isolates. The spatial and temporal phylogeny was reconstructed using a Bayesian Markov chain Monte Carlo approach, which estimated a mean evolutionary rate of 2.96 x 10^-4 (95%HPD=1.6 and 4.7 x 10^-4) substitutions/site/year for the analysed fragment. All of the isolates segregated into seven highly significant clades that correspond to the known geographical clades: in particular the two new isolates from northern Albania clustered significantly within the Europe 1 clade. Our phylogeographical reconstruction suggests that the global CCHFV clades originated about one thousand years ago from a common ancestor probably located in Africa. The virus then spread to Asia in the XV century and entered Europe on at least two occasions: the first in the early 1800s, when a still circulating but less or non-pathogenic virus emerged in Greece and Turkey, and the second in the early 1900s, when a pathogenic CCHFV strain began to spread in eastern Europe. The most probable location for the origin of this European clade 1 was Russia, but Turkey played a central role in spreading the virus throughout Europe. Given the close proximity of the infected areas, our data suggest that the movement of wild and domestic ungulates from endemic areas was probably the main cause of the dissemination of the virus in eastern Europe.     

Pericentric inversion of chromosome 19 in three families

Summary Pericentric inversion of chromosome 19 has been found in several members of three unrelated families from a restricted geographical region. In one of the families, an additional pericentric inversion of chromosome 9 was observed. Reproductive problems, multiple abortions in two families and a neonatal death in the third, were present. A review of previously described cases is included, and the genetic risk connected with this type of rearrangement is also discussed.     

A polysaccharide isolated from Cordyceps sinensis,a traditional Chinese medicine,protects PC12 cells against hydrogen peroxide-induced injury

Cordyceps sinensis, a well-known traditional Chinese medicine, possesses activities in anti-tumour, anti-oxidation and stimulating the immune system; however, the identity of active component(s) is not determined. By using anti-oxidation activity-guided fractionation, a polysaccharide of molecular weight approximately 210 kDa was isolated from cultured Cordyceps mycelia by ion-exchange and sizing chromatography. The isolated polysaccharide, having strong anti-oxidation activity, contains glucose, mannose and galactose in a ratio of 1 : 0.6 : 0.75. The pre-treatment of isolated polysaccharide on the cultured rat pheochromocytoma PC12 cells shows strong protective effect against hydrogen peroxide (H(2)O(2))-induced insult. Treatment of the cells with the isolated polysaccharide at 100 microg/ml prior to H(2)O(2) exposure significantly elevated the survival of PC12 cells in culture by over 60%. In parallel, the H(2)O(2)-induced production of malondialdehyde in cultured cells was markedly reduced by the polysaccharide treatment. Moreover, the pre-treatment of the isolated polysaccharide significantly attenuated the changes of glutathione peroxidase and superoxide dismutase activities in H(2)O(2)-treated cells in a dose-dependent manner. This is the first report in identifying a polysaccharide from Cordyceps, which protects against the free radical-induced neuronal cell toxicity.     

The HopF family of Pseudomonas syringae type III secreted effectors

Pseudomonas syringae is a bacterial phytopathogen that utilizes the type III secretion system to inject effector proteins into plant host cells. Pseudomonas syringae can infect a wide range of plant hosts, including agronomically important crops such as tomatoes and beans. The ability of P. syringae to infect such numerous hosts is caused, in part, by the diversity of effectors employed by this phytopathogen. Over 60 different effector families exist in P. syringae; one such family is HopF, which contains over 100 distinct alleles. Despite this diversity, research has focused on only two members of this family: HopF1 from P. syringae pathovar phaseolicola 1449B and HopF2 from P. syringae pathovar tomato DC3000. In this study, we review the research on HopF family members, including their host targets and molecular mechanisms of immunity suppression, and their enzymatic function. We also provide a phylogenetic analysis of this expanding effector family which provides a basis for a proposed nomenclature to guide future research. The extensive genetic diversity that exists within the HopF family presents a great opportunity to study how functional diversification on an effector family contributes to host specialization.     

Identification of three novel missense PKU mutations among Chinese.

Three novel missense mutations have been identified in the phenylalanine hydroxylase (PAH) genes of Chinese individuals afflicted with various degrees of phenylketonuria (PKU). A T-to-C transition was observed in exon 5 of the gene, resulting in the substitution of Phe161 by Ser161. Two substitutions, G-to-T and T-to-G, were observed in exon 7, resulting in the substitution of Gly247 by Val247 and Leu255 by Val255, respectively. Expression analysis demonstrated that these mutant proteins produced between 0 and 15% of normal PAH enzyme activity. Population screening of a Chinese sample population indicates that these mutations are quite rare, together accounting for only about 4% of all PKU alleles among the Chinese. The P161S and G247V mutations were each present on a single PAH RFLP haplotype 4 chromosome in patients form Northern China, while the L255V mutation was present on chromosomes of both haplotypes 18 and 21 in patients from Southern China. These results suggest that the remaining 30% of uncharacterized PKU alleles in the Chinese population may bear a large number of relatively rare PAH mutations.     

Generation of avian-derived anti-B7-H4 antibodies exerts a blockade effect on the immunosuppressive response

For highly conserved mammalian protein, chicken is a suitable immune host to generate antibodies. Monoclonal antibodies have been successfully targeted with immunity checkpoint proteins as a means of cancer treatment; this treatment enhances tumor-specific immunity responses through immunoregulation. Studies have identified the importance of B7-H4 in immunoregulation and its use as a potential target for cancer treatment. High levels of B7-H4 expression are found in tumor tissues and are associated with adverse clinical and pathological characteristics. Using the phage display technique, this study isolated specific single-chain antibody fragments (scFvs) against B7-H4 from chickens. Our experiment proved that B7-H4 clearly induced the inhibition of T-cell activation. Therefore, use of anti-B7-H4 scFvs can effectively block the exhaustion of immunity cells and also stimulate and activate T-cells in peripheral blood mononuclear cells. Sequence analysis revealed that two isolated scFv S2 and S4 have the same VH complementarity-determining regions (CDRs) sequence. Molecule docking was employed to simulate the complex structures of scFv with B7-H4 to analyze the interaction. Our findings revealed that both scFvs employed CDR-H1 and CDR-H3 as main driving forces and had strong binding effects with the B7-H4. The affinity of scFv S2 was better because the CDR-L2 loop of the scFv S2 had three more hydrogen bond interactions with B7-H4. The results of this experiment suggest the usefulness of B7-H4 as a target for immunity checkpoints; the isolated B7-H4-specific chicken antibodies have the potential for use in future cancer immunotherapy applications.