Development of a Whole Blood Paper-Based Device for Phenylalanine Detection in the Context of PKU Therapy Monitoring

Laboratory-based testing does not allow for the sufficiently rapid return of data to enable optimal therapeutic monitoring of patients with metabolic diseases such as phenylketonuria (PKU). The typical turn-around time of several days for current laboratory-based testing is too slow to be practically useful for effective monitoring or optimizing therapy. This report describes the development of a rapid, paper-based, point-of-care device for phenylalanine detection using a small volume (40 μL) of whole blood. The quantitative resolution and reproducibility of this device with instrumented readout are described, together with the potential use of this device for point-of-care monitoring by PKU patients.Keywords: whole-blood assay, phenylketonuria, phenylalanine detection, colorimetric readout, paper-based deviceKeywords: whole-blood assay, phenylketonuria, phenylalanine detection, colorimetric readout, paper-based devic

Okazaki Fragment Processing-independent Role for Human Dna2 Enzyme during DNA Replication

Dna2 is an essential helicase/nuclease that is postulated to cleave long DNA flaps that escape FEN1 activity during Okazaki fragment (OF) maturation in yeast. We previously demonstrated that the human Dna2 orthologue (hDna2) localizes to the nucleus and contributes to genomic stability. Here we investigated the role hDna2 plays in DNA replication. We show that Dna2 associates with the replisome protein And-1 in a cell cycle-dependent manner. Depletion of hDna2 resulted in S/G2 phase-specific DNA damage as evidenced by increased γ-H2AX, replication protein A foci, and Chk1 kinase phosphorylation, a readout for activation of the ATR-mediated S phase checkpoint. In addition, we observed reduced origin firing in hDna2-depleted cells consistent with Chk1 activation. We next examined the impact of hDna2 on OF maturation and replication fork progression in human cells. As expected, FEN1 depletion led to a significant reduction in OF maturation. Strikingly, the reduction in OF maturation had no impact on replication fork progression, indicating that fork movement is not tightly coupled to lagging strand maturation. Analysis of hDna2-depleted cells failed to reveal a defect in OF maturation or replication fork progression. Prior work in yeast demonstrated that ectopic expression of FEN1 rescues Dna2 defects. In contrast, we found that FEN1 expression in hDna2-depleted cells failed to rescue genomic instability. These findings suggest that the genomic instability observed in hDna2-depleted cells does not arise from defective OF maturation and that hDna2 plays a role in DNA replication that is distinct from FEN1 and OF maturation

Xpf and Not the Fanconi Anaemia Proteins or Rev3 Accounts for the Extreme Resistance to Cisplatin in Dictyostelium discoideum

Organisms like Dictyostelium discoideum, often referred to as DNA damage “extremophiles”, can survive exposure to extremely high doses of radiation and DNA crosslinking agents. These agents form highly toxic DNA crosslinks that cause extensive DNA damage. However, little is known about how Dictyostelium and the other “extremophiles” can tolerate and repair such large numbers of DNA crosslinks. Here we describe a comprehensive genetic analysis of crosslink repair in Dictyostelium discoideum. We analyse three gene groups that are crucial for a replication-coupled repair process that removes DNA crosslinks in higher eukarya: The Fanconi anaemia pathway (FA), translesion synthesis (TLS), and nucleotide excision repair. Gene disruption studies unexpectedly reveal that the FA genes and the TLS enzyme Rev3 play minor roles in tolerance to crosslinks in Dictyostelium. However, disruption of the Xpf nuclease subcomponent results in striking hypersensitivity to crosslinks. Genetic interaction studies reveal that although Xpf functions with FA and TLS gene products, most Xpf mediated repair is independent of these two gene groups. These results suggest that Dictyostelium utilises a distinct Xpf nuclease-mediated repair process to remove crosslinked DNA. Other DNA damage–resistant organisms and chemoresistant cancer cells might adopt a similar strategy to develop resistance to DNA crosslinking agents

Homologous Recombination Resolution Defect in Werner Syndrome

Werner syndrome (WRN) is an uncommon autosomal recessive disease whose phenotype includes features of premature aging, genetic instability, and an elevated risk of cancer. We used three different experimental strategies to show that WRN cellular phenotypes of limited cell division potential, DNA damage hypersensitivity, and defective homologous recombination (HR) are interrelated. WRN cell survival and the generation of viable mitotic recombinant progeny could be rescued by expressing wild-type WRN protein or by expressing the bacterial resolvase protein RusA. The dependence of WRN cellular phenotypes on RAD51-dependent HR pathways was demonstrated by using a dominant-negative RAD51 protein to suppress mitotic recombination in WRN and control cells: the suppression of RAD51-dependent recombination led to significantly improved survival of WRN cells following DNA damage. These results define a physiological role for the WRN RecQ helicase protein in RAD51-dependent HR and identify a mechanistic link between defective recombination resolution and limited cell division potential, DNA damage hypersensitivity, and genetic instability in human somatic cells

The Werner syndrome protein has separable recombination and survival functions

International audienceThe Werner syndrome (WS) protein WRN is unique in possessing a 3' to 5' exonuclease activity in addition to the 3' to 5' helicase activity characteristic of other RecQ proteins. In order to determine in vivo functions of the WRN catalytic activities and their roles in Werner syndrome pathogenesis, we quantified cell survival and homologous recombination after DNA damage in cells expressing WRN missense-mutant proteins that lacked exonuclease and/or helicase activity. Both WRN biochemical activities were required to generate viable recombinant daughter cells. In contrast, either activity was sufficient to promote cell survival after DNA damage in the absence of recombination. These results indicate that WRN has recombination and survival functions that can be separated by missense mutations. Two implications are that Werner syndrome most likely results from the loss of both activities and their associated functions from patient cells, and that $WRN$ missense mutations or polymorphisms could promote genetic instability and cancer in the general population by selectively interfering with recombination in somatic cells

Standardized proportionate incidence ratios (SPIRs) for malignancies in Japan-resident Werner syndrome patients.

*<p>statistically significant result (p<0.05).</p>**<p>includes WS patients with high WS diagnostic confidence (1965–2009, ages 10–69). Includes benign meningiomas diagnosed prior to 1988, but excludes non-melanoma skin neoplasms.</p>***<p>obtained using Osaka, Japan <i>CI5</i> volume case data (i.e., representative sample from 1970–2002).</p

Werner syndrome diagnostic confidence categories.

<p> <b><i>Diagnostic criteria and categorization notes:</i></b></p>*<p>WS diagnostic confidence categories were taken from the International Registry of Werner Syndrome: <a href="http://www.wernersyndrome.org/registry/diagnostic.html" target="_blank">www.wernersyndrome.org/registry/diagnostic.html</a> with the following modifications: 1. putative WS patients with known pathogenic mutations in both <i>WRN</i> alleles were also considered to be ‘Definite’/‘High confidence’; and 2. “mesenchymal neoplasms, rare neoplasms or multiple neoplasm” was not counted for any of the patients in the determination of Werner syndrome diagnostic confidence.</p>**<p>We had no case exclusions based on the onset of signs or symptoms prior to adolescence. Two Japan-resident cases were reported with voice changes prior to adolescence, and one Japan non-resident case was reported to have had premature greying of the hair at age 8 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059709#pone.0059709.s003" target="_blank">Table S1</a>).</p

Median ages and age ranges for cancer in Werner syndrome versus other cancer predisposition syndromes.

*<p>malignant cases only, except for meningiomas where benign cases were also included.</p>**<p>Japan-residents cases only, excluding tumor cases with ambiguous age at diagnosis.</p>***<p>BS = Bloom syndrome. Some discrepancies between data given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059709#pone-0059709-g002" target="_blank">Figure 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059709#pone-0059709-t004" target="_blank">Table 4</a> of reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059709#pone.0059709-German1" target="_blank">[23]</a>.</p>****<p>RTS = Rothmund-Thomson syndrome <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059709#pone.0059709-Siitonen1" target="_blank">[25]</a>. Note that adding 6 additional tumor cases reported in patients with RAPADILINO syndrome, which is also caused by mutations in <i>RecQL4</i>, has minimal effect (all sites estimate becomes 13 (2, 33)).</p>*****<p>Osaka population ages given by 5-year age groups, so medians are approximate. Data obtained from <i>CI5</i>plus online application (years 1963–2002); soft tissue and all sites data from <i>CI5</i> volumes 3–9 (years 1970–2002) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059709#pone.0059709-Ferlay1" target="_blank">[7]</a>. SEER data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059709#pone.0059709-Howlader1" target="_blank">[35]</a>.</p><p>na = not accessed.</p