Gene expression in nematode-infected plant roots

Summary A major pathogen of potato plants (Solanum tuberosum) is the potato cyst nematode (Globodera spp.), which induces localized redifferentiation of a limited number of host cells to form a specialized feeding-site termed the syncytium. A novel strategy utilizing the polymerase chain reaction (PCR) was employed to construct a cDNA library from dissected potato roots highly enriched in syncytial material. The library was differentially screened with cDNA probes derived from the infected root tissue from a compatible interaction and from healthy root tissue. Characterization of one gene identified by the library screen indicated an expression pattern that correlated with events in the immediate vicinity of the pathogen after syncytial establishment. The strategy for library construction and screening could be applicable to the study of gene expression in any plant-pathogen interaction in which the limited supply of cells at the interface of the two organisms precludes a more traditional approach.     

Fast muscle fibers are preferentially affected in Duchenne muscular dystrophy

We show that Duchenne muscular dystrophy (DMD) selectively affects a subset of skeletal muscle fibers specialized for fast contraction. Muscle fiber types were characterized immunohistochemically with monoclonal antibodies that distinguish isoforms of fetal and adult-fast or adult-slow myosin heavy chain present in the same fiber. Fetal myosin expression increased with patient age and was not due to arrested development but rather to de novo synthesis, which served as a sensitive indicator of muscle regeneration. A subset of fast fibers were the first to degenerate (type IIb). Extensive fast fiber regeneration occurred before slow fibers were affected. These results suggest that the DMD gene product has a specific function in a subpopulation of muscle fibers specialized to respond to the highest frequency of neuronal stimulation with maximal rates of contraction.     

FLT3 Mutations in Early T-Cell Precursor ALL Characterize a Stem Cell Like Leukemia and Imply the Clinical Use of Tyrosine Kinase Inhibitors

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subgroup of acute T-lymphoblastic leukemia (T-ALL) with a high rate of FLT3-mutations in adults. To unravel the underlying pathomechanisms and the clinical course we assessed molecular alterations and clinical characteristics in a large cohort of ETP-ALL (n = 68) in comparison to non-ETP T-ALL adult patients. Interestingly, we found a high rate of FLT3-mutations in ETP-ALL samples (n = 24, 35%). Furthermore, FLT3 mutated ETP-ALL was characterized by a specific immunophenotype (CD2+/CD5-/CD13+/CD33-), a distinct gene expression pattern (aberrant expression of IGFBP7, WT1, GATA3) and mutational status (absence of NOTCH1 mutations and a low frequency, 21%, of clonal TCR rearrangements). The observed low GATA3 expression and high WT1 expression in combination with lack of NOTCH1 mutations and a low rate of TCR rearrangements point to a leukemic transformation at the pluripotent prothymocyte stage in FLT3 mutated ETP-ALL. The clinical outcome in ETP-ALL patients was poor, but encouraging in those patients with allogeneic stem cell transplantation (3-year OS: 74%). To further explore the efficacy of targeted therapies, we demonstrate that T-ALL cell lines transfected with FLT3 expression constructs were particularly sensitive to tyrosine kinase inhibitors. In conclusion, FLT3 mutated ETP-ALL defines a molecular distinct stem cell like leukemic subtype. These data warrant clinical studies with the implementation of FLT3 inhibitors in addition to early allogeneic stem cell transplantation for this high risk subgroup.     

Inhibition of the herpes simplex virus type 1 DNA polymerase induces hyperphosphorylation of replication protein A and its accumulation at S-phase-specific sites of DNA damage during infection

The treatment of mammalian cells with genotoxic substances can trigger DNA damage responses that include the hyperphosphorylation of replication protein A (RPA), a protein that plays key roles in the recognition, signaling, and repair of damaged DNA. We have previously reported that in the presence of a viral polymerase inhibitor, herpes simplex virus type 1 (HSV-1) infection induces the hyperphosphorylation of RPA (D. E. Wilkinson and S. K. Weller, J. Virol. 78:4783-4796, 2004). We initiated the present study to further characterize this genotoxic response to HSV-1 infection. Here we report that infection in the presence of polymerase inhibitors triggers an S-phase-specific response to DNA damage, as demonstrated by induction of the hyperphosphorylation of RPA and its accumulation within viral foci specific to the S phase of the cell cycle. This DNA damage response occurred in the presence of viral polymerase inhibitors and required the HSV-1 polymerase holoenzyme as well as the viral single-stranded-DNA binding protein. Treatment with an inhibitor of the viral helicase-primase did not induce the hyperphosphorylation of RPA or its accumulation in infected cells. Taken together, these results suggest that the S-phase-specific DNA damage response to infection is dependent on the specific inhibition of the polymerase. Finally, RPA hyperphosphorylation was not induced during productive infection, indicating that active viral replication does not trigger this potentially detrimental stress response.     

Early Indicators of Fatal Leptospirosis during the 2010 Epidemic in Puerto Rico

Background

Leptospirosis is a potentially fatal bacterial zoonosis that is endemic throughout the tropics and may be misdiagnosed as dengue. Delayed hospital admission of leptospirosis patients is associated with increased mortality.

Methodology/Principal Findings

During a concurrent dengue/leptospirosis epidemic in Puerto Rico in 2010, suspected dengue patients that tested dengue-negative were tested for leptospirosis. Fatal and non-fatal hospitalized leptospirosis patients were matched 1:1–3 by age. Records from all medical visits were evaluated for factors associated with fatal outcome. Among 175 leptospirosis patients identified (4.7 per 100,000 residents), 26 (15%) were fatal. Most patients were older males and had illness onset during the rainy season. Fatal case patients first sought medical care earlier than non-fatal control patients (2.5 vs. 5 days post-illness onset [DPO], p < 0.01), but less frequently first sought care at a hospital (52.4% vs. 92.2%, p < 0.01). Although fatal cases were more often diagnosed with leptospirosis at first medical visit (43.9% vs. 9.6%, p = 0.01), they were admitted to the hospital no earlier than non-fatal controls (4.5 vs. 6 DPO, p = 0.31). Cases less often developed fever (p = 0.03), but more often developed jaundice, edema, leg pain, hemoptysis, and had a seizure (p ≤ 0.03). Multivariable analysis of laboratory values from first medical visit associated with fatal outcome included increased white blood cell (WBC) count with increased creatinine (p = 0.001), and decreased bicarbonate with either increased WBC count, increased creatinine, or decreased platelet count (p < 0.001).

Conclusions/Significance

Patients with fatal leptospirosis sought care earlier, but were not admitted for care any earlier than non-fatal patients. Combinations of routine laboratory values predictive of fatal outcome should be considered in admission decision-making for patients with suspected leptospirosis.     

The Use of Combining Ability Analysis to Identify Elite Parents for Artemisia annua F1 Hybrid Production

Artemisia annua is an important medicinal crop used for the production of the anti-malarial compound artemisinin. In order to assist in the production of affordable high quality artemisinin we have carried out an A. annua breeding programme aimed at improving artemisinin concentration and biomass. Here we report on a combining ability analysis of a diallel cross to identify robust parental lines for hybrid breeding. The parental lines were selected based on a range of phenotypic traits to encourage heterosis. The general combining ability (GCA) values for the diallel parental lines correlated to the positive alleles of quantitative trait loci (QTL) in the same parents indicating the presence of beneficial alleles that contribute to parental performance. Hybrids generated from crossing specific parental lines with good GCA were identified as having an increase in both artemisinin concentration and biomass when grown either in glasshouse or experimental field trials and compared to controls. This study demonstrates that combining ability as determined by a diallel cross can be used to identify elite parents for the production of improved A. annua hybrids. Furthermore, the selection of material for breeding using this approach was found to be consistent with our QTL-based molecular breeding approach.